张亚飞¹, 彭洁¹, 朱延松¹, 杨胜男¹, 王旭¹, 赵婉彤^1,2, 江东^,1,21 西南大学柑桔研究所,重庆 400712;
2 中国农业科学院柑桔研究所,重庆 400712

Genome Wide Identification of CCD Gene Family in Citrus and Effect of CcCCD4a on the Color of Citrus Flesh

ZHANG YaFei¹, PENG Jie¹, ZHU YanSong¹, YANG ShengNan¹, WANG Xu¹, ZHAO WanTong^1,2, JIANG Dong^,1,2 1 Citrus Research Institute, Southwest University, Chongqing 400712;
2 Citrus Research Institute of Chinese Academy of Agricultural Sciences, Chongqing 400712

通讯作者: 江东,E-mail：jiangdong@cric.cn

责任编辑: 赵伶俐
收稿日期:2019-01-10接受日期:2020-03-3网络出版日期:2020-05-16

基金资助:

国家重点研发计划.2018YFD1000101 教育部双一流学科建设项目.

Received:2019-01-10Accepted:2020-03-3Online:2020-05-16
作者简介 About authors
张亚飞,E-mail：1065120362@qq.com。









摘要
【目的】研究类胡萝卜素裂解双加氧酶（CCD）基因家族在柑橘基因组中的分布、结构及进化,对CcCCD4a在果肉颜色形成过程中的表达及其在不同果肉颜色的柑橘种质中的基因型进行研究,为开发用于果肉颜色的分子辅助育种标记奠定基础。【方法】根据已报道的CCD,采用同源比对法检索柑橘基因组中的CCD家族基因（CcCCD）。采用生物信息学软件构建系统进化树,进行亚细胞定位预测,预测蛋白质的相对分子质量与等电点（pI）等理化性质,预测保守motif,绘制家族基因Scaffold定位图。利用实时荧光定量PCR技术（qRT-PCR）分析CcCCD4a在柑橘果实颜色发育过程中的表达模式,利用测序技术鉴定30个柑橘品种的CcCCD4a基因型,采用Tassel软件进行单倍型分析。【结果】从克里曼丁橘（Citrus clementina）基因组中鉴定出14个CcCCD基因家族成员,可将其分为5个亚家族,即CcCCD1、CcCCD4、CcCCD7、CcCCD8和CcNCED。该家族蛋白理论等电点分布在6.05—8.53,编码氨基酸数目介于412—611个;亚细胞定位预测结果显示该基因家族成员主要位于叶绿体和细胞质中;聚类分析发现,CCD8亚家族与其他家族成员遗传距离较远,柑橘中各CCD均能在其他物种中找到同源基因;Scaffold定位分析发现,14个CCD家族成员成员分布在除5号Scafflod外的所有Scafflod上,且分布不均匀。对10个柑橘品种在4个时期的果肉色泽进行表型鉴定,随着果实趋于成熟,果肉的色调角（h）逐步下降,果肉颜色逐步加深;CcCCD4a在不同柑橘品种中相对表达量存在显著差异,果肉颜色为浓橙红色的品种CcCCD4a表达量显著低于果肉为橙色或浅橙黄色的品种(P<0.05),CcCCD4a相对表达量与色调角呈显著正相关(P<0.05);对30个柑橘品种进行测序分析,发现单倍型hap-1、hap-4和hap-5为果肉浓橙红色品种优势单倍型。【结论】‘克里曼丁’橘包含14个CCD基因家族成员,各成员均含有RPE65保守结构域,并定位于细胞的不同位置,分布在不同的Scaffold上。CcCCD4a参与柑橘果肉颜色的形成,其基因相对表达量与果肉色调角呈显著正相关,可作为潜在的柑橘果实颜色的辅助育种标记,尤其是单倍型hap-1、hap-4、hap-5与果肉红色的关联度较高,对颜色育种的早期杂种群体筛选有一定帮助。
关键词： 柑橘;CcCCD基因家族;CcCCD4a;果肉颜色;基因表达;单倍型

Abstract
【Objective】To reveal the distribution, structure and evolution of carotenoid cleavage dioxygenas gene family in the citrus genome (CcCCD), this study were performed to develope marker-assisted selection of flesh color in citrus breeding program, bioinformatics predication, expression analysis and genotype of CcCCD4a in flesh color development and different germplasm accessions. 【Method】The CCD gene family of Citrus clementina genome were identified by homologous search according to previously reported CCD in other plant species. Phylogenetic analysis, subcellular localization prediction, relative molecular weight, theoretical isoelectric point (PI), conserved motif prediction, and scaffold location were studied by bioinformatics methods. Real-time fluorescence quantitative PCR (qRT-PCR) was used to study the expression of CcCCD4a in 10 citrus accessions during the flesh color development period. Haplotype analysis was performed by Tassel software after sequencing of CcCCD4a in 30 citrus varieties. 【Result】Fourteen CCD family genes were found in the Citrus clementina genome, and these genes could be divided into five subfamily, namely, CcCCD1, CcCCD4, CcCCD7, CcCCD8 and CcNCED. Its theoretical isoelectric point were 6.05 to 8.53 and these CCD family genes encoded 412-611 amino acids. The subcellular localization prediction indicated that CcCCD genes mainly were located in chloroplast and cytoplasm. Phylogenetic analysis showed that CCD genes in citrus were also found in other plant species. Obviously, CCD8 subfamily had farther genetic distance with other CCD. Scaffold localization analysis showed that 14 CcCCD members were unevenly distributed in all scaffolds except scaffold 5. The phenotyping of flesh color in 10 citrus varieties demonstrated that the hue angle of flesh color was decreased along with fruit maturing. The relative expression of CcCCD4a in different citrus varieties was significantly different. The expression of CcCCD4a in the flesh color of orange red was significantly lower than that in the flesh color of orange or light orange yellow (P<0.05). There was a significant positive correlation between the relative expression of CcCCD4a and the hue angle during fruit ripening. Genotyping of CcCCD4a in 30 citrus varieties revealed that hap-1, hap-4 and hap-5 were dominant haplotype at orange red flesh varieties. 【Conclusion】The whole genome of Citrus clementina contained 14 members of CCD gene family. All these CCD gene family members contained RPE65 conserved domain, but they were located in different cell components and unevenly distributed at different scaffolds. CcCCD4a was involved in the development of citrus flesh color, and there was a significant positive correlation between its relative expression and the hue angle. Therefore, CcCCD4a could be used as potential marker for citrus fruit color breeding. Especially, hap-1, hap-4 and hap-5 had a high correlation with the phenotypes of orange red flesh, which might be helpful for selecting candidate hybrids in early stage of citrus breeding program.
Keywords：citrus;CCD gene family;CcCCD4a;flesh color;gene expression;haplotype


PDF (3881KB)元数据多维度评价相关文章导出EndNote|Ris|Bibtex收藏本文
本文引用格式
张亚飞, 彭洁, 朱延松, 杨胜男, 王旭, 赵婉彤, 江东. 柑橘CCD基因家族鉴定及CcCCD4a对果肉颜色的影响[J]. 中国农业科学, 2020, 53(9): 1874-1889 doi:10.3864/j.issn.0578-1752.2020.09.014
ZHANG YaFei, PENG Jie, ZHU YanSong, YANG ShengNan, WANG Xu, ZHAO WanTong, JIANG Dong. Genome Wide Identification of CCD Gene Family in Citrus and Effect of CcCCD4a on the Color of Citrus Flesh[J]. Scientia Acricultura Sinica, 2020, 53(9): 1874-1889 doi:10.3864/j.issn.0578-1752.2020.09.014

0 引言

【研究意义】柑橘是世界范围内最重要的经济作物之一,其艳丽的果肉、果皮色泽是吸引消费者的重要因素之一,也是育种学家所关注的重要性状。已有研究表明,类胡萝卜素裂解双加氧酶（CCD）基因广泛参与果实颜色的调控^[1]。利用生物信息学的方法对柑橘全基因组的CCD基因家族（CcCCD）进行全面分析,重点对CcCCD4a在柑橘果肉颜色发育过程中的基因表达和不同果肉颜色柑橘种质中CcCCD4a的基因型进行研究,对指导培育果肉为橙红色的柑橘品种具有重要意义。【前人研究进展】类胡萝卜素是一类在动植物中广泛存在的类异戊二烯色素。叶黄素（Xanthophylls）、紫黄质（Violaxanthin）是植物捕光蛋白复合物的关键组成部分^[2],番茄红素（Lycopene）、β-胡萝卜素（β-carotene）等是影响植物花和果实颜色的重要色素^[3,4],其含量高低决定了果肉色泽的深浅。高等植物中主要由类胡萝卜素裂解双加氧酶负责类胡萝卜素的氧化裂解,CCD是一类非血红素铁酶,均含有RPE65（Retinal pigment epithelial membrane protein）结构域,负责结合Fe^2+[5]。根据其底物是否含有环氧结构,可将其进一步划分为类胡萝卜素裂解双加氧酶（CCDs）和9-顺式环氧类胡萝卜素双加氧酶（NCEDs）^[6,7]。在拟南芥中共鉴定到9个CCD成员,其中4个CCDs成员（AtCCD1、AtCCD4、AtCCD7、AtCCD8）,5个NCEDs成员（AtNCED2、AtNCED3、AtNCED5、AtNCED6、AtNCED9）。CCD1在9'-10'双键裂解C40类胡萝卜素,形成C13、C14小分子挥发性物质,对植物香气的形成具有重要影响^[8,9,10]。CCD7和CCD8主要参与植物激素调节,其裂解产物是植物激素独角金内酯的重要前体^[11,12]。NCED以环氧类胡萝卜素为底物,是植物脱落酸（ABA）合成的限速酶,对植物抗逆性有重要作用^[13,14]。CCD4成员较为复杂,在拟南芥、水稻、蔷薇等都只鉴定到了一个成员^[1,7,15],而在番茄、葡萄、藏红花、菊花^{[16,17,18,19]}等鉴定到了多个CCD4,目前证据表明其可能参与花、果皮、果肉颜色的形成,柑橘CCD4b1的底物为β-隐黄质和玉米黄质,裂解位点在7'-8'双键,代谢产物为柑橘所特有的C30类胡萝卜素,使橘皮表现为橙红色^[20,21]。ZHENG等^[22]研究发现CCD4b1启动子上的一个MITE插入导致CCD4b1表达增强积累更多橙红色的C30类胡萝卜素。日本****MINAMIKAWA等^[23]利用柑橘资源及杂种群体的全基因组关联分析,发现与果肉颜色性状强关联的1个SNP位于CcCCD4a附近,暗示CcCCD4a可能直接或间接影响柑橘果肉颜色的形成。CCD由于具有多种生物学功能,在调节植物的生长发育、非生物胁迫和颜色形成等方面具有重要作用,因此倍受研究者的关注。【本研究切入点】CCD基因家族广泛参与植物生长发育和果实颜色形成,但目前对CCD基因家族的研究主要集中在拟南芥、番茄、水稻等模式植物中,对柑橘CCD基因家族进行全基因组鉴定尚未见报道。CcCCD4a可能参与调控柑橘果肉颜色的形成,但该基因在果实颜色形成过程中的表达与果肉颜色变化之间的关系尚不清楚,不同颜色柑橘品种的CcCCD4a基因型也未有研究。【拟解决的关键问题】本研究对CcCCD基因家族成员进行鉴定,并对该家族的基本信息、Scafflod定位、系统进化等进行预测分析;同时,选择10个柑橘品种,利用qRT-PCR技术测定果肉色泽形成期CcCCD4a在不同柑橘品种中的表达特征,同时通过对30个柑橘品种CcCCD4a的基因型鉴定,为探究CcCCD4a功能和开发基于CcCCD4a的分子标记奠定基础。

1 材料与方法

1.1 柑橘CCD基因家族成员鉴定

柑橘‘克里曼丁’橘（Citrus clementina）基因组和蛋白组^[24]数据下载自NCBI（https://www.ncbi.nlm. nih.gov/）。通过如下4种方法鉴定CcCCD家族成员：（1）下载RODRIGO等鉴定的5个CcCCD4^[21]（CcCCD4a、CcCCD4b1、CcCCD4b2、CcCCD4c、CcCCD4d）;（2）用柑橘中已鉴定到的5个CcCCD4和其他物种中已知的CCD家族基因（附表1）蛋白序列在克里曼丁橘蛋白质组数据库进行Blast搜索,获取柑橘中的同源序列;（3）在Pfam数据库（http://pfam.xfam.org/）中下载所有物种RPE65（PF03055）结构域序列,利用Hmmer2.3（http://hmmer.janelia.org/）构建隐马氏模型,在克里曼丁橘蛋白数据库中搜索含有RPE65结构域的序列;（4）合并（1）（2）（3）的结果,将得到的结果使用在线工具SMART（http://smart.embl- heidelberg.de/）分析结构域,去除不包含RPE65结构域的序列,最终得到CcCCD家族所有基因。

1.2 CcCCD家族生物信息学分析

对CcCCD蛋白的分子量和等电点使用ExPASy Proteomics Server（http://www.expasy.org/proteomics）进行预测。用在线软件MBC（http://cello.life.nctu.edu. tw）对CcCCD蛋白进行亚细胞定位预测。利用MEGA7（https://www.megasoftware.net/）中的MUSCL程序将鉴定到的CcCCD家族蛋白序列和其他物种的CCD家族蛋白进行多重序列比对,并利用近邻法（Neighbor- Joining,NJ）构建系统进化树（bootstrap=1 000）。柑橘中已有命名的CCD4名称不变,未命名的基因根据拟南芥中同源基因命名,没有同源基因的命名为CcCCD_like,同一个亚家族内的基因根据染色体上的位置按照小写字母依次排序。用在线工具MEME（http://meme-suite.org/）鉴定各家族成员motif。在phytozome数据库（https://phytozome.jgi.doe.gov/）中下载CcCCD成员的注释信息,用GSDS （http://gsds. cbi.pku.edu.cn/）制作基因结构图。同时获取基因的位置信息,用在线工具MG2C（http://mg2c.iask.in/ mg2c_v1.1/）展示Scafflod定位图。

1.3 试验材料

本试验用于基因分型的30份柑橘种质资源均来源于国家果树种质重庆柑橘资源圃（具体信息见表1）,除猴橙为甜橙外,大部分材料为宽皮柑橘及其杂种。2019年4月选取树势中等、树体健康的植株随机采集春梢叶片,液氮研磨后用于DNA提取。选取10株果肉色泽具有明显差异的柑橘植株,取果实进行基因表达分析（表1中编号1—10的材料）,从果实转色期开始,每15 d采样一次（2019年9月28日、10月12日、10月27日、11月13日）,直到充分转化为该品种固有色泽,拍照,进行色泽表型鉴定,取果肉液氮研磨用于RNA提取。

Table 1
表1
表1本研究30 份柑橘材料
Table 130 accessions of mandarin germplasms used in this study

编号 Code	材料名称 Name	种名 Specific name	果实成熟期 Ripen time	果皮颜色 Peel color	果肉颜色 Flesh color
1	丽红 Reikou	Citrus reticulata Blanco	11月下旬 Late Nov.	橙红色 Orange red	橙红色 Orange red
2	红橘 Hong ju	Citrus reticulata Blanco	11月中旬 Middle Nov.	红色 Red	橙红色 Orange red
3	美国糖橘 America tang ju	Citrus reticulata Blanco	11月上旬 Early Nov.	深红色 Deep red	橙红色 Orange red
4	椪柑 Ponkan	Citrus reticulata Blanco	11月下旬 Late Nov.	橙黄色 Orange yellow	橙红色 Orange red
5	茂谷柑 Murcott	Citrus reticulate Blanco × Citrus sinensis (L.) Osbeck	1月上旬 Early Jan.	橙红色 Orange red	橙红色 Orange red
6	沃柑 Orah	Citrus reticulata Blanco	1月上旬 Early Jan.	橙红色 Orange red	橙色 Orange
7	青橘 Qing Ju	Citrus reticulata Blanco	12月上旬 Early Dec.	橙黄色 Orange yellow	橙色 Orange
8	春见 Harumi	Citrus reticulata Blanco	12月上旬 Early Dec.	橙色 Orange	橙色 Orange
9	猴橙 Monkey Orange	Citrus sinensis (L.) Osbeck	11月下旬 Late Nov.	橙色 Orange	橙色 Orange
10	春香 Haruka	Citrus tamurana hort.ex Tanaka Spp.	12月中旬 Middle Dec.	浅橙黄色 Light orange yellow	浅橙黄色 Light orange yellow
11	朱红 Zhu hong ju	Citrus erythrosa Hort.ex Tan.	11月下旬 Late Nov.	红色 Red	橙红色 Orange red
12	汕头酸橘 Shan tou suan ju	Citrus sunki (Hayata) hort.ex Tanaka	12月上旬 Early Dec.	橙色 Orange	橙色 Orange
13	满头红 Man tou hong ju	Citrus reticulata Blanco	12月中旬 Middle Dec.	红色 Red	橙红色 Orange red
14	大红袍 Da hong pao ju	Citrus reticulata Blanco	11月中旬 Middle Nov.	红色 Red	橙红色 Orange red
15	宫本温州蜜柑 Miyamoto wase unshiu	Citrus unshiu Macf.	9月下旬 Late Sep.	橙黄色 Orange yellow	橙红色 Orange red
16	无核红橘 Seedless hong ju	Citrus reticulata Blanco	12月下旬 Late Dec	红色 Red	橙红色 Orange red
17	永春芦柑 Yong chun lu gan	Citrus reticulata Blanco	11月下旬 Late Nov.	橙黄色 Orange yellow	橙红色 Orange red
18	濑户佳实生 Seedling setoka	Citrus tangor	3月上旬 Early Mar.	橙黄色 Orange yellow	橙色 Orange
19	辉优 Hybrid fagllo 4-3	Citrus reticulata Blanco	11月中旬 Middle Nov.	红色 Red	橙红色 Orange red
20	立山椪柑 Li shan ponkan	Citrus reticulata Blanco	11月下旬 Late Nov.	橙色 Orange	橙红色 Orange red
21	肥之曙 Hinoakebono wase	Citrus unshiu Macf.	9月中旬 Middle Sep	橙色 Orange	橙红色 Orange red
22	红晕香柑 Hong yun xiang gan	Citrus tangor	3月上旬 Early Mar.	红色 Red	橙红色 Orange red
23	橘湘早 Ju xiang zao	Citrus unshiu Macf.	10月上旬 Early Oct.	橙黄色 Orange yellow	橙色 Orange
24	爱媛30号杂种3 Ehime No.30 new line	Citrus reticulata Blanco	11月下旬 Late Nov.	橙红色 Orange red	橙红色 Orange red
25	爱妃 Princess fairy	Citrus reticulata Blanco	10月中旬 Middle Oct.	橙红色 Orange red	橙红色 Orange red
26	牛肉红橘 Niu rou hong ju	Citrus reticulata Blanco	11月下旬 Late Nov.	深红色 Orange red	橙红色 Orange red
27	金秋砂糖橘 Gold autum sha tang ju	Citrus reticulata Blanco	10月下旬 Late Oct.	橙红色 Orange red	橙色 Orange
28	华美20号 Hua mei No.20	Citrus reticulata Blanco	10月中旬 Middle Oct.	红色 Red	橙色 Orange
29	华美4号 Hua mei No.4	Citrus reticulata Blanco	10月下旬 Late Oct.	红色 Red	橙红色 Orange red
30	华美41号 Hua mei No.41	Citrus reticulata Blanco	11月中旬 Middle Oct.	橙色 Orange	橙红色 Orange red

新窗口打开|下载CSV

1.4 果肉色泽测定

取不同时期样品果实沿赤道面横切,使用CR-400色差计（柯尼卡美能达）记录各样品颜色信息。采用Lab模型描述所测得的颜色数据。根据测量得到的a*、b*值按照公式h=tan^-1（b*/a*）获得色调角h,色调角h取值在0°—90°代表色泽介于红色与黄色,数值越小越偏向于红色,反之越偏向于黄色。本试验测定条件为：光源D65,标准观察角度2°,照明区域Φ50 mm。每个样品进行3次生物学试验重复,每次重复进行5次数据测定。

1.5 实时荧光定量PCR

使用RNAprep pure植物总RNA提取试剂盒（DP432,天根）提取植物总RNA。使用PrimeScript^TM RT Reagent Kit With gDNA Eraser Perfect Real Time（RR047,TaKaKa）试剂盒将RNA反转录成cDNA供荧光定量使用。使用Prime3（https://primer3plus. com）设计引物,引物信息见表2。内参基因为柑橘β-Actin,在CFX96 Touch^TM荧光定量PCR仪上对CcCCD4a在10个柑橘品种中不同时期的表达量进行分析。扩增体系含2 μL cDNA,上、下游引物各0.5 μL（10 μmol?L^-1）,SYBR 6.25 μL,ddH₂O 3.25 μL,总体系12.5 μL。反应程序为：95℃预变性30 s;95℃变性5 s,60℃退火30 s,95℃延伸15 s,40个循环,每个处理3次重复。以2^ΔΔCt法计算CcCCD4a相对表达量。

Table 2
表2
表2引物信息
Table 2Primers information

引物名称 Primer name	用途 Usage	引物序列 Primer sequence	片段大小 Length (bp)	循环数/退火温度 Cycles/Annealing temperature (℃)
CcCCD4a	扩增CcCCD4a Amplify CcCCD4a	F：ACTTGCCAGCCTTAAGCCGT	2465	35/56℃
		R：CAATATTGTGTGTGGTGGCC
CcCCD4aq	CcCCD4a荧光定量 qRT-PCR primers of CcCCD4a	F：TCTCTCAGCCTCAACCCAAG	220	40/60℃
		R：ACTACCTCACACTCCGTTGG
β-Actin	内参基因 Reference gene	F：CCCCATCGTTACCGTCCAG	150	40/60℃
		F：CGCCTTGCCAGTTGAATATCC

新窗口打开|下载CSV

1.6 基因型分析

取30份研磨后的叶片样品用CTAB小量法提取基因组DNA。根据NCBI上公布的克里曼丁橘基因组,使用Prime3设计CcCCD4a引物,引物序列见表2。使用DNA小量回收试剂盒（Magen D2111-03）纯化PCR产物。1—10号纯化样品委托深圳华大基因有限公司进行Sanger测序,每个样品重复3次,使用DNAsp（http://www.ub.edu/dnasp/）软件分析核苷酸多态性。另外,所有纯化后的PCR产物稀释到10 ng?μL^-1,放入核酸超声打断仪（宁波新芝）中进行打断处理（10 s超声,5 s间歇;35个循环）,使用二代测序建库试剂盒（天津诺禾）,构建插入片段为300 bp左右的illumina二代双端测序文库,经稀释、变性后使用Illumina MiniSeq平台测定30个柑橘品种CcCCD4a序列。测序结果经BWA（http://bio-bwa.sourceforge. net/）比对到‘克里曼丁’橘基因组,经Samtools（http:// www.htslib.org/）转换为bam文件后,再用Bcftools（http://www.htslib.org/doc/1.0/bcftools.html）检测核苷酸变异生成vcf（Variant call format）文件,vcf文件经Tassel软件（https://www.maizegenetics.net/tassel）分析编码区单倍型。

1.7 数据处理

利用SPSS18软件进行不同品种间基因表达差异的显著性分析,不同品种间色调角h差异显著性分析和色调角h与基因相对表达量的相关性分析。P<0.05表示差异显著。

2 结果

2.1 CcCCD基因家族成员信息

通过Blast比对及Hmmer搜索,SMART分析去除不含REP65结构域的序列,从柑橘基因组中共鉴定出14个CCD基因,如表3所示。通过ExPASy工具分析,柑橘中最长的CCD蛋白（CcCCD_like）包含611个氨基酸,分子量为69.18 kD,是14个蛋白质中分子量最大的。最短的CCD蛋白（CcCCD4d）包含412个氨基酸,分子量是14个蛋白中最小的。等电点范围为6.05（CcCCD1b）—8.53（CcCCD4c）。利用MBC软件对CcCCD家族成员进行亚细胞定位分析,结果显示该基因家族成员位于细胞不同位置,其中CcCCD1a、CcCCD1b、CcCCD4b2、CcCCD8a、CcCCD_like、CcNCED6定位于细胞质,其他成员定位于叶绿体。

Table 3
表3
表3柑橘基因组中的CCD基因
Table 3The CCD genes identified in citrus

基因名称 Gene name	基因组登录号 Gene accession No.	蛋白质大小 Protein length (aa)	RPE65位置 RPE65 domain location	分子量 Molecular mass (KD)	等电点 Isoelectric point (pI)	亚细胞定位 Subcellular localization
CcCCD1a	CICLEv10031014m	597	106—587	67.2	6.33	细胞质 Cytoplasm
CcCCD1b	CICLEv10031039m	589	100—579	66.67	6.05	细胞质 Cytoplasm
CcCCD4a	CICLEv10031003m	603	124—595	66.45	6.87	叶绿体 Chloroplast
CcCCD4b1	CICLEv10028113m	563	87—553	63.06	8.34	叶绿体 Chloroplast
CcCCD4b2	CICLE_v10030384m	508	38—504	56.91	6.56	细胞质 Cytoplasm
CcCCD4c	CICLEv10011335m	597	115—590	66.35	8.53	叶绿体 Chloroplast
CcCCD4d	CICLEv10013726m	412	93—403	45.86	7.25	叶绿体 Chloroplast
CcCCD7	CICLEv10027500m	590	43—580	66.51	6.07	叶绿体 Chloroplast
CcCCD8a	CICLEv10008050m	510	29—508	56.8	5.93	细胞质 Cytoplasm
CcCCD8b	CICLEv10010609m	556	76—554	61.965	5.98	叶绿体 Chloroplast
CcCCD_like	CICLEv10010551m	611	89—598	69.18	6.53	细胞质 Cytoplasm
CcNCED3	CICLEv10019364m	606	134—598	67.01	6.37	叶绿体 Chloroplast
CcNCED5	CICLEv10014639m	609	136—601	67.79	6.3	叶绿体 Chloroplast
CcNCED6	CICLEv10006710m	592	113—583	65.29	7.32	细胞质 Cytoplasm

新窗口打开|下载CSV

2.2 CCD基因家族系统进化分析

为研究CCD基因家族的遗传进化关系,对已鉴定到的CCD家族基因进行系统进化分析,选取柑橘（Citrus clementina）（14个）,拟南芥（Arabidopsis thaliana）（9个）,水稻（Oryza sativa）（9个）,番茄（Solanum lycopersicum）（6个）,葡萄（Vitis vinifera）（7个）,菊花（Chrysanthemum morifolium）、猕猴桃（Actinidiachinensis）、藏红花（Crocus sativus）（各3个）,欧洲越橘（Vaccinium myrtillus）、枸杞（Lycium chinense）、苹果（Malus×domestica）、突厥蔷薇（Rosa damascena）（各1个）共56个CCD蛋白（具体信息见附表1）构建系统进化树（图1）。根据亲缘关系可将56个CCD蛋白分为6个亚家族,分别为CCD1、CCD4、CCD7、CCD8、NCED、CCD_like。CCD8亚家族与其他CCD亚家族的遗传距离较远,CCD1、CCD4和NCED亚家族具有更近的遗传距离。

图1

新窗口打开|下载原图ZIP|生成PPT
图1CCD家族蛋白系统进化树

Fig. 1The phylogenetic tree of CCD proteins



由于CcCCD_like序列不完整,系统进化分析不能很好的确定其所归属的亚家族,因此分析了CcCCD家族成员的motif。在CcCCD家族蛋白中使用MEME工具找到了10个motif,如图2所示。CcCCD_like含有5个motif（4、5、6、7、10）,CcCCD8亚家族含有6个motif（3、4、5、6、7、10）,与CcCCD8亚家族相比,CcCCD_like只缺少第3个motif。

图2

新窗口打开|下载原图ZIP|生成PPT
图2柑橘CCD蛋白motif分析

Fig. 2Motif analysis of CCD proteins in citrus

2.3 CcCCD家族基因结构及染色体定位分析

利用GSDS软件对CcCCD家族各成员的基因结构进行分析（图3）。结果显示,CcCCD家族的基因结构存在较大的差异,外显子数目为1—13个,内含子数目为0—12个。对基因结构进一步分析发现,CcCCD4和CcNCED亚家族的基因结构较简单,除CcCCD4d含2个内含子外,其他基因均不含内含子;CcCCD7和CcCCD8成员中多数基因含有3—6个内含子;而CcCCD1成员的基因结构较为复杂,内含子数目较多,CcCCD1b、CcCCD_like含有12个内含子,CcCCD1a含有13个内含子。根据CcCCD家族在Scafflod的位置信息,利用MG2C工具获得了14个CcCCD在柑橘Scafflod上的分布图（图4）。由图可知,CcCCD家族成员在Scafflod上分布不均。1号、4号、6号、8号Scafflod上分布较多,2号、3号、7号、9号Scafflod上,只含有1个CcCCD家族成员,而5号Scafflod上不含CcCCD家族成员。进一步分析发现,CcCCD在Scafflod上呈现区域性分布。

图3

新窗口打开|下载原图ZIP|生成PPT
图3柑橘CCD家族基因结构

Fig. 3Gene structure of citrus CCD genes

图4

新窗口打开|下载原图ZIP|生成PPT
图4柑橘CCD基因在 Scafflod 上的相对位置

Fig. 4Scafflod locations of citrus CCD genes

2.4 果肉颜色表型分析

本试验将a*、b*两值换算为h值来表示果肉颜色。10个柑橘品种在4个时期中h值介于77.28°（11月13日,红橘）—100.80°（10月27日,春香）,色泽介于橙红色与浅橙黄色（图5、图6）,多数品种随着果实的成熟,h值不断下降,在10月27日至11月13日期间下降最明显。4个时期中橙色与浅橙黄色品种h值显著高于橙红色品种（图6）。橙红色品种中红橘色泽更深,在11月13日,其h值显著低于其他橙红色品种,‘丽红’在4个时期中h值下降缓慢,果肉色泽变化较小。橙色品种中‘青橘’的果肉色泽更为浓厚,其h值在最后一个时期也表现为显著低于其他橙色品种,‘春见’与‘猴橙’的h值在4个时期中始终保持较高水平,各时期果肉色泽差异不显著。浅橙黄色的‘春香’h值始终显著高于其他品种,在4个时期中差异不明显。

图5

新窗口打开|下载原图ZIP|生成PPT
图510个柑橘品种在4个时期的果肉色泽

Fig. 5Flesh color of 10 citrus varieties in 4 periods

图6

新窗口打开|下载原图ZIP|生成PPT
图610个柑橘品种4个时期果肉h值的变化

不同小写字母表示在P<0.05水平差异显著。下同
Fig. 6Changes in hue angle of 10 citrus varieties in 4 periods

Different lowercase letters indicate significant difference (P<0.05). The same as below

2.5 果实发育过程中CcCCD4a表达情况

在4个时期检测10个柑橘品种中CcCCD4a的表达水平,结果如图7。CcCCD4a在10个柑橘品种间的相对表达量差异较大,与9月27日‘红橘’相比,11月13日表达量最高的‘春香’达到52.40,而最低表达的‘丽红’仅为0.04。4个时期橙红色品种中‘茂谷柑’的表达量显著高于其他橙红色品种。橙色品种中‘猴橙’在第一个时期基因表达量显著低于其他橙色品种,而在后3个时期中显著高于其他橙色品种。浅橙黄色品种‘春香’始终保持较高水平表达,表达量显著高于其他品种。

图7

新窗口打开|下载原图ZIP|生成PPT
图7CcCCD4a在10个柑橘品种4个时期的相对表达量

Fig. 7Relative expression of CcCCD4a in 4 periods of 10 citrus varieties



10个品种除‘茂谷柑’外,CcCCD4a均表现出在果肉偏红色的品种中趋于中低表达水平,在果肉偏黄色的品种中趋于高表达水平,推测该基因表达水平高低与果肉颜色具有相关性。以色调角h和CcCCD4a相对表达量为随机变量进行相关分析,结果显示,随着时间的延长,CcCCD4a表达量与色调角h的相关性逐步增强,在最后一个时期相关系数为0.532（P<0.01）,去除‘茂谷柑’后,相关系数为0.765（P<0.01）,呈极显著相关。

2.6 CcCCD4a核苷酸多态性与单倍型分析

除‘茂谷柑’外,其余品种均符合CcCCD4a高表达果肉偏黄色,低表达偏红色的规律,为检测‘茂谷柑’在基因结构上与其他品种的差异,对所选10份材料进行Sanger测序分析其核苷酸多态性。在10个柑橘品种CcCCD4a的编码区共鉴定到31个SNP位点（图8）,其中同义突变17个,非同义突变14个,共有8个氨基酸突变位于RPE65结构域。‘茂谷柑’与‘克里曼丁’相比,所含有的SNP位点最多为21个,而‘椪柑’‘沃柑’最少。

‘茂谷柑’共有4个特有SNP位点,分别位于第294、913、1 137和1 657 bp。294和1 137 bp处为同义突变,其余两个为非同义突变且位于RPE65结构域上。913 bp变异导致第305位氨基酸由疏水性的丙氨酸突变为亲水性的苏氨酸,第1 657 bp处SNP导致第553位氨基酸由缬氨酸突变为异亮氨酸。

对10份柑橘材料的Sanger测序分析发现橙红色果肉的‘红橘’‘美国糖橘’具有完全相同的核苷酸序列（图8）,推测该基因的单倍型可作为柑橘果肉颜色的检测标记。因此,重新采集20份果肉偏红色的材料（表1）与原来的10份材料一起构建二代测序文库,测定CcCCD4a序列,用Tassel软件进行单倍型分析,分析结果见表4。30个柑橘品种的CcCCD4a具有9种单倍型,其中包含hap-1的12个品种均为宽皮柑橘中典型的橘类品种,且果肉均为橙红色的品种,在这些品种中,个别还包含有hap-4、hap-5单倍型,其中以‘牛肉红橘’和‘美国糖橘’的果肉颜色为最深,这两份材料在162 bp处均为M,在669 bp处均为Y,而在669 bp处为Y的品种均表现为果皮红艳。包含有hap-2单倍型的品种为‘温州蜜柑’或是含有‘温州蜜柑’血缘的杂柑品种,果肉也多为橙色至橙红色。包含有hap-6单倍型的品种为典型杂柑类品种,果肉颜色通常为橙色至橙红色,hap-7和hap-8为橙色至浅橙黄色品种,具有甜橙类基因的渗入,hap-9只包含‘茂谷柑’,该品种也是天然的橘橙杂种,明显具有‘甜橙’基因的渗入。

Table 4
表4
表430个柑橘品种的单倍型序列
Table 4The haplotype sequences of 30 citrus varieties

单倍型 Haplotype	序列 Sequence	材料份数 Number of accession	涉及材料 Related variety
hap-1	ATCGGTTACTCTCTATGTAGTCCGGTGTCTA	12	华美41号 Hua mei No.41、红橘 Hong ju、无核红橘Seedless hong ju、大红袍 Da hong pao、爱媛30杂种3 Ehime No.30 new line、牛肉红橘 Niu rou hong ju、永春芦柑 Yong chun lu gan、立山椪柑 Li shan ponkan、华美4号 Hua mei No.4、朱红 Zhu hong ju、满头红Man tou hong ju、美国糖橘 America tang ju
hap-2	ACCGGTTCCTCTCCAAGTATTCCGGCGTCTA	9	丽红 Reikou、春见 Harumi、爱妃 Princess fairy、红晕香柑 Hong yun xiang gan、华美20号 Hua mei No.20、金秋砂糖橘 Jin qiu sha tang ju、橘湘早Ju xiang zao、肥之曙 Hinoakebono wase、宫本温州Miyamoto wase unshiu
hap-3	ATCGGTTACTCTCCAAGTAGTCCGGCGTCTA	1	华美41号 Hua mei No.41
hap-4	ATCGGTTACTCTCCATGTAGTCCGGTGTCTA	6	红橘 Hong ju、无核红橘 Seedless hong、辉优 Hybrid fagllo 4-3、汕头酸橘 Shan tou suan ju、大红袍 Da hong pao ju、爱媛30杂种3 Ehime No.30 new line
hap-5	ATCGGTTCCTCTCCATGTAGTCCGGTGTCTA	1	牛肉红橘 Niu rou hong ju
hap-6	TTTACTTCCTCTCCATGTAGTCCGCCGCGAG	5	美国糖橘 America tang ju、椪柑 Ponkan、沃柑Orah、濑户佳实生 Seedling setoka、克里曼丁 Clementina
hap-7	TTCACACCCCATACGTGCAGCTAACCGCCTA	1	春香 Haruka、猴橙 Monkey orange
hap-8	ATCGGTTCCTCTCCATGTAGTCCGGCGTCTA	1	青橘 Qing ju
hap-9	TTCACACCTCAAACGTACGGCTAACCATCTA	1	茂谷柑 Murcott

新窗口打开|下载CSV

3 讨论

CCD基因家族是一类具有RPE65结构域的基因家族,其所编码蛋白参与类胡萝卜素代谢途径,通过裂解C40类胡萝卜素产生衍生代谢产物。从酵母到人类,几乎所有真核生物都含有CCD,尤其是植物存在大量该类基因,如拟南芥基因组中存在9个CCD^[6],水稻中鉴定出11个CCD^[1],番茄中鉴定出9个CCD^[16],葡萄中鉴定到19个^[17]。本研究从‘克里曼丁’基因组数据库中获得了14个CCD基因家族成员,与其他物种成员数不同,表明不同物种的CCD成员数存在差异,而这一差异主要表现在CCD4亚家族上,在拟南芥、水稻、马铃薯等颜色相对单一的植物中,CCD4一般只有1个,而在番茄、葡萄、藏红花、柑橘等颜色丰富且差异较大的植物中CCD4数目较多,这表明该基因在进化过程中可能存在基因扩增事件而通过选择得以保留。CcCCD基因家族的蛋白相对分子质量大小在45.86—69.18 kD,但大多数为65 kD左右,pI在5.98—8.53,表明CcCCD家族成员之间的蛋白质大小、pI等特征参数差异不大,这一结果与其他物种相似。motif分析显示CcCCD_like与CcCCD8亚家族更为相似,推测CcCCD_like属于CcCCD8亚家族。

本研究利用柑橘中的CCD家族成员与其他作物中报道的CCD家族成员进行系统演化分析,由于CCD基因的保守性,具有相似或者相同功能的基因常位于同一组,这为研究该基因家族相关基因的功能提供了重要依据。拟南芥中,AtCCD7与AtCCD8联合将β-胡萝卜素转变为独角金内酯的前体己内酯,继而使植物生长发育^[25],藏红花中CsCCD7、CsCCD8的表达通过影响独角金内酯的合成进而影响顶芽的分化^[18],类似的在猕猴桃^[26]、番茄^[27]、水稻^[28]等植物中,CCD7与CCD8参与了调控衰老、根的生长、分枝分蘖和花的发育等多种生命活动,由此推测柑橘CcCCD7、CcCCD8亚家族可能具有相同或相似的功能。NCED亚家族成员是ABA生物合成的限速酶,通过调节ABA的代谢,进而影响植物生命过程^[29,30]。KATO等^[31]曾在2006年通过Race扩增出柑橘CcNCED3与CcNCED5,通过重组蛋白表达发现CcNCED3与CcNCED5能在11'-12'位双键裂解紫黄质生成黄质醛（Xanthoxin）,这与拟南芥相似,而本研究鉴定到的CcNCED6与AtNCED6为同源基因,由此推测CcNCED6与AtNCED6具有相似的功能。

CCD4对类胡萝卜素的裂解与果肉、花器官的着色有关。过表达拟南芥AtCCD4的水稻中β-胡萝卜素和叶黄素分别降低74%和72%,β-紫罗酮增加了2倍^[32]。CCD4功能的缺失也会造成果实、花器官颜色的改变,比如杜鹃花瓣由黄色褪为白色^[33],洋橘梗浅黄色和白色花的形成^[34],以及百合花由黄变白^[35]等表型的改变和形成均由CCD4裂解类胡萝卜素功能改变所造成。不同于其他物种的CCD4在9'-10'双键裂解类胡萝卜素,柑橘中CcCCD4b1较为特殊,其裂解位置为7'-8'双键,产物为柑橘所特有的C30类胡萝卜素（β-citraurin,β-citraurinene等）,其高表达诱导柑橘果皮变红^[22]。CcCCD4b2由于在果实发育过程中表达量极低,在柑橘EST数据库（http://harvest.ucr.edu/）中缺乏EST序列,因此被认为是假基因^[21];CcCCD4c、CcCCD4d多在花瓣中表达,在果皮、果肉中均未检测到^[21],而前期的研究发现柑橘果肉颜色性状与CcCCD4a附近的SNP位点存在显著的相关性^[23],基于此研究结果,选择CcCCD4a作为本研究的重点。

本研究结果表明,随着果实的成熟,果肉颜色逐步由浅色调向深色调过渡,h值逐渐降低,颜色逐步加深,涉及的10个品种在11月13日果肉颜色差异最为明显,其后尽管颜色还会趋于加深,但果肉色泽与11月13日相比均没有显著差异,因此,以11月13日作为采样最终时间。本试验测定了10个柑橘品种在4个时期CcCCD4a的相对表达量和色调角h,发现CcCCD4a在不同品种中的表达量与色调角h显著正相关,说明在一定范围内,CcCCD4a高表达使果肉颜色偏向于更淡,低表达使果肉颜色偏向于更红。这一结果与ZHENG等^[22]关于CcCCD4b1的研究相反,该作者认为使果皮表现为红色的色素主要是高含量的C30类胡萝卜素,如β-柠乌素（β-citraurin）和β-香茅素（β-citronella）等,而MA^[36]和AGóCS^[37]等认为果肉表现的橙红色多由C40类胡萝卜素含量决定,如在橙红色的‘温州蜜柑’和‘丽红’果肉中含有大量的β-隐黄质等C40类胡萝卜素。在其他物种中,CcCCD4a同源基因功能的研究与本试验更为相似,比如葡萄中VvCCD4a的底物为红色的番茄红素,产物为浅橙色的6-甲基-5-庚烯-2-酮^[17];西葫芦中CpCCD4a在含低类胡萝卜素的种质中高表达,在高类胡萝卜素的种质中低表达^[38];RNA干扰（RNAi）菊花中CmCCD4a的表达,使白色菊花转变为黄色^[18];桃中通过病毒诱导的基因沉默（VIGS）抑制白肉桃CCD4表达,结果在白肉桃中诱导出了黄色^[39],白色矮牵牛编码区一段226 bp的MITE插入终止了ClCCD4的转录形成黄色突变^[40],甘蓝中也有类似报道^[41],这些研究结果均表明高表达的CCD4会使果肉的颜色偏浅。陶俊^[42]的研究也表明果肉偏红色的‘温州蜜柑’类胡萝卜素含量显著高于果肉偏黄色和浅橙色的‘甜橙’,由此进一步推测CcCCD4a的高表达降低了柑橘果肉中类胡萝卜素的含量,使果肉颜色偏黄。但‘茂谷柑’并不符合这一推测,因此,本研究对10个柑橘品种进行了测序分析。结果显示,10个柑橘品种中CcCCD4a并没有发生结构变异,也没有终止子突变,但‘茂谷柑’CcCCD4a编码区中含有较多的变异,其中A305T、V553I为其独有的氨基酸变异,这可能是导致‘茂谷柑’CcCCD4a的表达量与果肉颜色之间关系异常的原因,但还需要进一步的验证。对30个柑橘品种进行单倍型分析,发现果肉呈橙红色的品种中单倍型序列均以hap-1、hap-4、hap-5为优势单倍型,而含有hap-4、hap-5的品种果皮均呈现艳丽的红色。由此可见,根据CcCCD4a的基因分型结果,不仅能够为果肉颜色育种提供重要的分子标记,而且能够对30份柑橘种质进行清晰的系谱分类。‘美国糖橘’来源较为复杂,至今不清楚其具体亲本信息,在本试验中其两个单倍型分别为hap-1和hap-6,包含hap-1单倍型的种质多为中国系红橘品种,而含有hap-6单倍型的品种为杂柑类品种,由此推测‘美国糖橘’可能为红橘与杂柑类品种杂交而成。另外引人注意的是,hap-5仅有一个品种‘牛肉红橘’,其为‘朱红橘’突变体^[43],本试验中鉴定到其包含两种单倍型,相较于其他‘朱红橘’品种,‘牛肉红橘’的果肉为浓郁的橙红色,其果皮颜色也为深红色。结合在CcCCD4a中669 bp为Y的基因型均表现为果皮艳丽的红色,果肉为橙红色,669 bp位置的C/T基因型可作为潜在的柑橘颜色育种的分子标记。综合本试验结果,CcCCD4a的hap-1、hap-4、hap-5单倍型作为分子标记来预测柑橘果肉颜色,hap-4、hap-5和669 bp的C/T基因型来预测柑橘果皮颜色值得关注,下一步将通过对该单倍型遗传规律的深入研究予以进一步验证。

4 结论

本研究从柑橘全基因组中鉴定出14个CCD基因成员,其蛋白均含有RPE65保守结构域。亚细胞定位显示CcCCD定位于细胞质和叶绿体。CcCCD4a的表达水平影响柑橘果肉颜色的表现,高表达CcCCD4a的品种果肉颜色普遍较浅,而低表达品种果肉颜色普遍较深。因此,CcCCD4a可作为潜在的柑橘果实颜色的辅助育种标记,尤其是单倍型hap-1、hap-4和hap-5与果肉红色的关联度较高,对柑橘颜色育种的早期杂种群体筛选具有一定帮助。

参考文献 View Option 原文顺序
文献年度倒序
文中引用次数倒序
被引期刊影响因子

[1] VALLABHANENI R, BRADBURY L M, WURTZEL E T . The carotenoid dioxygenase gene family in maize, sorghum, and rice
Archives of Biochemistry and Biophysics, 2010,504(1):104-111.
DOI:10.1016/j.abb.2010.07.019URLPMID:20670614 [本文引用: 3]
Carotenoids and their apocarotenoid derivatives play essential physiological and developmental roles and provide plants tolerance to a variety of stresses. Carotenoid cleavage dioxygenases mediate the degradation of carotenoids to apocarotenoids. A better understanding of biosynthesis vs. degradation could be useful for controlling carotenoid levels leading to improved plant fitness and/or enhanced content of nutritionally valuable carotenoids. The Poaceae (grass) plant family contains many crops of agronomic value. Therefore this study focused on characterizing the carotenoid dioxygenase gene family in the grass species maize, rice, and sorghum with comparison made to newly identified gene families in two non-seed plants as well as an alga and previously identified eudicot genes. Genome analysis was used to map grass genes encoding the carotenoid dioxygenases to chromosome locations. Sequences of encoded proteins were phylogenetically compared. CCD8b was identified as a new class of cleavage dioxygenases that may play a specialized role in apocarotenoid biogenesis. A simple PCR assay was developed to measure CCD1 gene copy number which is known to vary in maize. Using a panel of maize inbred lines varying in carotenoid content, linear regression analysis revealed a statistically significant negative correlation between copy number of CCD1 and carotenoid content, an effect likely mediated through the resulting elevated levels of endosperm CCD1 transcripts in high copy number lines. The PCR assay adds to a growing toolbox for metabolic engineering of maize endosperm carotenoids. This new tool can be used to select maize lines that are less likely to promote endosperm carotenoid degradation, thus predicting optimal results in metabolic engineering of endosperm provitamin A and/or nonprovitamin A carotenoids.

[2] WOITSCH S, R?MER S . Expression of xanthophyll biosynthetic genes during light-dependent chloroplast differentiation
Plant Physiology, 2003,132(3):1508-1517.
DOI:10.1104/pp.102.019364URLPMID:12857831 [本文引用: 1]
In higher plants, etioplast to chloroplast differentiation is characterized by dramatic ultrastructural changes of the plastid and a concomitant increase in chlorophylls and carotenoids. Whereas the formation and function of carotenes and their oxygenated derivatives, the xanthophylls, have been well studied, little is known about the regulation of the genes involved in xanthophyll biosynthesis. Here, we analyze the expression of three xanthophyll biosynthetic genes (i.e. beta-carotene hydroxylase [bhy], zeaxanthin epoxidase [zep], and violaxanthin de-epoxidase [vde]) during de-etiolation of seedlings of tobacco (Nicotiana tabacum L. cv Samsun) under different light conditions. White-light illumination caused an increase in the amount of all corresponding mRNAs. The expression profiles of bhy and zep not only resembled each other but were also similar to the pattern of a gene encoding a major light-harvesting protein of photosystem II. This finding indicates a coordinated synthesis during formation of the antenna complex. In contrast, the expression pattern of vde was clearly different. Furthermore, the gene expression of bhy was shown to be modulated after illumination with different white-light intensities. The expression of all xanthophyll biosynthetic genes under examination was up-regulated upon exposure to red, blue, and white light. Gene expression of bhy and vde but not of zep was more pronounced under red-light illumination, pointing at an involvement of the phytochrome system. Expression analysis in the presence of the photosynthetic electron transport inhibitors 3-(3,4-dichlorophenyl)-1,1-dimethyl-urea and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone indicated a redox control of transcription of two of the xanthophyll biosynthetic genes (bhy and zep).

[3] WANG C N, QIAO A H, FANG X F, SUN L, GAO P, DAVIS A R, LIU S, LUAN F S . Fine mapping of lycopene content and flesh color related gene and development of molecular marker-assisted selection for flesh color in watermelon (Citrullus lanatus)
Frontiers in Plant Science, 2019,10:1240.
DOI:10.3389/fpls.2019.01240URLPMID:31649702 [本文引用: 1]
Lycopene content and flesh color are important traits determined by a network of carotenoid metabolic pathways in watermelon. Based on our previous study of genetic inheritance and initial mapping using F₂ populations of LSW-177 (red flesh) × cream of Saskatchewan (pale yellow flesh), red flesh color was controlled by one recessive gene regulating red and pale yellow pigmentation, and a candidate region related to lycopene content was detected spanning a 392,077-bp region on chromosome 4. To obtain a more precise result for further study, three genetic populations and a natural panel of 81 watermelon accessions with different flesh colors were used in this research. Herein, we narrowed the preliminary mapping region to 41,233 bp with the linkage map generated from F₂ populations of LSW-177 (red flesh) × cream of Saskatchewan (pale yellow flesh) with 1,202 individuals. Two candidate genes, Cla005011 and Cla005012, were found in the fine mapping region; therein Cla005011 was a key locus annotated as a lycopene β-cyclase gene. Phylogenetic tree analysis showed that Cla005011 was the closest relative gene in gourd. LSW-177 × PI 186490 (white flesh) and another BC₁ population derived from garden female (red flesh) × PI 186490 were generated to verify the accuracy of the red flesh candidate gene region. By analyzing the expression levels of candidate genes in different developmental stages of different color watermelon varieties, Cla005011 for the expression differences was not the main reason for the flesh color variation between COS and LSW-177. This indicated that the LCYB gene might regulate fruit color changes at the protein level. A new marker-assisted selection system to identify red and yellow flesh colors in watermelon was developed with flesh color-specific CAPS markers and tested in 81 watermelon accessions.

[4] ILAHY R, SIDDIQUI M W, TLILI I, MONTEFUSCO A, PIRO G, HDIDER C, LENUCCI M S . When color really matters: horticultural performance and functional quality of high-lycopene tomatoes
Critical Reviews in Plant Sciences, 2018,37(1):15-53.
[本文引用: 1]

[5] MESSING S A J, GABELLI S B, ECHEVERRIA I, VOGEL J T, GUAN J C, TAN B C, KLEE H J, MCCARTY D R, AMZEL L M . Structural insights into maize viviparous14, a key enzyme in the biosynthesis of the phytohormone abscisic acid
The Plant Cell, 2010,22(9):2970-2980.
DOI:10.1105/tpc.110.074815URLPMID:20884803 [本文引用: 1]
The key regulatory step in the biosynthesis of abscisic acid (ABA), a hormone central to the regulation of several important processes in plants, is the oxidative cleavage of the 11,12 double bond of a 9-cis-epoxycarotenoid. The enzyme viviparous14 (VP14) performs this cleavage in maize (Zea mays), making it a target for the rational design of novel chemical agents and genetic modifications that improve plant behavior through the modulation of ABA levels. The structure of VP14, determined to 3.2-? resolution, provides both insight into the determinants of regio- and stereospecificity of this enzyme and suggests a possible mechanism for oxidative cleavage. Furthermore, mutagenesis of the distantly related CCD1 of maize shows how the VP14 structure represents a template for all plant carotenoid cleavage dioxygenases (CCDs). In addition, the structure suggests how VP14 associates with the membrane as a way of gaining access to its membrane soluble substrate.

[6] AULDRIDGE M E, BLOCK A, VOGEL J T, DABNEY-SMITH C, MILA I, BOUZAYEN M, MAGALLANES-LUNDBACK M, DELLAPENNA D, MCCARTY D R, KLEE H J . Characterization of three members of the Arabidopsis carotenoid cleavage dioxygenase family demonstrates the divergent roles of this multifunctional enzyme family
The Plant Journal, 2006,45(6):982-993.
DOI:10.1111/j.1365-313X.2006.02666.xURLPMID:16507088 [本文引用: 2]
Arabidopsis thaliana has nine genes that constitute a family of putative carotenoid cleavage dioxygenases (CCDs). While five members of the family are believed to be involved in synthesis of the phytohormone abscisic acid, the functions of the other four enzymes are less clear. Recently two of the enzymes, CCD7/MAX3 and CCD8/MAX4, have been implicated in synthesis of a novel apocarotenoid hormone that controls lateral shoot growth. Here, we report on the molecular and genetic interactions between CCD1, CCD7/MAX3 and CCD8/MAX4. CCD1 distinguishes itself from other reported CCDs as being the only member not targeted to the plastid. Unlike ccd7/max3 and ccd8/max4, both characterized as having highly branched phenotypes, ccd1 loss-of-function mutants are indistinguishable from wild-type plants. Thus, even though CCD1 has similar enzymatic activity to CCD7/MAX3, it does not have a role in synthesis of the lateral shoot growth inhibitor. Rather, it may have a role in synthesis of apocarotenoid flavor and aroma volatiles, especially in maturing seeds where loss of function leads to significantly higher carotenoid levels.

[7] TAN B C, JOSEPH L M, DENG W T, LIU L J, LI Q B, CLINE K, MCCARTY D R . Molecular characterization of the Arabidopsis 9-cis epoxycarotenoid dioxygenase gene family
The Plant Journal, 2003,35(1):44-56.
DOI:10.1046/j.1365-313x.2003.01786.xURLPMID:12834401 [本文引用: 2]
A key regulated step in abscisic acid (ABA) biosynthesis in plants is catalyzed by 9-cis epoxycarotenoid dioxygenase (NCED), which cleaves 9-cis xanthophylls to xanthoxin, a precursor of ABA. In Arabidopsis, ABA biosynthesis is controlled by a small family of NCED genes. Nine carotenoid cleavage dioxygenase (CCD) genes have been identified in the complete genome sequence. Of these, five AtNCEDs (2, 3, 5, 6, and 9) have been cloned and studied for expression and subcellular localization. Although all five AtNCEDs are targeted to plastids, they differ in binding activity of the thylakoid membrane. AtNCED2, AtNCED3, and AtNCED6 are found in both stroma and thylakoid membrane-bound compartments. AtNCED5 is exclusively bound to thylakoids, whereas AtNCED9 remains soluble in stroma. A quantitative real-time PCR analysis and histochemical staining of promoter::GUS activity in transgenic Arabidopsis revealed a complex pattern of localized NCED expression in well-watered plants during development. AtNCED2 and AtNCED3 account for the NCED activity in roots, with localized expression in root tips, pericycle, and cortex cells at the base of lateral roots. Localized AtNCED2 and AtNCED3 expression in pericycle cells is an early marker of lateral initiation sites. AtNCED5, AtNCED6, AtNCED3, and AtNCED2 are expressed in flowers with very high AtNCED6::GUS activity occurring in pollen. AtNCED5::GUS, and to lesser degrees, AtNCED2::GUS and AtNCED3::GUS are expressed in developing anthers. AtNCED5, AtNCED6, AtNCED9, and AtNCED3 contribute to expression in developing seeds with high levels of AtNCED6 present at an early stage. GUS analysis indicates that AtNCED3 expression is confined to the base of the seed, whereas AtNCED5 and AtNCED6 are expressed throughout the seed. Consistent with the studies conducted by Iuchi and his colleagues in 2001, AtNCED3 is the major stress-induced NCED in leaves. Our results indicate that developmental control of ABA synthesis involves localized patterns of AtNCED gene expression. In addition, differential membrane-binding capacity of AtNCEDs is a potential means of post-translational regulation of NCED activity.

[8] YAHYAA M, BAR E, DUBEY N K, MEIR A, DAVIDOVICH R R, HIRSCHBERG J, ALY R, THOLL D, SIMON P W, TADMOR Y, LEWINSOHN E, IBDAH M . Formation of norisoprenoid flavor compounds in carrot (Daucus carota L.) roots: Characterization of a cyclic-specific carotenoid cleavage dioxygenase 1 gene
Journal of Agricultural & Food Chemistry, 2013,61(50):12244-12252.
DOI:10.1021/jf404085kURLPMID:24289159 [本文引用: 1]
Carotenoids are isoprenoid pigments that upon oxidative cleavage lead to the production of norisoprenoids that have profound effect on flavor and aromas of agricultural products. The biosynthetic pathway to norisoprenoids in carrots (Daucus carota L.) is still largely unknown. We found the volatile norisoprenoids farnesylacetone, α-ionone, and β-ionone accumulated in Nairobi, Rothild, and Purple Haze cultivars but not in Yellowstone and Creme de Lite in a pattern reflecting their carotenoid content. A cDNA encoding a protein with carotenoid cleavage dioxygenase activity, DcCCD1, was identified in carrot and was overexpressed in Escherichia coli strains previously engineered to produce different carotenoids. The recombinant DcCCD1 enzyme cleaves cyclic carotenes to generate α- and β-ionone. No cleavage products were found when DcCCD1 was co-expressed in E. coli strains accumulating non-cyclic carotenoids, such as phytoene or lycopene. Our results suggest a role for DcCCD1 in carrot flavor biosynthesis.

[9] BALDERMANN S, KATO M, KUROSAWA M, KUROBAYASHI Y, FUJITA A, FLEISCHMANN P, WATANABE N . Functional characterization of a carotenoid cleavage dioxygenase 1 and its relation to the carotenoid accumulation and volatile emission during the floral development of Osmanthus fragrans Lour
Journal of Experimental Botany, 2010,61(11):2967-2977.
DOI:10.1093/jxb/erq123URLPMID:20478967 [本文引用: 1]
Carotenoids are the precursors of important fragrance compounds in flowers of Osmanthus fragrans Lour. var. aurantiacus, which exhibit the highest diversity of carotenoid-derived volatiles among the flowering plants investigated. A cDNA encoding a carotenoid cleavage enzyme, OfCCD1, was identified from transcripts isolated from flowers of O. fragrans Lour. It is shown that the recombinant enzymes cleave carotenes to produce alpha-ionone and beta-ionone in in vitro assays. It was also found that carotenoid content, volatile emissions, and OfCCD1 transcript levels are subjected to photorhythmic changes and principally increased during daylight hours. At the times when OfCCD1 transcript levels reached their maxima, the carotenoid content remained low or slightly decreased. The emission of ionones was also higher during the day; however, emissions decreased at a lower rate than the transcript levels. Moreover, carotenoid content increased from the first to the second day, whereas the volatile release decreased, and the OfCCD1 transcript levels displayed steady-state oscillations, suggesting that the substrate availability in the cellular compartments is changing or other regulatory factors are involved in volatile norisoprenoid formation. Furthermore, the sensory evaluation of the aroma of the model mixtures suggests that the proportionally higher contribution of alpha-ionone and beta-ionone to total volatile emissions in the evening is probably the reason for the increased perception by humans of the scent emission of Osmanthus flowers.

[10] SUN Z K, HANS J, WALTER M H, MATUSOVA R, BEEKWILDER J, VERSTAPPEN F W A, ZHAO M, VAN ECHTELT E, STRACK D, BISSELING T, BOUWMEESTER H J . Cloning and characterisation of a maize carotenoid cleavage dioxygenase (ZmCCD1) and its involvement in the biosynthesis of apocarotenoids with various roles in mutualistic and parasitic interactions
Planta, 2008,228(5):789-807.
DOI:10.1007/s00425-008-0781-6URLPMID:18716794 [本文引用: 1]
Colonisation of maize roots by arbuscular mycorrhizal (AM) fungi leads to the accumulation of apocarotenoids (cyclohexenone and mycorradicin derivatives). Other root apocarotenoids (strigolactones) are involved in signalling during early steps of the AM symbiosis but also in stimulation of germination of parasitic plant seeds. Both apocarotenoid classes are predicted to originate from cleavage of a carotenoid substrate by a carotenoid cleavage dioxygenase (CCD), but the precursors and cleavage enzymes are unknown. A Zea mays CCD (ZmCCD1) was cloned by RT-PCR and characterised by expression in carotenoid accumulating E. coli strains and analysis of cleavage products using GC-MS. ZmCCD1 efficiently cleaves carotenoids at the 9, 10 position and displays 78% amino acid identity to Arabidopsis thaliana CCD1 having similar properties. ZmCCD1 transcript levels were shown to be elevated upon root colonisation by AM fungi. Mycorrhization led to a decrease in seed germination of the parasitic plant Striga hermonthica as examined in a bioassay. ZmCCD1 is proposed to be involved in cyclohexenone and mycorradicin formation in mycorrhizal maize roots but not in strigolactone formation.

[11] ALDER A, JAMIL M, MARZORATI M, BRUNO M, VERMATHEN M, BIGLER P, GHISLA S, BOUWMEESTER H, BEYER P, AL-BABILI S . The path from β-carotene to carlactone, a strigolactone- like plant hormone
Science, 2012,335(6074):1348-1351.
DOI:10.1126/science.1218094URLPMID:22422982 [本文引用: 1]
Strigolactones, phytohormones with diverse signaling activities, have a common structure consisting of two lactones connected by an enol-ether bridge. Strigolactones derive from carotenoids via a pathway involving the carotenoid cleavage dioxygenases 7 and 8 (CCD7 and CCD8) and the iron-binding protein D27. We show that D27 is a β-carotene isomerase that converts all-trans-β-carotene into 9-cis-β-carotene, which is cleaved by CCD7 into a 9-cis-configured aldehyde. CCD8 incorporates three oxygens into 9-cis-β-apo-10'-carotenal and performs molecular rearrangement, linking carotenoids with strigolactones and producing carlactone, a compound with strigolactone-like biological activities. Knowledge of the structure of carlactone will be crucial for understanding the biology of strigolactones and may have applications in combating parasitic weeds.

[12] BOOKER J, AULDRIDGE M, WILLS S, MCCARTY D, KLEE H, LEYSER O . MAX3/CCD7 is a carotenoid cleavage dioxygenase required for the synthesis of a novel plant signaling molecule
Current Biology, 2004,14(14):1232-1238.
DOI:10.1016/j.cub.2004.06.061URLPMID:15268852 [本文引用: 1]
Plant development is exquisitely environmentally sensitive, with plant hormones acting as long-range signals that integrate developmental, genetic, and environmental inputs to regulate development. A good example of this is in the control of shoot branching, where wide variation in plant form can be generated in a single genotype in response to environmental and developmental cues.

[13] SCHWARTZ S H, TAN B C, MCCARTY D R, WELCH W , ZEEVAART J A D. Substrate specificity and kinetics for VP14, a carotenoid cleavage dioxygenase in the ABA biosynthetic pathway
Biochimica et Biophysica Acta (BBA)-General Subjects, 2003,1619(1):9-14.
DOI:10.1016/j.bbagen.2017.02.015URLPMID:28216027 [本文引用: 1]

[14] 陈唯, 曾晓贤, 谢楚萍, 田长恩, 周玉萍 . 植物内源ABA水平的动态调控机制
植物学报, 2019,54(6):677-687.
[本文引用: 1]

CHEN W, ZENG X X, XIE C P, TIAN C E, ZHOU Y P . The dynamic regulation mechanism of the Endo-genous ABA in Plant
Chinese Bulletin of Botany, 2019,54(6):677-687. (in Chinese)
[本文引用: 1]

[15] VALLABHANENI R, BRADBURY L M T, WURTZEL E T . The carotenoid dioxygenase gene family in maize, sorghum, and rice
Archives of Biochemistry and Biophysics, 2010,504(1):104-111.
DOI:10.1016/j.abb.2010.07.019URLPMID:20670614 [本文引用: 1]
Carotenoids and their apocarotenoid derivatives play essential physiological and developmental roles and provide plants tolerance to a variety of stresses. Carotenoid cleavage dioxygenases mediate the degradation of carotenoids to apocarotenoids. A better understanding of biosynthesis vs. degradation could be useful for controlling carotenoid levels leading to improved plant fitness and/or enhanced content of nutritionally valuable carotenoids. The Poaceae (grass) plant family contains many crops of agronomic value. Therefore this study focused on characterizing the carotenoid dioxygenase gene family in the grass species maize, rice, and sorghum with comparison made to newly identified gene families in two non-seed plants as well as an alga and previously identified eudicot genes. Genome analysis was used to map grass genes encoding the carotenoid dioxygenases to chromosome locations. Sequences of encoded proteins were phylogenetically compared. CCD8b was identified as a new class of cleavage dioxygenases that may play a specialized role in apocarotenoid biogenesis. A simple PCR assay was developed to measure CCD1 gene copy number which is known to vary in maize. Using a panel of maize inbred lines varying in carotenoid content, linear regression analysis revealed a statistically significant negative correlation between copy number of CCD1 and carotenoid content, an effect likely mediated through the resulting elevated levels of endosperm CCD1 transcripts in high copy number lines. The PCR assay adds to a growing toolbox for metabolic engineering of maize endosperm carotenoids. This new tool can be used to select maize lines that are less likely to promote endosperm carotenoid degradation, thus predicting optimal results in metabolic engineering of endosperm provitamin A and/or nonprovitamin A carotenoids.

[16] WEI Y P, WAN H J, WU Z M, WANG R Q, RUAN M Y, YE Q J, LI Z M, ZHOU G Z, YAO Z P, YANG Y J . A comprehensive analysis of carotenoid cleavage dioxygenases genes in Solanum lycopersicum
Plant Molecular Biology Reporter, 2016,34(2):512-523.
[本文引用: 2]

[17] LASHBROOKE J G, YOUNG P R, DOCKRALL S J, VASANTH K, VIVIER M A . Functional characterisation of three members of the Vitis vinifera L. carotenoid cleavage dioxygenase gene family
BMC Plant Biology, 2013,13(1):156.
DOI:10.1186/1471-2229-13-156URLPMID:24106789 [本文引用: 3]
In plants, carotenoids serve as the precursors to C13-norisoprenoids, a group of apocarotenoid compounds with diverse biological functions. Enzymatic cleavage of carotenoids catalysed by members of the carotenoid cleavage dioxygenase (CCD) family has been shown to produce a number of industrially important volatile flavour and aroma apocarotenoids including β-ionone, geranylacetone, pseudoionone, α-ionone and 3-hydroxy-β-ionone in a range of plant species. Apocarotenoids contribute to the floral and fruity attributes of many wine cultivars and are thereby, at least partly, responsible for the &amp;quot;varietal character&amp;quot;. Despite their importance in grapes and wine; carotenoid cleavage activity has only been described for VvCCD1 and the mechanism(s) and regulation of carotenoid catabolism remains largely unknown.

[18] RUBIO-MORAGA A, AHRAZEM O, PéREZ-CLEMENTE R M, GóMEZ-CADENAS A, YONEYAMA K, LóPEZ-RáEZ J A, MOLINA R V, GóMEZ-GóMEZ L . Apical dominance in saffron and the involvement of the branching enzymes CCD7 and CCD8 in the control of bud sprouting
BMC Plant Biology, 2014,14(1):171.
DOI:10.1186/1471-2229-14-171URLPMID:24947472 [本文引用: 3]
In saffron (Crocus sativus), new corms develop at the base of every shoot developed from the maternal corm, a globular underground storage stem. Since the degree of bud sprouts influences the number and size of new corms, and strigolactones (SLs) suppress growth of pre-formed axillary bud, it was considered appropriate to investigate SL involvement in physiology and molecular biology in saffron. We focused on two of the genes within the SL pathway, CCD7 and CCD8, encoding carotenoid cleavage enzymes required for the production of SLs.

[19] OHMIYA A, KISHIMOTO S, AIDA R, YOSHIOKA S, SUMITOMO K . Carotenoid cleavage dioxygenase ( CmCCD4a) contributes to white color formation in Chrysanthemum petals
Plant Physiology, 2006,142(3):1193-1201.
DOI:10.1104/pp.106.087130URLPMID:16980560 [本文引用: 1]
The white petals of chrysanthemum (Chrysanthemum morifolium Ramat.) are believed to contain a factor that inhibits the accumulation of carotenoids. To find this factor, we performed polymerase chain reaction-Select subtraction screening and obtained a clone expressed differentially in white and yellow petals. The deduced amino acid sequence of the protein (designated CmCCD4a) encoded by the clone was highly homologous to the sequence of carotenoid cleavage dioxygenase. All the white-flowered chrysanthemum cultivars tested showed high levels of CmCCD4a transcript in their petals, whereas most of the yellow-flowered cultivars showed extremely low levels. Expression of CmCCD4a was strictly limited to flower petals and was not detected in other organs, such as the root, stem, or leaf. White petals turned yellow after the RNAi construct of CmCCD4a was introduced. These results indicate that in white petals of chrysanthemums, carotenoids are synthesized but are subsequently degraded into colorless compounds, which results in the white color.

[20] RODRIGO M J, ALQUéZAR B, ALóS E, MEDINA V, CARMONA L, BRUNO M, AL-BABILI S, ZACARíAS L . A novel carotenoid cleavage activity involved in the biosynthesis of Citrus fruit-specific apocarotenoid pigments
Journal of Experimental Botany, 2013,64(14):4461-4478.
DOI:10.1093/jxb/ert260URLPMID:24006419 [本文引用: 1]
Citrus is the first tree crop in terms of fruit production. The colour of Citrus fruit is one of the main quality attributes, caused by the accumulation of carotenoids and their derivative C30 apocarotenoids, mainly β-citraurin (3-hydroxy-β-apo-8'-carotenal), which provide an attractive orange-reddish tint to the peel of oranges and Mandarins. Though carotenoid biosynthesis and its regulation have been extensively studied in Citrus fruits, little is known about the formation of C30 apocarotenoids. The aim of this study was to the identify carotenoid cleavage enzyme(s) [CCD(s)] involved in the peel-specific C30 apocarotenoids. In silico data mining revealed a new family of five CCD4-type genes in Citrus. One gene of this family, CCD4b1, was expressed in reproductive and vegetative tissues of different Citrus species in a pattern correlating with the accumulation of C30 apocarotenoids. Moreover, developmental processes and treatments which alter Citrus fruit peel pigmentation led to changes of β-citraurin content and CCD4b1 transcript levels. These results point to the involvement of CCD4b1 in β-citraurin formation and indicate that the accumulation of this compound is determined by the availability of the presumed precursors zeaxanthin and β-cryptoxanthin. Functional analysis of CCD4b1 by in vitro assays unequivocally demonstrated the asymmetric cleavage activity at the 7',8' double bond in zeaxanthin and β-cryptoxanthin, confirming its role in C30 apocarotenoid biosynthesis. Thus, a novel plant carotenoid cleavage activity targeting the 7',8' double bond of cyclic C40 carotenoids has been identified. These results suggest that the presented enzyme is responsible for the biosynthesis of C30 apocarotenoids in Citrus which are key pigments in fruit coloration.

[21] 王莎莎, 栾雨婷, 徐昌杰 . 柑橘β-柠乌素积累及其调控研究进展
果树学报, 2018,35(6):760-768.
[本文引用: 4]

WANG S S, LUAN Y T, XU C J . Research progress in the regulation of β-citraurin accumulation in citrus fruits
Journal of Fruit Science, 2018,35(6):760-768. (in Chinese)
[本文引用: 4]

[22] ZHENG X J, ZHU K J, SUN Q, ZHANG W Y, WANG X, CAO H B, TAN M L, XIE Z Z, ZENG Y L, YE J L, CHAI L J, XU Q, PAN Z Y, XIAO S Y, FRASER P D, DENG X X . Natural variation in CCD4 promoter underpins species-specific evolution of red coloration in citrus peel
Molecular Plant, 2019,12(9):1294-1307.
DOI:10.1016/j.molp.2019.04.014URLPMID:31102783 [本文引用: 3]
Carotenoids and apocarotenoids act as phytohormones and volatile precursors that influence plant development and confer aesthetic and nutritional value critical to consumer preference. Citrus fruits display considerable natural variation in carotenoid and apocarotenoid pigments. In this study, using an integrated genetic approach we revealed that a 5' cis-regulatory change at CCD4b encoding CAROTENOID CLEAVAGE DIOXYGENASE 4b is a major genetic determinant of natural variation in C₃₀ apocarotenoids responsible for red coloration of citrus peel. Functional analyses demonstrated that in addition the known role in synthesizing β-citraurin, CCD4b is also responsible for the production of another important C₃₀ apocarotenoid pigment, β-citraurinene. Furthermore, analyses of the CCD4b promoter and transcripts from various citrus germplasm accessions established a tight correlation between the presence of a putative 5' cis-regulatory enhancer within an MITE transposon and the enhanced allelic expression of CCD4b in C₃₀ apocarotenoid-rich red-peeled accessions. Phylogenetic analysis provided further evidence that functional diversification of CCD4b and naturally occurring?variation of the CCD4b promoter resulted in the stepwise evolution of red peels in mandarins and their hybrids. Taken together, our findings provide new insights into the genetic and evolutionary basis of apocarotenoid diversity in plants, and would facilitate breeding efforts that aim to improve the nutritional and aesthetic value of citrus and perhaps other fruit crops.

[23] MINAMIKAWA M F, NONAKA K, KAMINUMA E, KAJIYA- KANEGAE H, ONOGI A, GOTO S, YOSHIOKA T, IMAI A, HAMADA H, HAYASHI T, MATSUMOTO S, KATAYOSE Y, TOYODA A, FUJIYAMA A, NAKAMURA Y, SHIMIZU T, IWATA H . Genome wide association study and genomic prediction in citrus: Potential of genomics-assisted breeding for fruit quality traits
Scientific Reports, 2017,7(1):4721.
DOI:10.1038/s41598-017-05100-xURLPMID:28680114 [本文引用: 2]
Novel genomics-based approaches such as genome-wide association studies (GWAS) and genomic selection (GS) are expected to be useful in fruit tree breeding, which requires much time from the cross to the release of a cultivar because of the long generation time. In this study, a citrus parental population (111 varieties) and a breeding population (676 individuals from 35 full-sib families) were genotyped for 1,841 single nucleotide polymorphisms (SNPs) and phenotyped for 17 fruit quality traits. GWAS power and prediction accuracy were increased by combining the parental and breeding populations. A multi-kernel model considering both additive and dominance effects improved prediction accuracy for acidity and juiciness, implying that the effects of both types are important for these traits. Genomic best linear unbiased prediction (GBLUP) with linear ridge kernel regression (RR) was more robust and accurate than GBLUP with non-linear Gaussian kernel regression (GAUSS) in the tails of the phenotypic distribution. The results of this study suggest that both GWAS and GS are effective for genetic improvement of citrus fruit traits. Furthermore, the data collected from breeding populations are beneficial for increasing the detection power of GWAS and the prediction accuracy of GS.

[24] GMITTER F G, CHEN C X, MACHADO M A, SOUZA A A, OLLITRAULT P, FROEHLICHER Y, SHIMIZU T . Citrus genomics
Tree Genetics & Genomes, 2012,8(3):611-626.
[本文引用: 1]

[25] BRUNO M, VERMATHEN M, ALDER A, WüST F, SCHAUB P, VAN DER STEEN R, BEYER P, GHISLA S, AL-BABILI S . Insights into the formation of carlactone from in-depth analysis of the CCD8 catalyzed reactions
FEBS Letters, 2017,591(5):792-800.
DOI:10.1002/1873-3468.12593URLPMID:28186640 [本文引用: 1]
Strigolactones are a new class of phytohormones synthesized from carotenoids via carlactone. The complex structure of carlactone is not easily deducible from its precursor, a cis-configured β-carotene cleavage product, and is thus formed via a poorly understood series of reactions and molecular rearrangements, all catalyzed by only one enzyme, the carotenoid cleavage dioxygenase 8 (CCD8). Moreover, the reactions leading to carlactone are expected to form a second, yet unidentified product. In this study, we used ¹³ C and ¹⁸ O-labeling to shed light on the reactions catalyzed by CCD8. The characterization of the resulting carlactone by LC-MS and NMR, and the identification of the assumed, less accessible second product allowed us to formulate a minimal reaction mechanism for carlactone generation.

[26] LEDGER S E, JANSSEN B J, KARUNAIRETNAM S, WANG T, SNOWDEN K C . Modified CAROTENOID CLEAVAGE DIOXYGENASE 8 expression correlates with altered branching in kiwifruit ( Actinidia chinensis)
The New Phytologist, 2010,188(3):803-813.
DOI:10.1111/j.1469-8137.2010.03394.xURLPMID:20659299 [本文引用: 1]
? CAROTENOID CLEAVAGE DIOXYGENASE (CCD) genes have been demonstrated to play an integral role in the control of branch development in model plants, including Arabidopsis, pea (Pisum sativum), petunia (Petunia hybrida) and rice (Oryza sativa). ? Actinidia chinensis is a woody perennial plant grown for commercial production of kiwifruit. CCD7 and CCD8 genes were isolated from A. chinensis and these genes are predominantly expressed in the roots of kiwifruit. AcCCD7 and AcCCD8 were able to complement the corresponding Arabidopsis mutants max3 and max4. The function of AcCCD8 in branch development was determined in transgenic kiwifruit plants containing an RNAi construct for AcCCD8. ? Reduction in expression of AcCCD8 correlated with an increase in branch development and delayed leaf senescence. ? The CCD pathway for control of branch development is conserved across a wide range of species, including kiwifruit, a woody perennial.

[27] VOGEL J T, WALTER M H, GIAVALISCO P, LYTOVCHENKO A, KOHLEN W, CHARNIKHOVA T, SIMKIN A J, GOULET C, STRACK D, BOUWMEESTER H J, FERNIE A R, KLEE H J . SlCCD7 controls strigolactone biosynthesis, shoot branching and mycorrhiza-induced apocarotenoid formation in tomato
The Plant Journal, 2010,61(2):300-311.
DOI:10.1111/j.1365-313X.2009.04056.xURLPMID:19845881 [本文引用: 1]
The regulation of shoot branching is an essential determinant of plant architecture, integrating multiple external and internal signals. One of the signaling pathways regulating branching involves the MAX (more axillary branches) genes. Two of the genes within this pathway, MAX3/CCD7 and MAX4/CCD8, encode carotenoid cleavage enzymes involved in generating a branch-inhibiting hormone, recently identified as strigolactone. Here, we report the cloning of SlCCD7 from tomato. As in other species, SlCCD7 encodes an enzyme capable of cleaving cyclic and acyclic carotenoids. However, the SlCCD7 protein has 30 additional amino acids of unknown function at its C terminus. Tomato plants expressing a SlCCD7 antisense construct display greatly increased branching. To reveal the underlying changes of this strong physiological phenotype, a metabolomic screen was conducted. With the exception of a reduction of stem amino acid content in the transgenic lines, no major changes were observed. In contrast, targeted analysis of the same plants revealed significantly decreased levels of strigolactone. There were no significant changes in root carotenoids, indicating that relatively little substrate is required to produce the bioactive strigolactones. The germination rate of Orobanche ramosa seeds was reduced by up to 90% on application of extract from the SlCCD7 antisense lines, compared with the wild type. Additionally, upon mycorrhizal colonization, C(13) cyclohexenone and C(14) mycorradicin apocarotenoid levels were greatly reduced in the roots of the antisense lines, implicating SlCCD7 in their biosynthesis. This work demonstrates the diverse roles of MAX3/CCD7 in strigolactone production, shoot branching, source-sink interactions and production of arbuscular mycorrhiza-induced apocarotenoids.

[28] KULKARNI K P, VISHWAKARMA C, SAHOO S P, LIMA J M, NATH M, DOKKU P, GACCHE R N, MOHAPATRA T, ROBIN S, SARLA N, SESHASHAYEE M, SINGH A K, SINGH K, SINGH N K, SHARMA R P . A substitution mutation in OsCCD7 cosegregates with dwarf and increased tillering phenotype in rice
Journal of Genetics, 2014,93(2):389-401.
DOI:10.1007/s12041-014-0389-5URLPMID:25189234 [本文引用: 1]
Dwarf plant height and tillering ability are two of the most important agronomic traits that determine the plant architecture, and have profound influence on grain yield in rice. To understand the molecular mechanism controlling these two traits, an EMS-induced recessive dwarf and increased tillering1 (dit1) mutant was characterized. The mutant showed proportionate reduction in each internode as compared to wild type revealing that it belonged to the category of dn-type of dwarf mutants. Besides, exogenous application of GA3 and 24-epibrassinolide, did not have any effect on the phenotype of the mutant. The gene was mapped on the long arm of chromosome 4, identified through positional candidate approach and verified by cosegregation analysis. It was found to encode carotenoid cleavage dioxygenase7 (CCD7) and identified as an allele of htd1. The mutant carried substitution of two nucleotides CC to AA in the sixth exon of the gene that resulted in substitution of serine by a stop codon in the mutant, and thus formation of a truncated protein, unlike amino acid substitution event in htd1. The new allele will facilitate further functional characterization of this gene, which may lead to unfolding of newer signalling pathways involving plant development and architecture.

[29] 王小龙, 刘凤之, 史祥宾, 王孝娣, 冀晓昊, 王志强, 王宝亮, 郑晓翠, 王海波 . 葡萄NCED基因家族进化及表达分析
植物学报, 2019,54(4):474-485.
[本文引用: 1]

WANG X L, LIU F Z, SHI X B, WANG X D, JI X H, WANG Z Q, WANG B L, ZHENG X C, WANG H B . Evolution and expression of NCED family genes in Vitis vinifera
Chinese Bulletin of Botany, 2019,54(4):474-485. (in Chinese)
[本文引用: 1]

[30] 巩檑, 宋继玲, 甘晓燕, 刘璇, 陈虞超, 郭志乾, 宋玉霞 . 模拟干旱胁迫下马铃薯StNCED1表达量及与ABA含量的相关性分析
植物遗传资源学报, 2018,19(3):561-567.
[本文引用: 1]

GONG L, SONG J L, GAN X Y, LIU X, CHEN Y C, GUO Z Q, SONG Y X . Correlation analysis of StNCED1 expression level and ABA content of potato under simulated drought stress
Journal of Plant Genetic Resources, 2018,19(3):561-567. (in Chinese)
[本文引用: 1]

[31] KATO M, MATSUMOTO H, IKOMA Y, OKUDA H, YANO M . The role of carotenoid cleavage dioxygenases in the regulation of carotenoid profiles during maturation in citrus fruit
Journal of Experimental Botany, 2006,57(10):2153-2164.
DOI:10.1093/jxb/erj172URLPMID:16714310 [本文引用: 1]
To investigate the relationship between a carotenoid profile and gene expression for carotenoid cleavage dioxygenases, three citrus varieties that exhibit different 9-cis-violaxanthin levels in their juice sacs, Satsuma mandarin (Citrus unshiu Marc.; a variety accumulating a low level of 9-cis-violaxanthin), Valencia orange (Citrus sinensis Osbeck; variety accumulating a high level of 9-cis-violaxanthin), and Lisbon lemon (Citrus limon Burm.f.; a variety accumulating an undetectable level of 9-cis-violaxanthin) were used. Three cDNAs (CitCCD1, CitNCED2, and CitNCED3) were cloned. The recombinant CitCCD1 protein cleaved beta-cryptoxanthin, zeaxanthin, and all-trans-violaxanthin at the 9-10 and 9'-10' positions and 9-cis-violaxanthin at the 9'-10' position. The recombinant CitNCED2 and CitNCED3 proteins cleaved 9-cis-violaxanthin at the 11-12 position to form xanthoxin, a precursor of abscisic acid (ABA). The gene expression of CitCCD1 increased in the flavedos and juice sacs of the three varieties during maturation. In Satsuma mandarin, the gene expression of CitNCED2 and CitNCED3 increased noticeably, accompanying a massive accumulation of ABA in the flavedo and juice sacs. In Valencia orange, the gene expression of CitNCED3 increased with a slight elevation of the ABA level in the flavedo, whereas neither the gene expression of CitNCED2 nor the ABA level increased noticeably in the juice sacs. In Lisbon lemon, the gene expression of CitNCED2 increased remarkably, accompanying increases in the ABA level in the flavedo and juice sacs. These results suggest that, in the juice sacs, the efficient cleavage reaction for ABA synthesis reduces the 9-cis-violaxanthin level in Satsuma mandarin and Lisbon lemon, whereas the low cleavage reaction maintains the predominant 9-cis-violaxanthin accumulation in Valencia orange.

[32] SONG M H, LIM S H, KIM J K, JUNG E S, JOHN K M M, YOU M K, AHN S N, LEE C H, HA S H . In planta cleavage of carotenoids by Arabidopsis carotenoid cleavage dioxygenase 4 in transgenic rice plants
Plant Biotechnology Reports, 2016,10(5):291-300.
DOI:10.1007/s11816-016-0405-8URL [本文引用: 1]

[33] URESHINO K, NAKAYAMA M, MIYAJIMA I . Contribution made by the carotenoid cleavage dioxygenase 4 gene to yellow colour fade in azalea petals
Euphytica, 2016,207(2):401-417.
[本文引用: 1]

[34] LIU H, KISHIMOTO S, YAMAMIZO C, FUKUTA N, OHMIYA A . Carotenoid accumulations and carotenogenic gene expressions in the petals of Eustoma grandiflorum
Plant Breeding, 2013,132(4):417-422.
[本文引用: 1]

[35] HAI N T L, MASUDA J I, MIYAJIMA I, THIEN N Q, MOJTAHEDI N, HIRAMATSU M, KIM J H, OKUBO H . Involvement of carotenoid cleavage dioxygenase 4 gene in tepal color change in Lilium brownii var
colchesteri. Journal of the Japanese Society for Horticultural Science, 2012,81(4):366-373.
[本文引用: 1]

[36] MA G, ZHANG L C, IIDA K, MADONO Y, YUNGYUEN W, YAHATA M, YAMAWAKI K, KATO M . Identification and quantitative analysis of β-cryptoxanthin and β-citraurin esters in Satsuma mandarin fruit during the ripening process
Food Chemistry, 2017,234(9):356-364.
DOI:10.1016/j.foodchem.2017.05.015URL [本文引用: 1]

[37] AGóCS A, NAGY V, SZABó Z, MáRK L, OHMACHT R, DELI J . Comparative study on the carotenoid composition of the peel and the pulp of different citrus species
Innovative Food Science and Emerging Technologies, 2007,8(3):390-394.
DOI:10.1016/j.ifset.2007.03.012URL [本文引用: 1]

[38] GONZáLEZ-VERDEJO C I, OBRERO á, ROMáN B, GóMEZ P . Expression profile of carotenoid cleavage dioxygenase genes in summer squash ( Cucurbita pepo. L)
Plant Foods for Human Nutrition, 2015,70(2):200-206.
DOI:10.1007/s11130-015-0482-9URLPMID:25861766 [本文引用: 1]
Carotenoids are important dietary components that can be found in vegetable crops. The accumulation of these compounds in fruit and vegetables is altered by the activity of carotenoid cleavage dioxygenases (CCDs) enzymes that produce their degradation. The aim of this work was to study the possible implication of CCD genes in preventing carotenoid storage in the horticultural crop summer squash (Cucurbita pepo L.). The relationship between the presence of these compounds and gene expression for CCDs was studied in three varieties showing different peel and flesh colour. Expression analysis for the CCD genes CpNCED1, CpNCED2, CpNCED3, CpNCED9, CpCCD1, CpCCD4a, CpCCD4b and CpCCD8 was carried out on different organs and at several fruit developmental stages. The results showed that the CpCCD4a and CpCCD4b genes were highly expressed in the variety with lowest carotenoid content suggesting a putative role in carotenoid accumulation pattern in summer squash fruit.

[39] BAI S L, PHAM A T, MIHO T, HIDEAKI Y, AKEMI O, CHIHIRO Y, TAKAYA M . Knockdown of carotenoid cleavage dioxygenase 4 (CCD4) via virus-induced gene silencing confers yellow coloration in peach fruit: Evaluation of gene function related to fruit traits
Plant Molecular Biology Reporter, 2016,34(1):257-264.
DOI:10.1007/s11105-015-0920-8URL [本文引用: 1]

[40] PHADUNGSAWAT B, WATANABE K, MIZUNO S, KANEKATSU M, SUZUKI S . Expression of CCD4 gene involved in carotenoid degradation in yellow-flowered Petunia×hybrida
Scientia Horticulturae, 2019:108916.
[本文引用: 1]

[41] ZHANG B, HAN F Q, CUI H L, LI X, REN W J, FANG Z Y, YANG Z Y, ZHUANG M, LU H H, LIU Y M . Insertion of a CACTA-like transposable element disrupts the function of the BoCCD4 gene in yellow-petal Chinese kale
Molecular Breeding, 2019,39(9):130.
DOI:10.1007/s11032-019-1008-1URL [本文引用: 1]

[42] 陶俊 . 柑橘果实类胡萝卜素形成及调控的生理机制研究
[D]. 杭州: 浙江大学, 2002.
[本文引用: 1]

TAO J . Physiological studies on carotenoid formation and regulation in citrus fruit
[D]. Hangzhou: Zhejiang University, 2002. (in Chinese)
[本文引用: 1]

[43] 谭萍, 谭奋勇, 王祖泽, 杨再英, 谭功亮 . 惠水独特优良品种─牛肉红金桔
江西柑桔科技, 1994(2):26-27.
[本文引用: 1]

TAN P, TAN F Y, WANG Z Z, YANG Z Y, TAN G L . Unique and excellent variety of Hui Shui-Niu rou hong ju
Jiangxi Citrus Technology, 1994(2):26-27. (in Chinese)
[本文引用: 1]