郭书磊^1,2, 鲁晓民¹, 齐建双¹, 魏良明¹, 张新¹, 韩小花¹, 岳润清¹, 王振华^,1, 铁双贵^,1, 陈彦惠^,21 河南省农业科学院粮食作物研究所/河南省玉米生物学重点实验室,郑州 450002
2 河南农业大学农学院,郑州 450046

Explore Regulatory Genes Related to Maize Leaf Morphogenesis Using RNA-Seq

GUO ShuLei^1,2, LU XiaoMin¹, QI JianShuang¹, WEI LiangMing¹, ZHANG Xin¹, HAN XiaoHua¹, YUE RunQing¹, WANG ZhenHua^,1, TIE ShuangGui^,1, CHEN YanHui^,2 1 Cereal Crops Institute, Henan Academy of Agricultural Sciences/Henan Provincial Key Lab of Maize Biology, Zhengzhou 450002
2 College of Agronomy, Henan Agricultural University, Zhengzhou 450046

通讯作者: 王振华,E-mail：wzh201@126.com铁双贵,E-mail：tieshuangg@126.com陈彦惠,E-mail：chy9890@163.com

责任编辑: 李莉
收稿日期:2019-05-20接受日期:2019-07-11网络出版日期:2020-01-01

基金资助:

中国博士后科学基金.2017M612404 河南省科技攻关计划项目.182102110122 河南省农业科学院优秀青年科技基金

Received:2019-05-20Accepted:2019-07-11Online:2020-01-01
作者简介 About authors
郭书磊,E-mail：guosl1309@163.com。












摘要
【目的】叶片宽度和长度等叶形特性是决定植株形态,进而影响种植密度的重要农艺性状,通过转录组测序技术筛选并挖掘玉米叶片形态建成相关的代谢路径及调控基因,为深入认识叶片发育的分子机理和鉴定叶宽、叶长候选基因奠定基础。【方法】以极端窄叶自交系NL409和宽叶自交系WB665为材料,利用RNA-Seq技术鉴定7叶期第七片叶近基部的差异表达基因（DEGs）,通过生物信息学分析,筛选与叶片发育密切相关的代谢通路,利用qRT-PCR验证不同激素路径叶形相关基因的表达结果,并结合启动子区域的序列差异挖掘叶形功能基因。【结果】分析对照（WB665）和样品（NL409）高通量测序结果,在叶宽形成关键部位共筛选出5 199个DEGs,其中,2 264（43.55%）个基因表达上调,2 935（56.45%）个基因下调表达,下调基因明显多于上调基因;GO功能富集分析表明,差异基因主要富集在细胞膜相关的细胞组分中,涉及代谢过程和细胞响应刺激;KEGG富集分析表明,差异基因主要参与到核糖体、植物激素信号转导、苯丙烷类代谢、乙醛酸和二羧酸代谢等过程,其中核糖体、植物激素信号转导、鞘脂类代谢下调表达基因较多的路径与叶片发育密切相关。核糖体路径富集到多个PRS（PRESSED FLOWER）基因,分析发现PRS13（PFL2）可能在调控窄叶发育过程中发挥重要作用。鞘脂代谢路径富集的基因几乎全部下调表达,引起抑制叶片发育的AP1（APETALA1）类和MAPK（Mitogen-Activated Protein Kinase）类基因上调,以及促进叶片发育的LFY（LEAFY）下调,与窄叶发育受抑制的表型一致。植物激素信号转导路径富集到的油菜素内酯（BR）响应基因和赤霉素（GA）代谢基因下调,细胞分裂素（CTK）和大部分生长素（Auxin）响应基因上调,与窄叶中DELLA蛋白基因上调表达,抑制GA并促进CTK基因表达的作用模式一致。通过qRT-PCR对18个叶片发育相关基因进行分析,结果表明,其表达趋势与转录组结果一致,分析发现BR相关的ROT3、Auxin相关的NAL7-like、AGO7-like以及TCP类转录因子CYC/TB1等基因与窄叶的形成密切相关。【结论】明确了一些与玉米叶片发育密切相关的代谢路径,还发现植物激素间的动态平衡对叶片发育有着重要影响,尤其是生长素与油菜素内酯、细胞分裂素与赤霉素之间的相互作用对调控叶片形态可能发挥重要作用。
关键词： 玉米;叶宽;叶长;RNA-Seq;形态建成;调控基因

Abstract
【Objective】Leaf shape characteristics are one of important agronomic traits that determine plant morphology and affect planting density. However, the molecular mechanism related to leaf shape remain unknown in maize. Here, transcriptome sequencing technology was used to screen and explore genome-wide analysis of regulatory genes and metabolic pathways involved in leaf morphogenesis. This study will lay the foundation for further understanding the regulator mechanism of leaf development in plant and identifying candidate genes of leaf shapes, such as leaf width and leaf length.【Method】 Extreme narrow-leaf inbred line NL409 and wide-leaf line WB665 were selected as the experimental materials. By RNA-Seq technology, the differentially expressed genes (DEGs) of the seventh leaf base between these two lines were identified during the 7th leaf stage. Furthermore, metabolic pathways closely related to leaf development were also analyzed using a series of bioinformatics analysis. qRT-PCR was used to validate the expression level of DEGs in different hormone pathways, and the further promoter analysis were performed to explore leaf-shape functional genes.【Result】By analyzing the high-throughput sequencing in WB665 and NL409, a total of 5 199 DEGs were obtained at the primary section of leaf width formation. Of which, 2 264 (43.55%) genes were up-regulated, whereas down-regulated genes were significantly more than up-regulated genes with 2 935 (56.45%) decreased genes. GO enrichment analysis showed that these DEGs were mainly enriched in cell membrane-associated function terms of cellular components, including metabolic process and cell stimulus response. KEGG enrichment analysis showed that these DEGs were mainly involved in ribosome, plant hormone signal transduction, sphingolipid metabolism pathways, phenylpropanoid biosynthesis, glyoxylate and dicarboxylate metabolism and other processes. among which ribosome, plant hormone signal transduction, sphingolipid metabolism pathways with more down-regulated genes were closely related to leaf development. One of PRS (PRESSED FLOWER) family genes, which were enriched in the ribosomal pathway in this study, PRS13 (PFL2) was identified to participate in regulating the development of narrow leaves. The expression pattern of genes enriched in sphingolipid metabolism pathway and its related MAP kinase, AP1-like, and LFY-like were consistent with the result of the inhibited development of narrow leaves. Notably, all of BR (Brassinosteroid) response genes and most of GA (Gibberellin) metabolic genes were down-regulated in plant hormone signal transduction pathway, while the expression level of all the CTK (Cytokinine) response genes and Auxin genes are mostly increased. The action of up-regulated expression of DELLA protein gene affecting the GA and CTK pathways was consistent with the phenotypic result of narrow leaves. Eighteen genes were validated by qRT-PCR. The result showed that the expression trend was consistent with the transcriptome data. Moreover, the BR-related ROT3, auxin-related NAL7-like, AGO7-like and TCP-like transcription factors CYC/TB1 were identified to be closely associated with the formation of narrow leaves.【Conclusion】Summarily, this study unveils several metabolic pathways closely related to leaf development in maize, and find the dynamic balance between plant hormones plays an important role in leaf development, especially the interaction between Auxin and BR as well as CTK and GA.
Keywords：maize (Zea mays);leaf width;leaf length;RNA-Seq;morphogenesis;regulatory gene


PDF (1786KB)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
郭书磊, 鲁晓民, 齐建双, 魏良明, 张新, 韩小花, 岳润清, 王振华, 铁双贵, 陈彦惠. 利用RNA-Seq发掘玉米叶片形态建成相关的调控基因[J]. 中国农业科学, 2020, 53(1): 1-17 doi:10.3864/j.issn.0578-1752.2020.01.001
GUO ShuLei, LU XiaoMin, QI JianShuang, WEI LiangMing, ZHANG Xin, HAN XiaoHua, YUE RunQing, WANG ZhenHua, TIE ShuangGui, CHEN YanHui. Explore Regulatory Genes Related to Maize Leaf Morphogenesis Using RNA-Seq[J]. Scientia Acricultura Sinica, 2020, 53(1): 1-17 doi:10.3864/j.issn.0578-1752.2020.01.001

0 引言

【研究意义】玉米作为增产潜力巨大的粮食作物,对于保障粮食安全,促进农业发展具有重要作用。近年来,中国玉米的种植密度维持在4.8—6.77万株/hm²,与美国8.5—10.95万株/hm²的种植密度存在显著差距,提高玉米的耐密植特性,成为实现中国玉米高产突破的重要措施^[1,2]。叶片是玉米进行光合作用和抗逆作用的主要营养器官,是耐密植理想株型的关键组成因素,对于植株干物质的积累及抗逆性有非常重要的影响。对于大面积密植的农作物,高密度条件下容易出现避荫综合症（shade avoidance syndrome,SAS）,迫使群体内个体将能量重新分配,用于叶片和植株伸长生长,从而获得更多的阳光,尽管避荫反应对单个植株的适应与生存是有利的,但是由于避荫反应会削弱植株叶片和储藏器官的发育^[3],对提高大面积种植的作物产量是不利的。高密度条件下,叶片宽大的群体冠层间通风透气性差,降低植株的抗病性,加剧病虫害的发生,导致生物量和籽粒产量降低。玉米耐密植株型的合理叶宽、叶长有利于改良理想株型,减少植株的避荫反应,使个体间竞争最小化,对提高种植密度具有重要的促进作用。叶宽、叶长等叶形特性是决定植株形态进而影响种植密度的重要农艺性状,因此,选育适宜高密度种植的合理叶宽、叶长玉米,是提高玉米产量的重要技术手段之一。虽然有大量研究揭示了玉米叶发育及形态建成的遗传基础,但是鲜有关于叶宽、叶长QTL克隆及调控网络的研究结果^[4],利用转录组（RNA-seq）筛选与叶宽、叶长发育相关的调控基因,并发掘其可能的调控路径,对叶宽、叶长主效基因研究及耐密株型设计育种具有重要意义。【前人研究进展】叶片的发育起始于茎顶端分生组织（shoot apical meristem,SAM）周边的初始细胞,主要包括叶原基的形成和叶极性轴向的建立^[5]。叶原基是由顶端分生组织中心“干细胞”在生长素诱导下形成成簇分布的外缘叶原初生细胞;叶原基形成后在自身遗传机制和环境因子作用下,建立基-顶轴（叶的基部-顶端）、腹-背轴（茎的近轴面-远轴面）、中-边轴（叶的主脉-边缘）3个极性轴向,这些过程中的发育异常将直接或间接影响叶片的发育和形态建成。玉米叶片的发育和形态建成是由多基因调控的复杂过程,主要由遗传因素控制,同时受环境影响,叶宽的遗传相比其他叶形性状受环境影响较小,而且其具有易观测的线性形态,更有利于研究叶片发育过程的形态变化和细胞学特征,是研究叶宽最为理想的模式植物^[4]。关于拟南芥、水稻、金鱼草等植物叶片发育的研究,取得了一系列重要进展,深入解析了叶片发育的调控机理,而玉米叶宽、叶长的研究多为遗传分析及QTL定位的报道,只有少数文献关于叶发育基因lg1、lg2、ns1、ns2、knotted1、rgd1/lbl1、rgd2、mwp1、AN3/GIF1和ZmGRF10遗传机理的分析^{[6,7,8,9,10,11,12,13,14]},揭示了叶宽、叶长发育形成的部分分子机理。高通量转录组测序技术,不仅能够检测全基因组所有基因的转录信息,还可以获得特定时期某一生物学过程显著富集的基因,以及特定组织或细胞中的多种转录本和优势表达基因,对研究特定组织的生物学功能具有重要的指导意义,利用转录组发掘玉米叶片发育相关基因,为叶片遗传机理的解析提供了新途径^[15,16]。【本研究切入点】关于叶宽、叶长等叶形主效基因克隆的研究相对有限,已有研究仅揭示了叶形发育的部分分子机理,叶宽、叶长候选基因及其调控路径有待进一步解析。【拟解决的关键问题】本研究通过RNA-seq测序探讨叶宽差异极显著自交系的转录表达特性,以期获得与叶形发育相关的关键基因及转录调控网络,更好地理解玉米叶片发育的分子机理,增加对窄叶形成机制的认识,进而为叶形候选基因的克隆及耐密分子育种提供参考。

1 材料与方法

1.1 植物材料

2017年夏极端窄叶自交系NL409（样品）和宽叶自交系WB665（对照）种植于河南省农业科学院现代农业科技示范基地,在田间正常生长至7叶期时（对生的叶耳包裹着新鲜的幼茎即将打开）,取第7片叶近基部叶片（近基部1/3叶长处,切取2.0 cm）,为了消除个体间差异,将相同基因型的材料3株混合取样,每个材料取3次生物学重复,样品经液氮速冻后-80℃保存,用于RNA提取。极端窄叶自交系NL409是欧洲杂交种连续自交选育而来,宽叶自交系WB665是昌7-2改良系,两者亲缘关系较远。

1.2 RNA样品的提取及转录组测序

委托北京安诺优达基因科技有限公司完成RNA提取、文库构建和转录组测序。首先,采用酚/氯仿法提取Total RNA,通过NanoDrop 2000微量分光光度计、Agilent 2100 Bioanalyzer和Agilent RNA 6000 Nano Kit检测RNA浓度、纯度和完整性;其次,检测合格样品利用Oligo（dT）富集mRNA,并通过Fragmentation Buffer使其短片段化,以短片段mRNA合成双链cDNA,纯化后进行末端修复、加碱基A、加测序接头处理;最后进行PCR扩增完成测序文库制备。采用Illumina NextSeq 550AR平台进行双末端测序,并对获得的原始序列（raw reads）进行过滤,去除含接头、低质量（Q<30）、含N比例大于5%的序列,得到高质量的Clean Reads用于后续分析。

1.3 基因表达数据分析

利用TopHat（v2.0.12）软件,将 6个样本过滤后的Clean Reads比对到B73基因组（AGPv3 版本）,通过FPKM（Fragments per Kilobase per Millon Mapped Fragments）对基因表达量进行标准化,以FPKM>1为表达标准,随后根据极端窄叶与宽叶的比较,用DEseq（|log2 FC（fold change）|≥1和P-value/FDR<0.05）确定差异表达基因（differentially expressed genes,DEGs）^[17]。利用Uniprot、Swissprot、COG、NR、GO和KEGG等数据库对DEGs进行功能注释,通过GO富集分析显示DEGs显著富集的功能分类^[18],用KEGG分析DEGs主要参与的代谢途径和信号通路（kyoto encyclopedia of genes and genomes,KEGG）。

1.4 实时荧光定量PCR（qRT-PCR）分析

选择与叶片发育相关的18个差异表达基因进行qRT-PCR验证。将转录组测序用到的RNA,通过反转录试剂盒（GoScript Reverse Transcription Kit购于promega生物技术有限公司）合成cDNA第一链,以其为模板,利用GoTaq qPCR Master Mix Kit（购于promega生物技术有限公司）在Bio-Rad CFX96实时荧光定量仪上进行qRT-PCR。以玉米中的18SrRNA作为内参基因,每个基因的定量分析设置3次生物学重复,3次技术重复,采用相对定量2^-ΔΔCt法分析结果^[19]。

2 结果

2.1 叶宽表型和RNA-Seq测序质量分析

极端窄叶自交系NL409和宽叶自交系WB665叶宽形态稳定时期（散粉后一周）的叶宽表型（图1）,以及第7叶期的叶宽达到极显著差异（表1）,在这两个时期,NL409的叶宽、叶长、叶面积均显著小于WB665。6个RNA-Seq文库原始测序过滤后分析结果（表2）,各样本生物学重复间的相关系数为0.984—0.995,其中约88%高质量序列被比对到外显子区域,表明测序结果良好,数据可用于进一步分析。

图1

新窗口打开|下载原图ZIP|生成PPT
图1自交系NL409（左）和WB665（右）稳定时期的叶片

Fig. 1Leaves of inbred lines NL409 (left) and WB665 (right) in the stable periods



Table 1
表1
表1自交系NL409和WB665第7叶期以及稳定时期的叶宽、叶长、叶面积
Table 1Leaf width, leaf length and leaf area of inbred lines NL409 and WB665 at the 7th leaf stage and in the stable period

材料1) Material	叶宽 Leaf width (cm)	叶长 Leaf length (cm)	叶面积 Leaf area (cm2)
NL409-7	2.49±0.13A	36.44±2.92A	66.12±8.59A
WB665-7	4.26±0.34B	29.67±1.67B	89.44±12.45B
NL409-S	4.67±0.27A	65.47±2.47A	223.31±13.11A
WB665-S	10.88±0.58B	59.89±2.57B	475.60±24.80B

¹⁾ 7表示第7叶期,S表示稳定时期。不同大写字母表示在P<0.01水平差异显著
¹⁾ 7 indicate the 7th leaf period, S indicate the stable period. Different uppercase letters indicate significant differences at P<0.01 levels

新窗口打开|下载CSV

Table 2
表2
表2样品reads分布情况
Table 2Overview of the sequencing reads

样品1) Sample	原始序列 Raw reads	过滤后序列 Clean reads	高质量序列 Clean reads (%)	比对到基因组序列 Mapped reads	锚定比例 Mapped (%)	单一位置序列 Unique mapped reads (%)	过滤后Q30比例 Q30 after filter (%)
NL409-71	48570184	47805714	98.43	38240306	79.99	77.12	93.19
NL409-72	45215054	44236906	97.84	34918002	78.93	76.23	92.06
NL409-73	46822340	46156716	98.58	37211749	80.62	77.79	95.02
WB665-71	45625722	44792028	98.17	34166045	77.96	73.34	92.53
WB665-72	43848894	43120278	98.34	33618206	78.80	75.21	94.68
WB665-73	47838874	47202952	98.67	37193868	78.02	76.00	95.38

¹⁾ 71、72、73分别代表7叶期的第1、2、3个样品
¹⁾ 71, 72 and 73 indicate the first, second and third samples of the 7th leaf period respectively

新窗口打开|下载CSV

2.2 基因表达数据分析

选取第7叶期窄叶自交系NL409（T7,样品）和宽叶自交系WB665（C7,对照）叶片发育最宽处进行转录组差异比较分析,发现5 199个差异表达基因,其中2 264（43.55%）个基因上调表达,2 935（56.45%）个基因下调表达。上（下）调1—3倍（fold change）的基因分别占差异表达上（下）调基因总数的29.37%（665）和21.69%（756）;上（下）调3—10倍的基因分别占43.86%（993）和29.24%（1 019）;上（下）调10—60倍的基因分别占20.76%（470）和12.82%（447）;上（下）调60倍以上的基因分别占6.01%（136）和24.29%（713）。从火山图中可以直观地看出对照组与比较样本组之间的表达水平具有显著差异,而且差异60倍以上的下调基因明显多于上调基因（图2）。

图2

新窗口打开|下载原图ZIP|生成PPT
图2差异表达基因火山图

横坐标为差异表达基因表达倍数变化,纵坐标为表达量变化的统计学显著程度
Fig. 2Volcano Plot of DEGs

The abscissa is the change in the expression fold of DGEs, the ordinate is the statistical significance of the change of expression level


GO功能分析表明,差异表达基因被注释到24个生物过程、22个细胞组分、19个分子功能类别中,差异基因主要富集在细胞膜相关的细胞组分中。其中生物过程显著富集在次生代谢、花粉雌蕊相互作用、花粉识别、防卫反应和细胞识别5个过程（图3）;细胞组分分析中差异基因显著富集在细胞外缘、质膜、细胞膜固有成分、细胞膜基本成分、膜部分、共质体、胞间接合和胞间连丝等10个组分类别（图4）;分子功能显著富集在单加氧酶活性、氧化还原活性、催化活性、纤维素合成酶活性、ADP结合、碳水化合物结合、铁离子结合、葡糖基转移酶活性等19个类别（图5）。

图3

新窗口打开|下载原图ZIP|生成PPT
图3差异表达基因显著富集的生物过程

Size代表圆点的大小,表示富集到该GO条目基因的数量,q-value表示该GO条目的富集程度,颜色越趋近于红色表示富集程度越高。下同
Fig. 3Biological process enriched analysis of DEGs

Size represents the size of the dot, indicating the number of genes enriched to the GO item, q-value indicates the enrichment degree of the GO item, and the closer the color is to red, the higher the enrichment. The same as below

图4

新窗口打开|下载原图ZIP|生成PPT
图4差异表达基因显著富集的细胞组分

Fig. 4Cellular component enriched analysis of DEGs

图5

新窗口打开|下载原图ZIP|生成PPT
图5差异表达基因显著富集的分子功能

Fig. 5Molecular function enriched analysis of DEGs



利用MapMan工具对差异表达基因进行pathway富集分析,差异基因被注释到125条代谢通路中,主要涉及生物合成、能量代谢、信号转导及次生代谢。以P-value≤0.05为筛选标准,发现这些基因显著富集到11条代谢路径（表3）,其中核糖体路径富集差异基因数目最多达到83个,37个基因表达上调,46个基因下调表达;其次,植物激素信号转导通路富集57个差异基因,24个基因上调,33个基因下调;苯丙烷类代谢路径18个基因上调,15个基因下调;乙醛酸和二羧酸代谢通路15个基因上调,10个基因下调;谷胱甘肽代谢路径13个基因上调,10个基因下调;鞘脂类代谢路径2个基因上调,11个基因下调（表4）;油菜素内酯生物合成路径4个基因上调,3个基因下调（表5）;类黄酮生物合成路径6个基因上调,2个基因下调;苯并恶唑嗪类生物合成和苯乙烯类、二芳庚酸类、姜酚类生物合成通路也富集到较多差异基因,分别有7个和5个基因下调表达。基于不同代谢通路中基因上下调比例,推断核糖体、植物激素信号转导、鞘脂类代谢这些基因下调表达较多的路径可能与叶片发育,尤其是窄叶的形成密切相关。

Table 3
表3
表3差异表达基因显著富集的代谢通路
Table 3Pathway enriched analysis of DEGs

代谢通路 Pathway	代谢通路ID Pathway ID	差异基因数目 Counts of DEGs	P值 P-value
油菜素内酯生物合成Brassinosteroid biosynthesis	map00905	7	0.00088
乙醛酸和二羧酸代谢Glyoxylate and dicarboxylate metabolism	map00630	25	0.00108
核糖体Ribosome	map03010	83	0.00464
苯丙烷类代谢Phenylpropanoid biosynthesis	map00940	33	0.00854
植物激素信号转导Plant hormone signal transduction	map04075	57	0.01099
苯并恶嗪类生物合成Benzoxazinoid biosynthesis	map00402	7	0.02726
谷胱甘肽代谢Glutathione metabolism	map00480	23	0.02847
鞘脂类代谢Sphingolipid metabolism	map00600	13	0.03916
二萜类生物合成Diterpenoid biosynthesis	map00904	6	0.04792
类黄酮生物合成Flavonoid biosynthesis	map00941	8	0.04716
苯乙烯类、二芳庚酸类和姜酚类生物合成Stilbenoid, diarylheptanoid and gingerol biosynthesis	map00945	7	0.04825

新窗口打开|下载CSV

Table 4
表4
表4鞘脂类代谢路径及其相关基因
Table 4Sphingolipid metabolism pathway and related genes

路径 pathway	基因编号 Gene ID	差异倍数 FC		基因描述/注释 Gene description/annotation
鞘脂类代谢 Sphingolipid metabolism	GRMZM2G165613	0.00	下调Down	丝氨酸软脂酰转移酶,长链碱基生物合成蛋白1b Serine palmitoyltransferase, Long chain base biosynthesis protein 1b
	GRMZM2G444378	0.00	下调Down	丝氨酸软脂酰转移酶,PKC激活蛋白磷酸酶1型抑制剂 Serine palmitoyltransferase, PKC-activated protein phosphatase-1 inhibitor
	GRMZM2G449763	0.00	下调Down	丝氨酸软脂酰转移酶,胺核黄素氧化还原酶 Serine palmitoyltransferase, Flavin containing amine oxidoreductase
	GRMZM2G076398	0.00	下调Down	丝氨酸软脂酰转移酶,长链碱基生物合成蛋白1b Serine palmitoyltransferase, Long chain base biosynthesis protein 1b
	GRMZM2G134248	0.24	下调Down	丝氨酸软脂酰转移酶,长链碱基生物合成蛋白1b Serine palmitoyltransferase, Long chain base biosynthesis protein 1b
	GRMZM2G144985	0.33	下调Down	丝氨酸软脂酰转移酶,长链碱基生物合成蛋白1b Serine palmitoyltransferase, Long chain base biosynthesis protein 1b
	GRMZM2G152888	0.30	下调Down	丝氨酸软脂酰转移酶,长链碱基生物合成蛋白2a Serine palmitoyltransferase, Long chain base biosynthesis protein 2a
	GRMZM2G357734	0.02	下调Down	丝氨酸软脂酰转移酶,胺核黄素氧化还原酶 Serine palmitoyltransferase, Flavin containing amine oxidoreductase
	GRMZM2G402368	0.01	下调Down	SUR2,鞘氨醇C4型单加氧酶2 SUR2, sphinganine C4-monooxygenase 2
	GRMZM2G030118	0.11	下调Down	SUR2,鞘氨醇C4型单加氧酶2 SUR2, sphinganine C4-monooxygenase 2
	GRMZM2G346455	0.46	下调Down	α-半乳糖苷酶,参与植物细胞壁 Alpha-galactosidase 1, involved in plant-type cell wall
	GRMZM2G095126	2.66	上调Up	α-半乳糖苷酶,参与植物细胞壁 Alpha-galactosidase 1, involved in plant-type cell wall
	GRMZM2G105922	2.27	上调Up	GBA2,非溶酶体葡糖神经酰胺酶 GBA2, non-lysosomal glucosylceramidase
MAP激酶 MAP kinase	GRMZM2G135904	13.57	上调Up	MAPK12,丝裂原激活蛋白激酶家族蛋白 MAPK12, MAP kinase family protein
	GRMZM2G459824	8.117	上调Up	MAPKKK2,丝裂原激活蛋白激酶激酶激酶ANP1 MAPKKK2, MAP kinase kinase kinase ANP1-like
	GRMZM5G878379	3.00	上调Up	MAPKK4,丝裂原激活蛋白激酶家族蛋白 MAPKK4, MAP kinase family protein
	GRMZM2G064601	2.49	上调Up	MAPKKK,丝裂原激活蛋白激酶激酶激酶YODA MAPKKK, MAP kinase kinase kinase YODA
	GRMZM2G062761	2.04	上调Up	MAPK17,丝裂原激活蛋白激酶家族蛋白 MAPK17, MAP kinase family protein
AP1类 AP1-like	GRMZM2G055782	17.58	上调Up	MADS-box型转录因子27 MADS-box transcription factor 27-like
	GRMZM2G147716	3.78	上调Up	AGL8/AP1,K-box和MADS-box型转录因子家族 AGL8/AP1, K-box region and MADS-box transcription factor family
	GRMZM2G070034	2.88	上调Up	MADS-box型转录因子56 MADS-box transcription factor 56-like
LFY 类LFY-like	GRMZM2G162814	0.25	下调Down	多叶,花分生组织蛋白,影响花和叶片的发育 Leafy, floral meristem identity protein, affect flower and leaf development

FC：表达量差异倍数（NL409/WB665）
FC：Differential multiple of expression (NL409/WB665)

新窗口打开|下载CSV

Table 5
表5
表5植物激素信号转导路径相关基因
Table 5Some genes related to plant hormone signal transduction

激素 Hormones	基因编号 Gene ID	差异倍数 FC（T7/C7）		基因描述/注释 Gene description/annotation
生长素信号 Auxin signaling	GRMZM2G030710	3.38	上调Up	ARF,类生长素响应因子15,调控转录 ARF, Auxin response factor 15-like, regulation of transcription
	GRMZM2G078274	2.54	上调Up	ARF,类生长素响应因子3,调控转录 ARF, Auxin response factor 3-like, regulation of transcription
	GRMZM5G874163	2.07	上调Up	ARF,类生长素响应因子3,激活生长素信号通路 ARF, Auxin response factor 3-like, auxin-activated signaling pathway
	GRMZM2G361376	0.00	下调Down	ARF,类生长素响应因子15,细胞响应生长素刺激 ARF, Auxin response factor 15-like, cellular response to auxin stimulus
	GRMZM5G853479	70.68	上调Up	生长素响应蛋白IAA4,细胞响应生长素刺激 Auxin-responsive protein IAA4, cellular response to auxin stimulus
	GRMZM2G104176	7.53	上调Up	生长素响应蛋白IAA2,激活生长素信号通路 Auxin-responsive protein IAA2, auxin-activated signaling pathway
	GRMZM2G077356	4.12	上调Up	生长素响应蛋白IAA13,激活生长素信号通路 Auxin-responsive protein IAA13, auxin-activated signaling pathwa
	GRMZM2G079957	3.86	上调Up	生长素响应蛋白IAA31,激活生长素信号通路 Auxin-responsive protein IAA31, auxin-activated signaling pathway
	GRMZM2G074427	0.12	下调Down	生长素响应蛋白IAA18,细胞响应生长素刺激 Auxin-responsive protein IAA18, cellular response to auxin stimulus
	GRMZM2G079200	0.01	下调Down	生长素响应蛋白IAA23,激活生长素信号通路 Auxin-responsive protein IAA23, auxin-activated signaling pathway
	GRMZM2G059544	0.02	下调Down	生长素响应蛋白IAA25,细胞响应生长素刺激 Auxin-responsive protein IAA25, cellular response to auxin stimulus
	GRMZM2G429254	4.12	上调Up	SAUR家族生长素响应蛋白SAUR32,生长素代谢 SAUR family auxin-responsive protein SAUR32, auxin metabolism
	GRMZM2G391596	3.46	上调Up	SAUR家族生长素响应蛋白SAUR36,生长素代谢 SAUR family auxin-responsive protein SAUR36, auxin metabolism
	GRMZM2G361993	0.34	下调Down	SAUR家族生长素响应蛋白SAUR32,生长素代谢 SAUR family auxin-responsive protein SAUR32, auxin metabolism
	GRMZM2G475683	0.24	下调Down	SAUR家族生长素响应蛋白SAUR12,生长素代谢 SAUR family auxin-responsive protein SAUR12, auxin metabolism
	GRMZM2G410567	2.35	上调Up	生长素响应GH3基因家族,响应生长素刺激 Auxin-responsive GH3 gene family, response to auxin stimulus
	GRMZM2G067022	4.17	上调Up	AUX1,类生长素转运蛋白1,跨膜转运体 AUX1, Auxin transporter-like protein 1, transmembrane transporter
	GRMZM2G127949	2.00	上调Up	AUX1,类生长素转运蛋白1,跨膜转运体 AUX1, Auxin transporter-like protein 1, transmembrane transporter
油菜素内酯信号BR signaling	GRMZM2G158775	0.39	下调Down	BRI1,油菜素内酯不敏感1,膜受体,激活BZR1 BRI1, brassinosteroid insensitive 1, membrane receptor, activation of BZR1
	AC194970.5_FG002	0.27	下调Down	BZR1,芸苔素唑拮抗1,油菜素内酯信号转导 BZR1, brassinazole-resistant 1, brassinosteroid signal transduction
	GRMZM2G127050	0.49	下调Down	BSK,油菜素内酯信号激酶,膜受体,BRI1激酶底物 BSK, BR-signaling kinase, membrane receptor, substrates of BRI1 kinase
	GRMZM2G164224	0.31	下调Down	BSK,油菜素内酯信号激酶,膜受体,BRI1激酶底物 BSK, BR-signaling kinase, membrane receptor, substrates of BRI1 kinase
	GRMZM2G054634	0.17	下调Down	BSK,油菜素内酯信号激酶,膜受体,BRI1激酶底物 BSK, BR-signaling kinase, membrane receptor, substrates of BRI1 kinase
	AC210669.3_FG001	0.05	下调Down	木葡聚糖转移酶TCH4,细胞伸长和扩展 Xyloglucosyl transferase TCH4, cell elongation and expansion
油菜素内酯合成BR biosynthesis	GRMZM2G103773	11.89	上调Up	油菜素内酯-6-氧化酶2,细胞膜固有成分 Brassinosteroid-6-oxidase 2, intrinsic component of membrane
	GRMZM2G107199	5.83	上调Up	D11,细胞色素P450,细胞膜固有成分,氧化还原活性 D11, cytochrome P450, integral component of membrane, oxidoreductase activity
	GRMZM2G143235	5.57	上调Up	D2,类固醇3-氧化酶,细胞膜部分,氧化还原活性 D2, steroid 3-oxidase, membrane part, oxidoreductase activity
	GRMZM2G032896	2.78	上调Up	D2,类固醇3-氧化酶,细胞膜固有成分,氧化还原活性 D2, steroid 3-oxidase, intrinsic component of membrane, oxidoreductase activity
	GRMZM2G065635	0.35	下调Down	DWF4,类固醇22-α-羟化酶,细胞膜固有成分 DWF4, steroid 22-alpha-hydroxylase, intrinsic component of membrane
	GRMZM2G162737	0.04	下调Down	CPD,细胞色素P450,细胞膜固有成分,氧化还原活性 CPD, cytochrome P450, integral component of membrane, oxidoreductase activity
	GRMZM5G872368	0.03	下调Down	BR6OX2,油菜素内酯-6-氧化酶2,细胞膜固有成分 BR6OX2, brassinosteroid-6-oxidase 2, intrinsic component of membrane
细胞分裂素信号CTK signaling	GRMZM5G884914	73.11	上调Up	双组分响应调节器ARR-B家族,响应刺激 Two-component response regulator ARR-B family, response to stimulus
	GRMZM2G409974	2.32	上调Up	双组分响应调节器ARR-B家族,响应刺激 Two-component response regulator ARR-B family, response to stimulus
	GRMZM2G013612	2.11	上调Up	类双组分响应调节器ARR-10,调控转录 Two-component response regulator ARR10-like, regulation of transcription
赤霉素信号GAs	GRMZM2G013016	6.80	上调Up	GAI1型DELLA蛋白,膜上细胞器,信号转导 DELLA protein GAI1-like, membrane-bounded organelle, signal transduction
赤霉素代谢 相关基因 GAmrg	GRMZM2G073779	0.38	下调Down	GRAS家族参与赤霉素代谢,调控植株生长与发育 GRAS family, as a player in GA metabolism, regulate plant growth and development
	GRMZM2G073805	0.29	下调Down	GRAS家族参与赤霉素代谢,调控植株生长与发育 GRAS family, as a player in GA metabolism, regulate plant growth and development
	GRMZM2G098517	0.23	下调Down	GRAS家族参与赤霉素代谢,调控植株生长与发育 GRAS family, as a player in GA metabolism, regulate plant growth and development
	GRMZM2G018254	0.04	下调Down	GRAS家族参与赤霉素代谢,调控植株生长与发育 GRAS family, as a player in GA metabolism, regulate plant growth and development
	GRMZM2G070371	0.00	下调Down	GRAS家族参与赤霉素代谢,调控植株生长与发育 GRAS family, as a player in GA metabolism, regulate plant growth and development
	GRMZM2G110579	3.02	上调Up	GRAS家族参与赤霉素代谢,调控植株生长与发育 GRAS family, as a player in GA metabolism, regulate plant growth and development
	GRMZM2G036340	0.42	下调Down	GA 3氧化酶1,赤霉素3-β-双加氧酶活性 GA 3-oxidase 1, gibberellin 3-beta-dioxygenase activity
	GRMZM2G006964	0.3	下调Down	GA 2氧化酶6,赤霉素2-β-双加氧酶活性 GA 2-oxidase 6, gibberellin 2-beta-dioxygenase activity
	GRMZM2G323504	0.37	下调Down	GA受体GID1L2,羟基亚甲丁酸吡喃葡糖转化酶 GA receptor GID1L2, tuliposide A-converting enzyme
	GRMZM2G049675	0.29	下调Down	GA受体GID1L2,羟基亚甲丁酸吡喃葡糖转化酶 GA receptor GID1L2, tuliposide A-converting enzyme
	GRMZM2G156310	0.09	下调Down	GA受体GID1L2,羟基亚甲丁酸吡喃葡糖转化酶 GA receptor GID1L2, tuliposide A-converting enzyme
	AC203966.5_FG005	0.21	下调Down	GA 20氧化酶1,调控叶形和矮化 GA 20 oxidase 1, regulate leaves shape and dwarfism

FC：表达量差异倍数（NL409/WB665）;BR：油菜素内酯Brassinosteroid;CTK：细胞分裂素Cytokinine;GAs：赤霉素信号Gibberellin signaling;GAmrg：赤霉素代谢相关基因Gibberellin metabolism related genes

新窗口打开|下载CSV

植物内源激素广泛参与到叶片发育过程,对叶片形态建成起到一定调节作用。进一步分析植物激素信号转导路径发现,在叶宽形成的关键部位检测到大量参与激素信号响应和转导的差异基因,涉及生长素、油菜素内酯、细胞分裂素、赤霉素、脱落酸、乙烯、茉莉酸等多种激素（图6）。分析不同响应路径（表5和图6）发现,生长素信号转导通路富集到18个差异基因,有12个响应基因表达上调;油菜素内酯信号响应路径富集到6个差异基因,全部下调表达;细胞分裂素响应路径富集的3个基因,表达均上调;赤霉素和水杨酸响应通路分别富集到1个差异基因,表达均上调;脱落酸路径富集到8个基因下调表达,4个上调表达基因;乙烯路径中4个基因上调表达,4个基因下调表达;茉莉酸路径中1个基因上调表达,7个基因下调表达。

图6

新窗口打开|下载原图ZIP|生成PPT
图6植物激素信号转导

Tryptophan metabolism：色氨酸代谢;Auxin：生长素;Ubiquitin mediated proteolysis：泛素介导的蛋白水解;Cell enlargement：细胞增大;Plant growth：植物生长;Zeatin biosynthesis： 玉米素的生物合成;Cytokinine：细胞分裂素;Cell division：细胞分裂;Shoot initiation：根的起始;Diterpenoid biosynthesis：二萜类化合物的生物合成;Gibberellin：赤霉素;Stem growth：茎的生长;Induced germination：诱导萌发;Carotenoid biosynthesis：类胡萝卜素的生物合成;Abscisic acid：脱落酸;Stomatal closure：气孔闭合;Seed dormancy：种子休眠;Cysteine and methionine metabolism：半胱氨酸和蛋氨酸代谢;Ethylene：乙烯;Endoplasmic reticulum(ER)：内质网;Fruit ripening：果实成熟;Senescence：衰老;Brassinosteroid biosynthesis：油菜素内酯生物合成;Brassinosteroid：油菜素甾醇;Proteasomal degradation：蛋白酶体降解;Cell elongation：细胞伸长;Alpha-Linolenic acid metabolism：α-亚麻酸代谢; Jasmonic acid：茉莉酸;Monoterpenoid biosynthesis类单萜生物合成;Indole alkaloid biosynthesis：吲哚生物碱生物合成;Stress response：应激反应;Phenylalanine metabolism：苯丙氨酸代谢;Salicylic acid：水杨酸;Disease resistance：抗病性。红色表示富集到的差异基因表达上调,绿色表示基因下调Red indicates that DEGs are up-regulated, and green indicates down-regulation of DEGs
Fig. 6Plant hormone signal transduction under leaf development for 7 leaf stage between narrow leaf sample and wide blade sample

2.3 荧光定量PCR验证RNA-Seq结果

研究表明多种转录因子参与叶细胞分化及叶片形态建成,如YABBY、MADS类转录因子决定叶原细胞命运向不同方向分化形成不同组织,GRF类调控特定部位细胞的增殖和生长;ARF、SBP、TCP、Myb、HB类调控叶细胞的分化和维管的形成;同时ARF、ARR、GRAS类在叶片发育过程分别参与生长素、细胞分裂素、赤霉素信号的响应和生物合成,影响叶片形态建成。为了进一步验证RNA-Seq结果的准确性,选取与叶片发育密切相关的18个不同的基因进行了qRT-PCR定量分析。对qRT-PCR与RNA-Seq结果进行相关性分析,发现两者的相关系数R²=0.962,说明RNA-Seq结果良好。

3 讨论

3.1 叶片发育密切相关的代谢通路

多条路径显著富集到大量与玉米叶片发育相关的基因。拟南芥PFL（POINTED FIRST LEAVES）和AE（Asymmetric Leaves Enhancer）分别编码不同亚族的核糖体蛋白（PRS）,与rRNA形成蛋白复合物促进叶片近轴组织的建立^[20,21]。本文核糖体路径显著富集到多个PFL和AE（电子附表1）,并鉴定出玉米PRS13（PFL2）启动子区域存在显著差异（电子附图1）,其在窄叶中下调表达的作用模式（图7）与拟南芥ae5、ae6缺失突变导致叶柄变长、叶片变窄的作用机理相似。鞘脂类（Sphingolipid）是细胞膜骨架及脂蛋白的重要组成成分,鞘脂代谢在细胞生长、不同功能细胞分化、细胞内以及细胞间信号传递发挥重要作用,鞘脂合成紊乱含量较低时,能够激活MAPK（mitogen-activated protein kinase）路径和转录因子AP1（APETALA1）,进而调控细胞增殖、分化^[22,23]。AP1抑制拟南芥花萼叶和茎叶形成,LFY（LEAFY）能够解除AP1的抑制作用,促进叶片发育^[24]。本文鞘脂代谢路径富集到的基因大部分下调表达（表4）,AP1类和MAPK类基因表达上调（表4）,LFY表达下调（表4）,与窄叶发育受抑制的表型一致。研究人员分析叶片发育的动态转录组学,不仅发现大量激素信号基因,细胞壁合成前体、纤维素合成酶、蛋白酶体、翻译后修饰基因也被大量检测到^[25]。BOLDUC等^[9]解析KN1调控网络,不仅在叶片和分生组织中发现大量激素合成和响应基因,而且富集到脂类代谢、次生代谢、细胞壁合成和调控RNA转录相关的基因。叶片发育过程不同部位、不同时期的蛋白质组学分析,也富集到大量的尿苷二磷酸-葡糖基/葡聚糖转移酶、细胞壁前体、细胞壁受体激酶、纤维素合成酶和RNA转录相关的基因^[26],本文显著富集的路径与前人的研究结果一致。由此可知,上述显著富集的代谢通路（表3）与叶片发育密切相关,这些代谢路径中的基因可能参与叶宽、叶长的形态建成。

图7

新窗口打开|下载原图ZIP|生成PPT
图7RNA-Seq与qRT-PCR结果比较

Fig. 7Comparison of results between RNA-Seq and qRT-PCR



本研究发现油菜素内酯信号转导路径显著富集的基因全部下调（表5）,不仅检测到前人已研究表达量降低的BZR类和BRI类基因^[9],还发现BR路径中BSK类、TCH类基因也下调表达。生长素信号转导路径富集到的基因大部分上调表达（表5）,其中ARF（GRMZM2G078274、GRMZM2G030710和GRMZM5G874163）、AUX-IAA（GRMZM2G104176、GRMZM2G077356、GRMZM2G079957、GRMZM2G074427）和Auxin transporter（GRMZM2G127949）在KN1调控叶片发育过程中发挥着重要作用^[9]。此外,DELLA高表达时,抑制GA信号及叶基部细胞的生长^[25],本文DELLA家族蛋白（GRMZM2G013016）在近基部窄叶中高表达（表5,图6）,且赤霉素代谢相关的基因几乎全部下调表达（表5）,这与窄叶发育受抑制的结果一致。由此推断油菜素内酯、生长素、赤霉素在叶片发育过程发挥重要作用。

3.2 不同激素途径发掘的叶形重要功能基因

植物器官发育过程中极性建立是器官形态建成的核心。叶片的发育起始于叶原基细胞的分裂增殖,伴随着一系列特定基因的精确调控,叶原基建立多维空间的极性轴向,促使叶原基细胞朝特定的方向分化增殖,并最终影响叶片的形态和大小。

TCP类转录因子通过AUX、BR、GA、CTK等多种激素调控叶片、花发育以及植株形态建成等生长过程。叶片发育过程TCP1可调控DWARF4影响拟南芥叶片及植株形态,而BRI是tcp1发挥功能所必需的;此外,多个AtTCP负向调控叶片边缘区细胞分化增殖,其中TCP3、TCP15分别负调控AUX1、PIN1和YUCCA1、YUCCA4影响生长素分布及含量^[9,27-28,31]。过量表达TCP II型基因LANCEOLATE (LA)使西红柿和拟南芥的叶片变小,或表现卷曲叶^[27,28],拟南芥TCP4的同源基因CYC/TB1（AC205574.3_FG006）在窄叶中上调表达的作用模式（图7,电子附表1）与前人研究结果一致。因此,推测CYC/TB1可能影响多种激素调控玉米叶片发育。

激素BR可影响玉米叶片形态建成。AtROT3编码一种细胞色素P450蛋白,作用于叶片纵向极性细胞的伸长生长,其功能缺失影响拟南芥叶柄变短,叶片变大变宽^[29],拟南芥BR合成相关的 P450家族det2和dwarf4突变后影响叶细胞变短变少^[30,31]。VP1（VIVIPAROUS1）、VAL（VP1/ABI3-LIKE）、RAV1（Related to ABI3/VP1）、RAVL1（Related to ABI3/ VP1-Like）分别编码不同的B3结构DNA结合蛋白,拟南芥val缺失突变后影响叶片变小且赤霉素含量降低^[32],RAV1作为BR受体负向调控拟南芥莲座叶的生长^[33],与本文中VAL-like下调和RAV-like表达上调（电子附表2）,以及赤霉素路径基因下调表达的结果一致（表5）。RAVL1通过E-box（CACCTG/ CACGTG/CACATG）结合位点激活BR响应和合成基因的表达,维持BR平衡对生长的调节作用^[34,35],本文中BR响应基因的启动子区域存在大量该元件（电子附图4—9）,说明植物激素BR与叶片发育密切相关。此外,ROT3（GRMZM2G143235）和VP1-like（GRMZM2G344521）在叶宽形成的关键部位上调表达（图7）,且位于已定位的叶宽区间内^[36],同时ROT3启动子区域的序列差异（电子附图2）,表明ROT3和VP1-like可能在叶长和叶宽发育过程中发挥着重要作用,由此推测BR通过某种途径影响玉米叶长、叶宽的形成。

生长素合成、分布异常影响叶片形态。玉米NAL7-like与水稻NAL7（OsYUC7）和NAL1（OsYUC1）同源,NAL1、NAL2、NAL3和 NAL7编码一种生长素合成酶,调控叶原基的形成,其功能缺失突变影响叶细胞的分裂和维管束的形成,导致水稻叶片变窄^{[37,38,39,40]},与本文NAL7-like在窄叶中下调表达（图7）的作用模式一致。RGD2编码一种AGO7型蛋白,是合成ta-siARF所必需的,RGD2通过ta-siARF调控ARF的转录,影响玉米叶片腹背面生长素分布,导致rgd2突变体叶片变窄卷曲^[41]。此外,NAL7-like位于叶宽研究的热点区域^[4],而且其启动子区域序列存在显著差异（电子附图3）。由此可知,NAL7-like（GRMZM2G480386）、AGO7-like（GRMZM5G892991）可能通过影响生长素调控窄叶的形成。

玉米叶片发育过程大量与生长素相关的基因参与其形成。玉米KN1的ChIP分析富集大量生长素AUX-IAA和ARF基因,转录组分析发现生长素基因（ARF12、ARF3）在玉米叶细胞朝不同功能区生长的过渡区上调表达^[9],与本研究中ARF12、ARF3（图7）表达结果一致。拟南芥ARF受叶原基近轴端特异表达tasiR-ARF的抑制,在近轴端不表达,在远轴端KAN1（KANAD1）与ARF3、ARF4相互作用的同时,影响YUC2、YUC5和YUC8表达上调,促进叶原细胞的分化及远轴组织的形成^[42,43];miR165/166抑制HD-ZIP lll类基因PHB、PHV、REV在叶原基远轴端的表达,促进近轴组织的形成^[44]。ARF3、KN1-like和KAN3（图7）位于已定位的叶宽位点内^[4,36]。此外,ARF12、GH5、REV-like（图7）分别受miR167、miR164、miR166调控并且与叶长、叶宽位点显著相关^[36],由此可知KN1-like（GRMZM2G017709）、ARF3（GRMZM5G874163）、KAN3（GRMZM2G175827）、ARF12（GRMZM2G078274）、GH5（GRMZM2G055585）、REV-like（GRMZM2G029692）在叶片发育过程可能发挥特定作用。

植物激素间的的相互作用可调节叶片发育。BR信号路径中的BIN2激酶能够磷酸化ARF2,导致其结合Auxin响应元件的功能丧失,影响拟南芥叶片等器官增大^[45,46],同时BR能够诱导BZR1靶基因ARF2的表达^[47]。BZR1与ARF6相互作用促进拟南芥下胚轴变长^[48]。Auxin能够诱导拟南芥DWARF4表达,同时DWARF4通过BR路径促进细胞分裂生长^[49]。此外,Auxin还可促进OsBRI1表达,加强BR信号转导,而拟南芥BRI1通过tcp影响生长素促进细胞膨大生长^[27],ARF能够特异结合Auxin响应元件（TGTCTC）^[50],本文BR响应基因启动子富含该元件（电子附图4—电子附图9）,而且BR响应基因全部下调,Auxin响应基因上调（表5,图7）,这暗示本文中ARF2-like（GRMZM2G030710）、ARF6-like（GRMZM2G078274）、DWARF4-like（GRMZM2G065635）、BRI1-like（GRMZM2G15877）（表5,电子附表1）可能介导Auxin和BR的平衡来调节植物器官以及叶片的发育。

HB类转录因子包含多个KNOX、bHLH和HD-ZIP lll基因。KNOTTED1（KN1）和KNOX7属于KNOX家族,在顶端分生组织特定表达,调控细胞分化向膨大生长的过渡,影响叶片多维空间的发育,尤其在基部-末端轴向的形成过程中,KN1与LG3、LG4、KNOX8相互作用调控叶片发育^[9]。KNOX蛋白能够促进细胞分裂素（CTK）合成基因上调,同时抑制GA3和GA20氧化酶合成,促进SAM细胞分裂形成叶原基^[9,51-52];提高GA含量可以解除DELLA对叶片发育的抑制作用^[52]。与本文中KNOX家族基因（KN1-like和KNOX7）（图7）、CTK路径基因（表5）、DELLA基因上调（表5）,GA氧化酶基因下调（表5）影响叶片变窄的结果一致。GA可通过DELLA蛋白负向调控CTK响应基因进而影响CTK变化^[53]。拟南芥GA信号的抑制因子SPY（SPINDLY）能够正向调控CTK促进植株及叶片的发育^[54]。由此可知KN1-like（GRMZM2G017709）和KNOX7（GRMZM2G433591）可能通过影响细胞分裂素与赤霉素的平衡参与玉米窄叶的形成。

生长调节因子GRF的互作因子AN3/GIF1作为叶片发育过程的调节开关,主要影响叶细胞横向分裂,分析其调控机理,在叶片基部分裂区富集到大量上调表达的GRF基因,在叶部伸长区仅发现GRF4和GRF10高量表达,其他GRF基因在叶部伸长区表达量逐渐降低,过量表达ZmGRF1和ZmGRF10使玉米叶片变小^[13,14],与本文中检测到GRF6（GRMZM2G041223）和GRF13（GRMZM2G018414）在窄叶叶片发育的过渡区域上调表达（图7）的趋势一致,同时GRF6和GRF13位于叶宽研究的热点区域^[4]。LG2（GRMZM2G143235）编码一种TGA结合域的碱性亮氨酸拉链蛋白,主要在SAM和叶片与叶鞘连接处表达,作用于叶基部叶耳、叶舌的形成,影响叶片的发育^[55]。ZmARF2（GRMZM2G082836）是ADP核糖基化转录因子,能够调控拟南芥叶面积和株高^[56]。由此推断GRF6、GRF13、LG2、ZmARF2（图7）在玉米叶片发育以及叶宽形成过程中可能发挥重要作用。

4 结论

明确了一些与玉米叶片发育密切相关的代谢路径,发现植物激素间的动态平衡对叶片发育有着重要影响,尤其是Auxin与BR、CKT与GA之间的相互作用对调控叶片形态可能发挥重要作用。

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] 明博, 谢瑞芝, 侯鹏, 李璐璐, 王克如, 李少昆 . 2005—2016年中国玉米种植密度变化分析
中国农业科学, 2017,50(11):1960-1972.
[本文引用: 1]

MING B, XIE R Z, HOU P, LI L L, WANG K R, LI S K . Changes of maize planting density in China
Scientia Agricultura Sinica, 2017,50(11):1960-1972. (in Chinese)
[本文引用: 1]

[2] 郭书磊, 陈娜娜, 齐建双, 岳润清, 韩小花, 燕树锋, 卢彩霞, 傅晓雷, 郭新海, 铁双贵 . 不同密度下玉米倒伏相关性状与产量的研究
玉米科学, 2018,26(5):71-77.
[本文引用: 1]

GUO S L, CHEN N N, QI J S, YUE R Q, HAN X H, YAN S F, LU C X, FU X L, GUO X H, TIE S G . Study on the relationship between yield and lodging traits of maize under different planting densities
Journal of Maize Sciences, 2018,26(5):71-77. (in Chinese)
[本文引用: 1]

[3] YANG C W, XIE F M, JIANG Y P, LI Z, HUANG X, LI L . Phytochrome A negatively regulates the shade avoidance response by increasing auxin/indole acidic acid protein stability
Developmental Cell, 2018,44(1):29-41.
[本文引用: 1]

[4] 郭书磊, 张君, 齐建双, 岳润清, 韩小花, 燕树锋, 卢彩霞, 傅晓雷, 陈娜娜, 库丽霞, 铁双贵 . 玉米叶形相关性状的Meta-QTL及候选基因分析
植物学报, 2018,53(4):487-501.
[本文引用: 5]

GUO S L, ZHANG J, QI J S, YUE R Q, HAN X H, YAN S F, LU C X, FU X L, CHEN N N, KU L X, TIE S G . Analysis of meta-quantitative trait loci and their candidate genes related to leaf shape in maize
Chinese Bulletin of Botany, 2018,53(4):487-501. (in Chinese)
[本文引用: 5]

[5] 崔晓峰, 黄海 . 叶发育的遗传调控机理研究进展
植物生理学报, 2011,47(7):631-640.
[本文引用: 1]

CUI X F, HUANG H . Recent progresses from studies of leaf development
Plant Physiology Journal, 2011,47(7):631-640. (in Chinese)
[本文引用: 1]

[6] MORENO M A, HARPER L C, KRUEGER R W, DELLAPORTA S L , FREELING M. liguleless1 encodes a nuclear-localized protein required for induction of ligules and auricles during maize leaf organogenesis
Genes & Development, 1997,11(5):616-628.
[本文引用: 1]

[7] WALSH J, WATERS C A, FREELING M . The maize geneliguleless2 encodes a basic leucine zipper protein involved in the establishment of the leaf blade-sheath boundary
Genes & Development, 1998,12(2):208-218.
[本文引用: 1]

[8] ZHANG X L, MADI S, BORSUK L, NETTLETON D, ELSHIRE R J, BUCKNER B, BUCKNER D J, BECK J, TIMMERMANS M, SCHNABLE P S, SCANLON M J . Laser microdissection of narrow sheath mutant maize uncovers novel gene expression in the shoot apical meristem
PLoS Genetics, 2007,3(6):1040-1052.
[本文引用: 1]

[9] BOLDUC N, YILMAZ A, MEJIA-GUERRA M K, MOROHASHI K, O'CONNOR D, GROTEWOLD E, HAKE S, . Unraveling the KNOTTED1 regulatory network in maize meristems
Genes & Development, 2012,26(15):1685-1690.
[本文引用: 8]

[10] TIMMERMANS M C, SCHULTES N P, JANKOVSKY J P, NELSON T . Leafbladeless1 is required for dorsoventrality of lateral organs in maize
Development, 1998,125(15):2813-2823.
[本文引用: 1]

[11] DOUGLAS R N, WILEY D, SARKAR A, SPRINGER N, TIMMERMANS MARJA C P, SCANLON M J, . Ragged seedling2 Encodes an ARGONAUTE7-like protein required for mediolateral expansion, but not dorsiventrality, of maize leaves
The Plant Cell, 2010,22(5):1441-1451.
[本文引用: 1]

[12] CANDELA H, JOHNSTON R, GERHOLD A, FOSTER T, HAKE S . The milkweed pod1 gene encodes a KANADI protein that is required for abaxial/adaxial patterning in maize leaves
The Plant Cell, 2008,20(8):2073-2087.
[本文引用: 1]

[13] NELISSEN H, EECKHOUT D, DEMUYNCK K, PERSIAU G, WALTON A, BEL M V, VERVOORT M, CANDAELE J, BLOCK J D, AESAERT S, LIJSEBETTENS M V, GOORMACHTIG S, VANDEPOELE K, LEENE J V, MUSZYNSKI M, GEVAERT K, INZE D, JAEGER G D . Dynamic changes in ANGUSTIFOLIA3 complex composition reveal a growth regulatory mechanism in the maize leaf
The Plant Cell, 2015,27(6):1605-1619.
[本文引用: 2]

[14] WU L, ZHANG D, XUE M, QIAN J J, HE Y, WANG S C . Overexpression of the maize GRF10, an endogenous truncated growth-regulating factor protein, leads to reduction in leaf size and plant height
Journal of Integrative Plant Biology, 2014,56(11):1053-1063.
[本文引用: 2]

[15] 吴庆飞, 秦磊, 董雷, 丁泽红, 李平华, 杜柏娟 . 玉米光合突变体hcf136(high chlorophyll fluorescence 136)的转录组分析
作物学报, 2018,44(4):493-504.
[本文引用: 1]

WU Q F, QIN L, DONG L, DING Z H, LI P H, DU B J . Transcriptome analysis on a maize photosynthetic mutant hcf136 (high chlorophyll fluorescence 136)
Acta Agronomica Sinica, 2018,44(4):493-504. (in Chinese)
[本文引用: 1]

[16] 邱化荣, 周茜茜, 何晓文, 张宗营, 张世忠, 陈学森, 吴树敬 . 基于转录组分析苹果水杨酸特异响应基因MdWRKY40的启动子鉴定
中国农业科学, 2017,50(20):3970-3990.
[本文引用: 1]

QIU H R, ZHOU Q Q, HE X W, ZHANG Z Y, ZHANG S Z, CHEN X S, WU S J . Identification of MdWRKY40 promoter specific response to salicylic acid by transcriptome sequencing
Scientia Agricultura Sinica, 2017,50(20):3970-3990. (in Chinese)
[本文引用: 1]

[17] TRAPNELL C, WILLAMS B A, PERTEA G, MORTAZAVI A, KWAN G, BAREN M J, SALZBERG S L , WOLD B JHTER L. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation
Nature Biotechnology, 2010,28(5):511-515.
[本文引用: 1]

[18] WANG L K, FENG Z X, WANG X, WANG X W, ZHANG X G . DEGseq: An R package for identifying differentially expressed genes from RNA-seq data
Bioinformatics, 2009,26(1):136-138.
[本文引用: 1]

[19] LIVAK K J, SCHMITTGEN T D . Analysis of relative gene expression data using real-time quantitative PCR and the 2 ^-ΔΔCT method
Methods, 2001,25(4):402-408.
[本文引用: 1]

[20] SCHIPPERS J H M, MUELLER-ROEBER B . Ribosomal composition and control of leaf development
Plant Science, 2010,179(4):307-315.
[本文引用: 1]

[21] YAO Y, LING Q H, WANG H, HUANG H . Ribosomal proteins promote leaf adaxial identity
Development, 2008,135(7):1325-1334.
[本文引用: 1]

[22] SPIEGEL S, MERRILL Jr A H, . Sphingolipid metabolism and cell growth regulation
The FASEB Journal, 1996,10(12):1388-1397.
[本文引用: 1]

[23] MERRILL Jr A H, SANDHOFF K . Metabolism and cell signaling//Biochemistry of Lipids, Lipoproteins and Mem-branes.
Amsterdam, Elsevier Science Press, 2002, 373-407.
[本文引用: 1]

[24] MONNIAUX M, MCKIM S M, CARTOLANO M, THEVENON E, PARCY F, MILTANTIS T, HAY A . Conservation vs divergence in LEAFY and APETALA1 functions between Arabidopsis thaliana and Cardamine hirsuta
New Phytologist, 2017,216(2):549-561.
[本文引用: 1]

[25] LI P H, PONNALA L, GANDOTRA N, WANG L, SI Y Q, TAUSTA S L, KEBROM T H, PROVART N, PATEL R, MYERS C R, REIDEL E J, TURGEON R, LIU P, SUN Q, NELSON T, BRUTNELL T P . The developmental dynamics of the maize leaf transcriptome
Nature Genetics, 2010,42(12):1060-1067.
[本文引用: 2]

[26] FACETTE M R, SHEN Z X, BJ?RNSDóTTIR F R, BRIGGS S P, SMITH L G . Parallel proteomic and phosphoproteomic analyses of successive stages of maize leaf development
The Plant Cell, 2013: 2798-2812.
[本文引用: 1]

[27] NICOLAS M, CUBAS P. The role of TCP transcription factors in shaping flower structure, leaf morphology, and plant architecture// Plant Transcription Factors: Evolutionary, Structural And Functional Aspects
Cambridge, MA: Academic Press, 2016: 249-267.
[本文引用: 3]

[28] MANASSERO N G U, VIOLA I L, WELCHEN E, GONZALEZ D H . TCP transcription factors: architectures of plant form
Biomolecular Concepts, 2013,4(2):111-127.
[本文引用: 2]

[29] KIM G T, TSUKAYA H, UCHIMIYA H . The ROTUNDIFOLIA3 gene of Arabidopsis thaliana encodes a new member of the cytochrome P-450 family that is required for the regulated polar elongation of leaf cells
Genes & Development, 1998,12(15):2381-2391.
[本文引用: 1]

[30] FUJIOKA S, LI J, CHOI Y H, SETO H, TAKATSUTO S, NOGUCHI T, WATANABE T, KURIYAMA H, YOKOT T, CHORY J, SAKURAI A . The Arabidopsis deetiolated2 mutant is blocked early in brassinosteroid biosynthesis
The Plant Cell, 1997,9(11):1951-1962.
[本文引用: 1]

[31] GUO Z, FUJIOKA S, BLANCAFLOR E B, MIAO S, GOU X P, LI J . TCP1 modulates brassinosteroid biosynthesis by regulating the expression of the key biosynthetic gene DWARF4 in Arabidopsis thaliana
The Plant Cell, 2010,22(4):1161-1173.
[本文引用: 2]

[32] SUZUK M, WANG H H Y, MCCARTY D R . Repression of the LEAFY COTYLEDON 1/B3 regulatory network in plant embryo development by VP1/ABSCISIC ACID INSENSITIVE 3-LIKE B3 genes
Plant Physiology, 2007,143(2):902-911.
[本文引用: 1]

[33] HU Y X, WANG Y H, LIU X F, LI J Y . Arabidopsis RAV1 is down-regulated by brassinosteroid and may act as a negative regulator during plant development
Cell Research, 2004,14(1):8.
[本文引用: 1]

[34] YUAN W Y, LUO X, LI Z C, YANG W N, WANG Y Z, LIU R, DU J M, HE Y H . A cis cold memory element and a trans epigenome reader mediate Polycomb silencing of FLC by vernalization in Arabidopsis
Nature Genetics, 2016,48(12):1527-1534.
[本文引用: 1]

[35] JE B I, PIAO H L, PARK S J, PARK S H, KIM C M, XUAN Y H, PARK S H, HUANG J, CHOI Y D, AN G, WONG H L, FUJIOKA S, KIM M C, SHIMAMOTO K, HAN C . RAV-Like1 maintains brassinosteroid homeostasis via the coordinated activation of BRI1 and biosynthetic genes in rice
The Plant Cell, 2010,22(6):1777-1791.
[本文引用: 1]

[36] TIAN F, BRADBURY P J, BROWN P J, HUNG H Y, SUN Q, FLINT-GARCIN S, ROCHEFORD T R, MCMULLEN M D, HOLLAND J B, BUCKLER E S . Genome-wide association study of leaf architecture in the maize nested association mapping population
Nature Genetics, 2011,43(2):159-165.
[本文引用: 3]

[37] ISHIWATA A, OZAWA M, NAGASAKI H, KATO M, NODA Y, YAMAGUCHI T, NOSAKA M, SHIMIZU-SATO S, NAGASAKI A, MAEKAWA M, HIRANO H Y, SATO Y . Two WUSCHEL-related homeobox genes, narrow leaf2 and narrow leaf3, control leaf width in rice
Plant and Cell Physiology, 2013,54(5):779-792.
[本文引用: 1]

[38] KUBO F C, YASUI Y, KUMAMARU T, SATO Y, HIRANO H Y . Genetic analysis of rice mutants responsible for narrow leaf phenotype and reduced vein number
Genes & Genetic Systems, 2016,91(4):235-240.
[本文引用: 1]

[39] JIANG D, FANG J J, LOU L, ZHAO J F, YUAN S J, YIN L, SUN W, PENG L X, GUO B T, LI X Y . Characterization of a null allelic mutant of the rice NAL1 gene reveals its role in regulating cell division
PLoS ONE, 2015,10(2):e0118169.
[本文引用: 1]

[40] FUJINO K, MATSUDA Y, OZAWA K, NISHIMURA T, KOSHIBA T, FRAAIJE M W, SEKIGUCHI H . NARROW LEAF 7 controls leaf shape mediated by auxin in rice
Molecular Genetics and Genomics, 2008,279(5):499-507.
[本文引用: 1]

[41] DOUGLAS R N, WILEY D, SARKAR A, SPRINGER N, TIMMERMANS M C P, SCANLON M J, . Ragged seedling2 encodes an ARGONAUTE7-like protein required for mediolateral expansion, but not dorsiventrality, of maize leaves
The Plant Cell, 2010: 1441-1451.
[本文引用: 1]

[42] DOTTO M C, PETSCH K A, AUKERMAN M J, BEATTY M, HAMMELL M, TIMMERMANS M C P . Genome-wide analysis of leafbladeless1-regulated and phased small RNAs underscores the importance of the TAS3 ta-siRNA pathway to maize development
PLoS Genetics, 2014,10(12):e1004826.
[本文引用: 1]

[43] XIE Y, STRAUB D, EGUEN T, BRANDT R, STAHL M, Jaime F MARTINEZ-GAECIA J F WENKEL S . Meta-analysis of Arabidopsis KANADI1 direct target genes identifies a basic growth-promoting module acting upstream of hormonal signaling pathways
Plant Physiology, 2015,169(2):1240-1253.
[本文引用: 1]

[44] EMERY J F, FLOYD S K, AIVAREZ J, ESHED Y, HAWKER N P, IZHAKI A, BAUM S F, BOWMAN J L . Radial patterning of Arabidopsis shoots by class III HD-ZIP and KANADI genes
Current Biology, 2003,13(20):1768-1774.
[本文引用: 1]

[45] OKUSHIMA Y, MITINA I, QUACH H L, THEOLOGIS A . AUXIN RESPONSE FACTOR 2 (ARF2): A pleiotropic developmental regulator
The Plant Journal, 2005,43(1):29-46.
[本文引用: 1]

[46] SCHRUFF M C, SPIELMAN M, TIWARI S, ADAMS S, FENBY N, SCOTT R J . The AUXIN RESPONSE FACTOR 2 gene of Arabidopsis links auxin signalling, cell division, and the size of seeds and other organs
Development, 2006,133(2):251-261.
[本文引用: 1]

[47] TIAN H Y, LV B S, DING T T, BAI M Y, DING Z J . Auxin-BR interaction regulates plant growth and development
Frontiers in Plant Science, 2018,8:2256.
[本文引用: 1]

[48] OH E, ZHU J Y, BAI M Y, ARENHART R A, SUN Y, WANG Z Y . Cell elongation is regulated through a central circuit of interacting transcription factors in the Arabidopsis hypocotyl
Elife, 2014,3:e03031.
[本文引用: 1]

[49] CHUNG Y, MAHARJAN P M, LEE O, FUJIOKA S, JANG S, KIM B, TAKATSUTO S, TSUJIMOTO M, KIM H, CHO S, PARK T, CHO H, HWANG I, CHOE S . Auxin stimulates DWARF4 expression and brassinosteroid biosynthesis in Arabidopsis
The Plant Journal, 2011,66(4):564-578.
[本文引用: 1]

[50] SAKAMOTO T, MORINAKA Y, INUKAI Y, KITANO H, FUJIOKA S . Auxin signal transcription factor regulates expression of the brassinosteroid receptor gene in rice
The Plant Journal, 2013,73(4):676-688.
[本文引用: 1]

[51] JASINSKI S, PIAZZA P, CRAFT J, HAY A, WOOLLEY L, RIEU I, PHILLIPS A, HEDDEN P, TSIANTIS M . KNOX action in Arabidopsis is mediated by coordinate regulation of cytokinin and gibberellin activities
Current Biology, 2005,15(17):1560-1565.
[本文引用: 1]

[52] NELISSEN H, RYMEN B, JIKUMARU Y, DEMUYNCK K, LIJSEBETTENS M V, KAMIYA Y, INZE D, BEEMSTER G T S . A local maximum in gibberellin levels regulates maize leaf growth by spatial control of cell division
Current Biology, 2012,22(13):1183-1187.
[本文引用: 2]

[53] FONOUNI-FARDE C, KISIALA A, BRAULT M, EMERY R J N, ANOUCK D, FLORIAN F . DELLA1-mediated gibberellin signaling regulates cytokinin-dependent symbiotic nodulation
Plant Physiology, 2017,175(4):1795-1806.
[本文引用: 1]

[54] GREENBOIM-WAINBERG Y, MAYMON I, BOROCHOV R, ALVAREZ J, OLSZEWSKI N, ORI N, ESHED Y, WEISS D . Cross talk between gibberellin and cytokinin: The Arabidopsis GA response inhibitor SPINDLY plays a positive role in cytokinin signaling
The Plant Cell, 2005,17(1):92-102.
[本文引用: 1]

[55] WALSH J, WATERS C A, FREELING M . The maize geneliguleless2 encodes a basic leucine zipper protein involved in the establishment of the leaf blade-sheath boundary
Genes & Development, 1998,12(2):208-218.
[本文引用: 1]

[56] WANG Q L, XUE X J, LI Y L, DONG Y B, ZHANG L, ZHOU Q, DENG F, MA Z Y, QIAO D H, HU C H, REN Y L . A maize ADP-ribosylation factor ZmArf2 increases organ and seed size by promoting cell expansion in Arabidopsis
Physiologia Plantarum, 2016,156(1):97-107.
[本文引用: 1]