,1,2Cloning and Expression Analysis of SERK2 Gene in Different Forms of Calli on Peach (Prunus persica L.)
TAN Bin1,2, CHEN TanXing1, HAN YaPing1, ZHANG YaRu1, ZHENG XianBo1,2, CHENG Jun1,2, WANG Wei1,2, FENG JianCan,1,2通讯作者:
收稿日期:2018-10-10接受日期:2018-11-17网络出版日期:2019-03-01
| 基金资助: |
Received:2018-10-10Accepted:2018-11-17Online:2019-03-01
作者简介 About authors
谭彬,E-mail：
摘要
关键词：
Abstract
Keywords：
PDF (7277KB)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
谭彬, 陈谭星, 韩亚萍, 张亚如, 郑先波, 程钧, 王伟, 冯建灿. 桃SERK2 的克隆及其在不同状态愈伤组织中的表达分析[J]. 中国农业科学, 2019, 52(5): 882-892 doi:10.3864/j.issn.0578-1752.2019.05.010
TAN Bin, CHEN TanXing, HAN YaPing, ZHANG YaRu, ZHENG XianBo, CHENG Jun, WANG Wei, FENG JianCan.
0 引言
【研究意义】植物体细胞胚胎发生指外植体的体细胞在离体条件下,经过特定培养,发生与合子胚相类似的发育途径,最终形成新个体的过程[1]。植物遗传转化的众多研究已经证明通过胚性愈伤组织诱导体细胞胚再生系统转化率较高且稳定,因此,胚状体再生系统是最为理想的遗传转化受体系统[2]。桃是重要的果树之一,目前,制约现代生物技术应用于桃上的关键环节就是缺乏成熟的离体再生和遗传转化体系[3]。基于此,开展桃胚性愈伤组织和体细胞胚的诱导及研究控制体细胞胚发生的相关基因对成功获得桃胚状体再生系统具有重要意义。【前人研究进展】已成功离体培养获得体细胞胚的植物,大多数为草本植物,木本植物中仅有核桃、落叶松、板栗、乌桕、柑橘、葡萄等体细胞胚诱导获得成功[4,5,6,7,8,9,10],其中,柑橘和葡萄还利用胚状体再生途径建立了稳定高效的遗传转化体系[11,12]。已经克隆的与植物体细胞胚发生相关的基因有BBM （BABY BOOM ）、LEC2 （LEAFY COTYLEDON2 ）、WUS （WUSCHEL ）和SERK （somatic embryogenesis receptor-like kinases,SERKs）等,BBM 、LEC2 和WUS 等能提高体细胞胚发生率或维持体细胞胚发生[13,14,15],在体细胞胚产生后才发挥作用,不能促使体细胞从营养生长向胚性生长的转化。SERK蛋白广泛存在于植物当中,SCHMIDT等[16]首先从胡萝卜组织中获得第一个DcSERK ,并发现它只在胚性细胞内表达。随后,SERK 相继在拟南芥[17]、玉米[18]、苜蓿[19]、向日葵[20]、水稻[21]、小麦[22]、棉花[23]等植物中获得,且在体胚发生过程中均可以检测到它们的表达。拟南芥AtSERK1 在花芽和发育初期的合子胚中表达显著高于叶、根和茎[17, 24]。ANDREA等[25]研究表明甘蓝型油菜SERK （SERK1 和SERK2 ）在小孢子培养1—5 d后BnSERK1 表达量明显上调,BnSERK2 在整个小孢子胚期（microspore-derived embryos,MDEs）表达量持续上升。PéREZNú?EZ等[26]研究表明CnSERK 的表达与椰子体细胞胚的产生有关,它在胚性愈伤组织中表达,而在非胚性愈伤组织中低表达或检测不到。相似的结果在香蕉和棉花中发现,其中香蕉体细胞胚的发生频率与MaSERK1 的表达量呈正相关[24];过量表达GhSERK1 使棉花体胚发生能力增强[27],而GhSERK2 在生殖器官中表达量明显高于营养器官,在体胚发生过程中在球形胚中的表达量最高[23]。以上结果表明SERK 不仅能促进体细胞胚的发生,还标志着体细胞从营养生长向胚性生长转变,预示SERK 可能会被发展成为体细胞胚发生的标记基因[16]。【本研究切入点】桃离体再生体系研究进展极其缓慢,有关体细胞胚的诱导及体细胞胚发生相关基因的研究报道较少。通过对桃SERK2 的克隆和表达分析,有助于从分子水平了解SERK2 在桃胚性愈伤组织及体胚发生过程中的作用,探究桃再生体系建立困难的原因。【拟解决的关键问题】本研究通过克隆桃PpSERK2 ,分析其在不同状态愈伤组织中的表达,揭示PpSERK2 在桃胚性愈伤组织及体细胞胚胎发生中的作用,为提高桃胚性愈伤组织的体细胞胚胎发生能力提供理论指导。1 材料与方法
1.1 植物材料
‘秋蜜红’（Prunus persica ‘Qiumihong’）茎尖取自河南农业大学果树种质资源圃,样品采集后迅速放入液氮速冻并带回实验室,-80℃保存。以‘秋蜜红’花药为外植体诱导愈伤组织,‘秋蜜红’花蕾采摘于河南农业大学果树种质资源圃,采回后取花药在无菌条件下进行培养,获得的不同状态的愈伤组织,用于后续的石蜡切片制作和SERK2 表达分析。
1.2 PpSERK2 的克隆
采用改良的CTAB法[28]提取桃茎尖及愈伤组织RNA,用1.5%琼脂糖凝胶电泳检测RNA的质量。利用反转录试剂盒（High Capacity cDNAReverse Transcription Kit,ABI）合成cDNA,-20℃保存备用。根据目标研究基因在GenBank（登录号为XP_007201734.1）中已知的cDNA序列,利用Vector NTI软件设计特异性引物（上游引物5′-ATGGAGAG CAAGGTAGGGAA-3′,下游引物5′-TCACCTTGGA CCAGATAATTCA-3′）,以桃茎尖RNA反转录合成的cDNA为模板进行PCR反应,PCR反应体系（20 μL）为1 μL cDNA、上下游引物（10 μmol·L-1）各1 μL、10 μL 2×Taq Plus Mster Mix（Dye Plus）,补ddH2O至20 μL。PCR反应条件为95℃ 4 min;95℃ 30 s,57℃ 30 s,72℃ 110 s,30个循环;72℃ 10 min。扩增产物经1.5%琼脂糖凝胶电泳检测,回收目的片段,连接至PMD 20-T载体,转化大肠杆菌DH5α感受态细胞,筛选阳性克隆送至生工生物工程（上海）股份有限公司进行测序。
1.3 PpSERK2 的生物信息学分析
所有序列均来自于NCBI（http://www.ncbi.nlm. nih.gov/）;运用Expy Protparatam（http://www.expasy. ch//tools/protparam.htmL）在线预测PpSERK2 编码蛋白质的理化性质;运用TMPred（http://embnet.vital-it. ch/software/TMPRED_form.html）进行PpSERK2跨膜区的预测;利用plant-mPloc（http://www.csbio.sjtu.edu. cn/bioinf/plant-multi/）预测亚细胞定位;采用InterProScan（http://www.ebi.ac.uk/InterProScan/）程序分析保守结构域;用DNAMAN软件对不同植物的氨基酸序列进行同源比对;利用MEGA 5.0软件对不同植物的SERK序列构建系统进化树。1.4 ‘秋蜜红’花药愈伤组织的诱导
将田间采集的‘秋蜜红’小花蕾期花蕾去除下部芽鳞片,在洗涤剂中浸泡15—30 min,流水冲洗0.5—1 h。在无菌条件下,首先将花蕾用75%酒精进行消毒,无菌水冲洗3次;然后用0.1%NaClO消毒处理5 min,无菌水冲洗5—6次;在超净工作台内将花药接种到NN69+6-BA 2.0 mg·L-1+2,4-D 1.0 mg·L-1+蔗糖60 g·L-1培养基,于25℃暗培养至愈伤组织长出,每4周继代一次。1.5 石蜡切片的制作及镜检
将需要固定的花药愈伤组织放入盛有FAA固定液的标本瓶中,盖上橡胶盖,用注射器缓慢抽气,除去材料表面的气泡直至材料沉于瓶子底部,不再有气泡冒出为止,4℃固定24—72 h。参照李和平[29]的方法制作石蜡切片,在正置光学显微镜（NIKON ECLIPSE E100,日本）下采集图像信息。1.6 实时荧光定量PCR分析
根据已得到的PpSERK2 全长,利用Primer 3.0在线工具（http://primer3.ut.ee/）设计实时荧光定量PCR引物（F：5′-TGTTGCTTGATTGGGTGAAA-3′;R：5′-CATTGGAGAACCTTGTGC-3′）,以桃TEF2 为内参基因（F：5′-AGCTGGTCATGTCAAAGTCCA-3′;R：5′-ACAAGCCAACTCGGAACT-3′）,引物由生工生物工程（上海）股份有限公司合成。以‘秋蜜红’不同状态的愈伤组织cDNA为模板,对PpSERK2 进行qRT-PCR分析。实时荧光定量PCR体系为SYBR Premix（2×）10.0 μL、上下游引物（10 μmol·L-1）各1.0 μL、cDNA 2.0 μL和DEPC H2O 6.0 μL。qRT-PCR反应条件参照韩亚萍[30]方法,每处理重复3次,以2-ΔΔCT法进行数据分析。1.7 数据统计与分析
运用SPSS17.0软件进行试验数据的统计学分析,采用邓肯式新复极差法进行处理间差异分析（P <0.05）。2 结果
2.1 PpSERK2 的克隆
用1.5%琼脂糖凝胶电泳检测‘秋蜜红’茎尖RNA,28S和18S条带清晰,且28S条带比18S的条带亮,结果表明,RNA的质量和浓度均可用于下一步的试验（图1-A）。以‘秋蜜红’茎尖cDNA为模板进行PCR扩增,获得一条约为1 881 bp的目的片段（图1-B）。图1
新窗口打开|下载原图ZIP|生成PPT图1PpSERK2 的克隆
A：‘秋蜜红’茎尖RNA;B：PpSERK2 的PCR扩增,M：DNA marker DL2000
Fig. 1Cloning of PpSERK2
A: RNA of shoot tip in ‘Qiumihong’; B: PCR amplification of PpSERK2 , M: DNA marker DL2000
2.2 PpSERK2 的生物信息学分析
PpSERK2 全长1 881 bp,编码626个氨基酸（图2）。运用Protparatam在线预测其蛋白质的理论分子量为68.99 kD,pI 为5.38,不稳定系数是41.73（不稳定）,分子式为C3084H4873N837O907S25,脂融指数为91.71,总平均疏水指数为-0.115,具疏水性。蛋白由22种氨基酸组成,其中亮氨酸（14.2%）含量最多,其次是甘氨酸（7.5%）,半胱氨酸（1.4%）含量最少。TMpred预测结果推测PpSERK2具备跨膜结构,属于跨膜蛋白。亚细胞定位预测结果显示PpSERK2定位于细胞膜。图2
新窗口打开|下载原图ZIP|生成PPT图2PpSERK2 的cDNA序列及其推导的氨基酸序列
Fig. 2The nucleotide sequence of PpSERK2 gene cDNA and its deduced amino acid sequence
用DNAMAN软件将PpSERK2氨基酸序列与已知不同植物的SERK序列进行同源性比对。结果显示,PpSERK2氨基酸序列与秘鲁番茄（Solanum peruvianu ）SpSERK1相似性最高,为92.71%,与温州蜜柑（Citrus unshiu ）CitSERK1次之,为92.01%,与拟南芥（Arabidopsis thaliana ）AtSERK5的最低,为67.88%（图3）。InterProScan分析PpSERK2结果显示,其含有多个保守区典型SERK结构,N端存在1个编码信号肽（signal peptide,SP）,紧接着是1个亮氨酸拉链结构（leu zipper,ZIP）、5个富亮氨酸重复序列（leu-rich repeat,LRR）、1个编码SPP（Ser-Pro-Pro）基序的富脯氨酸结构域、1个跨膜结构域（transmembrane region,TM）和11个亚区的激酶结构域（kinase domains）。此外,PpSERK2蛋白还存在1个C端结构域（图3）。
图3
新窗口打开|下载原图ZIP|生成PPT图3不同植物的SERK氨基酸序列比对
PpSERK2：桃Prunus persica ,XP_007201734.1;PsSERK2：牡丹Paeonia suffruticosa ,KY200849.1;CitSERK1：温州蜜柑Citrus unshiu ,AB115767.1;BdSERK2：二穗短柄草Brachypodium distachyon ,XP_003571417.2;SpSERK1：秘鲁番茄Solanum peruvianu ,EF623824.1;AcSERK1：菠萝Ananas comosus ,HM236375.2;AcSERK2：菠萝Ananas comosus ,HM236376.1;HvSERK1：大麦Prunus persica ,AK372118.1;OsSERK1：水稻Oryza sativa ,AB188247.1;OsSERK2：水稻Oryza sativa ,XP_015636497.1;ZmSERK1：玉米Zea mays ,AJ277702.1;ZmSERK2：玉米Zea mays ,AJ277703.1;AtSERK1：拟南芥Arabidopsis thaliana ,NP_177328.1;AtSERK2：拟南芥Arabidopsis thaliana ,AF384969.1;AtSERK3：拟南芥Arabidopsis thaliana ,AF384970.1;AtSERK4：拟南芥Arabidopsis thaliana ,NP_178999.2;AtSERK5：拟南芥Arabidopsis thaliana ,NM_126956.4。划线部分为保守结构域The conserved regions are marked with lines
Fig. 3The diagrams of SERK amino acid sequence in different plants
利用MEGA5.0软件对不同植物的SERK氨基酸序列构建系统进化树（图4）,结果显示,PpSERK2与SpSERK1、CitSERK1、PsSERK2、AtSERK1、AtSERK2等双子叶植物同源性较高;二穗短柄草（Brachypodium distachyon ）、菠萝（Ananas comosus ）、水稻（Oryza sativa ）、玉米（Zea mays ）等单子叶植物聚为一类,进化树结果与同源性比对结果一致;拟南芥AtSERK3、AtSERK4、AtSERK5独处于一个分支。
图4
新窗口打开|下载原图ZIP|生成PPT图4PpSERK2与其他植物的SERK蛋白系统进化树
Fig. 4Phylogenetic tree of PpSERK2 and SERK in other plants
2.3 PpSERK2 在不同状态‘秋蜜红’桃愈伤组织中的表达分析
将‘秋蜜红’花药接种到添加6-BA和2,4-D的NN69培养基,培养1周后,花药颜色逐渐变为褐色;培养15 d后,有的在其中部或端部的药室内形成裂缝,从中长出黄色的颗粒状愈伤组织。继续培养2周,对获得的愈伤组织进行形态学观察,可将花药培养获得的愈伤组织分为4类,黄色疏松状、绿色紧实状、浅黄色透明状和黄白色水渍状（图5）。组织细胞学观察发现黄色疏松状和绿色紧实状的愈伤组织细胞小、排列紧密且染色比较深（图5-E和图5-F）,分别出现球形胚和心形胚结构;浅黄色透明状愈伤组织细胞排列紧密、细胞体积小,靠近愈伤组织边缘的细胞排列比较紧密且细胞体积小（图5-G）,疑似鱼雷形胚结构尚未完全形成;而黄白色水渍状愈伤组织细胞体积大、排列不规则,细胞质稀且染色浅（图5-H）。根据形态学和组织学的鉴定结果,可知黄色疏松状、绿色紧实状和浅黄色透明状的3种状态愈伤组织均为胚性愈伤组织,而黄白色水渍状愈伤组织为非胚性愈伤组织。图5
新窗口打开|下载原图ZIP|生成PPT图5‘秋蜜红’不同状态愈伤组织形态学及细胞学观察
A：黄色疏松状胚性愈伤组织;B：绿色紧实状胚性愈伤组织;C：浅黄色透明状胚性愈伤组织;D：黄白色水渍状非胚性愈伤组织;E：黄色疏松状胚性愈伤组织细胞组织切片（箭头所示为球形胚结构）;F：绿色紧实状胚性愈伤组织细胞组织切片（箭头所示为心形胚结构）;G：浅黄色透明状胚性愈伤组织细胞组织切片（箭头所示为尚未完全形成的鱼雷形胚结构）;H：黄白色水渍状非胚性愈伤组织细胞组织切片。A—D：图标比例尺为1 mm;E—H：图标比例尺为200 μm
Fig. 5The morphological and cytological observation of different forms of calli from P. perscia ‘Qiumihong’
A: The yellow, loose embryogenic callus; B: The green, compact embryogenic callus; C: The pale yellow, transparent embryogenic callus; D: The yellow-white, dropsical non-embryonic callus; E: Microscope slide of the yellow, loose embryogenic callus (arrow indicated globular embryo); F: Microscope slide of the green, compact embryogenic callus (arrow indicated heart-shape embryo); G: Microscope slide of the pale yellow, transparent embryogenic callus (arrow indicated not yet fully formed torpedo-shape embryo); H: Microscope slide of the yellow-white, dropsical embryonic callus. A-D: Bar = 1 mm; E-H: Bar = 200 μm
通过对‘秋蜜红’4种状态愈伤组织中PpSERK2 的表达分析（图6）,结果表明,PpSERK2 在‘秋蜜红’3种状态胚性愈伤组织中的表达量均显著高于黄白色水渍状非胚性愈伤组织,且在3种胚性愈伤组织中,PpSERK2 在黄色疏松状愈伤组织中的表达量最高,结合组织细胞学观察发现黄色疏松状愈伤组织中有球形胚出现,其表达量明显高于绿色紧实状（心形胚）和浅黄色透明状（疑似鱼雷形胚出现）,球形胚为体细胞胚发育过程的第一个阶段,即PpSERK2 在桃体细胞胚发生的早期高表达。
图6
新窗口打开|下载原图ZIP|生成PPT图6PpSERK2 在‘秋蜜红’不同状态愈伤组织中的表达量
数据为平均值±标准误（n=3）;不同小写字母表示差异显著（P <0.05）
Fig. 6The relative of expression of PpSERK2 gene in different forms of calli of P. perscia ‘Qiumihong’
Data are means±standard error (n=3); Different small letters mean significant differences (P <0.05)
3 讨论
体细胞胚发生途径是植物离体再生的一条重要途径,而体细胞胚是研究植物胚胎发育和遗传转化的良好受体。在体细胞胚发生过程中,体细胞胚不仅生理生化及形态发生变化,它的基因表达方式也发生变化。3.1 PpSERK2 的克隆与分析
SERKs基因属于膜富亮氨酸重复序列类受体蛋白激酶（leucinerich repeat receptor-like kinases,LRR-RLKs）家族[31]。根据胞外LRR结构和数量的差异,LRR-RLKs可分为13个亚类,而SERK蛋白属于第2类[32]。SERK结构保守,具LRR结构域、富含丝氨酸-脯氨酸结构域（SPP）、跨膜结构域和胞内激酶活性结构域[33,34]。本研究从桃茎尖中克隆了SERK2 的cDNA全长,编码626个氨基酸,生物信息学分析表明其具有完整的SERK蛋白的保守结构域,因而能独立完成信号的接收、跨膜转导和胞内传递,将其命名为PpSERK2。PpSERK2与双子叶植物秘鲁番茄和温州蜜柑的SERK蛋白同源性较高,与单子叶植物的水稻、玉米、菠萝等同源性较低。3.2 桃花药胚性愈伤组织的诱导及鉴定
桃胚性愈伤组织诱导及体细胞胚发生获得成功的报道较少[35,36]。HAMMERSCHLAG等[35]从白色、紧密、节球状胚性愈伤组织中诱导出胚状体结构;刘航空等[36]以‘华光’叶片为外植体诱导出黄绿色疏松状胚性愈伤组织。以桃花药为外植体诱导获得胚性愈伤组织鲜有报道。本研究以‘秋蜜红’花药为外植体诱导出4种状态的愈伤组织,组织细胞学观察到2种细胞形态,其中黄色疏松状、绿色紧实状和浅黄色透明状3种状态的愈伤组织细胞小,排列紧密且染色深,为胚性愈伤组织;而黄白色水渍状愈伤组织细胞体积大,不规则排列,细胞质稀且染色浅,为非胚性愈伤组织。表明胚性愈伤组织和非胚性愈伤组织在形态上和细胞水平均表现明显差异。本研究诱导出的胚性愈伤组织状态与前人不同[35,36],这可能是由于基因型、外植体、培养基和培养条件不同导致的;而组织细胞学观察结果与前人研究结果一致[37]。胚性愈伤组织的成功获得是进行体细胞胚诱导的关键因素。体细胞胚发生一般经历球形胚、心形胚、鱼雷形胚和子叶形胚4个阶段。本研究获得的黄色疏松状、绿色紧实状和浅黄色透明状3种状态愈伤组织均为胚性愈伤组织,且分别出现球形胚、心形胚和尚未完全形成的鱼雷形胚结构,这3种状态胚性愈伤组织的获得为后续体细胞胚再生途径的建立提供保障。3.3 PpSERK2 在桃胚性愈伤组织和体细胞胚发生中的作用
目前,植物中已经分离出许多与体细胞胚胎发生相关的基因,如SERK[17]、LEC2[14]、BBM[13]、ARF5a（auxin response factor 15）[38]等,但只有SERK[16,39]是在体细胞由营养生长向胚性生长的转化中以及早期的体细胞胚胎发生中起作用。基于此,推测SERK 可能是一些植物体细胞胚胎发生的标记基因[31]。本研究中PpSERK2 在桃3种状态胚性愈伤组织中表达量均显著高于非胚性愈伤组织,且在体细胞胚发育的早期（黄色疏松状愈伤组织,出现球形胚）表达量最高,这与棉花[23]、椰子[26]、葡萄[40]、二穗短柄草[41]等研究结果相似。GhSERK2 在棉花生殖器官中表达量明显高于营养器官,且在体胚发生过程中在球形胚中的表达量最高[23];CnSERK 在椰子胚性愈伤组织中表达,而在非胚性愈伤组织中低表达或检测不到[26];SCHELLENBAUM等[40]研究发现VvSERK2 在葡萄花药诱导愈伤组织和体胚发生的过程中稳定表达;原位杂交结果表明BdSERK 的转录本在二穗短柄草胚状体整个发生过程均可检测到[41]。由此认为桃PpSERK2 在桃胚性愈伤组织中表达,而在非胚性愈伤组织中低表达,且在体细胞胚发育的早期发挥作用。4 结论
克隆获得PpSERK2 ,其cDNA全长1 881 bp,编码626个氨基酸。PpSERK2与秘鲁番茄和温州蜜柑相似性最高,分别为92.71%和92.01%,拥有SERK蛋白保守结构域。PpSERK2 在桃胚性愈伤组织中表达,在非胚性愈伤组织中低表达,且在体细胞胚发育的早期发挥作用。参考文献 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
DOI:10.1023/A:1015037920529URL [本文引用: 1]
Plants were regenerated from mature zygotic embryos of sandalwood ( Santalum album L.) through direct somatic embryogenesis. Somatic embryos were formed directly without any intervening callus phase on zygotic embryos plated on Murashige and Skoog (MS) medium containing thidiazuron or benzylaminopurine. Individual somatic embryos were then isolated and transferred to MS medium without cytokinin on which they formed secondary embryos in repetitive cycles with or without the addition of indole acetic acid to the medium. Conversion of somatic embryos into plantlets was achieved by isolating somatic embryos with distinct cotyledons and reculturing them onto half-strength MS medium with GA 3 (1.4 M). Recovered plantlets were acclimatised and grown in the greenhouse. This is the first report on in vitro regeneration via direct somatic embryogenesis of sandalwood.
DOI:10.3969/j.issn.1009-9980.2002.02.013URL [本文引用: 1]
现代生物技术如组织培养和遗传转化在植物育种方面有重要作用.综述了桃树的组织培养技术和桃遗传转化研究取得的成绩.文中侧重介绍了广泛应用于桃树研究的茎尖培养法、胚培养法两种主要的组织培养技术和农杆菌介导法、基因枪轰击法两种主要的遗传转化方法;详细说明了品种、外植体类型及其发育阶段、培养基类型、生长调节因子和培养环境等对桃组织培养的影响;初步分析了影响桃树遗传转化的关键问题,并对进一步的工作重点提出了建议.
DOI:10.3969/j.issn.1009-9980.2002.02.013URL [本文引用: 1]
现代生物技术如组织培养和遗传转化在植物育种方面有重要作用.综述了桃树的组织培养技术和桃遗传转化研究取得的成绩.文中侧重介绍了广泛应用于桃树研究的茎尖培养法、胚培养法两种主要的组织培养技术和农杆菌介导法、基因枪轰击法两种主要的遗传转化方法;详细说明了品种、外植体类型及其发育阶段、培养基类型、生长调节因子和培养环境等对桃组织培养的影响;初步分析了影响桃树遗传转化的关键问题,并对进一步的工作重点提出了建议.
.
[本文引用: 1]
DOI:10.3321/j.issn:1001-7488.2000.03.017URLMagsci [本文引用: 1]
总结了核桃体细胞胚发生的研究进展,列表统计已报道的核桃5个种和3个杂种体细胞胚发生的外植体与诱导条件,重点论述了影响核桃体细胞胚发生与次生胚发生的因素,介绍了核桃体细胞胚萌发与转化的方法。还总结核桃转基因研究的进展,提出了用核桃体细胞胚发生系统进行外源基因转移的操作模式。
DOI:10.3321/j.issn:1001-7488.2000.03.017URLMagsci [本文引用: 1]
总结了核桃体细胞胚发生的研究进展,列表统计已报道的核桃5个种和3个杂种体细胞胚发生的外植体与诱导条件,重点论述了影响核桃体细胞胚发生与次生胚发生的因素,介绍了核桃体细胞胚萌发与转化的方法。还总结核桃转基因研究的进展,提出了用核桃体细胞胚发生系统进行外源基因转移的操作模式。
DOI:10.11707/j.1001-7488.20161006URLMagsci [本文引用: 1]
<b>[目的]</b> 以长白落叶松的未成熟合子胚为外植体，进行胚性愈伤组织的诱导、增殖培养及体细胞胚胎发生的相关研究，揭示影响长白落叶松胚性愈伤组织诱导的关键因素，并探讨继代培养过程中添加不同种类、浓度的生长调节剂对胚性愈伤组织增殖及体胚发生的影响。<b>[方法]</b> 采集长白落叶松3个家系优良单株的种子，诱导胚性愈伤组织，比较外植体采集时间、家系、2，4-D浓度及基本培养基对胚性愈伤组织诱导的影响。诱导出的胚性愈伤组织继代于含不同种类、浓度生长调节剂的增殖培养基，通过体胚发生途径获得体细胞胚。选取发育正常的体胚进行萌发，待体胚生根后移栽。<b>[结果]</b> 不同时间采集的未成熟合子胚，其胚性愈伤组织诱导率存在较大差别，授粉后63天的合子胚诱导率为5.61%，授粉后70天诱导率为22.35%，而授粉后80天未诱导出胚性愈伤组织；‘长77-22’、‘长77-37’和‘长73-50’3个家系胚性愈伤组织的平均诱导率分别为6.69%，11.17%和3.11%；2，4-D浓度对长白落叶松胚性愈伤组织诱导具有一定影响，在一定范围内胚性愈伤组织的诱导率会随2，4-D浓度升高而升高，当其浓度为1.5 mg·L<sup>-1</sup>时胚性愈伤组织的诱导率最高，达到11.11%，而当2，4-D浓度超过1.5 mg·L<sup>-1</sup>时，胚性愈伤组织的诱导率则开始下降；BM，MS，S培养基均能诱导出胚性愈伤组织，其中，BM培养基的诱导率最高，S培养基的诱导率次之，MS培养基的诱导率最低。胚性愈伤组织在含2，4-D 0.3 mg·L<sup>-1</sup>、6-BA 0.1 mg·L<sup>-1</sup>及KT 0.1 mg·L<sup>-1</sup>的BM培养基上增殖15天可获得相对较多的胚性愈伤组织，增殖率为345.93%；在含2，4-D 1.5 mg·L<sup>-1</sup>、6-BA 0.5 mg·L<sup>-1</sup>及KT 0.5 mg·L<sup>-1</sup>的BM培养基上培养14天，再经诱导可获得较多的体细胞胚胎，每克愈伤组织平均诱导179.87个体胚，并且这些体胚的萌发率及植株再生率相对较高，分别为75.00%，66.67%；再生植株移栽成活率为27.08%。<b>[结论]</b> 不同长白落叶松家系的胚性愈伤组织诱导率不同，散粉后70天的合子胚适合诱导胚性愈伤组织，基本培养基为BM，添加2，4-D 1.5 mg·L<sup>-1</sup>。胚性愈伤组织长期继代在含高浓度生长调节剂的培养基上易丧失胚性，且增殖速度慢，但体胚的发生量及萌发率相对较高；适当降低生长调节剂浓度利于愈伤组织保持胚性及增殖，但体胚的发生量及体胚的萌发率有所下降；当培养基中生长调节剂浓度较低时，不利于愈伤组织的增殖，但仍能使其胚性长期保持；采用浓度为0.5 mg·L<sup>-1</sup>的NAA代替0.15 mg·L<sup>-1</sup>的2，4-D有助于胚性愈伤组织的增殖，并能在一定程度上提高体胚的萌发率。因此，可根据不同阶段的培养目的，选择添加不同种类和浓度生长调节剂的增殖培养基进行继代。
DOI:10.11707/j.1001-7488.20161006URLMagsci [本文引用: 1]
<b>[目的]</b> 以长白落叶松的未成熟合子胚为外植体，进行胚性愈伤组织的诱导、增殖培养及体细胞胚胎发生的相关研究，揭示影响长白落叶松胚性愈伤组织诱导的关键因素，并探讨继代培养过程中添加不同种类、浓度的生长调节剂对胚性愈伤组织增殖及体胚发生的影响。<b>[方法]</b> 采集长白落叶松3个家系优良单株的种子，诱导胚性愈伤组织，比较外植体采集时间、家系、2，4-D浓度及基本培养基对胚性愈伤组织诱导的影响。诱导出的胚性愈伤组织继代于含不同种类、浓度生长调节剂的增殖培养基，通过体胚发生途径获得体细胞胚。选取发育正常的体胚进行萌发，待体胚生根后移栽。<b>[结果]</b> 不同时间采集的未成熟合子胚，其胚性愈伤组织诱导率存在较大差别，授粉后63天的合子胚诱导率为5.61%，授粉后70天诱导率为22.35%，而授粉后80天未诱导出胚性愈伤组织；‘长77-22’、‘长77-37’和‘长73-50’3个家系胚性愈伤组织的平均诱导率分别为6.69%，11.17%和3.11%；2，4-D浓度对长白落叶松胚性愈伤组织诱导具有一定影响，在一定范围内胚性愈伤组织的诱导率会随2，4-D浓度升高而升高，当其浓度为1.5 mg·L<sup>-1</sup>时胚性愈伤组织的诱导率最高，达到11.11%，而当2，4-D浓度超过1.5 mg·L<sup>-1</sup>时，胚性愈伤组织的诱导率则开始下降；BM，MS，S培养基均能诱导出胚性愈伤组织，其中，BM培养基的诱导率最高，S培养基的诱导率次之，MS培养基的诱导率最低。胚性愈伤组织在含2，4-D 0.3 mg·L<sup>-1</sup>、6-BA 0.1 mg·L<sup>-1</sup>及KT 0.1 mg·L<sup>-1</sup>的BM培养基上增殖15天可获得相对较多的胚性愈伤组织，增殖率为345.93%；在含2，4-D 1.5 mg·L<sup>-1</sup>、6-BA 0.5 mg·L<sup>-1</sup>及KT 0.5 mg·L<sup>-1</sup>的BM培养基上培养14天，再经诱导可获得较多的体细胞胚胎，每克愈伤组织平均诱导179.87个体胚，并且这些体胚的萌发率及植株再生率相对较高，分别为75.00%，66.67%；再生植株移栽成活率为27.08%。<b>[结论]</b> 不同长白落叶松家系的胚性愈伤组织诱导率不同，散粉后70天的合子胚适合诱导胚性愈伤组织，基本培养基为BM，添加2，4-D 1.5 mg·L<sup>-1</sup>。胚性愈伤组织长期继代在含高浓度生长调节剂的培养基上易丧失胚性，且增殖速度慢，但体胚的发生量及萌发率相对较高；适当降低生长调节剂浓度利于愈伤组织保持胚性及增殖，但体胚的发生量及体胚的萌发率有所下降；当培养基中生长调节剂浓度较低时，不利于愈伤组织的增殖，但仍能使其胚性长期保持；采用浓度为0.5 mg·L<sup>-1</sup>的NAA代替0.15 mg·L<sup>-1</sup>的2，4-D有助于胚性愈伤组织的增殖，并能在一定程度上提高体胚的萌发率。因此，可根据不同阶段的培养目的，选择添加不同种类和浓度生长调节剂的增殖培养基进行继代。
[本文引用: 1]
[本文引用: 1]
.
DOI:10.1016/j.scienta.2015.09.040URL [本文引用: 1]
Sapium sebiferiumRoxb. is a highly valued ornamental plant species in tropical and subtropical regions. Here we present an efficient protocol for plant regeneration from immature zygotic embryos (IZEs) ofS. sebiferiumthrough direct somatic embryogenesis and shoot organogenesis for the first time. The results showed that mechanical damage, plant growth regulators (PGRs) and light regimes had great influence onin vitromorphogenesis. High frequency of somatic embryogenesis was obtained when the mechanical damaged IZEs were cultured on the Murashige and Skoog (MS) medium containing 1.0mgl鈭1indole-3-acetic acid (IAA) under continuous darkness for two weeks followed by a light/dark photoperiod cycle for four weeks. The induction frequency of somatic embryogenesis was up to 90.0%. Somatic embryos (SEs) at early cotyledonary stage showed highest proliferation frequency (81.7%) and 80.0% of secondary somatic embryos (SSEs) germinated on plant growth regulators (PGRs)-free MS medium. For direct shoot induction pathway, the best shoot regeneration rate (63.3%) was obtained on the medium supplemented with 1.0mgl鈭16-benzyladenine (6-BA). The plantlets derived from two ways were successfully acclimatized in the greenhouse with more than 90% survival rate. This study will be beneficial for the rescue of immature embryo, inheritance of heterosis as well as the genetic improvement ofS. sebiferium.
.
[本文引用: 1]
DOI:10.1007/s00344-017-9694-6URL [本文引用: 1]
The purpose of the study was to increase plant conversion efficiency and reduce waste of the abnormal somatic embryos; factors influencing both the embryo morphology in germination and the embryogenic callus reinduction rate in secondary embryogenesis were studied in Vitis vinifera L. ‘Thompson Seedless.’ Embryos having mono-, di-, and multiple-normal cotyledons, trumpet-shaped, fascicular, and fused abnormal cotyledons were observed during germination. Normal embryos of one, two, and multiple cotyledons have a relatively higher conversion rate (48.89–64.44%) than abnormal ones (2.22–22.22%). Histomorphology analysis of these embryos showed that normal somatic embryos had a better developed vasculature and shoot meristem than abnormal individuals. A lower percentage of abnormal embryos formed when they germinated on Murashige and Skoog’s medium (MS) and Woody Plant Medium (WPM) both of which contained 1502g/l sucrose and no plant growth regulators. Of all the abnormal embryos, only fused cotyledon embryos were suitable for recycling, and they were reinduced into the embryogenic callus. Cotyledons showed a higher reinduction rate (72.22%) than the other two parts: hypocotyls (4.44%) and roots (0%). In addition, recycled abnormal embryos produced many morphogenic types and this secondary embryogenesis could be used for embryo proliferation.
DOI:10.1371/journal.pone.0190413URLPMID:29293649 [本文引用: 1]
Genetic transformation of citrus has been mainly conducted via Agrobacterium tumefaciens using juvenile tissues of the epicotyl segments obtained from the in vitro germination of seeds. Transformation of the stem segments obtained from the mature material is an alternative to overcome or reduce the juvenile phase reported in perennial species such as citrus. However, some citrus species,... [Show full abstract]
.
[本文引用: 1]
DOI:10.1005/Tpc.001941URLPMID:12172019 [本文引用: 2]
The molecular mechanisms underlying the initiation and maintenance of the embryonic pathway in plants are largely unknown. To obtain more insight into these processes, we used subtractive hybridization to identify genes that are upregulated during the in vitro induction of embryo development from immature pollen grains of Brassica napus (microspore embryogenesis). One of the genes identified, BABY BOOM (BBM), shows similarity to the AP2/ERF family of transcription factors and is expressed preferentially in developing embryos and seeds. Ectopic expression of BBM in Arabidopsis and Brassica led to the spontaneous formation of somatic embryos and cotyledon-like structures on seedlings. Ectopic BBM expression induced additional pleiotropic phenotypes, including neoplastic growth, hormone-free regeneration of explants, and alterations in leaf and flower morphology. The expression pattern of BBM in developing seeds combined with the BBM overexpression phenotype suggests a role for this gene in promoting cell proliferation and morphogenesis during embryogenesis.
[本文引用: 2]
[本文引用: 1]
DOI:10.1016/S1386-1425(01)00499-1URLPMID:9169851 [本文引用: 3]
The first somatic single cells of carrot hypocotyl explants having the competence to form embryos in the presence of 2,4-dichlorophenoxyacetic acid (2,4-D) were identified using semi-automatic cell tracking. These competent cells are present as a small subpopulation of enlarged and vacuolated cells derived from cytoplasm-rich and rapidly proliferating non-embryogenic cells that originate from the provascular elements of the hypocotyl. A search for marker genes to monitor the transition of somatic into competent and embryogenic cells in established suspension cell cultures resulted in the identification of a gene transiently expressed in a small subpopulation of the same enlarged single cells that are formed during the initiation of the embryogenic cultures from hypocotyl explants. The predicted amino acid sequence and in vitro kinase assays show that this gene encodes a leucine-rich repeat containing receptor-like kinase protein, designated Somatic Embryogenesis Receptor-like Kinase (SERK). Somatic embryos formed from cells expressing a SERK promoter-luciferase reporter gene. During somatic embryogenesis, SERK expression ceased after the globular stage. In plants, SERK mRNA could only be detected transiently in the zygotic embryo up to the early globular stage but not in unpollinated flowers nor in any other plant tissue. These results suggest that somatic cells competent to form embryos and early globular somatic embryos share a highly specific signal transduction chain with the zygotic embryo from shortly after fertilization to the early globular embryo.
.
[本文引用: 3]
.
DOI:10.1007/s004250000471URLPMID:11523644 [本文引用: 1]
Genes encoding two novel members of the leucine-rich repeat receptor-like kinase (LRR-RLK) superfamily have been isolated from maize (Zea mays L.). These genes have been named ZmSERK1 and ZmSERK2 since features such as a putative leucine zipper (ZIP) and five leucine rich repeats in the extracellular domain, a proline-rich region (SPP) just upstream of the transmembrane domain and a C-terminal extension (C) after the kinase domain identify them as members of the SERK (somatic embryogenesis receptor-like kinase) family. ZmSERK1 and ZmSERK2 are single-copy genes and show 79% identity among each other in their nucleotide sequences. They share a conserved intron/exon structure with other members of the SERK family. In the maize genome, ZmSERK1 maps to position 76.9 on chromosome arm 10L and ZmSERK2 to position 143.5 on chromosome arm 5L, in regions generally not involved in duplications. ZmSERK1 is preferentially expressed in male and female reproductive tissues with strongest expression in microspores. In contrast, ZmSERK2 expression is relatively uniform in all tissues investigated. Both genes are expressed in embryogenic and non-embryogenic callus cultures.
.
URL [本文引用: 1]
.
DOI:10.1016/j.plaphy.2003.10.008URLPMID:15061082 [本文引用: 1]
Organogenesis or somatic embryogenesis can be induced on immature zygotic embryos (IZE) of sunflower depending on the culture conditions. Both morphogenic processes originate from the same group of cells and show identical kinetics. Using real-time PCR and in situ hybridisation, we showed that somatic embryogenesis receptor-like kinase ( SERK) transcripts accumulate early after the beginning of the culture in the morphogenic zone of IZE explants whatever the induction conditions used, i.e. organogenic, embryogenic or highly embryogenic conditions. Quantitative analyses failed to show any correlation between the SERK expression level during the period decisive for the orientation of the morphogenic pathway, i.e. the first 2 days of culture, and the type of morphogenesis induced. However, after 2 days of culture on the organogenic medium, the SERK gene expression level was severely down-regulated in the IZE explants. At 4 days of culture, SERK transcripts were no longer detectable by in situ hybridisation in the developing shoot structures whereas they still continued to accumulate in the embryonic structures induced on both embryogenic and highly embryogenic culture media. The significance of these expression analyses was addressed by transfer medium experiments. Results revealed that IZE cultured on the organogenic medium were able to form somatic embryos when transferred on the highly embryogenic medium as long as the SERK transcripts accumulated at a high level in their morphogenic zone, i.e. first 2 days of culture. Passt this delay, explants rapidly lost their embryogenic competence. Indeed, after 4 days of culture on the organogenic medium, IZE were definitely oriented towards shoot organogenesis. Taken together, these data suggest that reactive cells of IZE develop the competence to somatic embryogenesis during the first day of culture whatever the morphogenic induction conditions used.
.
DOI:10.1007/s00425-005-1534-4URLPMID:15968510 [本文引用: 1]
Here we report on the isolation and characterization of a somatic embryogenesis receptor-like kinase (OsSERK1) gene in rice (Oryza sativa). The OsSERK1 gene belongs to a small subfamily of receptor-like kinase genes in rice and shares a highly conserved gene structure and extensive sequence homology with previously reported plant SERK genes. Though it has a basal level of expression in various rice organs/tissues, as high expression level was detected in rice callus during somatic embryogenesis. Suppression of OsSERK1 expression in transgenic calli by RNA interference resulted in a significant reduction of shoot regeneration rate (from 72% to 14% in the japonica rice Zhonghuall). Overexpression of OsSERK1, however, increased the shoot regeneration rate (from 72% to 86%). Interestingly, OsSERK1 is significantly activated by the rice blast fungus, particularly during the incompatible interaction, and is associated with host cell death in Sekigushi lesion mimic mutants. This gene is also inducible by defense signaling molecules such as salicylic acid, jasmonic acid, and abscisic acid. Furthermore, constitutive overexpression of OsSERK1 in two rice cultivars led to an increase in host resistance to the blast fungus. Our data suggest that OsSERK1 may partially mediate defense signal transduction in addition to its basic role in somatic embryogenesis.
[本文引用: 1]
DOI:10.1016/S2095-3119(17)61726-XURL [本文引用: 4]
Somatic embryogenesis (SE) is one of the most important steps during regeneration of cotton, but the molecular mechanism of SE remains unclear. SOMATIC EMBRYOGENSIS RECEPTOR KINASE ( SERK ) gene is known to function in SE. A homolog GhSERK2 (accession number: JF430801) was cloned from Upland cotton and characterized for its functions in SE. GhSERK2 expressed in different tissues and showed higher expression level in floral organs than vegetative ones with the highest levels in ovule and anther. GhSERK2 expressed during SE with a high level at globular embryos stage. Upon treatment with indole-3-butytic acid (IBA), the transcription level of GhSERK2 was induced and promoted SE subsequently. A 2-day treatment of 2,4-dichlorophenoxyacetic acid (2,4-D) induced the expression of GhSERK2, but treatments of 2,4-D for longer periods sharply inhibited the GhSERK2 transcription level of embryogenic callus (EC). The levels of hormones, including 3-indoleacetic acid (IAA), abscisic acid (ABA), and brassinosteroid (BR), were increased in the initial calli induced from the over-expression of GhSERK2 cotton. Our results indicated that GhSERK2 expression was associated with induction of SE and closely related to hormone levels during tissue culture in Upland cotton, and the gene might play an important role in regeneration of cotton.
DOI:10.1007/s11105-009-0150-zURL [本文引用: 2]
A banana somatic embryogenesis receptor-like kinase ( SERK ) gene, designated as MaSERK1 , was isolated from Musa acuminata cv. Mas (AA). It encoded a protein of 628 amino acids with above 82% identities to reported SERKs of coconut, rice, maize, Arabidopsis , carrot, and Medicago truncatula . MaSERK1 was expressed weakly in male flower clusters, but not in male flower-derived nonembryogenic calli, but it was highly expressed in male flower-derived embryogenic calli and embryogenic cell suspensions (ECS). During subculture of ECS, MaSERK1 expression and frequency of somatic embryogenesis were influenced by the duration of subculture, wherein expression decreased within 0 to 6 days of subculture, increased to highest levels at 12 days following subculture, and dropped thereafter. The frequency of somatic embryogenesis of ECS positively correlated with MaSERK1 transcript levels. Moreover, MaSERK1 expression in leaves of Musa paradisiaca L. cv. Dongguan Dajiao (ABB), known to be resistant to Fusarium oxysporum f. sp. cubense race 4 (FOC race 4), was induced by exogenous salicylic acid (SA) or inoculation with FOC race 4. However, MaSERK1 expression levels in leaves of Mucus spp. cv. Pisang awak (ABB), known to be susceptible to FOC race 4, did not change following either treatment. These results suggested that MaSERK1 not only could serve as a molecular marker for banana somatic embryogenesis, but also played a role in disease resistance response in banana.
DOI:10.1007/s00299-015-1878-6URL [本文引用: 1]
KEY MESSAGE : The BnSERK1 and BnSERK2 are involved in the process of microspore embryogenesis induction, development, and plantlet regeneration. Little is known about regulatory role of somatic embryogenesis-related kinase (SERK) genes family in the induction of microspore embryogenesis, development and plant regeneration. In this study, the expression of two SERK genes (SERK1 and SERK2) was assessed during the microspore embryogenesis and plantlet regeneration in Brassica napus L. The BnSERK1 was severely up-regulated 1–502days following microspore culture and its expression drastically decreased in the globular-heart and also torpedo staged microspore-derived embryos (MDEs). In addition, high levels of BnSERK1 transcript were detected in the MDE maturation phase and in the roots and shoots of the regenerated plantlets which indicates a broader role(s) of BnSERK1 in the organ formation, rather than being specific to the embryogenesis. Results of partial sequencing indicated that the BnSERK1 shares a conserved serine-threonine kinase catalytic domain and exhibited 9502% similarity with AtSERK1, CsSERK1, BrSERK1, NaSERK1, and NbSERK1. A steady increase in the expression of BnSERK2 was observed during the MDE initiation and development so that, the highest expression was noted in the MDE maturation phase i.e., late cotyledonary MDEs. Our results also indicated low amounts of BnSERK2 transcript at the onset of rhyzogenesis but significantly higher expression in the developing roots. In contrast, the BnSERK202strongly up-regulated during the both initially and developed shoots. The BnSERK2 shares highly conserved LRR–RLK domain when compared with different species tested so that, high homology (10002%) was noticed with BrSERK2. Based on our findings, MDE formation and plantlet regeneration seem to be correlated with both BnSERK1 and BnSERK2 expression.
DOI:10.1007/s00299-008-0616-8URLPMID:18818928 [本文引用: 3]
Abstract Somatic embryogenesis involves different molecular events including differential gene expression and various signal transduction pathways. One of the genes identified in early somatic embryogenesis is S OMATIC E MBRYOGENESIS R ECEPTOR-like K INASE (SERK). Cocos nucifera (L.) is one of the most recalcitrant species for in vitro regeneration, achieved so far only through somatic embryogenesis, although just a few embryos could be obtained from a single explant. In order to increase efficiency of this process we need to understand it better. Therefore, the purpose of the present work was to determine if an ortholog of the SERK gene is present in the coconut genome, isolate it and analyze its expression during somatic embryogenesis. The results showed the occurrence of a SERK ortholog referred to as CnSERK. Predicted sequence analysis showed that CnSERK encodes a SERK protein with the domains reported in the SERK proteins in other species. These domains consist of a signal peptide, a leucine zipper domain, five LRR, the Serine-Proline-Proline domain, which is a distinctive domain of the SERK proteins, a single transmembrane domain, the kinase domain with 11 subdomains and the C terminal region. Analysis of its expression showed that it could be detected in embryogenic tissues before embryo development could be observed. In contrast it was not detected or at lower levels in non-embryogenic tissues, thus suggesting that CnSERK expression is associated with induction of somatic embryogenesis and that it could be a potential marker of cells competent to form somatic embryos in coconut tissues cultured in vitro.
[本文引用: 1]
DOI:10.3969/j.issn.1004-3268.2014.03.025URLMagsci [本文引用: 1]
<span id="ChDivSummary" name="ChDivSummary">为了从桃叶片中提取到高质量的RNA,以进行反转录、RT-PCR等后续分子生物学试验,选取了改良硼砂-CTAB法、改良CTAB法、改良SDS-苯酚法、试剂盒法、Trizol法,以及在试验中探索的改良Trizol法Ⅰ和改良Trizol法Ⅱ对桃不同发育时期的叶片总RNA进行提取,测定RNA样品的OD值,结合琼脂糖凝胶电泳检测总RNA样品的质量。结果表明,这些方法虽然都能获得桃叶片总RNA,但是或存在不同程度的DNA残留,或有蛋白、多糖等杂质污染,或降解严重;而改良的Trizol法提取总RNA的完整性和纯度较好;针对生长期和衰老期叶片的不同生理特性可分别采用改良Trizol法Ⅰ和改良Trizol法Ⅱ,提取的总RNA反转录后,通过RT-PCR检测,条带明亮清晰,与预期目的基因片段大小一致,说明这2种方法提取的总RNA能用于后期的分子生物学试验。 </span>
DOI:10.3969/j.issn.1004-3268.2014.03.025URLMagsci [本文引用: 1]
<span id="ChDivSummary" name="ChDivSummary">为了从桃叶片中提取到高质量的RNA,以进行反转录、RT-PCR等后续分子生物学试验,选取了改良硼砂-CTAB法、改良CTAB法、改良SDS-苯酚法、试剂盒法、Trizol法,以及在试验中探索的改良Trizol法Ⅰ和改良Trizol法Ⅱ对桃不同发育时期的叶片总RNA进行提取,测定RNA样品的OD值,结合琼脂糖凝胶电泳检测总RNA样品的质量。结果表明,这些方法虽然都能获得桃叶片总RNA,但是或存在不同程度的DNA残留,或有蛋白、多糖等杂质污染,或降解严重;而改良的Trizol法提取总RNA的完整性和纯度较好;针对生长期和衰老期叶片的不同生理特性可分别采用改良Trizol法Ⅰ和改良Trizol法Ⅱ,提取的总RNA反转录后,通过RT-PCR检测,条带明亮清晰,与预期目的基因片段大小一致,说明这2种方法提取的总RNA能用于后期的分子生物学试验。 </span>
[本文引用: 1]
[本文引用: 1]
[本文引用: 1]
[本文引用: 1]
DOI:10.1080/17429145.2017.1339836URL [本文引用: 2]
Abstract Somatic embryogenesis receptor-like kinases (SERKs), a subfamily of receptor-like kinases, showed important roles in plant response to abiotic and biotic stresses in addition to embryogenesis and organogenesis in numerous plant species. In the present study, three orthologs of SERK genes (HvSERK1/2/3) were isolated from barley, and their expression patterns during in vitro culture of microspores as well as their responses to different stresses including salinity and powdery mildew were characterized. Sequence analysis suggested that three HvSERK genes were highly conserved in the grass family. Subcellular localization showed the HvSERK1 protein located on the plasma membrane. The HvSERK1 transcript was up-regulated during the microspore culture period, suggesting its roles in microspore embryogenesis. HvSERK1 and HvSERK3 showed the highest expression level in the leaves; however no difference was detected for HvSERK2 expression in different plants tissues. Under salt stress, all three HvSERK genes were quickly induced in microspore-derived embryogenic calli, whereas only HvSERK1 was up-regulated in the roots of barley seedlings. Moreover, only HvSERK2 was induced in the barley leaves upon powdery mildew inoculation. These results suggest that barley SERK genes may participate in barley microspores development and plant response toward salt and fungal stress, and the function of them has some evolutionary changes.
.
[本文引用: 1]
DOI:10.1016/S1360-1385(98)01301-6URL [本文引用: 1]
Receptor protein kinases (RPKs) are components of signal transduction pathways that elicit cellular responses to extracellular information. These proteins play an essential role in animal development, but the traditional thinking has been that the cell wall is a barrier to this type of signaling in plants and that most intercellular communication must therefore occur via the plasmodesmata. However, recent evidence indicates that RPKs are also essential for a variety of plant processes, including development. A combination of molecular and genetic analysis has revealed the existence of several plant proteins that possess the characteristics of RPKs, although their function as a receptor has yet to be demonstrated.
DOI:10.1023/A:1007540610933URL [本文引用: 1]
Plant leucine-rich repeat receptor-like kinases were identified from databases using computer programs. Sequence comparison indicated that consensus sequences of the leucine-rich repeat motifs are highly conserved among these proteins. Multiple alignment of the catalytic kinase domains of all sequences displayed an overall amino acid sequence similarity. A phylogenetic analysis indicated that leucine- rich repeat receptor-like kinases form four subgroups in plants.
DOI:10.1007/BF00304907URLPMID:24252917 [本文引用: 3]
Peach plants were repeatedly regenerated from immature embryos but not from callus derived from mature embryos. A white, nodular, highly regenerative callus was obtained when friable, primary callus from immature embryos was transferred from medium containing 4.5 μM 2,4-dichlorophenoxyacetic acid and 0.44 μM benzyladenine (BA) to media containing 0.27 μM α-naphthaleneacetic acid (NAA) and 2.2 μM BA. This callus retained its morphogenetic potential for a minimum of three subcultures. Green nodular callus, that lacked regenerative capacity, was produced from primary callus derived from mature embryos. Maximum regeneration of shoots occurred when highly regenerative callus was transferred to a medium in which the NAA concentration was reduced five times and the BA concentration was increased two times. Regenerated shoots were rooted in the dark on a medium containing 28.5 μM indoleacetic acid. Cytogenetic analysis of regenerated plants indicated that all plants were diploid, 2n = 2x = 16. Phenotypic evaluation of regenerated plants, grown under field conditions, is now in progress.
DOI:10.3969/j.issn.1009-9980.2006.03.012 [本文引用: 3]
以早熟油桃华光和曙光为试材,对影响早熟油桃叶片产生胚性愈伤的基本培养基、外源激素、碳源、环境(光照强度,温度,暗培养时间)等因素进行研究,结果表明:碳源对两种油桃叶片胚性愈伤的诱导影响不显著,其他影响因子对油桃叶片胚性愈伤的诱导均显著.其中华光和曙光在暗培养21 d时,产生的有效愈伤较其他处理有极显著差异,有利于有效愈伤的产生;外源激素对诱导桃叶片产生胚性愈伤组织影响显著,NAA不是影响胚性愈伤产生的主效因子;基本培养基中,QL,1/2MS培养基有利于华光胚性愈伤的发育,QL,LP培养基有利于曙光胚性愈伤的发育;当温度在24～27.5℃时,光照强度为2000k,有利于胚性愈伤组织的诱导及生长;接种方式上,叶背横切,叶面贴于培养基表面的接种方法易产生胚性愈伤.实验中发现:叶背横切,叶面接于G+2.5 mg/L BA+0.5 mg/LIBA培养基上,暗培养21 d,转入1/2MS+2.0 mg/l TDZ+2.0 mg/L BA+0.5mg/L NAA的培养基中,温度控制在24～27.5℃,光照强度为20001k时,愈伤组织发育良好,获得高频胚性愈伤,进一步分化产生8%的不定芽.
DOI:10.3969/j.issn.1009-9980.2006.03.012 [本文引用: 3]
以早熟油桃华光和曙光为试材,对影响早熟油桃叶片产生胚性愈伤的基本培养基、外源激素、碳源、环境(光照强度,温度,暗培养时间)等因素进行研究,结果表明:碳源对两种油桃叶片胚性愈伤的诱导影响不显著,其他影响因子对油桃叶片胚性愈伤的诱导均显著.其中华光和曙光在暗培养21 d时,产生的有效愈伤较其他处理有极显著差异,有利于有效愈伤的产生;外源激素对诱导桃叶片产生胚性愈伤组织影响显著,NAA不是影响胚性愈伤产生的主效因子;基本培养基中,QL,1/2MS培养基有利于华光胚性愈伤的发育,QL,LP培养基有利于曙光胚性愈伤的发育;当温度在24～27.5℃时,光照强度为2000k,有利于胚性愈伤组织的诱导及生长;接种方式上,叶背横切,叶面贴于培养基表面的接种方法易产生胚性愈伤.实验中发现:叶背横切,叶面接于G+2.5 mg/L BA+0.5 mg/LIBA培养基上,暗培养21 d,转入1/2MS+2.0 mg/l TDZ+2.0 mg/L BA+0.5mg/L NAA的培养基中,温度控制在24～27.5℃,光照强度为20001k时,愈伤组织发育良好,获得高频胚性愈伤,进一步分化产生8%的不定芽.
DOI:10.16420/j.issn.0513-353x.2016-0495URLMagsci [本文引用: 1]
<p>以‘Red Lion’<span>朱顶红<span><span>（</span></span></span><em>Hippeastrum<span><span> vittatum</span></span></em><span>）</span>的鳞片为外植体进行体细胞胚诱导和形态学与组织学的胚性鉴定。结果表明，<span>鳞片在</span>MS + 2 mg · L<sup>-1 </sup>BA + 1 mg · L<sup>-1 </sup>NAA + 2 mg · L<sup>-1 </sup>TDZ + 30 g · L<sup>-1 </sup>蔗糖培养基上胚性愈伤组织诱导率可达90.63%，而不添加TDZ的培养基上胚性愈伤组织诱导率为0。胚性愈伤组织可呈白色松散、透明膨松、透明致密、微棕色松脆和翠绿色松脆等类型；非胚性愈伤组织呈微棕色致密状。胚性愈伤组织中可明显观察到球形胚、心形胚和<span>棒状胚</span>3个发育时期。体细胞胚在不添加植物生长调节剂的MS培养基上可发育形成芽和根，长成完整小植株。</p>
DOI:10.16420/j.issn.0513-353x.2016-0495URLMagsci [本文引用: 1]
<p>以‘Red Lion’<span>朱顶红<span><span>（</span></span></span><em>Hippeastrum<span><span> vittatum</span></span></em><span>）</span>的鳞片为外植体进行体细胞胚诱导和形态学与组织学的胚性鉴定。结果表明，<span>鳞片在</span>MS + 2 mg · L<sup>-1 </sup>BA + 1 mg · L<sup>-1 </sup>NAA + 2 mg · L<sup>-1 </sup>TDZ + 30 g · L<sup>-1 </sup>蔗糖培养基上胚性愈伤组织诱导率可达90.63%，而不添加TDZ的培养基上胚性愈伤组织诱导率为0。胚性愈伤组织可呈白色松散、透明膨松、透明致密、微棕色松脆和翠绿色松脆等类型；非胚性愈伤组织呈微棕色致密状。胚性愈伤组织中可明显观察到球形胚、心形胚和<span>棒状胚</span>3个发育时期。体细胞胚在不添加植物生长调节剂的MS培养基上可发育形成芽和根，长成完整小植株。</p>
DOI:10.7606/j.issn.1000-4025.2014.06.1075URL [本文引用: 1]
The full-length cDNA of one gene named DlARF5a(GenBank accession number:KF739401)was cloned from embryogenic callus of Dimocarpus longan by the RT-PCR combined with RACE method.Then the mRNA transcription level of the gene in the process of somatic embryogenesis was determined by qRT-PCR(real-time reverse transcription PCR) method.And the expression of ARF5a-GFP fusion protein in onion epidermal cells revealed its subcellular localization.The results indicated the complete cDNA sequence of DlARF5a was 3 322 bp containing 2 829 bp ORF encoding 942 amino acid,with 5 and 3 untranslated regions(UTR) of 212 bp and 258 bp,respectively.Bioinformatics analysis suggested that DlARF5a was probably a soluble protein,and functioned in the cytoplasm and consisted of a B3,Auxin-resp and AUX-IAA conservative district and structural function domain,and a Gln,Ser and Leu-rich middle region,which was considered as an activation domain.Phylogenetic analysis showed that protein encoded by DlARF5a had close genetic relationship with ARF5 in Citrus sinensis.The qPCR detection showed that DlARF5a had a remarkable rise in the expression levels at globular embryo and torpedo embryo stages,but the nadir mRNA transcription level of DlARF5a gene occurred at the cotyledonary stage(stage 6),demonstrating that DlARF5a was relevant to the torpedo embryo morphogenesis during longan somatic embryogenesis.The subcellular localization assays showed that the DlARF5a protein was located in the cytoplasm without adding the exogenous auxin IAA by confocal microscopy.Whereas,DlARF5a protein was located in the cell membrane,cytoplasm and nucleus under 1.0 mg/L IAA treatment,wich can be speculated that DlARF5a protein was able to change its spatial location in the cells in response to the external IAA.
DOI:10.7606/j.issn.1000-4025.2014.06.1075URL [本文引用: 1]
The full-length cDNA of one gene named DlARF5a(GenBank accession number:KF739401)was cloned from embryogenic callus of Dimocarpus longan by the RT-PCR combined with RACE method.Then the mRNA transcription level of the gene in the process of somatic embryogenesis was determined by qRT-PCR(real-time reverse transcription PCR) method.And the expression of ARF5a-GFP fusion protein in onion epidermal cells revealed its subcellular localization.The results indicated the complete cDNA sequence of DlARF5a was 3 322 bp containing 2 829 bp ORF encoding 942 amino acid,with 5 and 3 untranslated regions(UTR) of 212 bp and 258 bp,respectively.Bioinformatics analysis suggested that DlARF5a was probably a soluble protein,and functioned in the cytoplasm and consisted of a B3,Auxin-resp and AUX-IAA conservative district and structural function domain,and a Gln,Ser and Leu-rich middle region,which was considered as an activation domain.Phylogenetic analysis showed that protein encoded by DlARF5a had close genetic relationship with ARF5 in Citrus sinensis.The qPCR detection showed that DlARF5a had a remarkable rise in the expression levels at globular embryo and torpedo embryo stages,but the nadir mRNA transcription level of DlARF5a gene occurred at the cotyledonary stage(stage 6),demonstrating that DlARF5a was relevant to the torpedo embryo morphogenesis during longan somatic embryogenesis.The subcellular localization assays showed that the DlARF5a protein was located in the cytoplasm without adding the exogenous auxin IAA by confocal microscopy.Whereas,DlARF5a protein was located in the cell membrane,cytoplasm and nucleus under 1.0 mg/L IAA treatment,wich can be speculated that DlARF5a protein was able to change its spatial location in the cells in response to the external IAA.
DOI:10.2307/3869791URLPMID:8281041 [本文引用: 1]
The sulfhydryl (SH) modifying reagent N-ethylmaleimide (NEM) was applied to the internodal cells of Chara corallina to study the role of SH residues in the activity of the plasma membrane H+ pump. NEM (1 mu M) caused a marked depolarizing shift of the resting potential by 64+/-10 mV (n=7) together with depression of the conductance peak at around -200 mV, indicating a marked depression of the H+ pump activity. This effect of NEM was partly reversible, the membrane repolarized and the conductance peak was restored after extracellular washing. The H+ pump inhibitor, dicyclohexylcarbodiimide (DCCD), caused no additive membrane depolarization and/or depression of the H+ pump conductance, in the presence of NEM. This suggests that NEM blocks the H+ pump and that SH residues play a pivotal role in maintaining the H+ pump activity in Chara corallina.
[本文引用: 2]
DOI:10.1007/s00709-017-1089-9URL [本文引用: 2]
The wild grass species Brachypodium distachyon (L.) has been proposed as a new model for temperate grasses. Among the biotechnological tools already developed for the species, an efficient induction p
