太子参玉米黄质环氧化酶基因的克隆与表达分析(1)
[摘要] 玉米黄质环氧化酶(zeaxanthin epoxidase，ZEP)在植物脱落酸(abscisic acid，ABA)生物合成间接途径中发挥着重要调节作用。该研究根据太子参转录组数据，克隆获得太子参的Pseudostellaria heterophylla玉米黄质环氧化酶基因，命名PhZEP。对其序列进行生物信息学分析，结果表明PhZEP的开放阅读框(open reading frame，ORF)为1 263 bp，编码420个氨基酸，预测其蛋白的相对分子质量为47.34 kDa，等电点(theoretical pI)为6.64；具有脂质蛋白附着点和黄素蛋白单加氧酶的特征性结构域，且在N端含有分泌信号肽。实时荧光PCR分析发现，PhZEP在叶片中表达量最高，其次是茎和须根；PhZEP在盛花期后10，40 d块根中的表达量较高；ABA处理后PhZEP表达量与对照组相当，经氟啶酮处理后PhZEP的表达量显著高于对照组。该研究从太子参中分离鉴定了ZEP基因，对后续研究其基因的功能特点和解析ABA在太子参生长发育过程的遗传调控提供研究基础。
, 百拇医药
[关键词] 太子参； 玉米黄质环氧化酶； 克隆； 表达分析
[Abstract] Zeaxanthin epoxidase plays an important role in indirect pathway of plant abscisic acid biosynthesis. According to the data of Pseudostellaria heterophylla transcriptome， zeaxanthin epoxidase gene was isolated and named as PhZEP. The results of bioinformatics analysis showed that the coding sequence of PhZEP was 1 263 bp long and encoded 420 amino acids. The putative protein molecular weight was 47.34 kDa and its theoretical isoelectric point was 6.64. The characteristic structure domains were predicted， including binding site of lipoprotein and flavoprotein monooxyenase. A signal peptide was discovered at the N-terminal of amino acids. The Real-time PCR revealed that PhZEP had a higher expression level in leaves than other tissues of P.heterophylla. Highly expressed PhZEP was also observed at 10 d and 40 d tuberous root after flowering. PhZEP presented a different expression after treatment with ABA， fluridone and ABA +fluridone compared to the control. The expression of PhZEP in tuberous root after ABA treatment was close to that in control group， while PhZEP showed significant up-regulation in the fluridone treatment group. In this study， the PhZEP gene from P. heterophylla was cloned and this result has important significance for its functional identification. This research provides a basis for the further analysis on functional mechanism of ABA during development of P. heterophylla.
, 百拇医药
[Key words] Pseudostellaria heterophylla； zeaxanthin epoxidase； cloning； expression analysis
太子參为石竹科植物孩儿参Pseudostellaria heterophylla (Miq.) Pax ex Pax et Hoffm.的干燥块根，是我国传统的常用补益中药，具有益气健脾、生津润肺的功效。近年来，太子参的开发利用从临床药品不断延伸到功能性食品和化妆品。太子参块根的生长发育情况是直接影响药材的产量与品质的关键因素[1]，植物块根的形成和发育受多种因素影响，其中内源激素的调控是主要因素之一[2-3]。研究认为，脱落酸(abscisic acid，ABA)、细胞分裂素(cytokinin，CTK)、生长素(indole-3-acetic acid，IAA)等激素均有利于块根的膨大[4-6]。
ABA不仅在植物根的生长、块根类作物的物质运输以及种子休眠、果实成熟等生长发育过程中具有关键的调节作用，同时对植物干旱、低温等胁迫环境下的生理活动同样起着重要作用[7-8]。植物体内ABA的生物合成途径一般包括2种：C15直接途径和C40间接途径，高等植物主要以C40间接途径为主。在C40间接途径合成ABA的过程中，首先是C40类胡萝卜素的玉米黄素经过2次环氧化作用形成紫黄质，参与该步反应的关键调控酶基因是玉米黄素环氧化酶(zeaxanthin epoxidase，ZEP)基因[9]。ZEP基因除了在ABA生物合成途径中发挥着重要作用外，还在叶片、茎等叶绿体含量比较丰富的组织中参与植物的叶黄素循环[10]。Audran等[11]研究发现ZEP基因在植物的各组织均存在表达，但在不同组织中的表达具有显著性差异，当受到干旱胁迫诱导时，叶片和根中的ABA含量出现同时增加，对应的ZEP基因的转录水平只在根中增加，而叶片中转录水平受昼夜节律的调控呈周期性变化。因此，陶均等[12]认为ZEP基因可能不是参与ABA生物合成途径中的关键酶基因。, http://www.100md.com(郑伟 周涛 李军 龙登凯 江维克 丁铃)
, 百拇医药
[关键词] 太子参； 玉米黄质环氧化酶； 克隆； 表达分析
[Abstract] Zeaxanthin epoxidase plays an important role in indirect pathway of plant abscisic acid biosynthesis. According to the data of Pseudostellaria heterophylla transcriptome， zeaxanthin epoxidase gene was isolated and named as PhZEP. The results of bioinformatics analysis showed that the coding sequence of PhZEP was 1 263 bp long and encoded 420 amino acids. The putative protein molecular weight was 47.34 kDa and its theoretical isoelectric point was 6.64. The characteristic structure domains were predicted， including binding site of lipoprotein and flavoprotein monooxyenase. A signal peptide was discovered at the N-terminal of amino acids. The Real-time PCR revealed that PhZEP had a higher expression level in leaves than other tissues of P.heterophylla. Highly expressed PhZEP was also observed at 10 d and 40 d tuberous root after flowering. PhZEP presented a different expression after treatment with ABA， fluridone and ABA +fluridone compared to the control. The expression of PhZEP in tuberous root after ABA treatment was close to that in control group， while PhZEP showed significant up-regulation in the fluridone treatment group. In this study， the PhZEP gene from P. heterophylla was cloned and this result has important significance for its functional identification. This research provides a basis for the further analysis on functional mechanism of ABA during development of P. heterophylla.
, 百拇医药
[Key words] Pseudostellaria heterophylla； zeaxanthin epoxidase； cloning； expression analysis
太子參为石竹科植物孩儿参Pseudostellaria heterophylla (Miq.) Pax ex Pax et Hoffm.的干燥块根，是我国传统的常用补益中药，具有益气健脾、生津润肺的功效。近年来，太子参的开发利用从临床药品不断延伸到功能性食品和化妆品。太子参块根的生长发育情况是直接影响药材的产量与品质的关键因素[1]，植物块根的形成和发育受多种因素影响，其中内源激素的调控是主要因素之一[2-3]。研究认为，脱落酸(abscisic acid，ABA)、细胞分裂素(cytokinin，CTK)、生长素(indole-3-acetic acid，IAA)等激素均有利于块根的膨大[4-6]。
ABA不仅在植物根的生长、块根类作物的物质运输以及种子休眠、果实成熟等生长发育过程中具有关键的调节作用，同时对植物干旱、低温等胁迫环境下的生理活动同样起着重要作用[7-8]。植物体内ABA的生物合成途径一般包括2种：C15直接途径和C40间接途径，高等植物主要以C40间接途径为主。在C40间接途径合成ABA的过程中，首先是C40类胡萝卜素的玉米黄素经过2次环氧化作用形成紫黄质，参与该步反应的关键调控酶基因是玉米黄素环氧化酶(zeaxanthin epoxidase，ZEP)基因[9]。ZEP基因除了在ABA生物合成途径中发挥着重要作用外，还在叶片、茎等叶绿体含量比较丰富的组织中参与植物的叶黄素循环[10]。Audran等[11]研究发现ZEP基因在植物的各组织均存在表达，但在不同组织中的表达具有显著性差异，当受到干旱胁迫诱导时，叶片和根中的ABA含量出现同时增加，对应的ZEP基因的转录水平只在根中增加，而叶片中转录水平受昼夜节律的调控呈周期性变化。因此，陶均等[12]认为ZEP基因可能不是参与ABA生物合成途径中的关键酶基因。, http://www.100md.com(郑伟 周涛 李军 龙登凯 江维克 丁铃)