为探究棉花(Gossypium hirsutum L.)GhNCED3启动子的功能,从陆地棉新陆早22号中克隆GhNCED3启动子.利用Plant CARE在线软件分析GhNCED3启动子上的顺式作用元件,构建ProGhNCED3∷GUS-p1300融合表达载体.采用浸花法转化野生型拟南芥(Arabidopsis thaliana),对转基因阳性植株筛选验证.为进一步探究GhNCED3启动子的特性,在外源脱落酸处理下分析转ProGhNCED3∷GUS株系GUS基因表达量.同时对干旱胁迫处理前后的转基因株系进行GUS组织化学染色、GUS基因表达量检测及GUS酶活性测定.启动子序列分析结果表明,GhNCED3启动子上含有参与脱落酸信号转导途径的ABRE元件和响应干旱胁迫的HD-box元件.在T0 代转基因株系中扩增GhNCED3启动子片段结果表明,GhNCED3启动子已整合至拟南芥染色体中.T2 代转基因株系GUS组织化学染色及GUS基因表达量检测结果表明,克隆的1581 bp GhNCED3启动子具有转录活性,能够驱动GUS基因在拟南芥中稳定遗传表达.在不同浓度脱落酸处理下,转基因株系中GUS基因表达量上调.干旱胁迫下GhNCED3启动子转录活性鉴定结果表明,干旱处理后GUS染色区域、程度、GUS表达量及GUS酶活性均增加.表明1581 bp GhNCED3启动子的转录活性受脱落酸和干旱胁迫诱导.
Cloning and Preliminary Functional Analysis of GhNCED3 Gene Promoter in Gossypium hirsutum L.
This project aims to investigate the function of the GhNCED3 promoter in cotton by cloning the GhNCED3 promoter from upland cotton Xinluzao 22.The cis-acting elements on the GhNCED3 pro-moter were analyzed using Plant CARE online software and the ProGhNCED3∷GUS-p1300 fusion ex-pression vector was constructed.The wild-type Arabidopsis thaliana was transformed by flower-dipping method and the transgenic-positive plants were screened and verified.The characterization of the GhNCED3 promoter was further explored by analyzing GUS expression in trans-ProGhNCED3∷GUS lines under exogenous abscisic acid treatment.At the same time,GUS histochemical staining,GUS expression and GUS enzyme activity detected were performed on the transgenic lines before and after drought stress treatment.The results of promoter sequence analysis indicated that the GhNCED3 promoter contained ABRE elements involved in abscisic acid signal transduction pathway and HD-box elements in response to drought stress.Amplification of the GhNCED3 promoter fragment in the T0 transgenic lines showed that the GhNCED3 promoter was integrated into the Arabidopsis thaliana chromosome.The results of GUS histochemical staining and GUS expression detection in T2 transgenic lines showed that the cloned 1581 bp GhNCED3 promoter had transcriptional activity and could drive GUS stable genetic expression in Arabidopsis thaliana.GUS expression was up-regulated in transgenic lines under different concentrations of abscisic acid treatment.The transcriptional activity of GhNCED3 promoter under drought stress showed that GUS staining region,degree,GUS expression and GUS enzyme activity increased after drought treatment,indicating that the transcriptional activity of 1581 bp GhNCED3 promoter was induced by ab-scisic acid and drought stress.
万方数据
维普
