控制基因盐诱导且根系优势表达的小麦启动子Tasipro3克隆及功能分析

Cloning and Functional Analysis of Wheat Promoter Tasipro3 Controlling Genes Expression with Salt-inducible Root-preferential Pattern

在土壤盐胁迫下,小麦根系吸收水分和营养物质的功能受到抑制,从而影响植株的经济产量。因而,开展小麦耐盐育种,提高根系耐盐性是重要途径之一。使耐盐基因在根系中优势表达,并且在盐胁迫下增强表达,将显著提高根系耐盐性。而克隆和鉴定具有双重控制功能的启动子,是实现耐盐基因精准调控的基础。鉴于此,本研究利用Genevestigator在线生物信息学分析软件,筛选到425个盐诱导根系优势表达的探针,并从中选出2个候选探针,用于启动子验证。以1周龄小麦品种中国春的幼苗为材料,将其根系置于200 mmol/L的NaCl溶液中,分别于0 h、0.5 h、1 h、2 h、4 h和8 h进行根系取样,用于表达模式分析。结果表明,Ta.5463.1.A1_at探针的基因表达模式更符合生物信息学预测的结果,受盐胁迫诱导表达显著上调,且基因优势表达于根系。为进一步验证相应基因启动子的功能,对此探针对应的启动子区进行了克隆,并连接到启动子验证载体中,遗传转化获得转基因拟南芥植株。盐诱导后GUS染色的实验结果表明,该启动子使GUS报告基因在盐处理下表达量显著提高,且主要在根系表达。本研究成功克隆了耐盐遗传改良专用启动子,为小麦分子抗逆育种提供了优异资源。

Under salt stress from the soil,the economic yield of wheat will decrease due to reduced uptake of water and nutrients.The enhancement of root tolerance to salt will be one of the important ways in wheat breeding for salt-tolerance.It will significantly raise root salt-tolerance by driving salt-tolerance genes preferentially expressed in roots,greatly induced by salt stress,while isolation and characterization of the promoter with dual functions will be the basis of precise control of salt-tolerance genes.Hence,in this study,we screened and determined 425 probes,and selected 2 candidate probes from them for further promoter validation.The roots of one-week-old seedlings of wheat variety‘Chinese Spring’were placed in 200 mmol/L NaCl solution,and sampled at 0 h,0.5 h,1 h,2 h,4 h and 8 h for analysis of gene expression patterns.The results showed that the gene corresponding to Ta.5463.1.A1_at probe was fitting better into the expected bioinformatic results,which is preferentially expressed in roots and up-regulated by the induction of salt stress.To further verify the functions of its promoter,the promoter region was cloned and ligated into the promoter verification vector,and transgenic Arabidopsis plants were obtained.The results of salt induction and GUS staining showed that the promoter could regulate the expression of GUS reporter gene under salt treatment significantly,and the reporter gene was mainly expressed in roots.Our study successfully isolated specific promoter for salt tolerance,and provided an excellent resource for molecular breeding in wheat stress resistance.