大麦水孔蛋白基因HvTIP2;1及其启动子的表达特性分析  

Expression Patterns of a Root-specific Barley Aquaporin Gene HvTIP2;1 and Promoter

作　　者：徐登安 赵纯钦[1] 张赤红[1] 陈静[1] 

机构地区：[1]中国科学院成都生物研究所成都610041

出　　处：《中国生物工程杂志》2015年第7期15-21,共7页China Biotechnology

基　　金：国家自然科学基金(30871527);中国科学院院地合作项目(XBCD-2011-019);中国科学院知识创新工程重要方向项目(KSCX2-EW-J-22);四川省科技厅国际合作项目(2012HH0008)资助项目

摘　　要：组织特异性启动子作为基因工程的一种重要调控元件,在生物反应器、转基因新品种培育等领域有重要的应用前景。基于芯片数据的基因数字化差异显示分析,筛选到一个根特异表达的大麦水孔蛋白基因Hv TIP2;1,利用实时荧光定量RT-PCR方法了解该基因在不同组织和处理条件下的表达特性,并对基因启动子及功能进行了研究。结果表明,Hv TIP2;1表达具有根特异性并受植物生长发育的调控,其在成株期的表达量明显高于苗期。ABA激素处理组,Hv TIP2;1表现先上升,处理10h后又下降的表达变化趋势;铝、锰有毒金属离子处理组,Hv TIP2;1表达量明显高于对照组,但表现出上升-下降-上升的波动变化特征;盐胁迫导致Hv TIP2;1表达量持续下降,而干旱诱导Hv TIP2;1表达量不断升高,处理24h后表达量迅速下降,低于对照水平。利用PCR方法克隆位于基因编码区上游的启动子序列Hv1310p,该序列含有多个与根特异表达和胁迫响应有关的顺式作用元件。通过5'端缺失法分别构建514bp和1 258bp启动子的融合GUS报告基因表达载体,转基因烟草的GUS活性检测表明两个启动子片段都具有根特异性的启动活性。Plant tissue-specific promoters, as important regulatory elements, have promising application for bioreactor and crop breeding by genetic modification. Combined barley microarray data and gene digital differential display technology, a root-specific aquaporin gene HvTIP2;1 was obtained after real-time RT-PCR analysis for gene expression in different tissues and under ABA treatment and various abiotic stresses. Meanwhile, isolation and functional analysis of the HvTIP2;1 promoter was conducted. The results showed that HvTIP2; 1 was a root-specific gene regulated by plant growth and development, with significantly higher expression at adult stage than at seedling stage. ABA treatment caused up-regulated expression of HvTIP2;1 which decreased after 10h treatment. Under aluminum and manganese metal ion stresses, HvTIP2 ;1 expressed much higher than the control, with an up-down-up expression pattern. However, it was consistently down- regulated by salt stress. Drought induced HvTIP2 ;1 expression increase and then decrease dramatically after 24h treatment. A promoter named Hvl310p, upstream of HvTIP2;1 coding domains, was cloned, where some c/s- elements related to root-specific expression and plant response to various abiotic stresses were detected using biological software. Using 5＇ end deletion, two promoter fragments （514bp and 1 258bp） were fused with GUS gene and transformed into tobacco, respectively. Histochemistry staining and quantitative analysis of GUS activity showed that both can drive GUS gene to express specifically in roots.

关 键 词：大麦 水孔蛋白 基因表达 根特异性启动子 实时荧光定量RT-PCR 

分 类 号：Q78[生物学—分子生物学]