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中国生物工程杂志 2015.7 Vol 35, No.7     
大麦水孔蛋白基因HvTIP2;1及其启动子的表达特性分析
 
徐登安, 赵纯钦, 张赤红, 陈静
中国科学院成都生物研究所 成都 610041
 
Key Words:  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
 
 
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Abstract

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 HvTIP2;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, HvTIP2;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, HvTIP2;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, HvTIP2;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 HvTIP2;1 表达量持续下降,而干旱诱导 HvTIP2;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。
 
Abstract

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 Hv1310p, upstream of HvTIP2;1 coding domains, was cloned, where some cis-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.
 
 
 
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中国科学院遗传与发育生物学研究所
中国科学院遗
中国科学院遗传与发育生物学研究所 中国遗传学会
中国科学院遗传与发育生物学
中国科


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