水稻异淀粉酶基因ISA1及其启动子的表达特性分析

Expression Analysis of ISA1 Gene Encoding an Isoamylase-type Starch Debranching Enzyme in Rice

利用实时定量RT-PCR方法研究了水稻异淀粉酶基因ISA1在各组织及不同发育阶段籽粒中的表达模式,结果表明该基因只在发育籽粒中表达。同时,克隆了ISA1基因起始密码子上游1.1kb和2.1kb两个不同长度的启动子片段,并分别与GUS报告基因融合,经农杆菌介导转入水稻中。对转基因水稻植株中GUS活性的定性与定量结果表明,2.1kb的ISA1启动子具有很好的胚乳表达特异性,与内源基因定量表达分析的结果一致;而1.1kb的ISA1启动子则不具备该表达特性,它在胚乳、茎、茎节及谷壳中都有很高的表达。这可能是因为2.1kb启动子中含有抑制目的基因在茎等组织或器官中表达的顺式作用元件。

A quantitative real-time PCR （Q-PCR） analysis was performed to investigate the expression pattern of the rice ISA1 gene, encoding an isoamylase-type starch-debranehing enzyme, in different tissues as well as in grains at different developmental stages. The results showed that the ISA1 gene was strictly expressed in developing grains. In addition, the 1.1-kb and 2.1-kb promoter region upstream of the start codon of ISA1 gene were cloned and fused with the GUS reporter gene. Then, the GUS chimeric genes were subsequently introduced into rice via Agrobacteriurremediated transformation. The results of both histoehemieal staining and quantitative analysis of GUS （β-glueuronidase） activity showed that the 2.1-kb ISA1 pro- moter had an obvious endosperm-specific expression characters, which was consistent with the Q-PCR result of the endogenous gene in rice. However, the 1.1-kb ISA1 promoter presented different expression characters, which drove the GUS reporter gene at a high expression level not only in endosperm but also in culm, culm node and grain hull of transgenie rice. It was speculated that some functional elements which could restrain the expression of target gene in certain organs such as the stem existed in the 2.1 kb promoter.