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

Abstract

Abstract:
Abstract: 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debranching 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 Agrobacteriummediated transformation. The results of both histochemical staining and quantitative analysis of GUS (βglucuronidase) activity showed that the 2.1kb ISA1 promoter 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 transgenic 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.

Key words: rice (Oryza sativa), isoamylase 1, gene expression, promoter analysis, realtime reverse transcriptionpolymerase chain reaction, β-glucuronidase activity

摘要： 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1.1 kb和2.1 kb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，2.1 kb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1.1 kb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 2.1 kb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。

关键词: 水稻, 异淀粉酶1, 基因表达, 启动子分析, 实时定量聚合酶链式反应

LI Qianfeng , ,ZHANG Guiyun ,YU Hengxiu ,Samuel Saiming SUN ,GU Minghong ,LIU Qiaoquan . Expression Analysis of ISA1 Gene Encoding an Isoamylasetype Starch Debranching Enzyme in Rice[J]. Chinese Journal of Rice Science.

李钱峰张桂云,于恒秀,辛世文,顾铭洪,刘巧泉,. 水稻异淀粉酶基因ISA1及其启动子的表达特性分析[J]. 中国水稻科学.

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