摘要
水稻(Oryza sativa L.)苗期冷害是导致水稻减产的重要因素之一。为了发掘新的耐冷基因、诠释其耐冷分子机制并应用分子育种的手段提高水稻苗期耐冷性,前期从已构建的水稻苗期低温诱导表达正向抑制差减cDNA文库中,发现了一个受低温诱导上调表达的功能未知的C2H2型锌指结构域蛋白新基因(OsCOI)。本研究通过PCR的方法从水稻中克隆了该基因,构建其植物过表达和RNA干扰(RNAi)表达载体,并通过农杆菌介导转入水稻基因组中,获得了相应的转基因水稻株系。为进一步研究OsCOI的功能,探讨其在水稻耐冷性分子机制和分子育种中的应用价值奠定了基础。取得的主要研究结果有:
1.通过PCR技术克隆了OsCOI基因的cDNA,序列分析表明该cDNA片段全长为1235 bp,开放阅读框810 bp,编码269个氨基酸。核苷酸序列同源性分析表明,该基因与GenBank中已登记的AK072942的同源性高达100%。生物信息学分析表明,该基因编码一个含有两个C2H2锌指结构域的锌指蛋白。
2.分别构建了OsCOI基因的过表达载体pAHLG-OsCOI和RNAi表达载体p1300-OsCOIRNi1/2。采用电激法将两种表达载体导入根癌农杆菌中,并利用设计的两对特异性引物进行农杆菌菌落PCR检测,证明两种质粒均已导入农杆菌中。
3.通过农杆菌介导的遗传转化方法,用上述两种表达载体将OsCOI基因导入粳稻品种日本晴中。在含有潮霉素的培养基上筛选获得了3株过表达植株和12株RNAi表达植株。进一步利用PCR方法检测转基因植株,初步证明目的基因已经整合到T0代植株中。
Chilling injure in seedling period is one of major limiting factors for productivity of rice (Oryza sativa L.). To improve rice cold tolerance in seedling period, it is important to exploit novel cold-resistant genes, elucidate the underlying molecular mechanism and utilize the genes in cold-resistant molecular breeding. A novel cold-inducible gene encoding a putative C2H2-type zinc finger protein, designated OsCOI (Oryza sativa Cold-Inducible), has been identified in our former research. In this study, the gene was cloned by PCR method from rice. Plant binary over-expression and RNAi-expression vectors were construted and transformed into rice by Agrobacterium-mediated, and obtained some transgenic rice plant lines. The work established a basis for further studying the function and application potential of the gene in rice seedling cold tolerance. The main results were as following:
1. The cDNA of OsCOI gene was cloned by PCR method from rice. The sequence analysis showed that the total length of cDNA was 1235 bp with an open reading frame encoding 269 amino acids. Homology analysis of the nucleotide sequence showed that the identity was 100% to the published sequence AK072942. The results from bioinformatics software analysis showed that the gene wae a zinc finger protein with two C_2H_2domains.
2. Over-expression and RNAi-expression vectors were constructed and identificated by the enzymes digesting, and the different vectors were transformed into Agrobacterium tumefaciens by electroporation, which were proved by PCR assay with two pairs of specific primers of the gene.
3. The OsCOI gene was transformed into japonica rice variety Nipponbare using the two plant expression vectors by Agrobacterium-mediated approach. Three over-expression transgenic plants and twelve RNAi transgenic plants were obtained by screening in the media contained hygromycin. The results of PCR assay showed that the gene had been integrated into rice genome of T0 plants.
引文
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