辣椒COI1.2基因的克隆、表达分析和植物表达载体的构建

Cloning and Expression Analysis of COI1. 2 Gene of Chili Pepper and Plant Expression Cassettes Construction

利用RT-PCR技术,通过同源克隆从辣椒中分离到CaCOI1.2基因,采用生物学软件对序列进行生物信息学分析,采用实时定量RT-PCR技术分析基因的表达模式.结果表明：克隆到的CaCOI1.2基因长度为2041 bp,推测其读码框大小为1812 bp,编码603个氨基酸.在CaCOI1.2蛋白质N-端有一个F-box结构域,C-端有6个富含亮氨酸结构域.CaCOI1.2蛋白的氨基酸与巳知其他植物COI1蛋白序列有53.03 ％～93.37％的一致性.聚类分析结果显示：CaCOI1.2与番茄、烟草和葡萄等双子叶植物的亲缘关系较近,而和水稻、玉米、高粱等单子叶植物的亲缘关系较远.CaCOI1.2在辣椒的不同生长发育时期的组织中都能表达,在花和青熟期果实中表达水平较高,表明CaCOI1.2在花和果实的发育过程中起重要作用.构建植物表达载体pBI121-CaCOI1.2,并导入根癌农杆菌LBA4404中,得到植物转化工程菌,这为下一步用于辣椒等作物的转基因操作、研究CaCOI1.2基因在植物中的功能奠定了基础.

In this paper, the cDNA sequences of CaCOll. 2 from Chili pepper by homology cloning were cloned with RT-PCR, the bioinfor- marion of its sequences was analyzed by using biology software, and the expression of CaCOll. 2 was determined by real-rime quantitative RT- PCR. The results showed that CaCOI]. 2 was 2041 bp in length. CaCOI1.2 contained a 1812-bp open reading frame and encoded a putative polypeptide of 603 amino acids. CaCOI1.2 protein was predicted to possess an F-box at the N-tonninal and six leucine-rlch repeats domains at the C-terminal. Compared with other species in sequencing analysis, CaCOn. 2 protein sequence had 53.03 % -93.37 % identity in ami- no acids. The results of clustering analysis showed that CaCOI1.2 had closer relation with isolates from dicotyledon such as tomato, tobacco and grape than that from monocotyledon such as rice, maize and grain sorghum. The CaGOII gene was transcribed in different tissue organs of chili pepper, and the expression level of CaCOI1 in its flowers and mature green fruit were higher than that in other analyzed tissue organs, and these results indicated that CaCOI1 had an effect on the development of flower and fruit. After constructing the recombinant vector pBI121-CaCOll1.2, pBI121-CaCOI1.2 vector was introduced into A. tumefac/ens strain LBA4404, which lay the foundations for transformation of chili pepper and researching the function of CaCOI1.2.