拟南芥CYCD2;1基因植物表达载体的构建及在烟草中的遗传转化分析

Construction of Plant Expression Vector and Genetic Transformation Analysis of Arabidopsis thaliana CYCD2;1 Gene in Nicotiana tabacum

细胞分裂是生物的基本特征之一,在植物生长发育的过程中,发挥着极其重要的作用,细胞周期蛋白CYCD2;1基因作为一个调控因子,对调节细胞周期具有重要作用。本文以拟南芥细胞周期蛋白(At CYCD2;1基因)作为研究对象,利用PCR技术从拟南芥花序c DNA扩增出At CYCD2;1基因,构建植物表达载体(pROKIIAt CYCD2;1)并利用农杆菌介导的叶盘法转化野生型烟草。转基因植株的PCR检测结果表明,At CYCD2;1基因已经整合到了烟草基因组中。在T2代植株中,通过实时定量荧光PCR检测显示,At CYCD2;1在mRNA水平也均有表达。过量表达CYCD2;1的转基因烟草在花器官中存在明显表型,与野生型相比主要表现为转基因植株花冠宽度变大,花瓣和萼片长度变长,果实变大,上述结果表明At CYCD2;1基因影响花的发育。

Cell division is one of the basic characteristics of organisms, which plays an extremely important role in the plant growth and development. CYCD2 ; 1, as a regulatory factor, has been identified in regulation of cell cycle. In our study, CYCD2; 1 was amplified from Arabidopsis inflorescences cDNA by PCR. Plant overexpression vector （pROKII-AtCYCD2; 1 ） was constructed and transformed into wild-type tobacco by Agrobacterium-mediated leaf disc transformation. The transgenie plants were confirmed by PCR, and the result showed AtCYCD2 ; 1 gene was integrated into tobacco genome. The result of qRT-PCR detection of T2 generation plants showed the mRNA of AtCYCD2; 1 was transcribed in transgenic tobaccos. There were significant differences in the flower between the transgenic and wild type plant, such as the width of the corolla, the lengths of the petal and sepal, and increased volume of seed pod, which suggesting overexpression of AtCYCD2 ; 1 has an obvious influence on the development of flowers.