基于mCherry荧光蛋白的植物基因过表达遗传转化系统的初步研究  

A preliminary study on the genetic transformation system of plant gene overexpression based on a mCherry fluorescent protein vector

作　　者：高晓庆[1,2] 徐国娟[2] 袁正杰[2] 邹小维[2] 何海燕[2] 瞿绍洪[2] 

机构地区：[1]浙江师范大学化学与生命科学学院,浙江金华321004 [2]浙江省农业科学院病毒学与生物技术研究所,浙江杭州310021

出　　处：《浙江农业学报》2015年第11期1865-1871,共7页Acta Agriculturae Zhejiangensis

基　　金：转基因生物新品种培育重大专项(2012ZX08009001);国家重点基础研究发展计划前期项目(2012CB722504);浙江省农业科学院省部共建国家重点实验室培育基地开放基金资助项目(2010DS700124-KF1210)

摘　　要：过量表达是植物基因功能鉴定和农作物遗传改良的重要途径。为了提高转基因植株的研究效率,以m Cherry红色荧光蛋白为报告基因,构建了由玉米泛素启动子(P-Ubi)和豌豆T3A-poly A序列组成过量表达框的转基因载体。分别将4个水稻类受体激酶的编码序列插入表达框,通过农杆菌介导转化水稻品种泰粳394。转基因植株T1种子的m Cherry荧光检测结果表明,平均62.5%的T1株系呈3∶1分离。根据实时定量RT-PCR分析结果,过表达阳性植株的目的基因相对表达量显著高于阴性植株和未转化的泰粳394。运用该载体系统,能够进行转基因后代大规模筛选,获得目的基因过量表达的转基因株系,从而促进植物基因功能研究和转基因应用研究。Overexpression is an important approach for functional characterization of plant genes and genetic improvement of crop plants. To improve the efficiency of characterization of transgenic plants,a transformation vector with the maize（ Zea mays） ubiquitin-1 promoter（ P-Ubi） and the pea（ Pisum sativum） T3A-poly A sequence in the overexpression cassette was constructed using the m Cherry red fluorescent protein as a reporter. The coding sequences of four receptor-like kinases of rice（ Oryza sativa） were inserted into the overexpression cassette of the vector and then transformed into the rice cultivar Taijing-394,respectively. m Cherry fluorescence assay of the T1 transgenic seeds showed that on average,62. 5% of the T1 lines segregated with a 3∶ 1 ratio. Based on the results of real-time quantitative reverse-transcription polymerase chain reaction assays,the relative gene-expression levels of the overexpressed genes in the transgene-positive plants were significantly higher than those of the genes in the negative segregant and untransformed recipient cultivar. The vector system can be used for large-scale screening of transgenic progeny andobtaining of transgenic lines with target genes overexpressed,so as to promote the functional characterization of plant genes and the application of transgenic technology.

关 键 词：mCherry红色荧光蛋白 转基因 过量表达 水稻 

分 类 号：S184[农业科学—农业基础科学]