萼脊兰MADS-box基因的克隆及表达载体构建

摘要

为了研究"ABC"模型的B类PI基因,探究决定花瓣和雄蕊形成的基因特征.采用CTAB法提取萼脊兰花瓣总RNA,并通过RT-PCR法克隆萼脊兰PI基因的编码序列,该序列长度为633 bp,编码210个氨基酸,与小兰屿蝴蝶兰的氨基酸相似性最高.对PI所表达蛋白质的结构、亲水性和二级结构进行了分析,结果显示,该基因包含MADS-box和K-box保守结构域,属于MADS-box基因家族;该蛋白分子属于亲水性蛋白,包含56.19% α螺旋、13.81% 的延伸链以及30%的不规则折叠,实时荧光定量PCR结果显示,PI基因主要在花器官中表达,且表达量高,说明PI基因的表达具有组织特异性.构建植物表达载体的结果显示,目的基因和带启动子的片段已插入到表达载体pCAMBIA1301中,表明成功构建植物表达载体1301-PI,为最终获得新奇花型的萼脊兰奠定基础.%In order to study the B type PI gene of "ABC" model, and explore the gene characteristics of petals and stamen formation.In the study,using CTAB approach to extract the total RNA of the petal of Sedirea japonica,and cloning the part-length cDNA sequences of PI gene of Sedirea japonica by using RT-PCR approach.It found that the length of this gene sequence was 633 bp,it encoded 210 putative amino acid residues and the gene sequence had a high homology with Phalaenopsis equestris after sequence alignment.After analysis of the structure and the hydrophilic and the secondary structure of PI protein,it found that the gene sequence contained the MADS-box domain and K-box,it belonged to the MADS-box super family,PI was hydrophilic protein,and it contained 56.19% Alpha helix,13.81% extended strand and 30% random coil.The expression was analysed by Real-time fluorescent quantitative PCR,PI genes mainly expressed in floral organ,and high expression and PI gene expression had tissue specificity.During the construction of expression vector,it leads the sequence containing objective gene and promoter to the pCAMBIA1301 expression vector,it showed that the construction of expression vector 1301-PI was succeed,which laied the fundation for getting new varieties of Sedirea japonica.