重组des-pGlu1-Brazzein基因植物表达载体的构建及对人参果的遗传转化

Construction of Plant Expression Vector Verification with Recombinant des-pGlu1-Brazzein by Transgenic Pepino(Solanum muricatum)

本研究通过对茄科植物的E8基因的非编码区DNA序列进行亲缘关系及功能元件保守性分析,发现E8基因的非编码区包含ERE,茉莉酸和低温干旱应答等基序并存在较高的保守性,表明利用果实特异性E8启动子改善同属茄科茄属的人参果果实风味更具特异性;利用密码子偏爱性优化设计甜蛋白Brazzein,采用亚克隆技术成功获得甜蛋白des-pGlu1-Brazzein基因和E8启动子基因,经Blast对比均与GenBank中已报道的序列高度同源,并利用2KGQ模型分析des-pGlu1-Brazzein基因的空间结构特性;以中间载体pHANNIBAL及植物表达载体pCEPSP为基础,分别构建由E8启动子及35S启动子驱动的兼具重组甜蛋白及除草剂抗性的植物表达载体,并通过农杆菌介导法进行人参果的遗传转化,获得转化再生植株54株,经检测从中筛选出15株阳性植株,初步确定已经完成目的基因整合到人参果基因组中。

The genetic relatio n ship and CIS-regulatory elements conservative analysis of E8 gene non-coding region were analysised for Solanaceae plants in this study. The results showed that the non-coding region of E8 gene contain several extremely conserved motif, such as ethylene-responsive(ERE), the Me JA-responsiveness and drought-inducibility elements. Accordingly the fruit-specific promoter E8 genes were more specific to ginseng fruit flavor of Solanum muricatum Ait as we expected. Considering that codon preference, the sweet protein des-pGlu1-Brazzein was optimal designed. The sweet protein des-pGlu1-Brazzein and E8 promoter were subcloned by PCR, and the homology comparison results revealed that the cloned des-pGlu1-Brazzein and E8 promoter had higher homology with the genes reported in the Gen Bank. Also the spatial structure of Brazzein were analysised by model 2KGQ. The plant bivalent expression vectors were generated by intermediate vector p HANNIBAL and plant expression vector p CEPSPS. In addition, these plant expression vectors contain sweet protein Brazzein gene and herbicide resistance gene EPSPS which driven by fruit-specific E8 promoter and 35 S promoter. Fifty-four transgenic plants were obtained through Agrobacterium-mediated protocol and 15 positive plants were screened in pepino plantlets, in which traget genes were integrated into the genome in pepino.