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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。
In this study, the non-coding region of solanaceous plants E8 gene DNA sequence of the genetic relationship and conservative analysis of functional elements and found that the non-coding region of E8 gene contains ERE, jasmonic acid and low temperature drought response and other motifs and higher Conservative, indicating that the use of fruit-specific E8 promoter to improve the genus Gorgon fruit genus Gorgon fruit flavor more specific; the use of codon preference optimization design Brazzein protein, the use of subclone technology to successfully obtain des-pGlu1-Brazzein protein The gene and E8 promoter gene were highly homologous to the reported sequences in GenBank by Blast comparison. The spatial structural characteristics of des-pGlu1-Brazzein gene were analyzed by 2KGQ model. Based on the intermediate vector pHANNIBAL and the plant expression vector pCEPSP, The plant expression vector with both recombinant sweet protein and herbicide resistance driven by E8 promoter and 35S promoter was constructed respectively and the genetic transformation of ginseng fruit was carried out by Agrobacterium tumefaciens method to obtain 54 transformed and transformed plants, Fifteen positive plants were screened, and it was initially determined that the target gene had been integrated into the genome of ginseng fruit.