开花调控基因BpMADS4无抗性表达载体的构建

为了构建含有报告基因GFP基因和BpMADS4基因的无抗性双元表达载体pCAMBIA1302-GFP-Bp,参考已发表的欧洲白桦（Betula pendula）开花调控基因（BpMADS4）序列,利用逆转录-聚合酶链式反应（RT-PCR）从欧洲白桦的幼嫩花序中克隆得到促进开花的BpMADS4基因。将该基因替换pCAMBIA1302载体中的潮霉素抗性基因,构建含有报告基因GFP基因和BpMADS4基因的无抗性双元表达载体pCAM-BIA1302-GFP-Bp。结果表明：成功构建了无抗性双元表达载体pCAMBIA1302-GFP-Bp。该表达载体在苹果遗传转化中不使用抗生素筛选,可以解决抗生素抗性筛选降低苹果转基因转化效率的问题以及转基因苹果中选择标记基因造成的生物安全性问题,用该载体转化苹果可以缩短苹果童期,有效提高其育种效率。本研究为筛选对环境安全的、具有易成花特性的苹果新种质奠定了基础。

Construction of Non-resistance Plant Expression Vector of Flowering Regulatory Gene BpMADS4

To construct the non-resistance plant binary expression vector pCAMBIA1302-GFP-Bp containing GFP and BpMADS4 genes,according to the published flowering regulatory gene(BpMADS4) sequence of Betula pendula,we obtained BpMADS4 gene from the young inflorescence of B.pendula by RT-PCR method.The tide amphotericin resistance gene in pCAMBIA1302 vector was replaced by BpMADS4 gene.The results showed that PCR identification,restriction enzyme digestion and sequence analysis confirmed the non-resistance plant binary expression vector pCAMBIA1302-GFP-Bp was successfully constructed.Using pCAMBIA1302-GFP-Bp vector,antibiotic was not used in genetic transformation of apple.Some problems such as antibiotic resistance screening reducing the efficiency of apple transformation,and biological safety problems caused by using select marker genes in transgenic apple could be resolved.Transforming pCAMBIA1302-GFP-Bp vector into apple could shorten the juvenile stage,and effectively improve the breeding efficiency.This laid a foundation for screening new apple germplasm with the characteristics of safe to environment and early flowering.