Pina~m-Pinb~m-Gsp~m串联三基因表达载体的构建及其在普通小麦中的转化

籽粒硬度是小麦品质改良的重要目标性状之一,主要由5D短臂上的Hardness(Ha)位点的两个主效基因,即Puroindoline a(Pina-D1)和Puroindoline b(Pinb-D1)控制,同时受基因Grain Softness Protein-1(Gsp-1)的影响。本研究以一粒小麦(T.monococcum)DV92作为Pinam、Pinbm和Gspm基因的供体,将三基因各自完整的表达盒串联连接到载体pGEM-TEasy上,构建Pinam-Pinbm-Gspm三基因串联的表达载体。利用基因枪介导的转化方法转化普通小麦科农199幼胚,共轰击5608个小麦幼胚愈伤,经Biolaphos(化学除草剂)筛选,获得933株再生植株,经PCR检测,鉴定出11株阳性植株,有关转基因小麦的籽粒硬度还需进一步实验验证;本实验实现了串联三基因在普通小麦中的遗传转化,为利用基因枪介导的遗传转化方法改善小麦籽粒硬度提供了可行性。

Construction of Gene-bombarded Expression Vector Harboring Tandem Genes of Pina~m-Pinb~m-Gsp~m and Genetic Transformation in Common Wheat

The hardness is one of the most important characters of cereal grains to examine for wheat quality improvement.Hardness is mainly controlled by two genes,Puroindoline a,Puroindoline b with influence from Grain Softness Protein-1(Gsp-1) gene.All three genes are located on the Ha locus of the 5DS.In this study,the Triticum monococcum DV92 was used as the donor of the target genes,Pinam,Pinbm and Gspm.The expression vector for Pinam,Pinbm and Gspm tandem genes was constructed by connecting each complete expression cassette of three genes to pGEM-T Easy vector.Gene gun method was used to transform the three tandem genes into common wheat cultivar Kenong199.5 608 calluses derived from immature embryos were bombarded,and 933 regenerated plants were obtained after Biolaphos screening.In the end,11 transgenic plants were obtained after PCR identification with target gene specific primers.The hardness of transgenic wheat requires further validation.This study provided preliminary technology for transforming three tandem genes into common wheat and made it possible to improve wheat hardness by gene gun transformation method.