Pina和Pinb融合基因表达载体的构建及其在硬粒小麦中的转化

Construction of Expression Vector Harboring Pina & Pinb Fused Gene and Transformation into Durum Wheat

【目的】构建Pina、Pinb融合基因表达载体,验证Pina和Pinb基因的功能,为利用转基因技术改良小麦籽粒质地提供理论依据和基础材料。【方法】以软质小麦京411作为Pina和Pinb基因的供体,将Pina、Pinb基因融合后,与基础质粒pG4AB上的Ω和poly(A)等增强基因表达的调控序列及pAHC25上的Ubiqutin启动子和bar基因表达盒相连接,构建Pina、Pinb融合基因植物高效表达载体。利用基因枪转化硬粒小麦幼胚,并对硬粒小麦幼胚再生体系进行探索。【结果】在构建完成Pina、Pinb融合基因植物高效表达载体pFUBPaPb的基础上,转化硬粒小麦幼胚16000多枚,经biolaphos筛选、PCR鉴定和斑点杂交鉴定,得到再生植株2390株,35株为阳性植株。共得到T1代籽粒356粒,对种子量较多的16个单株籽粒进行蛋白表达分析,有2株检测到基因表达产物,其籽粒SKCS硬度值比受体品种分别降低了10和12。培养基SD2适于硬粒小麦幼胚愈伤组织的诱导,MS+8mg·L-1玉米素可获得较为理想的再生频率。【结论】Pina和Pinb的共同表达降低了硬粒小麦籽粒硬度,具有软化籽粒质地的功能。

[ Objective] Construction of expression vector harboring the fragment of fused Pina and Pinb, and verification of Pina and Pinb function. [Method] In the present study, a highly efficient expression vector （pFUPaPb） that harbors the fused fragment of Pina ＆Pinb flanked with regulation sequences of Ω and poly （A） was constructed, employing Ubiqutin as a promoter and bar gene as a selective marker. Common wheat variety Jing 411 is the donor of Pina and Pinb genes, pFUPaPb was trnsferred into durum wheat by biolistic method. [Result] After screening with bialophos and PCR identification with gene specific primers and dot blotting confirmation, 35 transplants were obtained. The expression of fused genes was detected in 2 of 16 transplants examined. The SKCS values of these two lines were decreased 10 and 12, respectively. Among the three medium used for callus inducing, SD2 medium was the comparatively efficient medium for callus induction of durum immature embryo. MS＋8 mg·L^-1 zeatin is good for callus regeneration. [Conclusion] Co-expression of Pina and Pinb decreased the endosperm texture of durum wheat.