小麦TaPHR1基因表达载体的构建

本研究以植物表达载体pROK2-Ubi为基础,设计带有酶切位点KpnⅠ和BamHⅠ的一对引物,从载体pAC25上扩增到目的基因TaPHR1。酶切回收后与同样双酶切的表达载体pROK2-Ubi连接,获得新的表达载体pTaPHR1,并将所构建的载体导入根瘤农杆菌LB4404菌株。另外以小麦品种济麦22为受体,利用农杆菌介导小麦成熟胚愈伤组织的遗传转化体系和构建的表达载体进行了初步的遗传转化。实验结果表明选择抗生素选择压Kan50mg/L,接种菌液浓度为OD600=0.6,侵染时间为60min时,抗性愈伤的获得率最高,达到4.08%,抗性愈伤组织的分化率达到3.28%。本研究为农杆菌介导缺磷响应基因TaPHR1的小麦遗传转化和小麦磷高效遗传改良研究提供了研究数据。

Construction on Expression Vector of TaPHR1 Gene in Wheat

In this study,Triticum aestivun L.gene TaPHR1 was amplified from vector pAC25 using specific primers containing restriction enzyme site(Kpn I and BamH I)designed based on expression vector pROK2-Ubi.The target gene and the expression vector pROK2-Ubi were digested with the same as restriction enzymes,and then ligated to construct the recombinant expression vector pTaPHR1,finally we introduced it into Agrobacterium tumefaciens strain LB4404.As well as we employed Jimai22 to be as interceptor for the genetic transformation of wheat mature embryo callus based on A.tumefaciens mediated.The results showed that at the condition of Kan 50 mg/L,cell density OD_(600)=0.6,and inoculation duration 60min,the transformation frequency reach the maximuna,account for 4.08%,with the differentiation frequency of resistant callus of 3.28%.This work would provide some research dams for the Agrobacterium-mediated genetic transformation of TaPHR1 into wheat and the efficient use of soil phosphorus in breeding research.