CAJ | 학술논문

本研究以植物表达载体pROK2-Ubi为基础,设计带有酶切位点Kpn I和BamH I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Kan 50 mg/L,接种菌液浓度为OD_(600)=0.6,侵染时间为60 min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.
본연구이식물표체재체pROK2-Ubi위기출,설계대유매절위점Kpn I화BamH I적일대인물,종재체pAC25상확증도목적기인TaPHR1.매절회수후여동양쌍매절적표체재체pROK2-Ubi련접,획득신적표체재체pTaPHR1,병장소구건적재체도입근류농간균LB4404균주.령외이소맥품충제맥22위수체,이용농간균개도소맥성숙배유상조직적유전전화체계화구건적표체재체진행료초보적유전전화.실험결과표명선택항생소선택압Kan 50 mg/L,접충균액농도위OD_(600)=0.6,침염시간위60 min시,항성유상적획득솔최고,체도4.08%,항성유상조직적분화솔체도3.28%.본연구위농간균개도결린향응기인TaPHR1적소맥유전전화화소맥린고효유전개량연구제공료연구수거.
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.