CAJ | 학술논문

[目的]构建青天葵器官大小调控基因———Erb3结合蛋白（Erb3-binding protein,EBP1）编码基因的原核表达载体,以期为通过原核表达体系鉴定该基因的功能奠定基础。[方法]将引入酶切位点的NfEBP1基因的PCR产物及表达载体pET-28、pET-16b分别进行双酶切,之后连接相应的酶切产物,热击转化到E.coli Top10细胞,通过菌落PCR、测序和酶切筛选阳性重组子。将确认的阳性重组子质粒转化至E.coli BL21表达宿主菌,并对其进行双酶切进行验证。[结果]构建了重组表达质粒pET-28-NfEBP1-1188和pET-16-NfEBP1-1188,其转化E.coliBL21表达宿主细胞后,获得含有2个载体的工程菌。[结论]该研究成功构建了青天葵EBP1基因的原核表达载体,为后续通过原核表达体系鉴定该基因的功能奠定了基础。
[목적]구건청천규기관대소조공기인———Erb3결합단백（Erb3-binding protein,EBP1）편마기인적원핵표체재체,이기위통과원핵표체체계감정해기인적공능전정기출。[방법]장인입매절위점적NfEBP1기인적PCR산물급표체재체pET-28、pET-16b분별진행쌍매절,지후련접상응적매절산물,열격전화도E.coli Top10세포,통과균락PCR、측서화매절사선양성중조자。장학인적양성중조자질립전화지E.coli BL21표체숙주균,병대기진행쌍매절진행험증。[결과]구건료중조표체질립pET-28-NfEBP1-1188화pET-16-NfEBP1-1188,기전화E.coliBL21표체숙주세포후,획득함유2개재체적공정균。[결론]해연구성공구건료청천규EBP1기인적원핵표체재체,위후속통과원핵표체체계감정해기인적공능전정료기출。
[Objective] To construct prokaryotic expression vectors encoding gene Erb3binding protein （EBP1）, which plays important roles in regulating plant organ size from Nervilia fordii （Hance） Schltr. [Methods] PCR products of NfEBP1 with particular restriction sites and expression vectors, pET-28 and pET-16b were digested. Ligation, transformation and selection were performed to construct the recombinant plasmids pET-28-NfEBP1 and pET-16-NfEBP1. The recombinant plasmids were transformed into E. coli BL21 using heat -shock transformation. [Results] Recombinant plasmids pET-28-NfEBP1-1188 and pET-16-NfEBP1-1188 were constructed and transformed into expressional host cells, E. coli BL21, and validated by colony PCR, sequencing and double digestion. [Conclusion] Prokaryotic expression vectors of EBP1 gene from N. fordii were successfully constructed, which laid the foundation for characterization of the gene function.