水稻OsRab7耐盐功能的初步鉴定及其表达载体的构建

Rab7是一个GTP结合蛋白,隶属于Rab家族,该家族在调控膜泡的运输、结合以及细胞内膜结构的组织中行使重要作用。本实验通过RT-PCR技术从盐敏感水稻品种‘日本晴’(‘Nipponbare’)获得了OsRab7基因全长序列,成功构建了原核表达载体pET-32a-OsRab7。在IPTG诱导下,该蛋白高效表达,并明显缓解了高盐环境(4.5% ～8.5% NaCl)对大肠杆菌的生长胁迫。为进一步研究该基因的功能,将OsRab7在大肠杆菌中的表达蛋白纯化,并利用p1301B(pCAMBIA1301改造载体)构建了植物表达载体,成功转化来源于水稻株系‘中花11’(盐敏感)的愈伤组织,为探讨其在水稻中的表达模式和功能分析及应用于水稻耐盐品种的改良奠定了一定的基础。

Preliminary Function Analysis of Rice OsRab7 in Salt Tolerance and Construction of Its Genetic Transformation Vector

Rab7 is a small GTP-binding protein belonging to the Rab family,which plays an important role in vesicle trafficking,fusion and membrane organization.Total RNA was extracted from the young leaves of ‘Nipponbare’,a salt sensitive rice variety(Oryza sativa L ssp.japonica)and specific PCR primers were designed.The sequence of OsRab7 was obtained by RT-PCR to construct the pET-32a-OsRab7 vector.Escherichina coli was used as a host organism to express and test the function of the protein in salt tolerance.The experiment has demonstrated that OsRab7 has largely improved the salt tolerance of E.coli in medium with high NaCl concentration(4.5%-8.5%).The plant genetic transformation vector was constructed using p1301B(a pCAMBIA1301 variation)and rice callus from ‘Zhonghua 11’(salt sensitive)was transformed via Agro-bacteria transformation.The expressed protein was purified as well for further research.