摘要
Na+/H+逆向转运蛋白是生物耐盐的关键因子,能够维持高盐胁迫下生物体的正常生长代谢。利用PCR技术从施氏假单胞菌(Pseudomonas stutzeri)中克隆质膜Na+/H+逆向转运蛋白基因nhaA。序列分析表明,该基因全长1167bp,编码388个氨基酸,含有12个跨膜结构域和保守的功能氨基酸残基位点:Asp-133、Asp-163、Asp-164、His-225、Gly-338。与大肠杆菌nhaA基因核苷酸和编码氨基酸序列的同源性分别为99%和99.2%。将该基因与pET-28a构建成原核表达载体pET-nhaA,转化E.coli BL21,经IPTG诱导后获得相对分子量约为41kD的蛋白。平板法和生长曲线法检测表明,重组菌在800mmol/LNaCl胁迫下仍能正常生长,耐盐能力显著提高。以上结果证实克隆到的基因是施氏假单胞菌nhaA基因家族的成员,具有盐胁迫下将Na+逆向转运至胞外的功能,为进一步利用该基因进行作物耐盐改良奠定了基础。该基因的GenBank登录号为EU545468。
Na ＋ /H ＋ antiporter is the key factor in the salt-stress tolerance in organism.It can maintain normal growth and metabolism of organism under high salt stress.Polymerase Chain Reaction （PCR） was performed to clone a plasma membrane Na ＋ /H ＋ antiporter gene nhaA from Pseudomonas stutzeri.Sequence analysis showed that the full length of the gene is 1167 bp,and it encodes a putative 388 amino acids polypeptide which contained twelve putative transmembrane domains and conservative functional amino acids residues,such as Asp-133,Asp-163,Asp-164,His-225,Gly-338.Nucleotide and amino acid sequence homology with nhaA of E.coli were 99% and 99.2%,respectively.The expression vector was constructed and the nhaA protein with the molecular weight about 41 kD could also be detected in E.coli BL21 protein expression system.Plate method and the growth curve analysis showed that the recombinant strains can survive under the salt stress of 800 mmol/L NaCl with increased salt tolerance.Results indicated that the cloned gene was a member of nhaA family,which they can antiport Na ＋,and further study in the salt-stress tolerance improvement of nhaA could base on these results.The gene has been accepted in GenBank by the accession number EU545468.
出处
《中国农学通报》
CSCD
北大核心
2010年第15期19-24,共6页
Chinese Agricultural Science Bulletin
基金
哈尔滨师范大学博士科研启动基金项目"利用基因工程技术拓宽大豆抗逆资源的研究"(KGB200903)
