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中国农学通报 ›› 2010, Vol. 26 ›› Issue (15): 19-24.

所属专题: 生物技术

• 生物技术科学 • 上一篇    下一篇

施氏假单胞菌Na+/H+逆向转运蛋白基因nhaA的克隆与表达分析

王全伟 张海玲 孟庆英 徐香玲 李新玲   

  • 收稿日期:2010-04-20 修回日期:2010-05-09 出版日期:2010-08-05 发布日期:2010-08-05
  • 基金资助:

    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”

Cloning and Expression Analysis of a Na+/ H+ Antiporter Gene(nhaA) from Pseudomonas Stutzeril

  • Received:2010-04-20 Revised:2010-05-09 Online:2010-08-05 Published:2010-08-05

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1167 bp,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41 kD的蛋白。平板法和生长曲线法检测表明,重组菌在800 mmol/L NaCl
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。

关键词: 植物景观, 植物景观, 生态型, 高校校园

Abstract:

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 genenhaA fromPseudomonas 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 withnhaA
ofE. coli were 99% and 99.2%, respectively. The expression vector was constructed and thenhaA protein with
the molecular weight about 41 kD could also be detected inE.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 ofnhaA
family, which they can antiport Na+, and further study in the salt-stress tolerance improvement ofnhaA could
base on these results. The gene has been accepted in GenBank by the accession number EU545468.