嗜碱单胞菌的新型羧基转移酶α亚基基因Aa-accA及其抗盐碱性能

A novel gene (Aa-accA) encoding acetyl-CoA carboxyltransferase α-subunit of Alkalimonas amylolytica N10 enhances salt and alkali tolerance of Escherichia coli and tobacco BY-2 cells

【目的】探讨解淀粉嗜碱单胞菌(Alkalimonas amylolytica)N10来源的羧基转移酶α亚基(Acetyl-coenzyme A carboxylase subunit alpha,AccA)基因Aa-accA对细菌及植物细胞耐盐碱性的作用。【方法】通过PCR方法从嗜碱菌N10基因组中扩增基因Aa-accA,并在大肠杆菌(Escherichia coli)K12中表达,通过测定工程菌及对照菌在不同盐浓度[0%,2%,4%,6%(W/V)NaCl]及不同碱性pH(8.0,8.5,9.0,9.5)的LB中生长12 h后的OD600值,以及二者在分别含6%(W/V)NaCl及pH 9的LB中的生长曲线,评价Aa-accA对大肠杆菌耐盐碱性的影响。同时以pPZP111为载体,构建了植物细胞重组表达载体,通过农杆菌介导方法将该基因转入烟草BY-2悬浮细胞表达,利用FDA染色方法测定经盐碱溶液处理后残存的活细胞数量评价该基因对植物细胞耐盐碱性的影响。【结果】PCR扩增得到基因Aa-accA,其ORF含957 bp,编码318个氨基酸的多肽,BLAST比对显示该基因为羧基转移酶α亚基(AccA)家族中的成员,其氨基酸序列与E.coli的AccA具有76%同源性;含有Aa-accA的E.coli K12相较于对照组在不同NaCl浓度及不同碱性pH的LB中表现出了明显的生长优势,特别是在6%(W/V)NaCl及pH 9的LB中培养12 h后,终OD600分别是对照菌的2.6倍和3.5倍;缺失体实验结果显示基因缺失的突变体E.coli K12ΔaccA在6%(W/V)NaCl及pH 9的LB中不能正常生长,而含有Aa-accA基因的重组质粒使得E.coli K12ΔaccA在同样条件下OD600值达到0.5和0.2;转入此基因的烟草BY-2细胞,经盐碱溶液处理后,其存活细胞比例高于野生型。【结论】本研究首次发现了Aa-accA基因与盐碱性的相关性,可提高大肠杆菌及烟草BY-2细胞的耐盐碱能力。

[ Objective l Acetyl-CoA carboxylase （ACC） catalyzes the first step of fatty acid synthesis. In most bacteria, ACC is composed of four subunits encoded by accA, accB, accC, and accD. Of them, accA encodes acetyl-CoA carboxyhransferase oL-suhunit. Our prior work on proteomics of Alkalimonas amylolytica N10 showed that the expression of the Aa-accA has a remarkable response to salt and alkali stress. This research aimed to find out the Aa-accA gene contributing to salt and alkali tolerance. [ Methods] The Aa-accA was amplified by PCR from A. amylolytica N10 and expressed in E. coli K12 host. The effects of Aa-accA expression on the growth of transgenic strains were examined under different NaC1 concentration and pH conditions. Transgenic tobacco BY-2 cells harboring Aa-accA were also generated via Agrobacterium-mediated transformation. The viability of BY-2 cells was determined with FDA staining method after salt and alkali shock. [ Results] The Aa-accA gene product has 318 amino acids and is homologous to the carboxyl transferase domain of acyl-CoA carboxylases. It showed 76% identity with AccA （acetyl-CoA carboxylase carboxyltransferase subunit alpha） from E. coll. Compared to the wild-type strains, transgenic E. coli K12 strain containing Aa-accA showed remarkable growth superiority when grown in increased NaC1 concentrations and pH levels. The final cell density of the transgenic strains was 2.6 and 3.5 times higher than that of the control type when they were cultivated in LB medium containing 6% （W/V） NaC1 and at pH 9, respectively. Complementary expression of Aa-accA in an accA-depletion E. coli can recover the tolerance of K12AaccA to salt and alkali stresses to some extent. Similar to the transgenic E. coli, transgenic tobacco BY-2 cells showed higher percentages of viability compared to the wild BY-2 cells under the salt or alkali stress condition. [ Conclusion] We found that Aa-accA from A. amylolytica N10 overexpression enhances the tolerance of both transgenic E. coli and tobacco BY-2 cells to NaC1 and alkali stresses.