| 摘要: |
| 【目的】对生防密旋链霉菌Act12中的四氢嘧啶合成基因簇进行鉴定及功能分析。【方法】对田间生防效果良好的密旋链霉菌Act12进行盐耐受能力检测，采用体积分数80%的乙醇抽提其胞内四氢嘧啶，分别通过TLC和HPLC进行定性及定量检测四氢嘧啶。通过Act12全基因组分析，克隆得到Act12中四氢嘧啶生物合成基因簇ectABC，将其与表达载体pET 28a连接，转化至大肠杆菌BL21（DE3）中，检测工程菌株的盐耐受能力。【结果】密旋链霉菌Act12可在NaCl质量分数为0%~10.0%的条件下生长，且能够生产相关耐盐相容性溶质四氢嘧啶。当NaCl质量分数为2.5%时，密旋链霉菌Act12胞内的四氢嘧啶积累量达到最大值，为72.39 mg/L。生物信息学分析结果表明,组成ectABC基因簇的3个基因ectA、ectB、ectC位于同一个操纵子上，分别编码二氨基丁酸乙酰基转移酶、二氨基丁酸氨基转移酶和四氢嘧啶合成酶。通过克隆得到长2 408 bp的ectABC基因簇，并使其成功在大肠杆菌中实现异源表达,通过Ni柱纯化得到了EctA蛋白。但含pET28a ectABC的大肠杆菌BL21(DE3)在本试验条件下，其盐耐受能力并无明显改善。【结论】生防链霉菌Act12中存在四氢嘧啶合成基因簇ectABC，且能够在大肠杆菌中成功异源表达。 |
| 关键词: 链霉菌 相容性溶质 四氢嘧啶 ectABC基因簇 |
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| 基金项目:中央高校基本科研业务费专项(Z109021426，Z109021432)；高校博士点基金项目(20120204120034，Z20120204120042)；陕西省农业科技创新与攻关项目（2015NY066） |
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| Identification and functional analysis of biosynthetic gene cluster of ectoine from Streptomyces pactum Act12 strain |
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GAO Bo,MA Yiming,HONG Yu,et al
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| Abstract: |
| 【Objective】The aim of this study was to identify ectoine biosynthesis genes and analyze their function in Streptomyces Act12 by molecular biology methods.【Method】The salt tolerance of Streptomyces pactum Act12 with excellent biological prevention and control effect in farmlands was detected and 80% ethanol was used for extracting ectoine.TLC and HPLC were then used for detecting the existence and accumulated amount of intracellular ectoine.Through whole genome analysis,the gene cluster ectABC was cloned and connected with expression vector pET 28a before being transformed into E.coli BL21(DE3).The salt tolerance of the engineering strain was also detected.【Result】Streptomyces pactum Act12 could grow in NaCl solution with mass fraction of 0%-10.0%,and produce ectoine.When mass fraction of NaCl was 2.5%,the accumulation of ectoine in cells of Streptomyces pactum Act12 reached the maximum of 72.39 mg/L.Bioinformatics analysis showed that three genes ectA,ectB and ectC of gene cluster ectABC were located in same operon and encoded diaminobutyric acid acetyltransferase,diaminobutyric acid aminotransferase,and ectoine synthase,respectively.The ectABC gene cluster of 2 408 bp was cloned and successfully expressed in E.coli,and the protein of EctA was purified by Ni column.However,the ability of salt tolerance of E.coli BL21(DE3) containing pET28a ectABC was not significantly improved under the experimental conditions.【Conclusion】The ectoine biosynthesis gene cluster of ectABC existed in Streptomyces Act12 and it could heterologous express in E.coli BL21(DE3) successfully. |
| Key words: Streptomyces compatible solutes ectoine ectABC gene cluster |
