摘要
【目的】获取低温淀粉酶基因,分析其相关功能,为工业生产上的应用提供参考。【方法】以气单胞菌(Aeromonas)LA77为出发菌株克隆低温α-淀粉酶基因,以BL-21(DE3)为宿主菌进行克隆。分析其它已知气单胞菌属低温α-淀粉酶基因同源性,设计特异引物,PCR扩增获得C13片段,将其克隆到pMAL-2X,转化到BL-21(DE3)中,筛选蓝白斑,验证PCR及EcoRⅠ和HindⅢ的双酶切,获得高效表达生物工程菌株pMAL-2X-C13。设计定点突变引物,以pMAL-2X-C13为模板,获得低温淀粉酶基因突变株三株C19、C29、C43。【结果】表达蛋白的分子大小为114kD,突变菌株C19蛋白表达量均高于pMAL-2X-C13。【结论】低温淀粉酶基因突变株C19比原始菌株pMAL-2X-C13淀粉酶基因蛋白表达量更高。
【Objective】In order to obtain the low-temperature amylase gene and its related functions,and better apply it in industrial production.【Methods】Cloning of the low temperatureɑ-amylase gene from the starting strain LA7 to BL-21(DE3).Other known low temperatureɑ-amylase gene was homology analysis,design specific primers,and C13 fragment was obtained by PCR amplification.Then clone it to pMAL-2X,Convert it to BL-21(DE3),and screening blue and white spots.Theɑ-amylase gene was verified by PCR and double enzyme digestion of EcoRⅠand HindⅢ,the highly expressed bioengineering strain pMAL-2X-C13was obtained.The site directed mutagenic primers was design,take pMAL-2X-C13 as the template,three mutant strains of low temperature amylase gene C19,C29 and C43 were obtained.【Results】SDS-PAGE test result display that the molecular size of the protein is 114 kD and the protein expression of mutant strain C19 was higher than that of strain pMAL-2X-C13.【Conclusion】The protein expressed of mutant strain C19 more than the original strain pMAL-2X-C13.It provides a scientific and theoretical basis for the future application of the engineering bacteria in industrial production.
作者
楚敏
史应武
顾美英
杨红梅
霍向东
张志东
CHU Min;SHI Yingwu;GU Meiying;YANG Hongmei;HUO Xiangdong;ZHANG Zhidong(Xinjiang Laboratory of Special Environmental Microbiology/Institute of Applied Microbiology,Xinjiang Academy of Agricultural Sciences,Urumqi 830091,China)
出处
《新疆农业科学》
CAS
CSCD
北大核心
2024年第2期479-484,共6页
Xinjiang Agricultural Sciences
基金
新疆维吾尔自治区重点研发任务专项(2022B02053-3)
第三次新疆综合科学考察(2022xjkk1204)
新疆农业科学院自主培育项目团队建设专项(nkyzztd-001)。
关键词
气单胞菌属
α-低温淀粉酶
基因改造
原核表达
Aeromonas
αcold-adapted amylase
genetically engineered
procaryotic expression
