农杆菌介导的出芽短梗霉遗传转化及高效筛选聚苹果酸高产菌株
Agrobacterium tumefaciens-mediated transformation of Aureobasidium pullulans and high-efficient screening for polymalic acid producing strain
投稿时间:2014-12-01  
DOI:  10.13345/j.cjb.140594
中文关键词:农杆菌,出芽短梗霉,遗传转化,高效筛选,聚苹果酸
英文关键词:Agrobacterium tumefaciens , Aureobasidium pullulans, transformation, high-efficient screening, polymalic acid
基金项目:国家高技术研究发展计划(863计划) (Nos. 2015AA021005, 2014AA021205),中央高校基本科研业务费专项资金 (Nos. XDJK2013B039, 2362014XK07),重庆大学“生物流变科学与技术”教育部重点实验室访问学者基金 (No. CQKLBST-2014-006) 资助。
作者单位E-mail
涂光伟 1 西南大学药学院重庆 4007152 重庆药物过程与质量控制工程技术中心重庆 400715  
王永康 1 西南大学药学院重庆 4007152 重庆药物过程与质量控制工程技术中心重庆 400715  
冯骏 1 西南大学药学院重庆 4007152 重庆药物过程与质量控制工程技术中心重庆 400715  
李晓荣 1 西南大学药学院重庆 4007152 重庆药物过程与质量控制工程技术中心重庆 400715  
郭美锦 3 华东理工大学生物反应器工程国家重点实验室上海 200237  
邹祥 1 西南大学药学院重庆 4007152 重庆药物过程与质量控制工程技术中心重庆 4007154 重庆大学 生物流变科学与技术教育部重点实验室重庆 400044 zhx1030@swu.edu.cn 
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中文摘要:
      建立根癌农杆菌介导的出芽短梗霉遗传转化方法及T-DNA突变库,高效筛选聚苹果酸高产菌株及功能基因。通过含潮霉素和草铵磷抗性基因的农杆菌转化出芽短梗霉,抗性压力筛选及PCR验证建立根癌农杆菌介导的出芽短梗霉遗传转化方法,结合发酵液pH与聚苹果酸含量响应变化,微孔板高效筛选高产聚苹果酸的T-DNA插入突变株,基因组步移确定T-DNA插入位点及功能基因。结果获得遗传稳定的抗性基因菌株,每107个细胞可获得80?120个转化子,出芽短梗霉H27 号T-DNA突变株聚苹果酸摇瓶发酵产量提高24.5%,基因组步移证实糖酵解途径磷酸甘油酸变位酶基因被破坏。成功建立了根癌农杆菌介导的出芽短梗霉遗传转化方法和T-DNA插入突变库,结合高效筛选方法为聚苹果酸合成功能基因挖掘及高产机制解析奠定基础。
英文摘要:
      To develop a genetic transformation method of Aureobasidium pullulans and T-DNA insertion for high-efficient screening of polymalic acid (PMA) producing strain. Agrobacterium tumefaciens-AGL1, containing the selection genes encoding hygromycin B phosphotase or phosphinothricin acetyltranferase, was used to transform Aureobasidium pullulans CCTCC M2012223 and transformants were confirmed by colony PCR method. Transferred DNA (T-DNA) insertional mutants were cultured in microwell plate, and screened for high-titer PMA producing strain according to the pH response model. DNA walking was used to detect the insertion sites in the mutant. Results show that the selection markers could stably generated in the transformants, and 80 to 120 transformants could be found per 107 single cells. A high-titer PMA mutant H27 was obtained, giving a good PMA production caused by the disruption of phosphoglycerate mutase, that increased by 24.5% compared with the control. Agrobacterium tumefaciens-mediated transformation and high-efficient screening method were successfully developed, which will be helpful for genetic transformation of Aureobasidium pullulans and its functional genes discovery.
涂光伟,王永康,冯骏,李晓荣,郭美锦,邹祥.农杆菌介导的出芽短梗霉遗传转化及高效筛选聚苹果酸高产菌株[J].生物工程学报,2015,31(7):1063~1072
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