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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2012, Vol. 32 Issue (01): 56-63    
技术与方法     
根癌农杆菌介导的黑曲霉遗传转化体系的建立及优化
黎明, 刘萌, 黄云雁, 周丽颖, 孙昕, 路福平
天津市工业微生物重点实验室 工业发酵微生物教育部重点实验室 天津科技大学生物工程学院 天津 300457
Establishment and Optimization of Agrobacterium tumefaciens-mediated Transformation System of Aspergillus niger
LI Ming, LIU Meng, HUANG Yun-yan, ZHOU Li-ying, SUN Xin, LU Fu-ping
Tianjin Key Laboratory of Industrial Microbiology, Key Laboratory of Industrial Fermentation Microbiology Ministry of Education, The College of Biotechnology, Tianjin University of Science & Technology,Tianjin 300457, China
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摘要：

基于根癌农杆菌介导的遗传转化方法的独特优点,研究黑曲霉转化过程中各主要影响因素,建立高效的黑曲霉遗传转化方法。构建双元载体pBI-hph,通过电转导入农杆菌LBA4404中,以黑曲霉TCCC41056为受体菌株,利用潮霉素B基因作为筛选标记,对影响转化效率的孢子悬液的新鲜程度及浓度、农杆菌菌液浓度、共培养时间、共培养温度这五个条件进行分析,建立根癌农杆菌介导的黑曲霉遗传转化体系。实验结果表明,上述条件对黑曲霉的转化效率有较大的影响,通过优化,黑曲霉转化效率可达83个转化子/107分生孢子;整合到黑曲霉基因组的外源基因可以稳定遗传,在转接10代后遗传性能仍保持稳定,并在众多转化子中筛选得到了糖化酶活力提高18%的黑曲霉突变株。根癌农杆菌介导的黑曲霉转化体系的建立,为进一步研究黑曲霉的功能基因以及开发黑曲霉表达系统提供了有力的手段。
关键词： 根癌农杆菌;  黑曲霉;  介导转化;  糖化酶    
Abstract:

In order to establish an efficient transformation method for Aspergillus niger, the effect of different conditions on transformation frequency which is based on the unique advantages of Agrobactrium tumefaciens-mediated system(ATMT) were studied. Binary vector, pBI-hph was constructed and transferred into A.tumefaciens LBA4404 by electroporation. Filamentous fungus A.niger TCCC41056 was used as the hose strain and hph gene was used as selective marker, the transformation system of A. niger mediated by A.tumefaciens was established. Besides, the factors affecting the transformation efficiency such as spores freshness,A.tumefaciens'concentration,spore concentration,co-culture temperature and time were analyzed. The result of PCR showed that the T-DNA was integrated into genome of the A.niger. All 8 randomly selected hygromycin B-resistant transformants tested were stable after 10 subcultures onto complete medium without hygromycin. Based on the above researches, the conditions of ATMT were optimized and much higher transformation efficiency was got:about 83 transformants/107 spores. From of numerous transformants, one A.niger mutant strain that high-yield of glucoamylase was screened. ATMT was applied in A.niger and this transformation system will provide a powerful tool for the further study on function genes of A. niger.
Key words: Agrobacterium tumefaciens    Aspergillus niger    Transformation    Glucoamylase
收稿日期: 2011-10-13 出版日期: 2012-01-25
ZTFLH:  Q78  
基金资助:

国家自然科学基金(21176190)、天津市科技支撑计划(11ZCKFSY00900)、天津市应用基础及前沿技术研究计划(11JCYBJC 09600)资助项目
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引用本文:

黎明, 刘萌, 黄云雁, 周丽颖, 孙昕, 路福平. 根癌农杆菌介导的黑曲霉遗传转化体系的建立及优化[J]. 中国生物工程杂志, 2012, 32(01): 56-63.

LI Ming, LIU Meng, HUANG Yun-yan, ZHOU Li-ying, SUN Xin, LU Fu-ping. Establishment and Optimization of Agrobacterium tumefaciens-mediated Transformation System of Aspergillus niger. China Biotechnology, 2012, 32(01): 56-63.

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https://manu60.magtech.com.cn/biotech/CN/    或    https://manu60.magtech.com.cn/biotech/CN/Y2012/V32/I01/56


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