农杆菌介导的马尔尼菲青霉基因转化技术的建立及优化

中国真菌学杂志　 2015, Vol. 10 　Issue (2): 88-91.

农杆菌介导的马尔尼菲青霉基因转化技术的建立及优化

李鑫垒¹, 李昕², 曹存巍¹

1. 广西医科大学第一附属医院皮肤性病科, 南宁 530021;
2. 常德市第一人民医院皮肤性病科, 常德 415000

收稿日期:2014-12-13 出版日期:2015-04-28 发布日期:2015-04-28
通讯作者: 曹存巍,E-mail:caocunwei@yeah.net E-mail:caocunwei@yeah.net
作者简介:李鑫垒,男(汉族),硕士研究生在读.E-mail:3024270@163.com
基金资助:
国家自然科学基金 (81060128,81271804);广西自然科学基金 (0991144,2012jjAA40175);广西卫生厅自筹课题 (Z2007082)

To establish and optimize the genetic transformation technology of Penicillium marneffei by Agrobacterium tumefaciens-mediated transformation

LI Xin-lei¹, LI Xin², CAO Cun-wei¹

1. Department of Dermatology and Venereology, The First Affiliated hospital ofGuangxi Medical University, Naning 530021, China;
2. Department of Dermatology and Venereology, The First People's Hospital of Changde, Changde 415000

Received:2014-12-13 Online:2015-04-28 Published:2015-04-28

摘要/Abstract

摘要：

目的建立农杆菌介导的马尔尼菲青霉 (PM)基因转化技术,并对该技术条件进行优化。方法以二元质粒pDHt/SK为载体,通过农杆菌介导将pyrG基因插入马尔尼菲青霉尿嘧啶缺陷株SPM4 (pyrG^-,niaD^-)中,在不含尿嘧啶的培养基中筛选阳性转化子。运用PCR验证重组子。进一步对影响转化效率的农杆菌类型、共培养浓度、转化媒介、共培养温度、共培养时间、乙酰丁香酮 (AS)等六个条件进行优化。结果 PCR验证pyrG基因成功的插入SPM4中,所得到转化子可稳定传代,通过条件优化,得到转化子约300个/10⁶个细胞。选用AGL-1,以农杆菌共培养浓度为OD₆₀₀=0.8,AS浓度为200 μmol/L,无膜IM固体共培养基为介质,25℃共培养48 h为最适转化条件。结论成功建立了农杆菌介导PM基因转化技术,简化并优化了转化条件,该方法可用于PM基因功能研究。

关键词: 马尔尼菲青霉, 农杆菌, 转化

Abstract:

Objective To construct and optimize the condition of Agrobacteriumtumefaciens-mediated transformattechology in Penicillium marneffei.Method A.tumefaciens transformed by Calcium chloride with pDHt/SK::pyrG plasmids insert pyrG gene into SPM4 (pyrG^-,niaD^-),medium without uracil screening positive transformat,Using PCR verification restructuring.It aimed at finding out the optimal protocol by changing some important effecting factors including A.tumefaciens strains,concentration,transformat medium,co-cultivation temperature,period,AS.The positive transformant growed well in the media without uracil,and PCR analysis showed that T-DNA was inserted into the transformants.Result The transformation efficiency was about 300 transformants/10⁶ spores by optimizing.The optimal condition for AGL-1,concentration of A.tumefaciens OD₆₀₀=0.8,the AS concentration of 200 μmol/L,without nitrocellulose filters,co-culture 48 h in 25℃.Conclusion A.tumefaciens-mediated transformattechology in P.marneffei.was successfully established,simplified and optimized.The method can be used for PM gene function research.

Key words: Penicillium marneffei, Agrobacterium tumefaciens, transformation

中图分类号:

R379.9

李鑫垒, 李昕, 曹存巍. 农杆菌介导的马尔尼菲青霉基因转化技术的建立及优化[J]. 中国真菌学杂志, 2015, 10(2): 88-91.

LI Xin-lei, LI Xin, CAO Cun-wei. To establish and optimize the genetic transformation technology of Penicillium marneffei by Agrobacterium tumefaciens-mediated transformation[J]. Chinese Journal of Mycology, 2015, 10(2): 88-91.

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