农杆菌介导的马尔尼菲青霉基因转化技术的建立及优化

摘要

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 medi⁃um,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.%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200μmol／ 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。