CAJ | 학술논문

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。
목적：건립농간균개도적마이니비청매（PM）기인전화기술，병대해기술조건진행우화。방법이이원질립pDHt／ SK 위재체，통과농간균개도장 pyrG 기인삽입마이니비청매뇨밀정결함주 SPM4（pyrG?，niaD?）중，재불함뇨밀정적배양기중사선양성전화자。운용 PCR 험증중조자。진일보대영향전화효솔적농간균류형、공배양농도、전화매개、공배양온도、공배양시간、을선정향동（AS）등륙개조건진행우화。결과 PCR 험증 pyrG 기인성공적삽입 SPM4중，소득도전화자가은정전대，통과조건우화，득도전화자약300개／106개세포。선용 AGL?1，이농간균공배양농도위 OD600＝0．8，AS 농도위200μmol／ L，무막 IM 고체공배양기위개질，25℃공배양48 h 위최괄전화조건。결론성공건립료농간균개도 PM 기인전화기술，간화병우화료전화조건，해방법가용우 PM 기인공능연구。
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.