白僵菌培养基继代培养对蛋白酶（cdep1）、几丁质酶（chit1）基因表达的影响

目的]对球孢白僵菌D6-2和L1-1菌株进行室内培养基继代培养,比较其不同世代之间毒力及毒力相关基因表达之间的差异.方法]对目的菌株进行室内生物测定,用LT50和LT90作为毒力评价标准来检测不同世代之间毒力的变化情况.为了明确继代培养对球孢白僵菌毒力相关酶基因表达情况的影响,以18S rDNA基因为内参,利用半定量RT-PCR对蛋白酶cdep1基因和几丁质酶chit1基因的表达情况进行检测.结果]在107孢子/mL供试浓度下,随着世代的更替,两个供试菌株F2代相比于F1代,其LT50和LT90降低,说明其对玉米螟的毒力有所增强.而两个供试菌株F2～F5代对玉米螟的毒力呈下降趋势.在内参基因稳定表达的情况下,两个供试菌株L1-1和D62的cdep1基因和chit1基因在F1和F2代表达稳定,随着继代次数的增多,供试菌株两个基因的表达量均呈下降趋势,这与对亚洲玉米螟毒力测试的结果呈正相关性.结论]F2代毒力和毒力酶基因表达最强,F1代与F2代基本相似或略低于F2代,F2～F5代毒力和毒力酶基因表达依次减弱.

Effects of Beauveria bassiana during subculturing on virulence and expression level of cdep1 and chit1 genes

Objective] To determine the impact of successive culture of Beauveria bassiana for 5 generations on virulence and expression level of virulence-related genes.Method] The conidia of two strains of B.bassiana,D6-2 and L1-1,were harvested from in vitro subcultures on SDAY medium.The expression level of virulence relative enzyme gene,cdep1 and chit1 of both strains tested,were detected by semi-quantitative RT-PCR,in which the housekeeping gene 18S rDNA was used as an internal control gene.Result] Bioassay results against Ostrinia furnacalis showed that the LT50 and LT90 of F2 generation of both strains were lower than those of the F1 generation during subculturing,which indicated that the virulence was increased,whereas the virulence from F2 to F5 generation declined.The expression of cdep1 and chit1 genes were not significantly different in F1 and F2 generations of both strains tested,but in the subculturing generations,declined.Conclusion] The expression levels of the two genes were positively correlated with the virulence against O.furnacalis in this study.