油菜黑胫病菌T-DNA插入诱变因素优化及突变体筛选  

Optimization of T-DNA insertion-mediated mutagenesis of Leptosphaeria biglobosa and mutant screening

作　　者：杨越寒 张静[1] 杨龙[1] 吴明德[1] 李国庆[1] YANG Yue-han;ZHANG Jing;YANG Long;WU Ming-de;LI Guo-qing(Hubei Key Laboratory of Crop Disease Monitoring and Safety Control,Plant Science and Technology College,Huazhong Agricultural University,Wuhan 430070,China)

机构地区：[1]湖北省作物病害监测与安全控制重点实验室,华中农业大学植物科学技术学院,湖北武汉430070

出　　处：《中国油料作物学报》2021年第6期1115-1125,共11页Chinese Journal of Oil Crop Sciences

基　　金：国家油菜产业技术体系油菜黑胫病防控岗位(CARS-12)。

摘　　要：油菜黑胫病是由Leptosphaeria biglobosa引起的一种真菌病害。这种病害在我国油菜产区广泛发生,并造成一定的经济损失。为通过获取突变体来研究L.biglobosa的生态适应性机制及致病机制,本文优化了影响农杆菌介导转化(ATMT)油菜黑胫病菌L.biglobosa菌株Lb731的因素,评估转化子质量,并筛选相关突变体。结果明确了农杆菌介导转化菌株Lb731的最佳因素:潮霉素B浓度为50μg/mL,转化受体(分生孢子)培养时间为15 d(20℃),浓度为2×10^(7-8)孢子/mL,农杆菌-受体共培养温度为25℃,共培养时间为72 h。在最适条件下的转化效率达到80个转化子/百万分生孢子。T-DNA插入基因组的频率为100%,单拷贝插入频率为72.7%,转化子抗潮霉素性状能稳定遗传。从2136个转化子中获得了11个菌丝生长减缓突变体,7个色素产生缺陷突变体和14个分生孢子产生缺陷突变体,并从这些突变体中鉴定出7个致病力丧失突变体。采用hiTAIL-PCR技术,从3个突变体中获得了T-DNA插入位点侧翼序列。上述结果为深入研究L.biglobosa的生态适应性机制及致病机制提供了材料和线索。Blackleg of oilseed rape(Brassica spp.)is caused by Leptosphaeria biglobosa,which has been found to occur widely in oilseed rape-producing areas in our country,resulting in a substantial seed yield loss in the industry of oilseed rape.However,the mechanisms for ecological adaptation and pathogenesis of L.biglobosa have not been elucidated.Therefore,it is necessary to conduct indepth studies on molecular mechanisms for ecological ad⁃aptation and pathogenesis of L.biglobosa.In order to fulfill this objective,this study was done to mutate L.biglobosa by Agrobacterium tumeficens-mediated transformation(ATMT).The contents included optimization of the factors affecting the ATMT,evaluation of the quality of the transformants,and screening of mutants.The results showed that the optimum ATMT factors for L.biglobosa Lb731 were:hygomycin B concentration at 50μg/mL,transformation re⁃cipients(conidia)from the 15-day-old cultures(20℃),conidial concentration at 2×10^(7-8) conidia/mL,co-incubation of A.tumeficens-recipients at 25℃for 72 h.The transformation efficiency reached 80 transformants per million conidia on average.The T-DNA insertion frequency reached up to 100%based on PCR assays with 72.7%single copy insertion among the transformants based on Southern blottings.Results also showed that the hygromycin-resis⁃tance trait in the transformants inherited stably through nuclear mitosis.A total of 32 mutants were screened from 2136 transformants,including 11 mutants with suppressed mycelial growth,7 mutants with deficiency in pigment biosynthesis,and 14 mutants with deficiency in conidial production.Meanwhile,7 out of the 32 mutants were identified to completely lose pathogenicity(virulence)on oilseed rape.Using the hiTAIL-PCR technique,three DNA sequences flanking the T-DNA-insertion sites were obtained from three mutants.Overall,these results laid a solid foundation for further elucidation of the molecular mechanisms for ecological adaptation and pathogenesis in L.big⁃lobosa.

关 键 词：油菜黑胫病菌 农杆菌介导遗传转化 T-DNA插入诱变 突变体筛选 

分 类 号：Q785[生物学—分子生物学] S565.4[农业科学—作物学]