致病疫霉PITG_02860基因沉默及功能鉴定  

PITG_02860 Gene Silencing and Functional Identification in Phytophthora infestans

作　　者：王娜 张杨倩 谭晨 徐裴 罗展宏 高建莉[3] 唐唯[1,2] 刘晶 WANG Na;ZHANG Yang-Qian;TAN Chen;XU Pei;LUO Zhan-Hong;GAO Jian-Li;TANG Wei;LIU Jing(School of Life Science,Yunnan Normal University,Kunming 650500,China;Joint Academy of Potato Science,Yunnan Normal University,Kunming 650500,China;Wenshan Academy of Agriculture Sciences,Wenshan 663099,China;Yunnan Yinmore Modern Agriculture Co.,Ltd.,Kunming 650500,China)

机构地区：[1]云南师范大学生命科学学院,昆明650500 [2]云南师范大学马铃薯科学研究院,昆明650500 [3]文山州农业科学院,文山663099 [4]云南英茂现代农业有限公司,昆明650500

出　　处：《农业生物技术学报》2024年第6期1404-1414,共11页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(31660503);云南省基础研究计划(202001AU070091,202201AT070026)。

摘　　要：交配型是卵菌重要性状,致病疫霉(Phytophthora infestans)效应子PITG_02860和A1交配型性状分离连锁。为进一步探索PITG_02860基因功能,本研究首先改良了致病疫霉原生质体转化技术,以碗豆汁培养72 h的菌丝,采用复合酶(裂解酶,10 mg/m L;纤维素酶,5 mg/m L)于26℃裂解45 min,原生质体制备率最高,为(31.5±2.36)个/μL;原生质体在黑麦培养基上再生率可达(3.34±0.20)%。依托该转化系统,以载体p TOR-m RFP为骨架,构建PITG_02860基因沉默载体并转化致病疫霉88069菌株,对2个阳性沉默子进行功能验证,发现与野生型相比,沉默子有性生殖产生的卵孢子数量显著下降且卵孢子畸形率显著增高(P<0.05),菌丝生长受阻,游动孢子数量显著下降(P<0.05),对马铃薯(Solanum tuberosum)叶片致病能力显著降低(P<0.05)。上述结果表明,PITG_02860不仅是致病疫霉有性生殖正常发生的关键基因,也是侵染马铃薯的重要毒性功能基因。本研究为明确致病疫霉有性生殖发生和解析致病疫霉对马铃薯的毒性机制提供了新思路。Mating system is an important trait in oomycetes,effector PITG_02860 was segregated and linked with the mating type of A1 of Phytophthora infestans.In order to investigate the function of the P.infestans PITG_02860 gene,protoplasts transformation protocol was optimized under the conditions of the mycelium cultured with soybean juice for 72 h,and then lysed by the complex enzyme(10 mg/mL lysing enzyme,5 mg/mL cellulase)at 26℃for 45 min,resulted in the highest rate of preparation of protoplasts,which was up to(31.5±2.36)protoplasts/μL;and the regeneration of the protoplasts in the medium of rye could reach to(3.34±0.20)%.Based on this transformation system,the PITG_02860 gene silencing vector was constructed with the vector pTOR-mRFP as the backbone and transformed into strain 88069.Functional verification of the 2 silenced strains revealed that,compared to the wild-type,the number of oospores produced during sexual reproduction significantly decreased and the rate of oospore deformity significantly increased(P<0.05),the growth of hyphae was hindered,the number of zoospores significantly decreased(P<0.05),and the pathogenicity to potato(Solanum tuberosum)leaves was significantly reduced(P<0.05).The above results indicated that PITG_02860 was not only a key gene for the normal development of sexual reproduction of P.infestans,but also an important virulence function gene when infecting potatoes.The study provides new ideas for sexual reproduction occurrence and virulence mechanism of P.infestans.

关 键 词：致病疫霉 原生质体 转化 基因沉默 有性生殖 

分 类 号：S432.1[农业科学—植物病理学]