农杆菌介导苹果炭疽病菌的遗传转化及转化子鉴定  

Transformation of Agrobacterium tumefaciens-Mediated Colletotrichum gloeosporioides and Identification of Transformants

作　　者：王海艳[1] 李保华[1,2] 张清明[3] 李桂舫[1,2] 董向丽[1,2] 王彩霞[1,2] 

机构地区：[1]青岛农业大学农学与植物保护学院,山东青岛266109 [2]山东省植物病虫害综合防控重点实验室,山东青岛266109 [3]青岛农业大学化学与药学院,山东青岛266109

出　　处：《中国农业科学》2013年第9期1799-1807,共9页Scientia Agricultura Sinica

基　　金：国家现代农业产业技术体系(CARS-28);国家自然科学基金项目(31000891;31272001);山东省科技攻关计划(2010GNC10918);山东省"泰山学者"建设工程

摘　　要：【目的】优化农杆菌(Agrobacterium tumefaciens)介导转化苹果炭疽病菌(Colletotrichumgloeosporioides)的技术体系,并对转化子进行筛选鉴定,比较其生物学特性和致病性差异。【方法】以苹果炭疽病菌LXS010101分生孢子为转化受体,利用携带重组双元载体的农杆菌进行转化;对获得的转化子进行遗传稳定性测定、PCR检测及Southern blot分析;选取一定数量的转化子,比较其菌落形态、生长速率及分生孢子发育等主要生物学性状及致病性的差异。【结果】苹果炭疽病菌的最优转化体系为:病菌分生孢子悬浮液浓度1×105个/mL,共培养温度和时间分别为22℃和24 h,共培养基中添加200μmol.mL-1乙酰丁香酮,其转化效率达到1×105个孢子可获得439个转化子。随机选取30个转化子进行鉴定,发现T-DNA已整合进炭疽病菌基因组中,在所检测的转化子中都是以单拷贝的形式整合,且能够稳定遗传。与野生型菌株相比,转化子的菌落形态没有发生明显变化,但23.33%的菌株生长速率显著减低;转化子ATJ-3和ATJ-15不能产生分生孢子,在28个产孢菌株中,分生孢子萌发率显著减低的菌株占39.29%,附着胞形成比例显著减低和形态异常的菌株占25.00%。致病性测定结果显示,11个转化子的致病力显著降低,其中菌株ATJ-19完全丧失致病能力。【结论】优化的苹果炭疽病菌遗传转化技术体系,可用于炭疽病菌致病相关基因的克隆及致病分子机理的研究。[Objective] The objective of this study is to optimize the Agrobacterium tumefaciens-mediated transformation （ATMT） technology system of Colletotrichum gloeosporioides, screen and identify the transformants and compare the difference of biological characteristics and pathogenicity between wild-type strain and transformants. [Method ] Using the conidia of C. gloeosporioides strain LXS010101 as transformation recipients, A. turnefaciens strain EHA105 carrying plasmid pBIG3C harboring the hygromycin B phosphotransferase gene （hph） was transformed into C. gloeosporioides. The obtained transformants were screened and identified by hygromycin B resistance, PCR and Southern blot analysis. Partial transformants were selected randomly and analyzed on the colony morphology, mycelia growth rate, conidia development and pathogenicity. [Result] Successful transformation of C. gloeosporioides was performed and the highest efficiency reached on 439 transformants per 1 × 10^5 spores. The optimal transformation conditions were that 1 × 10^5 spores per milliliter of C. gloeosporioides spore suspension were co-cultured withAgrobacterium cells at 22℃for 24 h, in the presence of co-culture medium containing aeetosyringone （AS） at 200 μmol.mL^-1. The transformants were stable when grown on PDA medium without hygromycin B for five times and were verified by PCR amplification with the hph primers and by Southern blot analysis with the hph probe. The results showed that all the detected transformants could be amplified the target bands and all the T-DNA were single-copy inserted into the genome of C. gloeosporioides Compared with the wild-type strain LXS010101, most of the transformants did not change on the colony morphologies, however, in which 23.33% of the transformants decreased the mycelia growth rate significantly and two strains ATJ-3 and ATJ-15 could not produce conidia. In the other 28 transformants, 39.29% of the strains reduced the spore germination rate and 25.00% of the strains changed appressorium formati

关 键 词：苹果炭疽病菌 遗传转化 转化子鉴定 致病性 

分 类 号：S436.611[农业科学—农业昆虫与害虫防治]