柑橘溃疡病抗性相关转录因子CitMYB20的功能  

Function of Citrus Bacterial Canker Resistance-Related Transcription Factor CitMYB20

作　　者：姚利晓[1] 范海芳 张庆雯 何永睿[1] 许兰珍[1] 雷天刚[1] 彭爱红[1] 李强[1] 邹修平[1] 陈善春[1] YAO LiXiao;FAN HaiFang;ZHANG QingWen;HE YongRui;XU LanZhen;LEI TianGang;PENG AiHong;LI Qiang;ZOU XiuPing;CHEN ShanChun(National Center for Citrus Variety Improvement,Citrus Research Institute,Southwest University/Chinese Academy of Agricultural Sciences,Chongqing 400712)

机构地区：[1]西南大学/中国农业科学院柑桔研究所国家柑桔品种改良中心,重庆400712

出　　处：《中国农业科学》2020年第10期1997-2008,共12页Scientia Agricultura Sinica

基　　金：国家现代农业产业技术体系建设专项资金(CARS-26);重庆市基础与前沿研究(一般)项目(cstc2018jcyjAX0247);中央高校基本业务费专项资金(XDJK2019B018)。

摘　　要：[目的]了解柑橘不同品种CitMYB20对柑橘溃疡病菌(Xanthomonas citri subsp.citri,Xcc)和外源植物激素的应答情况,评价转基因植株对柑橘溃疡病的抗性,验证CitMYB20的生物学功能.[方法]根据柑橘基因组序列设计引物,PCR法同源克隆柑橘溃疡病抗性品种金弹金柑(Fortunella japonica)和敏感品种枣阳小叶枳(Poncirus trifoliata)的CitMYB20基因序列和启动子序列,利用在线软件PlantCARE对启动子序列进行顺式作用元件预测.以针刺法对金弹金柑和枣阳小叶枳离体叶片接种柑橘溃疡病菌,用外源激素水杨酸、茉莉酸甲酯和乙烯利处理金弹金柑和枣阳小叶枳的叶片,在处理的不同时期收集材料,利用实时荧光定量PCR(qRT-PCR)检测CitMYB20的表达水平.分别构建CitMYB20的过表达载体和抑制表达载体,根癌农杆菌法转化枳上胚轴,随机摘取转基因株系成熟叶片进行柑橘溃疡病离体抗性评价.[结果]金弹金柑和枣阳小叶枳中CitMYB20(GenBank登录号分别为MN689609和MN689608)序列高度保守,相似度达到99%;两者的启动子序列(GenBank登录号分别为MN689611和MN689610)虽然都具有脱落酸和茉莉酸甲酯的响应元件,但金弹金柑中还具有水杨酸响应的顺式作用元件,而枣阳小叶枳中缺少该元件.在体外接种柑橘溃疡病菌5 d时,金弹金柑成熟叶片中CitMYB20表达量显著上升,达到对照的2.5倍;而枣阳小叶枳叶片CitMYB20在整个接菌周期内表达量未发生显著变化.外源激素处理时,CitMYB20对水杨酸和茉莉酸甲酯的响应相似,即在金弹金柑中呈现出先升高后降低的趋势,而在枣阳小叶枳中表达量降低,随后又恢复正常.乙烯利处理时,CitMYB20在金弹金柑中表达量略有下降,在枣阳小叶枳中表达量先升后降.遗传转化共获得7株过表达植株和5株抑制表达植株,转基因植株与对照植株相比在形态发育上未见明显差异.CitMYB20过表达植株能够在一定程度上减弱柑橘溃�【Objective】The objective of this study is to reveal the response of CitMYB20 to Xanthomonas citri subsp.citri(Xcc)and exogenous phytohormones in different citrus varieties,evaluate the resistance of transgenic plants to citrus bacterial canker(CBC),and to verify the biological function of CitMYB20.【Method】The primer pairs were designed according to the Citrus genome,the open reading frame sequences and promoter sequences of CitMYB20 were cloned from CBC resistant variety Fortunella japonica and CBC sensitive variety Poncirus trifoliata by PCR.The cis-acting elements of promoter sequences were predicted using online software PlantCARE.The detached mature leaves from F.japonica and P.trifoliata were inoculated with Xcc by acupuncture method,and collected at 0,1,3,and 5 d after inoculation.The citrus leaves were collected after being treated with exogenous salicylic acid,methyl jasmonate,and ethephon respectively for 0,12,24,36,and 48 h.The expression of CitMYB20 was detected by qRT-PCR using the above treated leaves.The over-expressed vector and RNA interference vector of CitMYB20 were constructed through inserting the target sequences into the pLGNe plasmid.The transgenic plants were obtained through Agrobacterium tumefaciens,and the mature leaves of transgenic lines were randomly selected to conduct resistance evaluation against CBC【.Result】The open reading frames of CitMYB20 in F.japonica(GenBank number MN689609)and P.trifoliata(GenBank number MN689608)were highly conservative,with a similarity of 99%.There were abscisic acid-responsive and methyl jasmonate-responsive cis-acting elements in the promoter sequence of CitMYB20 from two varieties of citrus.However,there was a salicylic acid-responsive element in the promoter sequence from F.japonica(GenBank number MN689611),but not in the sequence from P.trifoliata(GenBank number MN689610).At the 5th day of Xcc in vitro inoculation,the expression of CitMYB20 in mature leaves of F.japonica was increased significantly,reaching 2.5 times of the healthy cont

关 键 词：柑橘黄单胞杆菌柑橘亚种 柑橘溃疡病 R2R3-MYB转录因子 过表达 RNA干扰 抗病性 

分 类 号：S436.66[农业科学—农业昆虫与害虫防治]