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

[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疡病的症状,而抑制CitMYB20表达植株对柑橘溃疡病菌的敏感性增强.[结论]CitMYB20在柑橘溃疡病抗性品种中受柑橘溃疡病菌的诱导表达;CitMYB20是防御柑橘溃疡病菌入侵的一种正向调控转录因子,其抗柑橘溃疡病功能可能需要水杨酸和茉莉酸信号途径的作用;CitMYB20可作为柑橘抗溃疡病分子育种的候选基因.

【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 control,while the expression of CitMYB20 in P.trifoliata did not show significantly change during the whole infection period.During exogenous hormone treatment,the response of CitMYB20 to salicylic acid and methyl jasmonate was similar,that is,it showed the trend of first increasing and then decreasing in F.japonica,while the expression of CitMYB20 in P.trifoliata was decreased firstly and then returned to normal.When the leaves were treated with exogenous ethephon,the expression of CitMYB20 in F.japonica was decreased slightly,while in P.trifoliata,it showed the trend of first increasing and then decreasing.A total of 7 over-expressed plants and 5 RNA-interfered ones were obtained through genetic transformation.There was no significant difference in morphological development between transgenic plants and control plants.The results of disease resistance evaluation showed that the CitMYB20 over-expressed plants could reduce the symptom of CBC,while the RNA-interfered plants were more sensitive to Xcc.【Conclusion】The expression of CitMYB20 was induced by Xcc in CBC resistant variety.CitMYB20 is a positive regulatory transcription factor against Xcc,its function against CBC may require salicylic acid and jasmonic acid signaling pathway.CitMYB20 can be used as a candidate gene for genetically modified breeding against CBC.