| csi-miR399响应柑橘溃疡病菌侵染的表达模式及其抗病性分析 |
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| 引用本文： | 王兆昊,郭兴茹,张乐欢,何永睿,陈善春,姚利晓.csi-miR399响应柑橘溃疡病菌侵染的表达模式及其抗病性分析[J].中国农业科学,2023,56(8):1484-1493. |
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| 作者姓名： | 王兆昊 郭兴茹 张乐欢 何永睿 陈善春 姚利晓 |
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| 作者单位： | 西南大学柑桔研究所/国家柑桔工程技术研究中心/国家柑桔品种改良中心，重庆 400712 |
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| 基金项目： | 国家重点研发计划(2021YFD1400800); 国家重点研发计划(2021YFD1600800); 国家现代农业（柑橘）产业技术体系(CARS-26) |
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| 摘 要： | 【目的】明确csi-miR399响应柑橘溃疡病菌（Xanthomonas citri subsp. citri,Xcc）侵染的表达模式，筛选其靶基因，进而分析csi-miR399与寄主Xcc抗性的相关性，为柑橘溃疡病抗性种质的创制打下基础。【方法】分别以柑橘溃疡病抗性品种四季橘（Citrus microcarpa）和感病品种纽荷尔脐橙（Citrus sinensis）为试材，通过茎环qPCR方法分析csi-miR399在叶片离体注射Xcc 1、3和5 d时的表达量变化，明确抗/感品种中csi-miR399响应Xcc侵染的表达模式；利用在线软件psRNATarget预测csi-miR399的靶基因，并通过qPCR分析候选靶基因在接种Xcc柑橘叶片和瞬时表达csi-miR399叶片中表达量的变化；克隆csi-miR399前体基因序列，通过同源重组方法构建病毒表达载体pCLBV202-MIR399，根癌农杆菌介导的真空浸润法接种尤力克柠檬（Citrus limon），通过qPCR分析csi-miR399表达量；进而采用离体叶片针刺法接种Xcc，观察发病症状，统计病情指数，分析csi-miR3...
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| 关 键 词： | csi-miR399 柑橘溃疡病 靶基因 柑橘病毒表达载体 生物胁迫 |
| 收稿时间： | 2023-01-20 |
Expression Pattern of csi-miR399 in Response to Xanthomonas citri subsp. citri Infection and Its Disease Resistance Analysis |
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| WANG ZhaoHao,GUO XingRu,ZHANG LeHuan,HE YongRui,CHEN ShanChun,YAO LiXiao.Expression Pattern of csi-miR399 in Response to Xanthomonas citri subsp. citri Infection and Its Disease Resistance Analysis[J].Scientia Agricultura Sinica,2023,56(8):1484-1493. |
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| Authors: | WANG ZhaoHao GUO XingRu ZHANG LeHuan HE YongRui CHEN ShanChun YAO LiXiao |
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| Institution: | Citrus Research Institute, Southwest University/National Citrus Engineering Technology Research Center/National Center for Citrus Varieties Improvement, Chongqing 400712 |
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| Abstract: | 【Objective】The objective of this study is to identify the expression pattern of csi-miR399 in response to the infection of citrus canker bacteria (Xanthomonas citri subsp. citri, Xcc), screen its target genes, analyze the correlation between csi-miR399 and Xcc resistance in host plants, and to lay a foundation for the creation of citrus canker resistant germplasms.【Method】In order to clarify the expression pattern of csi-miR399 in response to Xcc infection, Xcc-resistant variety Calamondin (Citrus microcarpa) Xcc-sensitive variety Newhall Navel Orange (Citrus sinensis) were used as materials, and changes in the relative expression of csi-miR399 were analyzed by stem-loop qPCR after their leaves were injected with Xcc at 1, 3 and 5 d in vitro. The online software psRNATarget was used to predict the target genes of csi-miR399, which were further confirmed by qPCR in citrus leaves infected with Xcc and transiently over-expressed with csi-miR399. The viral expression vector pCLBV202-MIR399 was constructed by in-fusion cloning through csi-miR399 precursor sequence being inserted into pCLBV202, and transferred into Eureka Lemon (Citrus limon) by Agrobacterium tumefaciens-mediated vacuum infiltration. The lemon over-expressed with csi-miR399 was evaluated for resistance against Xcc through being stab-inoculated with the pathogen and investigated disease index.【Result】After inoculation with Xcc, the expression of csi-miR399 in Calamondin showed a downward trend and then an upward trend, while that in Newhall Navel Orange continued to decrease. At 5 d, the expression of csi-miR399 in Calamondin and Newhall Navel Orange was 4.64 times and 7.61% as its expression in healthy leaves, respectively, preliminary indicating that csi-miR399 was related to citrus canker resistance. Thirteen predicted target genes were screened from citrus genome. Three of them were confirmed because of the opposite expression trends with csi-miR399, which were Cs2g06030 (PHO2), Cs7g03830 (unknown protein), and Cs8g18800 (laccase). Three lemon strains (Y37, Y41 and Y57) with over-expressed csi-miR399 were obtained. Comparing with L35 (empty vector pCLBV202), csi-miR399 was significantly up-regulated in the Y37, Y41 and Y57 strains. The area of canker lesions in Y37, Y41 and Y57 was also significantly reduced, and the disease index was significantly decreased after inoculation with Xcc (P<0.01). It indicated that overexpression of csi-miR399 significantly enhanced the resistance to citrus canker.【Conclusion】csi-miR399 is closely related to the resistance of citrus to canker disease. Overexpression of csi-miR399 significantly improves the resistance, which can be applied to the molecular breeding of citrus against canker disease. |
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| Keywords: | csi-miR399 citrus canker target gene over-expression vector based on citrus virus biotic stress |
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