csi-miR399响应柑橘溃疡病菌侵染的表达模式及其抗病性分析

doi:10.3864/j.issn.0578-1752.2023.08.005

摘要/Abstract

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-miR399过表达对Xcc抗性的影响。【结果】接种Xcc后，csi-miR399在抗病品种四季橘中的表达呈现先下降再上升的趋势，而在感病品种纽荷尔脐橙中的表达呈持续下降趋势。接种Xcc 5 d时，csi-miR399在四季橘与纽荷尔脐橙中的表达量分别是健康对照的4.64倍和7.61%，初步表明csi-miR399与柑橘的溃疡病抗性相关。从13个预测靶基因中筛选鉴定了csi-miR399的3个靶基因Cs2g06030、Cs7g03830、Cs8g18800，分别编码泛素偶联酶PHO2、未知蛋白和漆酶。利用构建的病毒表达载体pCLBV202-MIR399获得过表达csi-miR399的柠檬植株（Y37、Y41和Y57），与空载体对照pCLBV202接种植株（L35）相比，Y37、Y41和Y57中csi-miR399表达量显著增加，接种Xcc后的溃疡病病斑面积显著减小，病情指数显著降低（P<0.01），表明csi-miR399过表达显著提高了柠檬的溃疡病抗性。【结论】csi-miR399与柑橘的溃疡病抗性密切相关，过表达csi-miR399显著提高柑橘对溃疡病的抗性，可应用于柑橘抗溃疡病的分子育种。

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

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.

Key words: csi-miR399, citrus canker, target gene, over-expression vector based on citrus virus, biotic stress

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.

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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引物名称 Primer name	核苷酸序列 Nucleotide sequence (5′-3′)
csi-Actin-F	CATCCCTCAGCACCTTCC
csi-Actin-R	CCAACCTTAGCACTTCTCC
miR399-RT	CTCAACTGGTGTCGTGGAGTCCGGCAATTCAGTTGAGCAGGGCAA
miR399-F	ACACTCCAGCTGGGTGCCAAAGGA
Universal-miR	AACTGGTGTCGTGGAG
Cs2g02870F	AATGCCGCTATTTCTGTCCCA
Cs2g02870R	GCTCCTCAGCCAGGTTTGTT
Cs2g06030F	GAGCTTGTGGAGGAGCACTT
Cs2g06030R	CCCACTGAACATCCGTTGGA
Cs2g14510F	AGGAATTCAGGCAGCCCATC
Cs2g14510R	CGACTTGGCTTCTTCCCCTT
Cs3g09820F	CGGCAACTTGAGCAAAGCAT
Cs3g09820R	GAACGCTTGGAAGGCTCAAC
Cs4g04510F	TCAAATCCCCAAGCCTTTGC
Cs4g04510R	AACAGAACAAGTCAGCGAGT
Cs7g03830F	ATAGCCGGTGTGCTCATTGT
Cs7g03830R	TCCCAGTACACTCGCAAGGA
Cs7g08000F	TATTCCGGTGCGTGTGATCG
Cs7g08000R	ATTTGACACAACCGCAAGGC
Cs7g22930F	AGAGGTGGGCCTCAATTTGG
Cs7g22930R	AGCTTGCCTTAGGTCACCAAT
Cs8g05410F	ACCACCTGATGCTCCCCTTA
Cs8g05410R	TGATCCGACGGCAGATAGTG
Cs8g18800F	ACACAGCCGATTTCCCACAA
Cs8g18800R	ATGAAGAGGGTGGCTTTCGG
1.1g025013mF	CGTCAAACTCCAACGTGCTT
1.1g025013mR	ATCATACCCCTGGCGAAGTC
1.1t01536F	GGTCTTGCAACCAGGGATCA
1.1t01536R	ACTGCCTGTGATGATCTGCC
1.1t02010F	CCACCTGGATCTAAGGGCAA
1.1t02010R	CATTTGCTGGAAAAGCGCCA

引物名称 Primer name	核苷酸序列 Nucleotide sequence (5′-3′)
pLGN-MIR399-F	GGGTACCCGGGGATCAAAGCAGTTTTAGGGCACCTCTT
pLGN-MIR399-R	TTAAAGCAGGGAATTGAAGCAGTCACAGGGCAACTCT
pCLBV202-MIR399-F	AGATTGAGAAAACCCAAAGCAGTTTTAGGGCACC
pCLBV202-MIR399-R	CAGAATTCGGGACCCGAAGCAGTCACAGGGCAAC
pClbv-s	CTGATGGGAGCGTTAGACTGA
pClbv-r	TGTAAAGTCCTGGCCCACC