查尔酮合成酶基因在葡萄抗灰霉病和霜霉病中的作用

doi:10.3864/j.issn.0578-1752.2022.06.007

摘要/Abstract

摘要：

【目的】克隆不同抗性葡萄品种中查尔酮合成酶（chalcone synthase）基因CHS1,明确该基因在葡萄灰霉病菌（Botrytis cinerea）和葡萄霜霉病菌（Plasmopara viticola）侵染下的表达模式,并对CHS1进行亚细胞定位和功能验证,为探究CHS1的广谱抗病作用机理提供依据。【方法】分别以毛葡萄（Vitis quinquangularis）‘野酿二号’、欧亚种（Vitis vinifera）‘无核白’和山葡萄(Vitis amurensis）‘双红’叶片cDNA为模板,克隆CHS1,分析不同品种中CHS1的序列差异,并对CHS1蛋白的理化性质进行生物信息学分析;利用qRT-PCR检测不同葡萄品种受到灰霉病菌和霜霉病菌侵染后CHS1的表达模式;构建表达载体,利用农杆菌介导的瞬时表达技术,分别在烟草和‘无核白’组培苗叶片中进行亚细胞定位和抗病功能验证。【结果】CHS1在不同葡萄品种中高度保守,ORF全长均为1 182 bp,编码393个氨基酸,预测编码的蛋白分子量为42.92 kD,为稳定的亲水蛋白。受灰霉病菌侵染后,毛葡萄VqCHS1和欧亚种VvCHS1的表达模式变化类似,但灰霉病抗性品种‘野酿二号’中VqCHS1的表达水平整体显著偏高;受霜霉病菌侵染后,山葡萄VaCHS1和欧亚种VvCHS1表现出不同的表达模式变化,霜霉病抗性品种‘双红’中VaCHS1的表达量不断增加,而VvCHS1的表达量逐渐下降。亚细胞定位结果表明,CHS1蛋白在细胞膜质、细胞核中都有分布,但以细胞核定位为主。人工接种灰霉病菌和霜霉病菌后,与阴性对照相比,过表达VaCHS1的‘无核白’叶片具有更强的灰霉病和霜霉病抗性。【结论】抗性品种中查尔酮合成酶基因CHS1的表达量比感病品种高,过表达CHS1能够提高感病葡萄品种对灰霉病和霜霉病的抗性。

关键词: 查尔酮合成酶, 葡萄, 灰霉病菌, 霜霉病菌, 抗病性

Abstract:

【Objective】The objective of this study is to clone the chalcone synthase gene CHS1 in different resistant grape varieties, clarify the expression pattern under the infection of Botrytis cinerea and Plasmopara viticola, verify the subcellular localization and function of CHS1, and to provide a basis for exploring the broad-spectrum resistance mechanism of CHS1.【Method】CHS1 genes were cloned from the leaf cDNAs of Vitis quinquangularis ‘Yeniang-2’, Vitis vinifera ‘Thompson Seedless’ and Vitis amurensis ‘Shuanghong’. The differences in the nucleic acid sequence and physicochemical properties of CHS1 protein were analyzed by bioinformatics. Expression patterns of CHS1 in different varieties during B. cinerea and P. viticola infection were also detected by qRT-PCR. The expression vector was constructed, and the transient expression technology mediated by Agrobacterium was used to verify the subcellular localization and disease resistance function in the leaves of tobacco and ‘Thompson Seedless’ tissue culture seedlings.【Result】The CHS1 is highly conserved among different grape varieties. The full length of ORF is 1 182 bp, which encodes 393 amino acids. The predicted molecular weight of the encoded protein is 42.92 kD, which is a stable hydrophilic protein. After being infected by B. cinerea, the expression patterns of VqCHS1 and VvCHS1 were similar, but the expression level of VqCHS1 in the resistant variety ‘Yejiang-2’ was significantly higher. After being infected by P. viticola, VaCHS1 and VvCHS1 showed different expression pattern changes. The expression level of VaCHS1 in the resistant variety ‘Shuanghong’ continued to increase, while the expression level of VvCHS1 gradually decreased. The results of subcellular localization showed that CHS1 protein is distributed in the cytoplasm and nucleus, but it is mainly localized in the nucleus. Compared with the negative control, the ‘Thompson Seedless’ leaves overexpressing VaCHS1 showed stronger resistance to gray mold and downy mildew after artificial inoculation. 【Conclusion】The expression level of CHS1 in resistant varieties is higher than that in susceptible varieties, and overexpression of CHS1 can increase the resistance of susceptible grape varieties to gray mold and downy mildew.

Key words: chalcone synthase (CHS), grape, Botrytis cinerea, Plasmopara viticola, disease resistance

郭泽西,孙大运,曲俊杰,潘凤英,刘露露,尹玲. 查尔酮合成酶基因在葡萄抗灰霉病和霜霉病中的作用[J]. 中国农业科学, 2022, 55(6): 1139-1148.

GUO ZeXi,SUN DaYun,QU JunJie,PAN FengYing,LIU LuLu,YIN Ling. The Role of Chalcone Synthase Gene in Grape Resistance to Gray Mold and Downy Mildew[J]. Scientia Agricultura Sinica, 2022, 55(6): 1139-1148.

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参考文献 28

[1]	王佳媛. 海南龙血树查尔酮合酶基因(DcCHS)的克隆及其启动子功能分析[D]. 海口: 海南大学, 2014.
	WANG J Y. Cloning of chalcone synthase gene from Dracaena cambodiana and functional identification of its promoter[D]. Haikou: Hainan University, 2014. (in Chinese)
[2]	王志彬. 柑橘查尔酮合成酶基因遗传多样性及其功能研究[D]. 重庆: 西南大学, 2019.
	WANG Z B. Genetic diversity and functional study of chalcone synthase gene in citrus[D]. Chongqing: Southwest University, 2019. (in Chinese)
[3]	ZHAO C Y, WANG F, LIAN Y H, XIAO H, ZHENG J K. Biosynthesis of citrus flavonoids and their health effects. Critical Reviews in Food Science and Nutrition, 2020,60(4):566-583. doi: 10.1080/10408398.2018.1544885
[4]	ZHANG Y, DOU H T, LI H J, HE Z F, WU H J. The citrus flavonoid nobiletin inhibits proliferation and induces apoptosis in human pancreatic cancer cells in vitro. Food Science and Biotechnology, 2014,23(1):225-229. doi: 10.1007/s10068-014-0031-6
[5]	TERASHIMA M, KAKUNO Y, KITANO N, MATSUOKA C, MURASE M, TOGO N, WATANABE R, MATSUMURA S. Antioxidant activity of flavonoids evaluated with myoglobin method. Plant Cell Reports, 2012,31(2):291-298. doi: 10.1007/s00299-011-1163-2
[6]	KROL P, IGIELSKI R, POLLMANN S, KEPCZYNSKA E. Priming of seeds with methyl jasmonate induced resistance to hemi-biotroph Fusarium oxysporum f. sp. lycopersici in tomato via 12-oxo- phytodienoic acid, salicylic acid, and flavonol accumulation. Journal of Plant Physiology, 2015,179:122-132. doi: 10.1016/j.jplph.2015.01.018
[7]	ZUK M, DZIATO M, RICHTER D, DYMINSKA L, MATULA J, KOTECKI A, HANUZA J, SZOPA J. Chalcone synthase (CHS) gene suppression in flax leads to changes in wall synthesis and sensing gene, cell wall chemistry and stem morphology parameter. Frontiers in Plant Science, 2016,7:894.
[8]	薛保国, 谢丽华, 杨丽荣, 孙润红, 全鑫, 杨艳艳, 孙虎, 雷振生, 赵献林. 小麦查尔酮合成酶基因及其启动子序列的克隆与分析. 植物病理学报, 2017,47(1):50-60.
	XUE B G, XIE L H, YANG L R, SUN R H, QUAN X, YANG Y Y, SUN H, LEI Z S, ZHAO X L. Cloning and functional analysis of TaCHS and its promoter in wheat. Acta Phytopathologica Sinica, 2017,47(1):50-60. (in Chinese)
[9]	陈帅. 烟草类黄酮代谢途径中关键酶CHS基因与R2R3 MYB类转录抑制因子功能研究[D]. 成都: 四川农业大学, 2017.
	CHEN S. Functional analysis of CHS genes and R2R3 MYB repressors related to flavonoid biosynthesis pathway in Nicotiana tabacum[D]. Chengdu: Sichuan Agricultural University, 2017. (in Chinese)
[10]	LU Y F, CHEN Q, BU Y F, LUO R, HAO S X, ZHANG J, TIAN J, YAO Y C. Flavonoid accumulation plays an important role in the rust resistance of Malus plant leaves. Frontiers in Plant Science, 2017,8:1286. doi: 10.3389/fpls.2017.01286
[11]	梁先利, 张丽清, 柯丽萍, 孙玉强. 彩色棉查尔酮合成酶基因GhCHS1的克隆和功能分析. 农业生物技术学报, 2019,27(6):993-1002.
	LIANG X L, ZHANG L Q, KE L P, SUN Y Q. Cloning of chalcone synthase gene GhCHS1 and its function analysis in coloured cotton (Gossypium hirsutum). Journal of Agricultural Biotechnology, 2019,27(6):993-1002. (in Chinese)
[12]	DAO T T, LINTHORST H J M, VERPOORTE R. Chalcone synthase and its functions in plant resistance. Phytochemistry Reviews, 2011,10(3):397-412. doi: 10.1007/s11101-011-9211-7
[13]	LEI K J, ZHANG L, DU X Y, AN Y, CHANG G H, AN G Y. A chalcone synthase controls the verticillium disease resistance response in both Arabidopsis thaliana and cotton. European Journal of Plant Pathology, 2018,152:769-781. doi: 10.1007/s10658-018-1521-9
[14]	尹玲. 葡萄霜霉病抗性基因MrRPV1结构域及霜霉菌效应分子功能研究[D]. 北京: 中国农业大学, 2015.
	YIN L. Functional analysis of domains of grape downy mildew resistance gene MrRPV1 and effectors of Plasmopara viticola[D]. Beijing: China Agricultural University, 2015. (in Chinese)
[15]	BASKER V, VENKATESH R, RAMALINGAM S. Flavonoids (antioxidants systems) in higher plants and their response to stresses//Antioxidants and Antioxidant Enzymes in Higher Plants. Springer, Cham, 2018: 253-268.
[16]	YU H N, WANG L, SUN B, GAO S, CHENG A X, LOU H X. Functional characterization of a chalcone synthase from the liverwort Plagiochasma appendiculatum. Plant Cell Reports, 2015,34(2):233-245. doi: 10.1007/s00299-014-1702-8
[17]	何春艳, 甘露, 闫蒙举, 张兰, 苏浩天, 尹淑霞. 草地早熟禾查尔酮合成酶基因PpCHS1克隆、功能与表达分析. 中国草地学报, 2018,40(4):8-15.
	HE C Y, GAN L, YAN M J, ZHANG L, SU H T, YIN S X. Cloning, functional and expression analysis of chalcone synthase gene PpCHS1 in Kentucky bluegrass. Chinese Journal of Grassland, 2018,40(4):8-15. (in Chinese)
[18]	许明, 林世强, 倪冬昕, 伊恒杰, 刘江洪, 杨志坚, 郑金贵. 藤茶查尔酮合成酶基因AgCHS1的克隆及功能鉴定. 中国农业科学, 2020,53(24):5091-5103.
	XU M, LIN S Q, NI D X, YIN H J, LIU J H, YANG Z J, ZHENG J G. Cloning and function characterization of chalcone synthase gene AgCHS1 in Ampelopsis grossedentata. Scientia Agricultura Sinica, 2020,53(24):5091-5103. (in Chinese)
[19]	周军, 陶建敏, 彭日荷, 熊爱生, 蔡斌, 徐锦涛, 金晓芬, 张斌, 高峰, 高建杰, 章镇, 姚泉洪. 巨峰葡萄查尔酮合成酶基因4的克隆及表达特性的RT-PCR分析. 南京农业大学学报, 2010,33(2):39-44.
	ZHOU J, TAO J M, PENG R H, XIONG A S, CAI B, XU J T, JIN X F, ZHANG B, GAO F, GAO J J, ZHANG Z, YAO Q H. Cloning and expression analysis of CHS4 of Kyoho grape by RT-PCR. Journal of Nanjing Agricultural University, 2010,33(2):39-44. (in Chinese)
[20]	MA W L, WU Y, WU M, REN Z M, ZHONG Y. Cloning, characterization and expression of chalcone synthase from medicinal plant Rhus chinensis. Journal of Plant Biochemistry and Biotechnology, 2015,24(1):18-24. doi: 10.1007/s13562-013-0231-9
[21]	LI L L, CHENG H, YUAN H H, XU F, CHENG S Y, CAO F L. Functional characterization of the Ginkgo biloba chalcone synthase gene promoter in transgenic tobacco. Genetics and Molecular Research, 2014,13(2):3446-3460. doi: 10.4238/2014.April.30.6
[22]	WANG J Y, DAI H F, LI H L, GUO D, MEI W L, PENG S Q. Molecular and functional characterization of the chalcone synthase gene (DcCHS1) promoter in response to hormones. Plant Omics Journal, 2015,8(5):398-404.
[23]	CHEN L J, GUO H M, LIN Y, CHENG H M. Chalcone synthase EaCHS1 from Eupatorium adenophorum functions in salt stress tolerance in tobacco. Plant Cell Reports, 2015,34(5):885-894. doi: 10.1007/s00299-015-1751-7
[24]	VANNOZZI A, DRY I B, FASOLI M, ZENONI S, LUCCHIN M. Genome-wide analysis of the grapevine stilbene synthase multigenic family: Genomic organization and expression profiles upon biotic and abiotic stresses. BMC Plant Biology, 2012,12:130. doi: 10.1186/1471-2229-12-130
[25]	CIAFFI M, PAOLACCI A R, PAOLACCI M, ALICANDRI E, BIGINI V, BADIANI M, MUGANU M. Transcriptional regulation of stilbene synthases in grapevine germplasm differentially susceptible to downy mildew. BMC Plant Biology, 2019,19:404. doi: 10.1186/s12870-019-2014-5
[26]	QIAO Y, MENG X J, JIN X X, DING G H. Genes expression of key enzymes in phenylpropanes metabolism pathway in cucumber with RT-PCR. Advanced Materials Research, 2013,746:53-57. doi: 10.4028/www.scientific.net/AMR.746
[27]	KORTEKAMP A. Expression analysis of defence-related genes in grapevine leaves after inoculation with a host and a non-host pathogen. Plant Physiology and Biochemistry, 2006,44(1):58-67. doi: 10.1016/j.plaphy.2006.01.008
[28]	黄启秀. 海岛棉类黄酮代谢途径中与枯萎病抗性相关基因的克隆及功能验证[D]. 乌鲁木齐: 新疆农业大学, 2017.
	HUANG Q X. Cloning and functional verification of genes related to Fusarium wilt resistance in flavonoid metabolism pathway of island cotton (Gossypium baradense L.)[D]. Urumqi: Xinjiang Agricultural University, 2017. (in Chinese)

引物名称 Primer name	序列Primer sequence (5′-3′)	用途Purpose
CHS1-F	ATGGTGTCAGTGGGGGAAAT	基因扩增 Gene amplification
CHS1-R	TGCTACACAATCGACTCAC	基因扩增 Gene amplification
CHS1-qF	CGTTCTGAGCGAGTATGGGA	实时荧光定量PCR Real-time fluorescence quantitative PCR
CHS1-qR	TGTGGTGCCCTTTCCTTCTT	实时荧光定量PCR Real-time fluorescence quantitative PCR
EF-1α-F	AATTTTGACCAAGATCGACAGG	葡萄内参基因 Reference gene in grape
EF-1α-R	CAGCAACAGTTTGACGCATG	葡萄内参基因 Reference gene in grape
CHS1-2F	GCTCTAGAATGGTGTCAGTGGGGGAAATC	亚细胞定位 Subcellular localization
CHS1-2R	GCCCCGGGATGTGAGTCGATTGTGTAGCA	亚细胞定位 Subcellular localization