刺葡萄VdMAPK7参与炭疽病胁迫响应的功能分析  

Functional analysis of VdMAPK7 from Chinese wild spine grape(Vitis davidii F?ex)in response to Colletotrichum viniferum

作　　者：雷龑[1] 陈婷[1] 刘鑫铭[1] 谢倩[2] 陈清西[2] LEI Yan;CHEN Ting;LIU Xinming;XIE Qian;CHEN Qingxi(Fruit Research Institute,Fujian Academy of Agricultural Sciences,Fuzhou 350013,Fujian,China;College of Horticulture,Fujian Ag-riculture and Forestry University,Fuzhou 350002,Fujian,China)

机构地区：[1]福建省农业科学院果树研究所,福州350013 [2]福建农林大学园艺学院,福州350002

出　　处：《果树学报》2022年第11期2036-2045,共10页Journal of Fruit Science

基　　金：福建省人民政府中国农业科学院农业高质量发展超越“5511”协同创新工程(XTCXGC2021006);福建省农业科学院科技创新团队建设(CXTD2021009-2);财政部和农业农村部:国家现代农业产业技术体系(CARS-29)。

摘　　要：【目的】克隆刺葡萄VdMAPK7基因,并对其参与炭疽病胁迫响应的功能进行分析。【方法】结合前期转录组数据,以刺葡萄福安(Vitis davidii‘Fu’an’)为试材,通过实时荧光定量PCR(quantitative real time PCR,qRT-PCR)分析VdMAPK7基因在炭疽菌侵染后不同时间点表达水平变化,通过聚类分析VdMAPK7蛋白与其他物种相关MAPK蛋白的系统发育关系。利用烟草叶片亚细胞定位技术分析VdMAPK7蛋白在细胞中的位置。在番茄中异源表达VdMAPK7基因后,番茄果实接种尖孢炭疽菌(Colletotrichum acutatum),验证其对炭疽病胁迫的响应。【结果】刺葡萄VdMAPK7基因响应炭疽菌诱导后表达量逐渐升高,在接种第7天达到高峰。VdMAPK7蛋白与欧洲葡萄、番茄、马铃薯、茶树、珙桐、烟草聚为一大类;亚细胞定位发现,VdMAPK7蛋白定位在细胞质和细胞核中;VdMAPK7转基因番茄植株矮于野生型植株,果实变小。转基因番茄果实接种尖孢炭疽菌后,相较于野生型番茄,VdMAPK7基因过表达番茄果实发病较轻;q RT-PCR结果显示,VdMAPK7基因过表达番茄植株中响应水杨酸信号通路的基因SlPR1和SlPR2上调表达。【结论】VdMAPK7基因在番茄中过量表达均可增强对尖孢炭疽菌的抗性,推测VdMAPK7基因参与了葡萄对炭疽菌的胁迫响应。【Objective】Mitogen activated protein kinase(MAPK)cascade pathway,a major signal transduction pathway widely distributed in eukaryotes,has an important function in plant responses to various biotic and abiotic stresses.Transcriptome analysis of Chinese wild spine grape(Vitis davidii F?ex)infected by Colletotrichum viniferum was performed in a previous study.The infected grape fruits at 7 dpi showed significant changes in gene expression,and that the up-regulated genes were enriched for those MAPK cascade,calcium ion binding and serine/threonine kinase.The VdMAPK7,selected from transcriptome data,showed highly up-regulated expression during C.viniferum infection,indicating that the VdMAPK7 was responsible for the resistance to C.viniferum.The main objective of this study was to clone and verify the function of the VdMAPK7 in response to C.viniferum.【Methods】Chinese wild spine grape(V.davidii‘Fu’an’)was used as the experimental materials and the VdMAPK7 was selected based on the transcriptome data for further study.The grape berries were inoculated by C.viniferum(strain FJ017)then the exocarps were collected after inoculation at different infection time points(0 d,1 d,3 d and 7 d)for further analysis.The RNA from C.viniferum infected grape exocarp were extracted by lithium chloride precipitation method and reverse transcription and cDNA synthesis were performed using PrimeScriptTMRT kit with gDNA Eraser.The expression patterns of the VdMAPK7 at different time points after C.viniferum infection were analyzed by qRT-PCR using GAPDH(CB973647)as reference gene with 2-ΔΔCtmethod.CDS sequence of the VdMAPK7 were cloned and the amino acid sequence of the VdMAPK7 was compared based on NCBI database,then other related proteins with high homology were screened.The phylogenetic relationship between the VdMAPK7 protein and other related proteins were analyzed by cluster analysis using Phylogeny.fr platform.The fulllength CDS sequence of the VdMAPK7 was obtained by sequencing,and the amino acid sequence of the Vd

关 键 词：刺葡萄 炭疽病 MAPK基因 

分 类 号：S663.1[农业科学—果树学]