茶树CsWAK8克隆及其在响应冷胁迫过程中的功能分析  

Cloning and Functional Analysis of CsWAK8 Gene from Camellia sinensis during Cold Stress

作　　者：焦小雨 吴琼 刘丹丹 孙明慧 阮旭 王雷刚 王文杰 JIAO Xiao-yu;WU Qiong;LIU Dan-dan;SUN Ming-hui;RUAN Xu;WANG Lei-gang;WANG Wen-jie(Tea Research Institute,Anhui Academy of Agricultural Sciences,Hefei 230001)

机构地区：[1]安徽省农业科学院茶叶研究所,合肥230001

出　　处：《生物技术通报》2025年第2期210-220,共11页Biotechnology Bulletin

基　　金：国家自然科学基金联合基金项目(U23A20213);安徽省茶叶产业技术体系(AHCYJSTX-11);安徽省农业科学院成果转化项目(2024YL041)。

摘　　要：【目的】细胞壁关联蛋白激酶(wall associated kinase,WAK)是一类特殊的类受体激酶(receptor like kinase,RLK),其在调节植物生长和应对生物或非生物胁迫等方面发挥着重要作用。探究CsWAK8在响应冷胁迫过程中的功能,为今后解析茶树抗寒机理提供理论依据。【方法】从茶树叶片中克隆了CsWAK8。采用实时荧光定量PCR(qPCR)分析CsWAK8在不同组织以及越冬期不同抗寒性茶树品系中的表达模式。通过农杆菌介导法在拟南芥中异源表达CsWAK8,并对转基因植株进行冷处理表型观察、酶活测定和冷响应相关基因表达检测。【结果】CsWAK8的CDS全长2307 bp,编码768个氨基酸,具有WAK家族特征保守结构域。CsWAK8在茶树成熟叶片中高表达,在越冬期,冷敏感型茶树品系叶片和根中CsWAK8表达量多显著高于冷耐受型茶树品系。通过异源过表达获得了9株转CsWAK8拟南芥纯合株系,对其中3个株系进行了耐冷性分析,发现在冷胁迫下,转基因株系的根长和存活率显著低于野生型,盆栽苗莲座叶的枯死程度较野生型更高,且转基因株系L48在冷冻处理6 h时的MDA含量显著高于野生型。此外,q PCR分析结果显示,在冷胁迫下,转CsWAK8拟南芥中AtCBFs的相对表达量大多显著低于野生型。【结论】转CsWAK8拟南芥相较于野生型拟南芥对冷处理更敏感。CsWAK8可能通过CBF介导的冷信号途径在响应和耐受冷胁迫过程中起负调节作用。【Objective】The wall-associated kinase(WAK)is a unique class of receptor-like kinase(RLK)that plays an important role in regulating plant growth and responding to both biotic and abiotic stresses.Exploring the function of CsWAK8 in responding to cold stress may provide a theoretical basis for further analysis of the cold resistance mechanisms in Camellia sinensis.【Method】The CsWAK8 gene was cloned from the leaves of C.sinensis.The quantitative real-time PCR method was used to analyze the expression pattern of the CsWAK8 gene in different tissues and different cold-resistant tea tree varieties during the wintering period.An agrobacterium-mediated method was used to heterologously express the CsWAK8 gene in Arabidopsis thaliana.The cold-treated phenotypic observation,enzyme activity determination,and cold-response-related gene expression detection of transgenic plants were also carried out.【Result】The CDS of CsWAK8 gene is 2307 bp,and encodes a protein of 768 amino acids,which contained a conserved domain unique to the WAK family.CsWAK8 was highly expressed in the mature leaves of C.sinensis,and its expression in the leaves and roots of cold-sensitive tea tree varieties was significantly higher than that in cold-tolerant ones during the wintering period.Nine transgenic lines of A.thaliana were acquired through the heterologous over-expressions of the CsWAK8 gene,and three of these lines were analyzed for their cold resistances.Under cold stress,the root lengths and survival rates of the transgenic lines were significantly lower than those of the wild type.Additionally,the degree of wilting in the rosette leaves of potted seedlings was higher in the transgenic lines compared to the wild type,and the MDA content in the transgenic variety L48 was significantly higher than that of the wild type after 6 h of freezing treatment.Furthermore,qPCR analysis revealed that the relative expressions of AtCBFs in the CsWAK8 gene transgenic A.thaliana under cold treatment were significantly lower than those in the wild

关 键 词：茶树 细胞壁关联蛋白激酶(WAK) 基因克隆 功能验证 冷胁迫 

分 类 号：S57[农业科学—作物学]