山葡萄COR413家族基因克隆及其参与低温胁迫的表达分
析
丁小玲;张宁波;焦淑珍;申威;史文婷;徐伟荣
【期刊名称】《农业生物技术学报》 【年(卷),期】2017(025)003
【摘要】冷适应蛋白(cold-regulated,COR413)基因作为植物特有的一类低温胁迫响应功能基因,在抗寒过程中发挥重要作用.为了鉴定葡萄属(Vitis)植物COR413基因参与低温胁迫响应的功能特点,本研究基于欧洲葡萄基因组数据库挖掘,采用同源克隆法获得了抗寒种质山葡萄(Vitis amurensis) COR413基因家族成员的4条序列,分别命名为VaCOR413-PM2A、-PM1X1、-IM1与-PM2B.经序列分析表明,所克隆的4条山葡萄COR413基因家族成员与欧洲葡萄相应基因的核苷酸与氨基酸序列高度同源,其核苷酸序列的差异仅为个别单核苷酸的替代.所克隆的4个山葡萄冷适应蛋白家族基因均含有WCOR413保守结构域、跨膜结构域与丝/苏/酪氨酸磷酸化位点,其中仅VaCOR413-PM1X1含有一个预测的N端信号肽序列,仅VaCOR413-PM2B包含1个糖基化磷脂酰肌醇锚点.源于2个葡萄种质中COR413基因家族成员可以聚类为2个明显的簇,COR413-PM2A,-PM1X1与-PM2B聚类在一簇,而COR413-IM1分布在另外一簇.半定量RT-PCR组织特异性检测结果表明,VaCOR413-PM2A、-PM1X1、-IM1与-PM2B在山葡萄不同组织和器官中表达存在差异,且不同于欧洲葡萄相应基因的表达模式;其中山葡萄VaCOR413-PM2A、-PM1X1、-IM1与-PM2B在所有检测的组织中均表达且强度不同,VaCOR413-PM2A与-PM1X1在嫩梢、卷须、花絮以及幼叶中表达量较高.在低温胁迫条件下,山葡萄
COR413基因家族4个成员参与低温胁迫的表达模式分为2类,而欧洲葡萄中相应成员有3种模式;山葡萄VaCOR413-IM1受低温胁迫24h表达量最高,为对照的9.57倍,而欧洲葡萄VvCOR413-IM1在低温胁迫3h后的表达量较高,约为对照的3.65倍.低温胁迫下的表达数据表明,山葡萄COR413基因家族成员中聚类为一簇的VaCOR413-PM2A,-PM1X1与-PM2B响应低温总体呈下调表达,其表达幅度均低于欧洲葡萄相应基因的表达量,而聚类在另外一簇的VaCOR413-IM1对低温胁迫(24 h)响应诱导表达显著;研究结果为探明该基因家族成员在葡萄响应低温胁迫过程中的可能功能和作用机制提供了参考依据,同时为葡萄属植物抗寒(冻)性分子改良提供了理论依据.%The COR413 (cold-regulated) gene,which encodes a plant-specific cold responsive protein,plays a critical role in cold acclimation.To determine the functional characterization of COR413 genes in response to cold stress,integrated grapevine (Vitis vinifera) genomic approaches of data mining and homologous cloning were used to obtain a total of 4 COR413
gene
family
members
from
cold-tolerant
Vitis
amurensis,designated as VaCOR413-PM2A、-PM1X1、-IM1 and-PM2B,respectively.Sequence analysis revealed that the 4 COR413 family members from V.amurensis showed significant nucleotide (97.2％～100％) and amino acid (99.5％～100％) similarity with those from V.vinifera,and the nucleotide differences were several single nucleotide substitutions.All COR413 proteins from V.amurensis contained the conserved domain WCOR413,several transmembrane motifs and
Serine/Threonine/Tyrosine phosphorylation sties,where a predicted N-terminal signal peptide was only found in VaCOR413-PM1X1 and a glycosylphosphatidylinositol (GPI) anchor was only observed in VaCOR413-PM2B.The 4 members of COR413 from 2 grapevine species can be grouped into 2 distinct clusters,where COR413-PM2A,-PM1X1 and-PM2B were grouped together and-IM1 fall into another cluster.Moreover,tissue-specific expression profile of COR413 gene family members and their responses to cold stress were analyzed in both V.amurensis and V.vinifera cv.Cabernet Sauvignon.The results showed that VaCOR413-PM2A,-PM1X1,-IM1 and-PM2B displayed different tissue-specific features that were also not similar to those from V.vinifera.Compared to the corresponding gene expression profiles in V.vinifera,VaCOR413-PM2A,-PM1X1,-IM1 and-PM2B were detected in all tissues of V.amurensis with different expression intensities,among which VaCOR413-PM2A and-PM1X1 showed a relatively high expression in shoots,tendrils,flowers and young leaves,whereas no signals of VvCOR413-PM1X1 and-IM1 were detected in V.vinifera.Analysis of the gene expression profiles during the time-course of cold stress demonstrated the existence of 2 groups with different expression profiles in V.amurensis and 3 groups in V.vinifera.In one group of V.amurensis containing VaCOR413-PM2A,-PM2B and-PM1X1,a similar expression pattern was observed in with a decreased tendency
山葡萄COR413家族基因克隆及其参与低温胁迫的表达分析
