葡萄休眠相关基因DAMs的克隆分析及对单氰胺的响应

Cloning and Analysis of Dormancy Related Genes DAMs and Their Response to Cyanamide in Grapevine

DAM基因是真核生物转录因子之一,在木本果树的芽休眠中起着重要的调控作用,且在不同物种间数量不同。以‘水晶’葡萄为试材,于冬芽休眠期间采集枝条后均匀涂抹2.5%单氰胺,在处理后的不同时间采集冬芽,用于克隆VvDAMs基因序列,对其进行生物信息学和荧光定量分析,并测定冬芽激素含量。结果表明,克隆获得的VvDAM1、VvDAM2和VvDAM3基因的开放阅读框的长度分别为690、696、702 bp,分别编码229、231、233个氨基酸和1个终止密码子,相对分子量分别为25.96、26.76、25.99 kD,理论等电点分别为7.71、10.49、9.10;三者的二级结构形式均呈现α-螺旋>无规则卷曲>延伸链的规律;VvDAM1、VvDAM2、VvDAM3氨基酸序列上有一定的同一性,VvDAM1与欧洲李PsDAM1、VvDAM2与樱桃李PcDAM2、VvDAM3与李PsDAM3之间的同源性均达到98%以上;此外,冬芽休眠解除期间,VvDAM 1、VvDAM 2、VvDAM 3基因的表达量逐渐下降,IAA、GA3+4、ZR的含量和GA3+4/ABA逐渐上升,ABA含量和IAA/ZR逐渐下降,单氰胺处理后使其变化提前且幅度增大,从而使芽提前萌发,为葡萄冬芽破眠、早熟栽培等提供理论依据和实践意义。

DAM gene is one of the transcription factors in eukaryotes,which plays an important role in the regulation of bud dormancy in woody fruit trees,and the number of DAM gene is different among different species.Using'Shuijing'grapevine as the test material,branches were collected during the dormancy period of winter buds.2.5%cyanamide was evenly applied to the branches and buds.Winter buds were collected at different times after treatment,and used to clone VvDAMs gene series.Bio-informatics andfluorescence quantification analysis were performed on them,and the hormone content of winter buds was measured.The results showed that the length of the open reading frame of the cloned VvDAM1,VvDAM2 and VvDAM3 genes was 690,696,702 bp,respectively;encoding 229,231,233 amino acids and a stop codon,with relative molecular weights of 25.96,26.76,25.99 kD,and theoretical isoelectric point of 7.71,10.49 and 9.10,respectively.All three have two levels of structureα-spiral>irregular curl>extended chain.It could be seen that there was a certain degree of identity in the amino acid sequences of VvDAM1,VvDAM2,and VvDAM3.The homology between VvDAM1 of'Shuijing'grapevine and Prunusdomestica PsDAM1,VvDAM2 and Prunuscerasifera PcDAM2,VvDAM3 and Prunussalicina PsDAM3 respectively reaches more than 98%.In addition,during the period of dormancy release of winter buds,the expression levels of VvDAM1,VvDAM2,and VvDAM3 genes gradually decrease with the passage of dormancy release time.The content of IAA,GA3+4,ZR,and GA3+4/ABA gradually increase,while the content of ABA and IAA/ZR gradually decrease.After cyanamide treatment,this change was accelerated and the amplitude increases,leading to early germination of buds,providing theoretical basis and practical significance for grape winter bud dormancy breaking and early maturing cultivation.