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钙依赖蛋白激酶(calcium-dependent protein kinase,CDPK)是一类能响应低温等非生物胁迫的蛋白激酶,目前已从拟南芥(Arabidopsis thaliana)、水稻(Oryza sativa)和杨树(Populus trichocarpa)等植物中分离出多个CDPK基因。为了研究铁皮石斛(Dendrobium officinale)钙依赖蛋白激酶的结构和功能,探寻其在逆境胁迫下的作用,从铁皮石斛中分离出1个DoCDPK6基因(Gen Bank No.KY682702)及其启动子,通过生物信息学分析序列特征、结构及进化关系,结果表明,DoCDPK6基因的cDNA全长1 976 bp,开放阅读框1 725 bp,编码574个氨基酸,分子式为C2845H4500N766U858S24,分子量为63.9 k D;具有CDPK典型的保守结构:可变区、蛋白激酶区、连接区和钙调控区;进化分析表明,DoCDPK6与同科植物蝴蝶兰(Phalaenopsis amabilis)亲缘关系最近,符合植物学分类地位;在DoCDPK6基因1 500 bp的上游序列含有核心启动子元件TAAT-box和CAAT-box,并含有光、温、水杨酸、脱落酸、生长素和赤霉素等逆境信号相关的响应元件;通过q RT-PCR发现,铁皮石斛DoCDPK6主要在叶片中表达,其次是茎和根;在4℃低温、脱落酸(abscisic acid,ABA)和Na Cl胁迫处理后,DoCDPK6基因表达量均有不同程度的上调,因此可以推测,DoCDPK6基因在低温等非生物胁迫中起到调控作用,该研究结果可为DoCDPK6基因及其启动子在非生物胁迫中的调控机制和信号传导途经提供信息。
The expression of calcium-dependent protein kinase (CDPK), a type of protein kinase that can respond to abiotic stresses such as low temperature, has been isolated from Arabidopsis thaliana, Oryza sativa and Populus trichocarpa And other plants isolated a number of CDPK genes. In order to study the structure and function of Dendrobium officinale calcium-dependent protein kinase and to explore its role under stress, a DoCDPK6 gene (Gen Bank No. KY682702) and its promoter were isolated from Dendrobium candidum. The results showed that the cDNA of DoCDPK6 gene was 1 976 bp in length and 1 725 bp in open reading frame encoding 574 amino acids with molecular formula of C2845H4500N766U858S24 and molecular weight of 63.9 kD. Conserved structure: variable region, protein kinase region, junction region and calcium regulatory region. Phylogenetic analysis showed that DoCDPK6 was closest to the Phalaenopsis amabilis family, The upstream sequence contains the core promoter elements TAAT-box and CAAT-box, and contains response elements related to the adversity signal such as light, temperature, salicylic acid, abscisic acid, ghrelin and gibberellin. By q RT-PCR, Dendrobium DoCDPK6 was mainly expressed in leaves, followed by stems and roots. DoCDPK6 gene expression levels were both lower at 4 ℃, abscisic acid (ABA) and NaCl stress Degrees of increase, it can be speculated that, DoCDPK6 gene play a regulatory role in abiotic stresses such as low temperature, the results can DoCDPK6 gene and its promoter and regulatory mechanisms in abiotic stress signal transduction via the provided information.