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目的克隆铁皮石斛(Dendrobium officinale)钙依赖蛋白激酶(calcium-dependent protein kinases,CDPKs)基因并进行分子特征分析。方法 利用反转录PCRC(RT-PCR)和CDNA末端快速扩增(RACE)技术,从铁皮石斛叶cDNA中分离CDPKs基因,并进行编码蛋白等电点、分子量、保守结构域、信号肽以及跨膜域等生物信息学分析;采用DNASTAR和MEGA4软件进行氨基酸多序列比对和进化树构建分析;借助实时荧光定量PCR技术检测基因组织表达模式。结果 分离到两个铁皮石斛钙依赖蛋白激酶基因DoCPK2和DoCPK3(GenBank注册号JX219469和JX219470),全长为2 119和2 418 bp,各编码一条由541和536个氨基酸组成的肽链,相对分子质量分别为60.46×103和60.30×103,等电点6.14和6.32;两个基因编码的蛋白不含信号肽,均具有19个氨基酸的跨膜域(分别为248～266和243～261位)和CDPKs蛋白家族保守的丝氨酸/苏氨酸蛋白激酶的结构域及Ca2+结合EF-hand基元。两个基因与植物CDPKs基因同源性很高(67%～81%),与小麦和水稻等单子叶植物CDPKs基因亲缘关系较近;DoCPK2和DoCPK3基因在组织中为组成型表达,前者在根和原球茎中表达量较高,后者在茎和种子中的表达量较低。结论分离到两个铁皮石斛钙依赖蛋白激酶基因DoCPK2和DoCPK3,为进一步揭示其在植物生长发育和逆境胁迫等过程中的生物学功能奠定基础。
Objective To clone the cDNA of Dendrobium officinale calcium-dependent protein kinases (CDPKs) and analyze its molecular characteristics. Methods The cDNA of CDPKs was isolated from cDNA of Dendrobium officinale with reverse transcription PCR (RT-PCR) and rapid amplification of cDNA ends (RACE), and the deduced amino acid sequences of the isoelectric point, molecular weight, conserved domain, Membrane and other bioinformatics analysis; DNASTAR and MEGA4 software for amino acid sequence alignment and evolutionary tree construction analysis; real-time fluorescent quantitative PCR detection of gene expression patterns. Results Two calcium-dependent protein kinase genes DoCPK2 and DoCPK3 (GenBank accession numbers JX219469 and JX219470) were isolated and their full length was 2 119 and 2 418 bp, each encoding a peptide chain consisting of 541 and 536 amino acids. The relative molecular (60.46 × 103 and 60.30 × 103), and isoelectric points (6.14 and 6.32). The two proteins encoded no signal peptide and all had 19 amino acid transmembrane domains (248-266 and 243-261, respectively) And the domain of the conserved serine / threonine protein kinase of the CDPKs family of proteins and the Ca2 + binding EF-hand motif. The two genes are highly homologous to plant CDPKs (67% -81%), closely related to the CDPKs gene of monocotyledonous plants such as wheat and rice. The DoCPK2 and DoCPK3 genes are constitutively expressed in tissues, And protocorm showed higher expression, which was lower in stem and seed. Conclusion Two calcium-dependent protein kinase genes DoCPK2 and DoCPK3 from Dendrobium officinale were isolated and laid the foundation for further revealing their biological functions in plant growth and development and stress tolerance.