中文摘要：

目的克隆铁皮石斛（Dendrobium officinale）钙依赖蛋白激酶（calcium—dependentproteinkinases，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2119和2418bp，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60．46×10^3和60．30×10^3，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Ca^2＋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．

英文摘要：

OBJECTIVE To clone and characterize calcium-dependent protein kinases （CDPKs） genes in Dendrobium officinale Kimura et Migo. METHODS Reverse transcription PCR （RT-PCR） and rapid-amplification of cDNA ends （RACE） methods were used to isolate CDPKs genes from the leaf eDNA of D. officinale. Characteristics ineluding the molecular weight, theoretical pI （ isoelectric point） , conserved domain, transmembrane structure, signal peptide, and subcellular localization of the deduced proteins were analyzed using serials of bioinformatics algorithms. The analyses of multiple alignment and phylogenetic tree were respectively performed using DNASTAR and MEGA4. Tissue specific expression patterns were determined using real-time quantitative PCR （qPCR） analyses. RESULTS Two full length genes DoCPK2 and DoCPK3 （ GenBank accessions JX219469 and JX219470 ） , 2 119 and 2 418 bp in length, respectively, were obtained. DoCPK2 was deduced to a 541 aa （ amino acid） protein with a molecular weight of 60. 46 ×103 and a pI of 6. 14, while DoCPK3 encoded a 536 aa protein with a molecular weight of 60. 30 × 103 and a pI of 6. 32. The two deduced proteins, without signal peptide, both contained the conserved caniocal serine/threonineprotein kinase catalytic domain, Ca2＋ binding EF hand motifs, and a 19 aa transmembrane structure at 248 -266 and 243 -261 aa position, respectively. They were highly homologues （67% -81% ） to the plant CDPKs genes, and were mostly close to monocots CDPKs genes from wheat and rice. DoCPK2 and DoCPK3 were constitutively expressed among the five tissues. DoCPK2 transcripts were more abundant in the roots and PLBs （ protocorm-like bodies） , while the transcription levels of DoCPK3 were repressed in the seeds and stems. CONCLUSION Two calciumdependent kinases genes DoCPK2 and DoCPK3 are identified from the valuable herb D. officinale, which will be useful for further functional determination of the genes involving in the growth, development, biotic and abiotic responses in D. o