目的 克隆铁皮石斛蔗糖非酵解1型相关蛋白激酶2（SnRK2）家族基因，并进行生物信息学和表达分析。方法 通过RT-PCR、RACE克隆铁皮石斛SnRK2（DoSRK2E）基因cDNA全长，利用生物信息学软件预测基因编码蛋白的相对分子质量、等电点、结构域、跨膜结构、信号肽及亚细胞定位等；利用DNASTAR和MEGA进行多序列比对和系统发育关系重建分析；利用实时荧光定量PCR（qRT-PCR）技术检测基因组织表达模式。结果 获得铁皮石斛DoSRK2E基因（GenBank登录号API65110），cDNA全长1 795 bp，包含1个1 086 bp的完整开放阅读框，编码蛋白相对分子质量40 850，等电点4.80，无信号肽和跨膜域，包含1个蛋白激酶结构域、1个ATP结合位点和1个Ser/Thr蛋白激酶活化位点，预测定位在内质网膜上；DoSRK2E蛋白与植物SnRK2蛋白序列高度一致，系统发育位置位于SnRK2亚家族的分支Ⅲ，与拟南芥AtSnRK2.6的亲缘关系最近；DoSRK2E基因在铁皮石斛根中表达量最高，茎中次之，叶中最低。结论 首次从珍稀濒危药用植物铁皮石斛中克隆得到SnRK2家族基因DoSRK2E，对其分子特性与组织表达模式进行了分析研究，为进一步揭示该基因在铁皮石斛逆境胁迫中的作用机制提供基础。

Objective To clone the SnRK2 gene in Dendrobium officinale and investigate its characteristics and expression pattern. Methods RT-PCR and RACE techniques were used to clone the full-length cDNA of DoSRK2E, with the aids of a series of online bioinformatic software, characteristics including molecular mass, isoelectric point, conserved domain, transmembrane structure, signal peptide, and subcellular localization of the deduced protein were analyzed. Besides, the sequence of the deduced protein was aligned with those of other plant SnRK2 by DNASTAR, and phylogenetic relationships were reconstructed utilizing MEGA. Finally, tissue specific expression pattern of DoSRK2E was tested by quantitative real-time PCR (qRT-PCR). Results The full-length cDNA of DoSRK2E (GenBank accession API65110) is 1 795 bp with a 1 086 bp complete open reading frame (ORF). The predicted molecular mass and isoelectric point of the deduced protein DoSRK2E were 40 850 and 4.80, respectively. No signal peptide nor transmembrane region were detected, this protein contains one protein kinase domain, one ATP binding site, and one Ser/Thr active site, which was predicted most likely to be located in the endoplasmic reticulum membrane. DoSRK2E protein showed high similarity with those from other plant SnRK2, and its phylogenetic location was in Group Ⅲ of SnRK2 subfamily, and phylogenetically closest to AtSnRK2.6 from Arabidopsis. In addition, qPCR analysis revealed that DoSRK2E showed the highest expression level in root, followed by stem and leaf. Conclusion A SnRK2 family gene DoSRK2E was cloned from the rare and endangered medicinal plant D. officinale for the first time. The Characteristics and expression pattern of this gene were analyzed. This study will provide a basis for further exploration of the regulation mechanisms of DoSRK2E in D. officinale under stress conditions.

陕西省教育厅科学研究计划项目（18JK0221）；陕西中医药大学2017年博士科研启动经费（104080001）；陕西省高校青年杰出人才支持计划项目；咸阳市中青年科技领军人才项目