大花杓兰钙依赖蛋白激酶基因克隆及植物表达载体构建  

Cloning and Expressing Vector Construction of Calcium-Dependent Protein Kinase Gene in Cypripedium macranthum

作　　者：付亚娟[1,2] 侯荟玲 乔洁 耿晓进[1] 王聪艳 侯晓强 FU Ya-juan;HOU Hui-ling;QIAO Jie;GENG Xiao-jin;WANG Cong-yan;HOU Xiao-qiang(College of Life Science,Langfang Normal University,Langfang Hebei 065000;Technical Innovation Center for Utilization of Edible and Medicinal Fungi Resources in Hebei Province,Langfang 065000)

机构地区：[1]廊坊师范学院生命科学学院,河北廊坊065000 [2]河北省食药用菌资源高值利用技术创新中心,廊坊065000

出　　处：《植物遗传资源学报》2019年第6期1613-1620,共8页Journal of Plant Genetic Resources

基　　金：河北省教育厅青年拔尖人才项目(BJ2016045);国家自然科学基金(31100314);廊坊师范学院博士基金项目(LSLB201405);河北省高等学校遗传学重点发展学科项目(201221)~~

摘　　要：RT-PCR结合RACE技术,克隆到1个全长2079 bp的大花杓兰钙依赖蛋白激酶基因CmCDPK,cDNA为1491 bp,编码496个氨基酸。CmCDPK是1个具有CDPKs典型的Ser/Thr蛋白激酶保守结构域、含1个跨膜结构域、无信号肽、稳定的亲水性蛋白。CmCDPK二级结构主要由α-螺旋和无规卷曲构成。相对于其他植物CDPKs,CmCDPK与小兰屿蝴蝶兰和铁皮石斛的亲缘关系更近。通过DNA重组技术将CmCDPK片段克隆到pBI121质粒上。PCR、酶切及DNA测序的结果表明,重组质粒pBI-CmCDPK包含1个1491 bp的CmCDPK片段,且核苷酸序列及插入方向完全正确。本研究首次克隆了大花杓兰CmCDPK基因,并成功构建了植物过表达载体pBI-CmCDPK,为CmCDPK基因在烟草中实现遗传转化和功能研究奠定基础。A calcium-dependent protein kinase gene(CDPK)was isolated from the Cypripedium macranthum Sw.roots using reverse transcription-PCR(RT-PCR)and rapid amplification of cDNA ends(RACE)techniques.The full-length fragment of CmCDPK gene was 2079 bp,with a complete open reading frame of 1491 bp,which encodes for 496 amino acids.CmCDPK was predicted to be a stable hydrophilic protein,possessing a typical and conserved serine/threonine protein kinase domain and a transmembrane structure domain.Secondary structure of CmCDPK is abundant inα-helices and random coils.By phylogenetic tree analysis,CmCDPK were clustered with CDPKs from Orchidaceae,such as Phalaenopsis equestris(Schauer)Rchb.f.and Dendrobium catenatum Lindl.The fragment of interest was subsequently cloned into the pBI121 vector,which was verified by colony PCR,restriction enzyme digestion and Sanger sequencing.Taken together,this work isolated a CDPK gene from C.macranthum Sw.and generated the plant binary expression vector,which might provide the possibility for making transformation in tobacco and further illustrating the biological function of CmCDPK.

关 键 词：大花杓兰 钙依赖蛋白激酶 RT-PCR RACE 植物表达载体 

分 类 号：Q94[生物学—植物学]