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大花杓兰钙依赖蛋白激酶基因克隆及植物表达载体构建

花匠小妙招
2024-09-13 13:38

作者:

付亚娟 1,2 侯荟玲 1 乔洁 1,2 耿晓进 1 王聪艳 1 侯晓强 1,2

作者单位:

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

基金项目:

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)。

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

Author:

FU Ya-juan 1,2

FU Ya-juan

1College of Life Science, Langfang Normal University, Hebei Langfang 065000; 2Technical Innovation Center for High Value Utilization of Edible and Medicinal Fungi Resources in Hebei Province, Langfang 065000
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HOU Hui-ling 1

HOU Hui-ling

1College of Life Science, Langfang Normal University, Hebei Langfang 065000;
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QIAO Jie 1,2

QIAO Jie

GENG Xiao-jin 1

GENG Xiao-jin

1College of Life Science, Langfang Normal University, Hebei Langfang 065000;
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WANG Cong-yan 1

WANG Cong-yan

1College of Life Science, Langfang Normal University, Hebei Langfang 065000;
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HOU Xiao-qiang 1,2

HOU Xiao-qiang

Affiliation:

1College of Life Science, Langfang Normal University, Hebei Langfang 065000;2Technical Innovation Center for High Value Utilization of Edible and Medicinal Fungi Resources in Hebei Province, Langfang 065000

Fund Project:

Talented Youth Project of Hebei Education Department (BJ2016045); National Science Foundation of China (31100314); Doctoral Foundation of Langfang Normal University (LSLB201405); Key Development Project of Genetics in Hebei Province (201221).

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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。

Abstract:

A calcium-dependent protein kinase gene (CDPK) was isolated from the Cypripedium macranthum 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 in α-helices and random coils. By phylogenetic tree analysis, CmCDPK were clustered with CDPKs from Orchidaceae, such as Phalaenopsis equestris and Dendrobium catenatum. 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 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.

引用本文

付亚娟,侯荟玲,乔洁,等.大花杓兰钙依赖蛋白激酶基因克隆及植物表达载体构建[J].植物遗传资源学报,2019,20(6):1613-1620.

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