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中国农学通报

中国农学通报 ›› 2014, Vol. 30 ›› Issue (25): 148-153.doi: 10.11924/j.issn.1000-6850.2014-0400

所属专题： 生物技术

• 林学 园艺 园林 • 上一篇    下一篇

柚CCoAOMT基因的克隆与超表达载体构建

徐玉 潘腾飞 潘东明   

  • 收稿日期:2014-02-21 修回日期:2014-05-24 出版日期:2014-09-05 发布日期:2014-09-05
  • 基金资助:
    国家科技支撑计划项目“台湾农业新品种、新技术引进创新研究与示范”(2007BAD07B01)；福建省种业创新与产业化工程项目、福建农林大学创新团队项目“园艺植物种质与高优生产技术创新”(cxtd12013)。

Cloning of Caffeoyl-coenzyme A O-methyltransferase Gene from Citrus maxima (Burm.) Merr. and Its Plant Expressing Vector Construction

  • Received:2014-02-21 Revised:2014-05-24 Online:2014-09-05 Published:2014-09-05

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摘要： 为探明咖啡酰辅酶A-O-甲基转移酶（CCoAOMT）在柚[Citrus maxima (Burm.) Merr.]果实发育中对木质素单体合成的影响，克隆了柚CCoAOMT基因ORF序列并构建超表达载体转化至农杆菌。采用Trizol法提取柚汁胞总RNA，经反转录得到cDNA，根据已知柚CCoAOMT片段序列，设计其ORF引物。PCR扩增得到柚CCoAOMT基因ORF序列，长度为744 bp，编码247个氨基酸，与龙眼(Dimocarpus longan)、麻疯树(Jatropha curcas)、杨树(Populus balsamifera)的CCoAOMT基因同源性大于90%。经软件预测，该酶定位于质膜的可能性最大，不具有跨膜结构。将柚CCoAOMT基因正向导入植物过表达载体p1301，位置在CaMV35S启动子的之后，并将载体转化至农杆菌EHA105。本研究得到的基因片段包含柚CCoAOMT基因ORF，成功构建植物超表达载体p1301-cmCCoAOMT，并导入农杆菌。含p1301-cm CCoAOMT的农杆菌株可转化多种植物受体，为深入研究该基因功能奠定了基础。

关键词: 抑制机制, 抑制机制

Abstract: To probe the effects of the caffeoyl-coenzyme A 3-O-methyltransferase on lignin biosynthesis in granulated pummelo juice sac, a cmCCoAOMT gene was cloned from pummelo juice sac and constructed into plant expressing vector, and then was transformed into Agrobacterium tumefaciens. Total RNA was obtained by Trizol method, and was reverse transcribed as cDNA. cmCCoAOMT gene was cloned by PCR with a primer pair designed from a CCoAOMT gene sequence of ORF. The open reading frame (ORF) contained 744 base pairs coding 247 amino acids. The sequence homology with Populus balsamifera, Jatropha curcas and Dimocarpus longan exceeded 90% . Prediction result indicated that cmCCoAOMT was located on ER with possibility of 0.595 and it was not a transmembrane protein. The over expression vector obtained by inserting the ORF of cmCCoAOMT cDNA into p1301 under the control of 35S promoter was transformed into Agrobacterium tumefaciens. As a conclusion, the ORF of cmCCoAOMT gene was cloned and constructed into plant expression vector p1301 in this study. Agrobacterium tumefaciens contained the vector can infect many plants and be used as the base for gene function study.