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作物学报 ›› 2016, Vol. 42 ›› Issue (10): 1569-1574.doi: 10.3724/SP.J.1006.2016.01569

• 研究简报 • 上一篇    

海马齿甜菜碱醛脱氢酶基因克隆、高效表达及酶学特性分析

喻时周1,4,杨成龙2,*,郭建春3,段瑞军3   

  1. 1贵州省油菜研究所，贵州贵阳550008；2贵州省亚热带作物研究所,贵州兴义562400；3中国热带农业科学院热带生物技术研究所/农业部热带作物生物学与遗传资源利用重点实验室,海南海口571101;4贵州禾睦福种子有限公司,贵州贵阳550008
  • 收稿日期:2016-02-19 修回日期:2016-07-11 出版日期:2016-10-12 网络出版日期:2016-07-28
  • 通讯作者: 杨成龙,E-mail:yangchenglong208@163.com,Tel:0859-3911295
  • 基金资助:

    本研究由国家农业科技成果转化资金项目(2014GB2F200240),贵州省农业科学院自主创新专项[(2014)014],贵州省科研机构服务企业行动计划项目黔科合服企[(2015)4001],贵州省科技厅重大专项[黔科合重大专项字(2013)6005],贵州省农业科学院专项[黔农科院院专项(2015)16],贵州省科技厅‘百层次’人才项目[黔科合人才(2015)4018]和贵州省农业科学院人才团队项目[黔农科合CR合字(2014)64]资助。

Cloning, Expression, and Enzymatic Characteristics of Betaine Aldehyde De-hydrogenase Gene in Sesuvium portulacastrum L.

YU Shi-Zhou1,4,YANG Cheng-Long2,*,GUO Jian-Chun3,DUAN Rui-Jun3   

  1. 1 Guizhou Rape Institute, Guiyang 550008, China; 2 Guizhou Institute of Subtropical Crops, Xingyi 562400, China; 3 Institute of Tropical Bioscience and Biotechnology / Key Laboratory of Biology and Genetic Resources of Tropical Crops, Ministry of Agriculture / Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, China; 4 Guizhou Hemufu Seed Co. Ltd, Guiyang 550008, China
  • Received:2016-02-19 Revised:2016-07-11 Published:2016-10-12 Published online:2016-07-28
  • Contact: 杨成龙,E-mail:yangchenglong208@163.com,Tel:0859-3911295
  • Supported by:

    ThisstudywassupportedbyNationalAgriculturalScienceandTechnologyAchievementsTransformationFundProject(2014GB2F200240),theSpecialProjectofIndependentInnovationProject[(2014)014],theProjectofGuizhouProvinceforServiceEnterprisebyScientificResearchInstitution[(2015)4001],theKeySpecialProjectforGuizhouScienceandCooperation[(2013)6005],theSpecialProjectofGuizhouAcademyofAgriculturalSciences[(2015)16],GuizhouTop100TalentsHierarchyProjectofScienceandTechnologyDepartmentofGuizhouProvince[(2015)4018],andtheTalentTeamProjectofGuizhouAcademyofAgriculturalSciences[CR(2014)64].

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摘要：

在许多渗透调节剂中，甜菜碱是最理想的有机小分子渗透调节物质。甜菜碱在植物体内大量积累不会带来危害，同时能提高植物对环境胁迫的抗性。将海马齿中克隆到的甜菜碱醛脱氢酶基因构建到表达载体pET-28a(+)上，获得重组载体pET-SpBADH并将其成功地转化到BL21(DE3)中得到重组工程菌，经IPTG诱导能高效表达55 kD目的蛋白，表达量可以达到301 μg mL–1。酶学特征分析表明，该蛋白最适pH值为7.2，在偏碱条件下能维持较高的催化活性；SpBADH蛋白对高温敏感，且温度对催化活性影响较大，超过55℃时酶活性只有20%，最适酶催化活性温度为37℃；而有机小分子醇类对酶的催化活性有保护作用，可以通过自身特征维持酶催化活性的微环境。

关键词: 海马齿, 甜菜碱醛脱氢酶, 原核表达, 酶活测定

Abstract:

Among many osmotic materials, glycine betaine is a best organic micro-molecular, and functionally works for osmotic regulation in plants, which is non-toxic to plant growth. A lot of glycine betaine accumulated in plant can enhance the resistance of plants to environmental stresses. In the study, a full-length sequence of betaine aldehyde dehydrogenase gene from Sesuvium portulacastrum was ligated with the vector pET-[28a](+), named pET-SpBADH, and successfully transformed into BL21(DE3) to obtain the corresponding recombinant engineering bacteria, which could highly express 55 kD protein induced by IPTG, with the expression level to 301 μg mL–1. The purified protein was obtained, showing the optimum pH value of 7.2, and maintain high catalytic activity the enzyme under slightly alkaline conditions. SpBADH protein very sensitive to high temperature effected the enzyme activity, with the optimum temperature to 37℃. The enzyme activity was only 20% when temperature was over 55℃. The small organic molecules of the reveral compounds of alcohol had a protective effect on the catalytic activity of the enzyme. The microenvironment of catalytic activity could be maintained by its own characteristics.

Key words: SesuviumportulacastrumL., Betainealdehydedehydrogenase, Prokaryoticexpression, Enzymeactivityassay

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