导入nodD-lba串联基因的根瘤菌工程菌株的构建

生物技术通报 ›› 2013, Vol. 0 ›› Issue (8): 119-123.

导入nodD-lba串联基因的根瘤菌工程菌株的构建

李珊珊, 李海英, 于冰

（黑龙江大学生命科学学院黑龙江省普通高等学校分子生物学重点实验室，哈尔滨 150080）

收稿日期:2013-03-12 修回日期:2013-08-11 出版日期:2013-08-11 发布日期:2013-09-02
作者简介:李珊珊，女，硕士研究生，研究方向：分子生态学；E-mail：446540970@qq.com
基金资助:
哈尔滨市科技创新人才研究专项资金项目（RC2009XK001004），黑龙江省科技攻关项目（GA08B101），黑龙江大学高层次人才（创新团队）支持计划（Hdtd2010-05）

The Construction of Rhizobia Engineering Strain of Introduction a Tandem Gene nodD-lba into Sinorhizobium fredii

Li Shanshan, Li Haiying, Yu Bing

(Key Laboratory of Molecular Biology, College of Life Sciences, Heilongjiang University, Harbin 150080）

Received:2013-03-12 Revised:2013-08-11 Published:2013-08-11 Online:2013-09-02

摘要/Abstract

摘要：

旨在利用同源序列克隆法，克隆出结瘤相关基因nodD和大豆血红蛋白基因lba。以串联的方式将nodD和lba基因连接到lac启动子的下游，通过DNA重组技术构建出带有发光酶基因luxAB的重组载体pTR-P_lac-nodD-lba。通过三亲本杂交方法构建转基因费氏中华根瘤菌(Sinorhizobium fredii)，Western blot试验证明导入基因能够进行表达。将初始菌株和基因工程菌株侵染大豆幼苗后进行固氮酶活的测定。测定结果表明，转基因工程菌株均能够提高固氮酶效率，导入串联基因的工程菌株在提高固氮酶活性上比导入lba和nodD基因的工程菌株高出4%和15.1%。

关键词: 结瘤基因nodD, 大豆血红蛋白基因lba, 费氏中华根瘤菌, 三亲本杂交

Abstract:

Abstract: In this study, homology-based cloning was used to clone hemoglobin gene of lba and nodulation gene of nodD. The two genes were ordered joined into downstream of lac promoter and connected by the DNA recombinant technique to constructed expression vector pTR-P_lac-nodD-lba marked by luxAB gene. The vector was transformed into Sinorhizobium fredii with the method of triparental hybridization to construct genetic engineering strain. Western blot indicated the genes we used can express normally. Strains were inoculated soybean seedlings in order to get nitrogenase activity. The result showed engineering strains can improve the nitrogenase activity and the engineering strain with tandem genes can enhance nitrogenase activity 4% and 15.1%, respectively, by contrast with engineering strain with lba or nodD gene.

Key words: Nodulation gene nodD, Glycine max leghemoglobin gene lba, Sinorhizobium fredii, Triparental hybridization

李珊珊, 李海英, 于冰. 导入nodD-lba串联基因的根瘤菌工程菌株的构建[J]. 生物技术通报, 2013, 0(8): 119-123.

Li Shanshan, Li Haiying, Yu Bing. The Construction of Rhizobia Engineering Strain of Introduction a Tandem Gene nodD-lba into Sinorhizobium fredii[J]. Biotechnology Bulletin, 2013, 0(8): 119-123.

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