大豆血红蛋白基因lba转化根瘤菌工程菌株的构建  

Construction of Genetic Engineering Strain of Introduction Glycine max Leghemoglobin Gene of lba into Native Rhizobium japonicum

作　　者：于海鹏[1,2,3] 潘钰[1,2] 蒙明明[1,2] 李海英[1,2] 

机构地区：[1]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 [2]黑龙江大学农业微生物技术教育部工程研究中心,哈尔滨150500 [3]中国农业科学院农业信息研究所,北京100081

出　　处：《生物技术通报》2012年第2期69-74,共6页Biotechnology Bulletin

基　　金：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)

摘　　要：以土著大豆根瘤菌接种大豆幼苗45 d后获得的根瘤为材料,提取其总RNA并反转录成cDNA,采用同源序列克隆法扩增大豆血红蛋白基因lba编码区序列。利用DNA重组技术,将lba基因连到lac启动子的下游,利用带有发光酶标记基因luxAB的质粒载体pTR102构建表达载体pTR-Plac-lba。采用三亲本杂交的方式,将表达载体pTR-Plac-lba及作为对照的空载体pTR102分别转化土著大豆根瘤菌,获得根瘤菌工程菌株SFH(pTR-Plac-lba)和SFH(pTR102)。盆栽试验发现,接种SFH(pTR-Plac-lba)的大豆植株各生理指标明显高于接种SFH(pTR102)、土著根瘤菌以及未接菌的大豆植株各生理指标。试验证明,导入大豆血红蛋白基因lba的根瘤菌工程菌株SFH(pTR-Plac-lba)对于提高大豆根瘤的固氮酶活性,增加大豆产量起到显著效果。The nodules were obtained from soybean seedlings which were inoculated by native Rhizobium japonicum for 45 days.Moreover,the total RNA of soybean nodule was extracted and reversed transcript into cDNA,which was used to clone Glycine max leghemoglobin gene lba by Homology-based cloning.The lba gene was inserted into the downstream of lac promoter,and expression vector pTR-Plac-lba was constructed using plasmid pTR102 with marker gene luxAB by the DNA recombinant technique.The expression vector pTR-Plac-lba and empty vector plasmids pTR102 were respectively transformed into native Rhizobium japonicum with method of triparental hybridization to construct engineering strains SFH（pTR-Plac-lba） and SFH（pTR102）.Pot experiment results showed that the physiological indexes of soybean plants infected by SFH（pTR-Plac-lba） were significantly higher than that infected by SFH（pTR102）,native Rhizobium japonicum and that were not infected.The results showed that engineering strains SFH（pTR-Plac-lba） which was imported Glycine max leghemoglobin gene lba had a significant effect on increaseing the soybean root nodule nitrogenase activity and soybean production.

关 键 词：土著大豆根瘤菌 大豆血红蛋白基因lba DNA重组技术 工程菌株 三亲本杂交 

分 类 号：S144.3[农业科学—肥料学]