BvM14-GAI基因的克隆及亚细胞定位  

BvM14-GAI Gene: Cloning and Subcellular Localization

作　　者：马春泉 孙培琳[1,2] 李海英 Ma Chunquan;Sun Peilin;Li Haiying(Engineering Research Center of Agricultural Microbiology Technology,Ministry of Education,Heilongjiang University,Harbin 150500;Key Laboratory of Molecular Biology,College of Heilongjiang Province/School of Life Sciences,Heilongjiang University,Harbin 150080)

机构地区：[1]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 [2]黑龙江大学生命科学学院/黑龙江省普通高校分子生物学重点实验室,哈尔滨150080

出　　处：《中国农学通报》2020年第16期28-33,共6页Chinese Agricultural Science Bulletin

基　　金：国家自然科学基金资助项目“甜菜M14品系抗氧化酶系统响应盐胁迫应答过程的研究”(31471552);国家自然科学基金资助项目“甜菜M14品系乙二醛酶I基因的转录因子参与响应盐胁迫的调控机制研究”(3167100696);黑龙江大学研究生创新科研项目“盐胁迫下转录因子BvM14-GAI功能研究”(YJSCX2019-211HLJU)。

摘　　要：GAI蛋白属于GRAS家族中的DELLA亚家族,参与植物发育、光合、抗逆等重要的植物生长过程。实验室前期在盐胁迫的转录组数据中发现甜菜M14品系GAI基因(BvM14-GAI)受盐胁迫上调表达,为了进行该基因生物学功能的研究,本研究以甜菜M14品系为材料,通过PCR技术获得BvM14-GAI基因cDNA全长序列,并进行生物信息学以及亚细胞定位分析。研究成功获得了BvM14-GAI基因cDNA序列;生物信息学分析显示,BvM14-GAI基因开放阅读框为1824 bp(包括终止密码子),编码607个氨基酸,理论分子量为66 kDa,等电点为5.37;蛋白多重序列比对和系统进化树分析显示,BvM14-GAI蛋白与菠菜(Spinacia oleracea)和藜麦(Chenopodium quinoa)中GAI蛋白亲缘关系最近;亚细胞定位预测显示定位在细胞核。进一步将BvM14-GAI基因与pCAMIA2300-eYFP载体重组,构建亚细胞定位载体,利用冻融法转化农杆菌EHA105,进行烟草注射,结果显示BvM14-GAI蛋白定位在细胞核。本研究成功获得BvM14-GAI基因的cDNA全长,并确定该基因的烟草亚细胞定位在细胞核中,结果为后续该基因功能的研究提供了重要参考。GAI protein belongs to the DELLA subfamily of the GRAS family, and is involved in plant growth processes such as development, photosynthesis and stress tolerance. In the early research of the authors’ laboratory, it was found that the GAI gene of sugar beet M14 line(BvM14-GAI) was up-regulated under salt stress. To investigate the functions of BvM14-GAI, BvM14 was selected as experimental material, and the fulllength of BvM14-GAI gene was amplified by reverse transcription polymerase chain reaction(RT-PCR). In addition, the bioinformatics analysis and subcellular localization were conducted. The results showed that the full length cDNA of BvM14-GAI gene was obtained, and the results of bioinformatics analysis revealed that the full-length of BvM14-GAI gene was 1824 bp which encoded 607 amino acids with the predicted molecular mass of 66 kDa and the pI of 5.37. Multiple sequence alignment of BvM14-GAI and other GAI proteins showed that BvM14-GAI protein was highly homologous with the GAI proteins in spinach(Spinacia oleracea)and quinoa(Chenopodium quinoa). Subcellular localization prediction showed that BvM14-GAI protein was localized in the nucleus. Subsequently, the BvM14-GAI gene was ligated with the pCAMIA2300-eYFP vector,and the recombinant plasmids were transformed into Agrobacterium EHA105 and injected into tobacco. Finally the results exhibited that the BvM14-GAI protein was localized in the nucleus. In conclusion, the full-length cDNA of the BvM14-GAI gene is successfully obtained in this experiment, and subcellular location analysis confirms that the BvM14-GAI is localized in the nucleus. This work lays an important foundation for studying the function of BvM14-GAI gene.

关 键 词：甜菜M14品系 BvM14-GAI基因 生物信息学分析 亚细胞定位 

分 类 号：S566.3[农业科学—作物学]