BvM14-GAI基因的克隆及亚细胞定位

doi:10.11924/j.issn.1000-6850.casb20191200907

中国农学通报 ›› 2020, Vol. 36 ›› Issue (16): 28-33.doi: 10.11924/j.issn.1000-6850.casb20191200907

所属专题：生物技术

BvM14-GAI基因的克隆及亚细胞定位

马春泉¹^,²(), 孙培琳¹^,², 李海英¹^,²^,^*()

^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

收稿日期:2019-12-04 修回日期:2020-01-09 出版日期:2020-06-05 发布日期:2020-05-20
通讯作者: 李海英
基金资助:
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)

BvM14-GAI Gene: Cloning and Subcellular Localization

Ma Chunquan¹^,²(), Sun Peilin¹^,², Li Haiying¹^,²^,^*()

^4.Engineering Research Center of Agricultural Microbiology Technology, Ministry of Education, Heilongjiang University, Harbin 150500
^2.Key Laboratory of Molecular Biology, College of Heilongjiang Province/School of Life Sciences, Heilongjiang University, Harbin 150080

Received:2019-12-04 Revised:2020-01-09 Online:2020-06-05 Published:2020-05-20
Contact: Li Haiying

摘要/Abstract

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。

关键词: 甜菜M14品系, BvM14-GAI基因, 生物信息学分析, 亚细胞定位

Abstract:

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 full-length 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.

Key words: sugar beet M14 line, BvM14-GAI gene, bioinformatics analysis, subcellular localization

中图分类号:

S566.3

马春泉, 孙培琳, 李海英. BvM14-GAI基因的克隆及亚细胞定位[J]. 中国农学通报, 2020, 36(16): 28-33.

Ma Chunquan, Sun Peilin, Li Haiying. BvM14-GAI Gene: Cloning and Subcellular Localization[J]. Chinese Agricultural Science Bulletin, 2020, 36(16): 28-33.

图/表 8

图1. 甜菜M14叶片总RNA的1%琼脂糖凝胶电泳结果图

图2. 甜菜BvM14-GAI基因PCR产物1%琼脂糖凝胶电泳结果图

图3. BvM14-GAI蛋白的二级结构预测结果

图4. BvM14-GAI蛋白三级结构预测结果

图5. BvM14-GAI蛋白系统发育树

图6. BvM14-GAI蛋白亚细胞定位预测结果

图7. 植物表达载体pCAMIA2300-eYFP-BvM14-GAI图示

图8. BvM14-GAI蛋白在烟草叶片中共聚焦显微镜观察结果

参考文献 18

[1]	伍国强, 刘海龙, 李智强 . 甜菜组织培养与植株再生体系的建立[J]. 中国糖料, 2018,40(6):14-18.
[2]	郭德栋, 康传红, 刘丽萍 . 异源三倍体甜菜(VVC)无融合生殖的研究[J]. 中国农业科学, 1999,32(40):1-5.
[3]	李海英 . 甜菜无融合生殖品系M14花期特异表达基因的研究[A].中国植物生理学会.2006年拟南芥研究学术研讨会论文集[C].中国植物生理学会, 2006: 1.
[4]	王玮, 管利萍, 张静 , 等. 拟南芥DELLA蛋白编码基因RGA和GAI的原核表达和多克隆抗体制备[J]. 兰州大学学报:自然科学版, 2016,52(3):422-428.
[5]	Bolle C . The role of GRAS proteins in plant signal transduction and development[J]. Planta, 2004,218(5):683-692. doi: 10.1007/s00425-004-1203-z URL
[6]	Chen Y Q, Tai S S, Wang D W , et al. Homology-based analysis of the GRAS gene family in tobacco[J]. Genetics and Molecular Research, 2015,14(4):15188-15200. doi: 10.4238/2015.November.25.7 URL
[7]	Zhang B, Liu J, Yang Z E , et al. Genome-wide analysis of GRAS transcription factor gene family in Gossypium hirsutum L.[J]. BMC Genomics, 2018,19(1):348. doi: 10.1186/s12864-018-4722-x URL
[8]	Guo Y, Wu H, Li X , et al. Identification and expression of GRAS family genes in maize (Zea mays L.)[J]. PLoS One, 2017,12(9):e0185418. doi: 10.1371/journal.pone.0185418 URL
[9]	Niu Y, Zhao T, Xu X , et al. Genome-wide identification and characterization of GRAS transcription factors in tomato (Solanum lycopersicum)[J]. Peer J, 2017,5:e3955.
[10]	Grimplet J, Agudelo-Romero P, Teixeira R T , et al. Structural and functional analysis of the GRAS gene family in Grapevine indicates a role of GRAS proteins in the control of development and stress responses[J]. Frontiers in Plant Science, 2016,7:353.
[11]	张文霞, 刁志娟, 吴为人 . 植物GRAS蛋白研究的新进展[J]. 分子植物育种, 2016,14(5):1159-1165.
[12]	Ito T, Okada K, Fukazawa J , et al. DELLA-dependent and -independent gibberellin signaling[J]. Plant Signaling & Behavior, 2018,13(3):e1445933.
[13]	Goh T, Toyokura K, Wells D M , et al. Quiescent center initiation in the Arabidopsis lateral root primordia is dependent on the SCARECROW transcription factor[J]. Development, 2016,143(18):3363-3371. doi: 10.1242/dev.135319 URL
[14]	Yuan Y, Fang L, Karungo S K , et al. Overexpression of VaPAT1, a GRAS transcription factor from Vitis amurensis, confers abiotic stress tolerance in Arabidopsis[J]. Plant Cell Reports, 2016,35(3):655-666. doi: 10.1007/s00299-015-1910-x URL
[15]	Miguel A, Milhinhos A, Novák O , et al. The SHORT-ROOT-like gene PtSHR2B is involved in Populus phellogen activity[J]. Journal of Experimental Botany, 2016,67(5):1545-55. doi: 10.1093/jxb/erv547 URL
[16]	陈英杰, 詹杰鹏, 王志江 , 等. 棉花DELLA蛋白GhGAI2b基因的克隆和功能初步分析[J]. 石河子大学学报:自然科学版, 2014,32(5):635-640.
[17]	Liu S, Xuan L, Xu L A, et al. Molecular cloning, expression analysis and subcellular localization of four DELLA genes from hybrid poplar[J]. Springer Plus, 2016, 5(1):1129.0 doi: 10.1186/s40064-016-2728-x URL
[18]	李雯瑞, 白朕卿, 刘景玲 , 等. 丹参转录因子SmGRAS3的克隆、亚细胞定位和表达分析[J]. 中国中药杂志, 2019, 44(22):4830-4836.

BvM14-GAI基因的克隆及亚细胞定位

BvM14-GAI Gene: Cloning and Subcellular Localization

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