Cloning of an RNA-binding Protein Gene from Alfalfa and Expression in Tobacco

doi:10.11733/j.issn.1007-0435.2013.02.026

›› 2013, Vol. 21 ›› Issue (2): 379-387.DOI: 10.11733/j.issn.1007-0435.2013.02.026

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Cloning of an RNA-binding Protein Gene from Alfalfa and Expression in Tobacco

WANG Hui-min¹, LONG Rui-cai³, SHEN Yi-xin¹, KANG Jun-mei², ZHANG Tie-jun², ZHANG Yu¹, YANG Qing-chuan^1,2

1. College of Animal Science and Technology, Nanjing, Agriculture University, Nanjing, Jiangsu Province 210095, China;
2. Institute of Animal Science, CAAS, Beijing 100094, China;
3. Institute of Agriculture and Life Science, Chongqing University, Chongqing 400044, China

Received:2012-11-02 Revised:2012-12-10 Online:2013-04-15 Published:2013-04-23

紫花苜蓿MsRBP基因的克隆、分析及烟草的遗传转化

王慧敏¹, 龙瑞才³, 沈益新¹, 康俊梅², 张铁军², 张瑜¹, 杨青川^1,2

1. 南京农业大学动物科技学院, 江苏南京 210095;
2. 中国农业科学院北京畜牧兽医研究所, 北京 100094;
3. 重庆大学农学及生命科学研究院, 重庆 400044

通讯作者: 杨青川,qchyang66@yahoo.com.cn
作者简介:王慧敏(1986-),女,山东聊城人,硕士研究生,研究方向为牧草及草坪草育种,E-mail:maohai_1201@163.com
基金资助:
"十二五"国家科技支撑计划项目(2011BAD17B01-01-3)资助

Abstract

Abstract: Based on an expressed sequence tag of Medicago sativa L., a full length of 1551 bp cDNA was isolated using RT-PCR and RACE-PCR techniques. The gene was named MsRBP (GenBank accession No. JN986878.1). Sequence analysis revealed that MsRBP contained a maximum open reading frame of 1230 bp and encoded a 409 amino acids protein. The putative amino acid sequence had high identity with RNA binding proteins from Medicago truncatula, Glycine max, and Arabidopsis thaliana. Real-time quantitative PCR showed that the transcription level of MsRBP was up-regulated in NaCl, ABA, and PEG stress treatments. It indicated that MsRBP probably played an important role in the tolerance of abiotic stress. MsRBP gene was transferred into tobacco with leaf disc method by Agrobacterium LBA4404 and expressed under the control of CaMV 35S promoter. The transgenic plants were selected on MS medium supplemented with kanamycin and analyzed by PCR, RT-PCR and GUS assay. These results showed that MsRBP was integrated into tobacco genome and expressed in transgenic tobacco.

Key words: Alfalfa, RNA binding protein(RBP), Real-time RT-PCR, Expression analysis, Tobacco transformation

摘要： 采用RACE技术以一段紫花苜蓿(Medicago sativa)盐诱导基因的EST(expressed sequence tag)序列为模板设计引物克隆此基因的全长序列。序列分析结果表明,该基因全长1551 bp,包含一个1230 bp的最大开放阅读框,编码409个氨基酸,包含3个RNA结合蛋白结构域(RRM domain),命名为MsRBP (GenBank accession No. JN986878.1)。亚细胞定位分析表明此基因编码蛋白定位于细胞核。经同源比对和进化树分析,MsRBP基因编码的氨基酸序列与截形苜蓿(Medicago truncatula)、大豆(Glycine max)、拟南芥(Arabidopsis thaliana)等物种中的某些RNA结合蛋白的氨基酸序列具有很高的相似性。RT-PCR (Real-time PCR)分析结果表明,在NaCl,ABA和PEG胁迫下MsRBP基因的表达水平均上调,推测该基因可能在紫花苜蓿逆境胁迫调控中发挥重要作用。经构建植物超表达载体pBI21-MsRBP,采用农杆菌介导法对烟草(Nicotiana tabacum)进行遗传转化,获得了抗性转化再生植株。通过PCR,RT-PCR及GUS组织化学染色分析表明:MsRBP基因在烟草的基因组中能够进行转录和表达。本研究为该基因的功能鉴定与调控机制研究奠定了基础。

关键词: 紫花苜蓿, RNA结合蛋白, 实时定量RT-PCR, 表达分析, 烟草转化

CLC Number:

Q943.2

WANG Hui-min, LONG Rui-cai, SHEN Yi-xin, KANG Jun-mei, ZHANG Tie-jun, ZHANG Yu, YANG Qing-chuan. Cloning of an RNA-binding Protein Gene from Alfalfa and Expression in Tobacco[J]. , 2013, 21(2): 379-387.

王慧敏, 龙瑞才, 沈益新, 康俊梅, 张铁军, 张瑜, 杨青川. 紫花苜蓿MsRBP基因的克隆、分析及烟草的遗传转化[J]. , 2013, 21(2): 379-387.

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Cloning of an RNA-binding Protein Gene from Alfalfa and Expression in Tobacco

紫花苜蓿MsRBP基因的克隆、分析及烟草的遗传转化

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