Cloning and Characterization of a DExD/H box RNA Helicase Gene from Medicago sativa L.

doi:10.11733/j.issn.1007-0435.2013.03.026

›› 2013, Vol. 21 ›› Issue (3): 581-589.DOI: 10.11733/j.issn.1007-0435.2013.03.026

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Cloning and Characterization of a DExD/H box RNA Helicase Gene from Medicago sativa L.

GAO Yan-li^1,2, LONG Rui-cai³, KANG Jun-mei², ZHANG Tie-jun², YANG Qing-chuan^1,2, DONG Kuan-hu¹

1. College of Animal Science and Technology, Shanxi Agriculture University, Taigu, Shanxi Province 030801, China;
2. Institute of Animal Science, CAAS, Beijing 100193, China;
3. School of Life Science, Chongqing University, Chongqing 400044, China

Received:2012-11-23 Revised:2013-01-12 Online:2013-06-15 Published:2013-06-05

紫花苜蓿DExD/H box RNA解旋酶基因的克隆与分析

高燕丽^1,2, 龙瑞才³, 康俊梅², 张铁军², 杨青川^1,2, 董宽虎¹

1. 山西农业大学动物科技学院, 山西太谷 030801;
2. 中国农业科学院北京畜牧兽医研究所, 北京 100193;
3. 重庆大学生命科学院, 重庆 400044

通讯作者: 杨青川
作者简介:高燕丽(1987- ),女,山西忻州人,硕士研究生,研究方向为牧草种质资源与育种,E-mail:gaoyanli.025@163.com
基金资助:
"十二五"国家科技支撑计划项目(2011BAD17B01-01-3);国家牧草产业体系岗位科学家资助

Abstract

Abstract: Based on an EST in the SSH library of Medicago sativa L., a full length of 1678 bp cDNA was isolated by RACE. The cDNA sequence was predicted to contain a 1281 bp ORF and code a protein of 426 amino acids, which is homology to Arabidopsis thaliana DEAD-box ATP-dependent RNA helicase 56(RH56) and RH15. The gene was predicted to be a DExD/H box RNA helicase gene and named as MsRH (GenBank accession No. JX508648). Transient expression of MsRH-GFP fusion in onion epidermis cell indicated that the MsRH localized in nucleus. To investigate the function of MsRH, the plant over-expression vector pBI-MsRH was constructed and transferred into tobacco by Agrobacterium LBA4404. After PCR and RT-PCR analysis of five kanamycin resistant plants, 35S::MsRH was confirmed to insert into tobacco genome and transcribe mRNA successfully. After treated with 250 mM NaCl for seven days, the proline contents of transgenic plants were lower than those of control plants (WT), and the malondialdehyde contents and relative electric conductivity were higher than those of control tobacco (WT). Taken together, these results demonstrated that MsRH increased the salt sensitivity of tobacco plants under salt stress.

Key words: Medicago sativa L., RNA helicase, Subcellular localization, Tobacco transformation

摘要： 基于紫花苜蓿(Medicago sativa L.)盐胁迫抑制消减杂交文库中的一条EST序列,使用RACE技术克隆得到一条1678 bp的cDNA序列。核酸序列分析表明该cDNA序列包含一个1281 bp的最大开放阅读框,编码426个氨基酸,与拟南芥(Arabidopsis thaliana)RNA解旋酶RH15和RH56的氨基酸序列相似性在90%以上,将该基因命名为MsRH(GenBank序列号:JX508648)。对此基因编码蛋白进行结构域预测表明其含有明显的DEXDc和HELICc结构域,预测其为DExD/H box RNA解旋酶。洋葱(Allium cepa)表皮细胞亚细胞定位分析表明MsRH定位于细胞核。为进一步研究此基因的功能,构建了MsRH基因的超表达载体pBI-MsRH,使用农杆菌介导法转化烟草(Nicotiana tobacum),筛选得到5株具有卡那霉素抗性再生烟草植株。 PCR和RT-PCR检测结果表明MsRH基因成功插入烟草基因组中并表达。使用250 mM NaCl处理7 d后,转基因烟草中脯氨酸含量低于野生型烟草,而丙二醛和相对电导率则高于野生型烟草。初步试验结果表明转MsRH基因烟草对盐敏感性升高。

关键词: 紫花苜蓿, 解旋酶, 亚细胞定位, 烟草转化

CLC Number:

Q943.2

GAO Yan-li, LONG Rui-cai, KANG Jun-mei, ZHANG Tie-jun, YANG Qing-chuan, DONG Kuan-hu. Cloning and Characterization of a DExD/H box RNA Helicase Gene from Medicago sativa L.[J]. , 2013, 21(3): 581-589.

高燕丽, 龙瑞才, 康俊梅, 张铁军, 杨青川, 董宽虎. 紫花苜蓿DExD/H box RNA解旋酶基因的克隆与分析[J]. , 2013, 21(3): 581-589.

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Cloning and Characterization of a DExD/H box RNA Helicase Gene from Medicago sativa L.

紫花苜蓿DExD/H box RNA解旋酶基因的克隆与分析

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