全部 标题 作者
关键词 摘要

OALib Journal期刊
ISSN: 2333-9721
费用:99美元

查看量下载量

相关文章

更多...
棉花学报  2014 

棉花GhEPSPS基因的原核表达载体的构建及诱导表达

DOI: 1002-7807(2014)06-0563-06, PP. 563-568

Keywords: 棉花,GhEPSPS基因,原核表达,SDS-PAGE

Full-Text   Cite this paper   Add to My Lib

Abstract:

目的在于构建棉花GhEPSPS基因的原核表达载体,并在宿主大肠杆菌中成功诱导表达。利用RT-PCR技术从陆地棉苏棉18中克隆获得GhEPSPS基因的开放阅读框序列,连接到pEASY-Blunt平端载体,构建获得重组载体pEASY-Blunt-GhEPSPS;以该重组载体为模板,PCR获得两端含有EcoRⅠ和XhoⅠ酶切位点的GhEPSPS基因CDS序列,通过这2个酶切位点插入到pET32a载体,构建获得原核表达重组载体pET32a-GhEPSPS,转化到宿主菌株BL21(DE3)中,经IPTG诱导后,SDS-PAGE检测目的基因的表达情况。研究结果表明,成功克隆获得了棉花GhEPSPS基因的CDS序列,并成功构建了该基因的原核表达载体,目的基因能够在宿主菌中被诱导大量表达。该研究结果将为深入研究棉花EPSPS酶的结构、功能以及酶学特性提供重要的研究基础。

References

[1]  Hermann K M, Weaver L M. The shikimate pathway[J]. Annual Review of Plant Physiology and Plant Molecular Biology, 1999(50): 473-503.
[2]  Schonbrunn E, Eschenburg S, Shuttleworth W A, et al. Interaction of the herbicide glyphosate with its target enzyme 5-enolpyruvylshikimate-3-phosphate synthase in atomic detail[J]. Proceedings of the National Academy of Sciences of the United States of America, 2001(98): 1376-1380.
[3]  Funke T, Yang Y, Han H, et al. Structural basis of glyphosate resistance resulting from the double mutation Thr 97 →Ile and Pro 101 →Ser in 5-enolpyruvylshikimate-3-phosphate synthase from Escherichia coli[J]. Journal of Biological Chemistry, 2009, 284(15): 9854-9860.
[4]  童旭宏, 吴玉香, 祝水金. 陆地棉EPSPS基因的克隆及组织特异性表达分析[J]. 棉花学报, 2009, 21(4): 259-264.
[5]  Tong Xuhong, Wu Yuxiang, Zhu Shuijin. Cloning of 5-enolpyruvylshikimate-3-phosphate synthase genes EPSPS from the upland cotton and its expression in different cotton tissues[J]. Cotton Science, 2009,21(4):259-264.
[6]  巩元勇, 郭书巧, 束红梅, 等. 陆地棉水通道蛋白GhNIP6.1基因的克隆及表达分析[J]. 棉花学报, 2013, 25(1): 1-8.
[7]  Gong Yuanyong, Guo Shuqiao, Shu Hongmei, et al. Cloning of aquaporin gene GhNIP6.1 from the upland cotton and its expression in different cotton tissues[J]. Cotton Science, 2013, 25(1): 1-8.
[8]  胡根海, 喻树迅. 利用改良的CTAB法提取棉花叶片总RNA[J]. 棉花学报, 2007, 19(1): 69-70.
[9]  Hu Genhai, Yu Shuxun. Extraction of high-quality total RNA in cotton leaf with improved CTAB method[J]. Cotton Science, 2007, 19(1): 69-70.
[10]  郭尧君. 蛋白质电泳实验技术[M]. 北京: 科学出版社, 1999:138.
[11]  Guo Yaojun. Protein electrophoresis technology[M]. Beijing: Science Press, 1999:138.
[12]  Studier F W, Moffatt B A. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes [J]. Journal of Molecular Biology, 1986, 189(1): 113-130.
[13]  Studier F W, Rosenberg A H, Dunn J J, et al. Use of T7 RNA polymerase to direct expression of cloned genes[J]. Methods in Enzymology, 1990(185): 60-89.
[14]  Wang Zefang, Feng Shuren, Huang Yujian, et al. Prokaryotic expression, purification, and polyclonal antibody production of a hydrophobin from Grifola frondosa[J]. Acta Biochimica et Biophysica Sinica, 2010, 42(6): 388-395.
[15]  郝梦雨, 郎明林, 杨学举. 印度芥菜BjJ10-2基因原核表达载体构建及抗盐功能的鉴定[J]. 核农学报, 2011, 25(2): 247-252.
[16]  Hao Mengyu, Lang Minglin, Yang Xueju. Construction of recombinant prokaryotic expression vector for Brassica juncea BjJ10-2 and identification of its function for high salinity tolernance[J]. Journal of Nuclear Agricultural Sciences, 2011, 25(2): 247-252.
[17]  张婷婷, 王春丽, 韩蕊莲, 等. 丹参C4H基因pET32a原核表达载体的构建[J]. 西北农林科技大学学报: 自然科学版,2011, 39(7): 156-162.
[18]  Zhang Tingting, Wang Chunli,Han Ruilian, et al. Construction of a vector conferring herbicide and pest resistance in tobacco plant[J]. Journal of Northwest A&F University :Nature Science Edition, 2003, 39(7):156-162.
[19]  Shuttleworth W A, Pohl M E, Helms G L, et al. Site-directed mutagenesis of putative active site residues of 5-enolpyruvylshikimate-3-phosphate synthase[J]. Biochemistry, 1999, 38(1): 296-302.
[20]  Ng C H, Wickenswary R, Salmijah S, et al. Gene polymorphisms in glyphosate-resistant and -susceptible biotypes of Eleusine indica from Malaysia[J]. Weed Research, 2003(43):108-115.

Full-Text

Contact Us

service@oalib.com

QQ:3279437679

WhatsApp +8615387084133