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棉花学报 ›› 2016, Vol. 28 ›› Issue (1): 17-26.doi: 10.11963/issn.1002-7807.201601003

• 研究与进展 • 上一篇    下一篇

棉花EPSPS基因一个可变剪接体的克隆及功能分析

巩元勇1,倪万潮1*,郭书巧1,束红梅1,帕尔哈提·买买提2,何林池3   

  1. 1. 江苏省农业科学院经济作物研究所/农业部长江下游棉花和油菜重点实验室,江苏 南京 210014;2. 国家棉花工程技术研究中心, 新疆 乌鲁木齐830091;3. 江苏沿江地区农业科学研究所,江苏 如皋226541
  • 收稿日期:2015-04-01 出版日期:2016-01-15 发布日期:2016-01-15
  • 通讯作者: nwchao2002@aliyun.com
  • 作者简介:巩元勇(1982―),男,博士,副研究员,gongyuanyong1982@163.com
  • 基金资助:
    国家自然科学基金(31301682);江苏省农业科技自主创新基金(CX(14)5009);国家转基因重大专项(2013ZX08005);国家科技支撑计划课题(2014BAD11B02)

Cloning and Functional Analysis of an Alternative Splicing Sequence of Cotton EPSPS Gene

Gong Yuanyong1, Ni Wanchao1*, Guo Shuqiao1, Shu Hongmei1, Paerhatio Maimaiti2, He Linchi3   

  1. 1. Institute of Industrial Crops, Jiangsu Academy of Agricultural Sciences/Key Laboratory of Cotton and Rapeseed, Ministry of Agriculture, Nanjing, Jiangsu 210014, China; 2. Research Center of National Cotton Engineering and Technology, Urumqi 830091, China; 3. Institute of Agricultural Sciences of Jiangsu Changjiang River Bank District, Rugao, Jiangsu 226541, China
  • Received:2015-04-01 Online:2016-01-15 Published:2016-01-15

摘要: 可变剪接是生物体基因在转录过程中存在的普遍现象,该现象是导致蛋白质功能多样性的重要原因之一,但是在棉花中关于功能基因可变剪接事件的报到相对较少。本文在克隆棉花EPSPS(5-enolpyruvylshikimate 3-phosphate synthase)基因的过程中,发现了该基因的一条可变剪接序列。运用生物信息学的方法研究发现,该序列比正常的EPSPS基因的cDNA序列少152 bp,这造成了终止密码子在该序列的提前出现;正常的EPSPS基因内含子的剪切都遵循常见的“GT-AG”剪切规律,而该可变剪接序列最后一个内含子的剪切是按照 “AT-AC”规则进行;该可变剪接序列预测的三维结构模型同正常的EPSPS基因的三维结构存在显著差异。将该可变剪接序列插入到原核表达载体pET32a,随后将构建好的原核表达重组载体转化入大肠杆菌菌株BL21(DE3)ΔaroA,通过在M9基本培养基中的生长研究发现该可变剪接序列不具备正常EPSPS基因所具有的功能。本研究结果丰富了棉花EPSPS基因转录本存在的形式,为深入研究棉花EPSPS基因的转录机制打下了基础。

关键词: 可变剪接; 棉花; EPSPS基因; 原核表达

Abstract: Alternative splicing is a universal phenomenon in gene transcription to increase protein functional diversity, but it has not been widely reported in functional cotton genes. While cloning the cotton 5-enolpyruvylshikimate 3-phosphate synthase gene(EPSPS), we found an alternative splicing variant. Bioinformatic techniques analyses showed that the cDNA sequence of this alternative splicing sequence is 152 bp shorter than normal EPSPS cDNA, and would result in the introduction of an early stop codon. All introns in the normal EPSPS genomic sequence follow the commonsplicing rule of "GT-AG", while the last intron in the alternative splicing sequence follows the "AT-AC" splicing rule. Notable differences were identified between the predicted three-dimensional structure of the normal EPSPS cDNA sequence and the alternative splicing sequence. To determine the function of this alternative sequence, we constructed a prokaryotic expression vector in which the sequence was inserted into the pET32a vector, then transformed into host bacteria Escherichia coli BL21(DE3) ΔaroA.The alternative splicing sequence was shown to lack the function of the normal EPSPS cDNA sequence. This study enriches what is known about cotton EPSPS transcriptsand lays the foundation for further research into the transcriptional mechanisms of cotton EPSPS.

Key words: alternative splicing; cotton; EPSPS; prokaryotic expression

中图分类号: 
  • S562.01