棉花EPSPS基因一个可变剪接体的克隆及功能分析

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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c DNA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p ET32a,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)Δaro A,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。 Variable splicing is a common phenomenon in the transcriptional process of biological genes. This phenomenon is one of the important reasons leading to the diversity of functional proteins. However, there are relatively few reports of alternative splicing events of functional genes in cotton. In this study, a cloned cotton EPSPS (5-enolpyruvylshikimate 3-phosphate synthase) gene was found in an alternative splicing sequence of this gene. Using bioinformatics methods, it was found that this sequence was 152 bp less than the c DNA sequence of the normal EPSPS gene, which resulted in the early appearance of the stop codon in the sequence. The normal EPSPS gene intron cleavage followed the common The “GT-AG” cleavage rule of the variable splicing sequence is based on the “AT-AC” rule. The predicted three-dimensional structural model of the alternative splicing sequence is the same as the normal There are significant differences in the three-dimensional structure of the EPSPS gene. The variable splicing sequence was inserted into the prokaryotic expression vector p ET32a, and then the recombinant prokaryotic expression vector was transformed into Escherichia coli BL21 (DE3) Δaro A, which was found by the growth study in M9 minimal medium The sequence does not have the function of the normal EPSPS gene. The results of this study enriched the form of cotton EPSPS gene transcripts and laid the foundation for further study of the transcriptional mechanism of cotton EPSPS gene.
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