陆地棉GhUGP1基因的克隆与表达分析

Cloning and Expression Analysis of GhUGP1 Gene from Gossypium hirsutum

为进一步了解UGPase在棉花中的作用,根据全基因组测序结果,筛选且克隆了在陆地棉纤维发育过程中的关键基因GhUGP1。利用生物信息学方法对其核苷酸和氨基酸序列进行分析,通过同源重组技术,将GhUGP1基因的编码序列构建到原核表达载体pMAL-C4x上,通过IPTG诱导蛋白表达来确定IPTG诱导蛋白的最佳条件,最后利用Western Blot鉴定重组蛋白。结果显示,GhUGP1(XP_040931642.1)全长序列6572 bp,编码区1404 bp,编码467个氨基酸,预测分子质量约为51.493 ku,等电点为5.86。氨基酸序列比对分析发现在陆地棉、亚洲棉、木槿等不同物种间UGP序列的相似率为90.63%。进化树分析结果显示GhUGP1蛋白与亚洲棉UGP蛋白亲缘关系最近,同源性最高并在一个分支上。亚细胞定位结果显示该蛋白为核膜共定位。蛋白诱导时由于IPTG浓度梯度结果差别不明显,选择IPTG终浓度为0.3 mmol/L,而温度梯度和时间梯度结果差异明显,确定最佳诱导温度为28℃,最佳诱导时间为6 h,蛋白溶解及纯化的温度和时间为28℃诱导6 h。Western Blot结果表明重组蛋白的大小正确,最终成功获得了大小为95.9 ku的pMAL-C4x-GhUGP1重组蛋白,为后期对GhUGP1功能深度解析提供帮助。

This study aimed to understand the role of UGPase in cotton further.GhUGP1,a key gene in fibre development of Gossypium hirsutum,was screened and cloned based on whole genome sequencing results.Bioinformatics was then employed to analyze GhUGP1 nucleotide and amino acid sequences,and its coding sequences were subsequently inserted into a prokaryotic expression vector pMAL-C4x through the homologous recombination technique.IPTG-induced protein expression was used to determine the optimal conditions for IPTG-induced protein.Finally,Western Blot was used to identify the recombinant protein.The results showed that the full-length sequence of GhUGP1(XP_040931642.1)is 6572 bp,with a coding region of 1404 bp,encoding 467 amino acids,a predicted molecular mass of about 51.493 ku,and an isoelectric point of 5.86.Amino acid sequence comparison analysis revealed that the similarity rate of UGP sequences among different species,such as Gossypium hirsutum,Gossypium arboreum,and Hibiscus syriacus was 90.63%.The results of evolutionary tree analysis showed that the GhUGP1 protein was closest to the UGP protein from Gossypium arboreum,with the highest homology,and on the same branch.The results of subcellular localization showed that the protein was co-localized at the nuclear membrane.The results of protein induction were not obvious due to the IPTG concentration gradient;thus,we chose the condition that the final concentration of IPTG was 0.3 mmol/L.The results of the temperature gradient and time gradient were obvious:the optimal induction temperature was 28℃,the optimal induction time was 6 h,and the temperature and time of protein solubilization and purification were 28℃with induction for 6 h.The Western Blot results showed that the size of the recombinant protein was correct,and finally,the pMAL-C4x-GhUGP1 recombinant protein with a size of 95.9 ku was successfully obtained,which will help to analyze the function of GhUGP1 in depth at a later stage.