棉花内切-β-1,4-葡聚糖酶(EG17)基因的克隆及生物信息学分析

Cloning and Bioinformatics Analysis of Endo-β-1,4-glucanase(EG17)Gene in Cotton

EG17基因在植物器官的的脱落过程中发挥一定作用。根据已公布的拟南芥EG17基因序列的CDS设计引物,从陆地棉‘新陆早50号’中克隆一个同源基因GhEG17(Endoglucanse 17-like,GenBank登录号:NM_001327069.1)核苷酸序列,并利用qRT-PCR技术研究脱吐隆悬浮剂(TDZ)处理后叶托组织中GhEG17基因的表达模式。结果表明,获得的GhEG17基因开放阅读框(ORF)为1533 bp,编码510个氨基酸。通过结构域分析,发现Gh EG17中含有一个GH9家族特有的保守域结构,是GH9家族基因所特有的Glyco_hygro_9(GH9)结构域。GhEG17蛋白二级结构预测显示,α-螺旋占27.65%,延伸链占20.00%,无规则卷曲占52.35%。系统进化树分析结果表明GhEG17和GaEG17亲缘关系较近。qRT-PCR表明,GhEG17基因在‘新陆早50号’和‘XND1378’棉花6个时期棉花叶托组织中的表达量均在18 h中最高,但在品种‘新陆早50号’响应TDZ处理诱导胁迫比‘XND1378’更强烈。利用基因工程技术挖掘并利用棉花脱落相关基因,培育转基因机械采收棉花新品种,以促进机采棉育种的不断完善,具有十分重要的理论价值和现实价值。

EG17 gene plays a role in the exfoliation of plant organs.A pair of primers were designed based on the published CDS sequence of Arabidopsis EG17 gene.A homologous gene GhEG17(Endoglucanse 17-like,GenBank landing number:NM001327069.1)nucleotide sequence was cloned from upland cotton’Xinluzao 50’and’XND-1378’.The expression pattern of GhEG17 gene in the leaves after TDZ treatment was studied by qRT-PCR.The results showed that the open reading frame(ORF)of GhEG17 gene was 1533 bp encoding 510 amino acids.Through domain analysis,we found that GhEG17 contains a conserved domain structure unique to the GH9 family,which is the Glyco hygro9(GH9)domain unique to GH9 family genes.Prediction of secondary structure of GhEG17 protein showed that alpha-helix accounted for 27.65%,extended chain accounted for 20.00%,and irregular curl accounted for 52.35%.Phylogenetic tree analysis showed that GhEG17 and GaEG17 were closely related.QRT-PCR showed that the expression of GhEG17 gene was the highest at 18 h in cotton leaf tissue at six stages of’Xinluzao50’.The highest expression of GhEG17 was f ound in the stipul e tissues of cotton at six stages of’Xinluzao50’and’XND1378’,but GhEG17 responded to TDZ treatment more strongly than’XND1378’in the early’Xinluzao50’.It is of great theoretical and practical value to use genetic engineering technology to excavate and use cotton to exfoliate related genes and cultivate new varieties of transgenic mechanical cotton to promote the continuous improvement of mechanical cotton breeding.