陆地棉基因GhMIPS1A的克隆及功能分析

Cloning and functional analysis of GhMIPS1A gene in upland cotton(Gossypium hirsutum L.)

【目的】对肌醇-1-磷酸合酶(myo-inositol-1-phosphate synthase,MIPS)基因GhMIPS1A进行功能分析,探究其在陆地棉纤维发育及抗逆过程中的作用。【方法】通过系统发育分析、基因结构分析、保守基序分析、荧光定量分析、烟草瞬时转化、拟南芥过表达试验、病毒诱导的基因沉默(virus-induced gene silencing,VIGS)技术等对GhMIPS1A基因进行研究。【结果】从陆地棉TM-1中克隆获得GhMIPS1A基因,通过生物信息学分析发现该基因编码蛋白中存在4个高度保守的序列,分别为GWGGNNG、LWTANTERY、NGSPQNTFVPGL和SYNHLGNNDG。亚细胞定位结果显示,GhMIPS1A蛋白位于细胞膜。在拟南芥中过表达GhMIPS1A基因可促使拟南芥根长显著增加,肌醇含量提高1倍以上。空间表达模式分析表明,GhMIPS1A基因在根、茎、叶、纤维中优势表达,且在高衣分品种新陆早18号中的表达量显著高于其在低衣分品种德字棉531中的表达量;表达模式分析表明,Gh MIPS1A基因在纤维发育早期阶段表达量较高。利用VIGS技术沉默TM-1的GhMIPS1A基因,3个株系的棉纤维密度显著降低,衣分分别降低4.48、4.93、3.95百分点,肌醇含量分别降低31.83%、32.90%、29.46%。在干旱处理或盐处理下,GhMIPS1A基因的表达量均表现出先升高后降低的趋势。【结论】GhMIPS1A在棉纤维发育中发挥积极作用,并且能够响应干旱胁迫和盐胁迫,可以为培育优质高产、耐盐耐旱的棉花新品种提供基因资源和遗传基础。

[Objective] The purpose of this study is to analyze the function of myo-inositol-1-phosphate synthase gene GhMIPS1A in fiber development and stress resistance of upland cotton(Gossypium hirsutum L.). [Method] Phylogenetic analysis, gene structure analysis, conserved motif analysis, quantitative real-time polymerase chain reaction(qRT-PCR), tobacco transient transformation over-expression in Arabidopsis, and virus-induced gene silencing(VIGS) were performed to study the functions of GhMIPS1A. [Result] GhMIPS1A gene was cloned from upland cotton TM-1. Bioinformatics analysis showed that there were four highly conserved motifs, which were GWGGNNG, LWTANTERY, NGSPQNTFVPGL, and SYNHLGNNDG.GhMIPS1A was localized to the cell membrane. Overexpression of GhMIPS1A gene in Arabidopsis thaliana resulted in elongation of root, and a more than 1-fold increase of inositol content. The temporospatial expression pattern analysis showed that GhMIPS1A gene was highly expressed in root, stem, leaf, and fiber, and the expression level in Xinluzao 18 with high lint percentage was significantly higher than that of in Delfos 531 with low lint percentage. And GhMIPS1A gene had a high expression level at the initial stage of fiber development. Compared with the wild type, the cotton fiber density of three GhMIPS1A-silenced lines significantly decreased, and the lint percentage decreased by 4.48, 4.93, and 3.95 percentage points,and the inositol content decreased by 31.83%, 32.90%, and 29.46%, respectively. Under drought treatment or salt treatment, the expression level of Gh MIPS1A gene increased first and then decreased. [Conclusion] GhMIPS1A gene plays an positive role in cotton fiber development and can respond to drought stress and salt stress, which can provide genetic resources and genetic basis for the breeding of new cotton varieties with high yield, fine fiber, and salt and drought tolerance.