陆地棉低磷胁迫应答基因GhGDPD1的克隆与表达分析

Cloning and expression analysis of low phosphorus stress response geneGhGDPD1 in Gossypium hirsutum

【目的】基于前期陆地棉‘新陆早19’Gossypium hirsutum‘Xinluzao 19’根部低磷胁迫基因表达谱芯片差异表达序列数据分析,挖掘相关基因,并对其克隆与表达分析。【方法】克隆陆地棉‘新陆早19’GhGDPD1基因并进行基因组DNA与cDNA测序分析,借助生物信息学方法分析GhGDPD1的基因结构和进化关系;采用半定量RT-PCR技术与实时荧光定量PCR (RT-qPCR)的方法检测该基因于根、茎、叶、花等4个组织的基因表达量的变化以及低磷胁迫下的表达模式。【结果】成功克隆GhGDPD1基因,开放阅读框序列长度为1 149 bp,共编码382个氨基酸,属于GDPD家族,其中存在一个保守结构域,名为GDPD_GDE5_like_1_plant。半定量RT-PCR和RT-qPCR试验结果均显示:GhGDPD1基因主要表达于根,中量表达于花和茎,微量表达于叶。该基因在受到低磷胁迫的刺激后,立即会对低磷胁迫做出应答,胁迫4 h相对表达量达到最高值。【结论】首次成功克隆到陆地棉‘新陆早19’GhGDPD1基因,获得了GhGDPD1基因的组织表达以及低磷胁迫下的表达模式,为深入解析棉花GhGDPD1基因的生物学功能以及培育棉花磷高效利用新种质提供科学参考。

[Objective]Based on the analysis of differential expression sequence data of genome-wide expression profile of Gossypium hirsutum‘Xinluzao 19’seedlings under low phosphorus stress in our previous research,this study aims to explore the related genes,and to clone and analyze their expression,so as to provide a scientific reference for further research on the biological functions of GhGDPD1 gene and the cultivation of new cotton germplasm with the efficient utilization of phosphorus.[Method]GhGDPD1 gene was cloned from‘Xinluzao 19’.Genomic DNA and cDNA sequence of the gene were performed,and the genetic structure and evolutionary relationship of GhGDPD1 were analyzed by bioinformatics method.Semi-quantitative RT-PCR and fluorescence quantitative PCR(RT-qPCR)were used to detect the changes of gene expression in root,stem,leaf and flower tissues,as well as the expression pattern under low phosphorus stress.[Result]The GhGDPD1 gene was cloned,with an open reading frame sequence length of 1149 bp,encoding 382 amino acids.This gene sequence belonged to the GDPD family,with a conservative structure domain named GDPD_GDE5_like_1_plant.Semi-quantitative RT-PCR and RT-qPCR showed that GhGDPD1 gene was mainly expressed in roots,moderately expressed in flowers and stems,and slightly expressed in leaves.After being stimulated by low phosphorus stress,the gene would immediately respond to low phosphorus stress and its expression level reached the highest value after 4 hours of stress.[Conclusion]The GhGDPD1 gene of‘Xinluzao 19’is successfully cloned for the first time.The tissue expression of GhGDPD1 and its expression pattern under low phosphorus stress are obtained.