陆地棉磷高效基因GhMGD3的克隆与表达分析

Cloning and expression of phosphorus efficient gene GhMGD3 in Gossypium hirsutum

【目的】基于前期陆地棉Gossypium hirsutum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GhMGD3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GhMGD3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)的方法检测该基因在根、茎、叶、花4个组织中的基因表达量的变化以及低磷胁迫下的表达模式。【结果】成功克隆了陆地棉GhMGD3基因。GhMGD3基因的编码序列全长为681 bp,共编码226个氨基酸,存在3个内含子,分子量是26 610.54 Da,等电点为8.74,是稳定的亲水碱性蛋白。二级结构以α-螺旋和无规则卷曲为主。该蛋白不存在信号肽、跨膜结构域、N-糖基化位点,但含有多个磷酸化位点。亚细胞定位结果显示:该基因编码的蛋白定位于叶绿体。GhMGD3蛋白与木槿Hibiscus syriacus氨基酸序列相似性较高,其亲缘关系最近。半定量RT-PCR和RT-qPCR试验结果均表示:GhMGD3基因主要表达于根,中量表达于茎,微量表达于叶和花,在低磷胁迫72 h时其相对表达量达到最高值。【结论】首次成功克隆到了陆地棉GhMGD3基因,获得了GhMGD3基因的组织表达以及低磷胁迫下的表达模式,GhMGD3基因在棉花磷高效利用信号调控中具有重要作用。图7表1参27。

[Objective] Based on the genome-wide expression profile of cotton seedlings under low phosphorus stress in the early stage of our research group, related genes were excavated and their preliminary expression analysis was conducted. [Method] Genomic DNA and cDNA sequence of the gene were cloned and 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. The expression patterns of GhMGD3 under low phosphorus stress were analyzed by RT-qPCR technology. [Result] GhMGD3 gene was cloned. The coding sequence of GhMGD3 was 681 bp, encoding 226 amino acids and containing 3 introns. The molecular weight is 26 610.54 Da, isoelectric point is 8.74, it is a stable hydrophilic alkaline protein. The secondary structure is dominated by α-helix and random crimp. The protein does not have signal peptide, transmembrane domain and N-glycosylation site, but contains multiple acidification sites. Subcellular localization showed that the gene encoded a protein in chloroplast. GhMGD3 protein and Hibiscus syriacus protein amino acid sequence similarity is high, the most recent genetic relationship. The results of semiquantitative RT-PCR and RT-qPCR showed that GhMGD3 gene was mainly expressed in root, medium expression in stem, and trace expression in leaf and flower,and its expression level reached the highest value at 72 h of low phosphorus stress. [Conclusion] The GhMGD3 gene was preliminarily obtained. The expression patterns of GhMGD3 in different tissues and under low phosphorus stress were analyzed. GhMGD3 gene has an important function in regulation of the efficient utilization of phosphorus in cotton. [Ch, 7 fig. 1 tab. 27 ref.]