低磷胁迫大豆RT-qPCR内参基因的筛选及谷胱丙肽合成代谢酶的表达

Selection of reference genes for RT-qPCR and expression of genes involved in homoglutathione anabolism in soybean under stress of phosphorus deficiency

关键词:  大豆, 内参基因, 实时荧光定量PCR, 谷胱丙肽合成代谢, 低磷

Key words:  soybean, reference gene, homoglutathione anabolism, RT-qPCR, phosphorus deficiency

摘要:

谷胱丙肽在作物耐低磷中起重要作用,而选择稳定的内参基因是保证实时荧光定量PCR(RT-qPCR)结果准确的前提。该研究检测了低磷和正常条件下两个不同磷效率的大豆品种根叶中15个内参基因的表达,GeNorm、NormFinder、BestKeeper、ΔCt对比和RefFinder分析表明PSC、TUB和18sRNA的稳定性最好; 同时以这3个基因为内参,检测了不同磷胁迫时期谷胱丙肽合成代谢中两个关键基因谷氨酸半胱氨酸合成酶( γ-ECS)和谷胱丙肽合成酶(hGSHS)的表达水平。结果表明:低磷胁迫下,磷低效品种桂香1号(GX1)根叶中γ-ECS和hGSHS的相对表达量(Fold change, FC)都随着胁迫时间延长而减少; 磷高效品种桂夏2号(GX2)根叶γ-ECS以及根中的hGSHS的相对表达量都随着胁迫时间延长而增加,而叶片中hGSHS的相对表达量随胁迫时间增加而降低。谷胱丙肽合成酶基因表达差异可能是品种磷效率差异的原因。该研究结果为大豆低磷抗性分子机理研究提供了理论依据。

Abstract:

Selection of stable reference gene(s)is the prerequisite for RT-qPCR analysis. Homoglutathion plays an important roles in utilization of phosphate in plants. In this study, the expression of fifteen candidate reference genes were determined in two soybean varieties differing in phosphorus efficiency under normal and stress of low phosphorus availability. Fifteen candidate reference genes were validated by GeNorm, NormFinder, BestKeeper and ΔCt comparision and RefFinder analysis, and out of those, the three most stable genes, PSC, TUB and 18sRNA, were used to normalize the expression of two genes, γ-glutamylcysteine synthetase (γ-ECS)and homoglutathione synthetase(hGSHS), involved in homoglutathion anabolism. In the low P efficient soybean variety GX1, the expression fold changes(FC, as compared to normal)of γ-ECS and hGSHS in roots and leaves all decreased with the rise of stress duration; while in the high P efficient variety GX2, the FCs of γ-ECS in roots and leaves, as well as hGSHS in roots increased with the increase of stress duration; except that hGSHS in leaves decreased. The results suggest that the difference of gene expression might account to the phosphorus utilization efficiency.