白三叶质子焦磷酸酶基因TrVP1生物信息学分析及定量表达

Bioinformatics Analysis and Quantitative Expression of Proton Pyrophosphatase Gene TrVP1 from Trifolium repens cv. ‘Ladino'

质子焦磷酸酶(H^+-PPase)在提高植物抗逆性方面发挥着重要的作用。本试验以白三叶品种‘拉丁诺’(Trifolium repens cv.‘Ladino’)c DNA为模板克隆出了白三叶H^+-PPase基因TrVP1的全长序列,并利用相关分析软件和网站对其进行生物信息学分析,并研究它在非生物胁迫及信号物质诱导下的表达模式。结果表明,TrVP1全长为2 594 bp,开放阅读框(ORF)为2 304 bp,共编码767个氨基酸;其编码蛋白为亲水性跨膜蛋白,分子量80.49 k D,等电点p I 5.18,多序列比对及系统进化树分析显示该基因属于Ⅰ型VP家族。采用荧光定量(qRT-PCR)方法分析TrVP1的表达模式发现,重金属(CdSO_4)、低温(4℃)、高温(35℃)、干旱(PEG-6000)和盐(NaCl)胁迫均显著上调了白三叶根系中TrVP1的表达量,但下调了叶片中TrVP1的表达量。此外,白三叶根系中TrVP1的表达受到Ca^(2+)信号的显著诱导,而叶片中的TrVP1表达则受到抑制。与此同时,NO和H_2O_2信号对白三叶根系和叶片中TrVP1的表达均起到抑制作用。本试验为深入研究质子焦磷酸化酶基因,提高白三叶抗逆性的功能及其应用提供了理论依据。

Proton pyrophosphatase （H^＋-PPase） plays a key role in the improvement of tolerance to stress in plants. In this study, we cloned the complete TrVP1 sequence from the H^＋-PPase gene of white clover based on the template of the cDNA of white clover cultivar （Trifolium repens cv.‘Ladino＇）. Bioinformatics analysis was applied to the sequence by relevant analysis software and websites to study the expression pattern under abiotic stress and signal material induction. The result showed that the full length of TrVP1 was 2594 bp with an open reading frame of 2304 bp, encoding 767 amino acids. The encoded protein was a hydrophilic transmembrane protein with a molecular mass of 80.49 kD and a pI of 5.18. Multiple sequence alignment and phylogenetic tree analysis indicated that the TrVP1 gene belonged to the VP family of type I. Fluorescent quantitative analysis （qRT-PCR） of TrVP1 expression pattern revealed that TrVP1 expression level of white clover roots were significantly increased by heavy metal （CdSO4）, low temperature （4℃）, high temperature （35℃）, drought （PEG-6000） and salt （NaCl） stress, but the TrVP1 expression in leaf was decreased under the same situations. In addition, expression of TrVP1 in white clover roots was remarkably induced by Ca^2＋ signal, while was inhibited by the signal in leaf. At the same time, NO and H2O2 signal could both inhibit the TrVP1 expression in leaf and root of whit clover. The study could provide a theoretical basis for the further research on proton pyrophosphorylase gene and the improvement of the function and application of stress tolerance of white clover.