摘要：

为了进一步分析粉红单端孢中TrPLD家族基因的生物学特性和在致病过程中的表达特性,通过对粉红单端孢全基因组测序得到的4个TrPLD基因,利用SMART、MEGA 7、ProtScale、SOPMA等软件对TrPLD基因家族进行生物信息学分析;应用RT-qPCR技术分析粉红单端孢在模拟侵染甜瓜果实过程中不同TrPLD基因的表达情况。系统发育树分析表明,TrPLD1与炭疽菌亲缘关系最近,同源性为63.89%;TrPLD2与淡紫拟青霉同源性为74.57%;TrPLD3和TrPLD4分别与禾谷镰刀菌和铜绿假单胞菌亲缘关系最近,同源性分别为65.38%和55.45%。生物信息学分析表明,TrPLD1、TrPLD2、TrPLD3和TrPLD4蛋白均具有PLD的2个保守结构域HKD基序,除HKD结构域外,TrPLD3还包含PX和PH结构域,且4种蛋白均属于亲水性不稳定蛋白,不含信号肽和跨膜结构,属于非分泌蛋白,4种蛋白包含不同数量的磷酸化位点,其中TrPLD3包含的数量最多;通过亚细胞定位预测发现,TrPLD1和TrPLD3主要定位于细胞核上,TrPLD2和TrPLD4主要定位于质膜上;RT-qPCR分析表明,在粉红单端孢模拟侵染甜瓜果实过程中,以CK表达量为对照,TrPLD1、TrPLD2、TrPLD3和TrPLD4的基因表达量随侵染过程的发生显著上调,其中TrPLD3基因表达量显著高于TrPLD1、TrPLD2和TrPLD4。综上表明,4个TrPLD具有不同的结构和生物学功能,其中,TrPLD3在粉红单端孢侵染甜瓜果实过程中发挥重要的调控作用。

关键词: 粉红单端孢, 磷脂酶D, 生物信息学分析, 表达分析

Abstract:

To further analyze the biological characteristics and expression of TrPLD family genes in Trichothecium roseum,four TrPLD genes were obtained by the whole-genome sequencing of Trichothecium roseum,the online tools of SMART,MEGA 7,ProtScale,SOPMA and other software were used to conduct bioinformatics analysis of TrPLD genes and their encoded proteins;RT-qPCR technology was employed to analyze TrPLD genes expression during the pathogen infection muskmelon fruit.Phylogenetic tree analysis showed that TrPLD1 had the closest genetic relationship with Colletotrichum aenigma with a homology of 63.89%;TrPLD2 had 74.57% homology with Purpureocillium lilacinum;TrPLD3 and TrPLD4 had the closest genetic relationships with Fusarium graminearu and Paecilomyces lilacinus,with homology of 65.38% and 55.45%,respectively.Bioinformatics analysis showed that the proteins of TrPLD1,TrPLD2,TrPLD3 and TrPLD4 all had two conserved domain HKD motifs.TrPLD3 contained PX and PH domains,besides the HKD structure.The four proteins belonged to unstable hydrophilic proteins,without signal peptide and transmembrane structure,and belonged to non-secretory proteins.The four proteins contained different numbers of phosphorylation sites,among which,TrPLD3 contained the most phosphorylation sites.Subcellular localization prediction showed that TrPLD1 and TrPLD3 were mainly located in the nucleus,and TrPLD2 and TrPLD4 were mainly located in the plasma membrane.RT-qPCR analysis showed that,during of simulation the pathogen infecting muskmelon fruit,taking the control expression as a control,the gene expressions of TrPLD1,TrPLD2,TrPLD3 and TrPLD4 increased significantly with the infection process,and the expression of TrPLD3 gene was significantly higher than other genes during the whole infection process.In summary,the four TrPLDs have different structures and biological functions,among which TrPLD3 plays an important regulatory role during the process of T.roseum infecting muskmelon fruit.

Key words: Trichothecium roseum, Phospholipase D, Bioinformatics analysis, Expression analysis