Identification of TrPLD Gene Family of Trichothecium roseum and Gene Expressions During the Pathogen Infection Muskmelon Fruit

doi:10.7668/hbnxb.20193186

Abstract

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

LIU Qili, ZHANG Qianqian, XUE Huali, BI Yang, ZONG Yuanyuan, PENG Hui. Identification of TrPLD Gene Family of Trichothecium roseum and Gene Expressions During the Pathogen Infection Muskmelon Fruit[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(6): 63-71. doi: 10.7668/hbnxb.20193186.

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Figures/Tables 9

Tab.1 The primers used for Real-time quantitative

基因名称 Gene name	引物序列(5' —3') Primer sequences(5'— 3')
TrPLD1	F: CACACCGACAACCGCTATCA
	R: AAGTACGAGGCACCGTCAACA
TrPLD2	F:GTATGCCGTGGCACGATGT
	R:CCAACGCAACACGAAATGTT
TrPLD3	F: TGTACAAAGATCCCCGAACCA
	R: CGTGATCCACAACGCAGATTT
TrPLD4	F:ACGCCCTGGGAGGTGTTCTTC
	R:CGTAGCGGTCGCAGTGGATTT
TRACTIN	F: CAGACCAACTGCGATCAGGT
	R: AAAGTCCTTAGGCGTGCGAA

Fig.1 Genetic structures of TrPLD1,TrPLD2,TrPLD3 and TrPLD4

Fig.2 Domain analysis of TrPLD1,TrPLD2,TrPLD3 and TrPLD4 PX.Phox homology domain;PH. Pleckstrin homology domain;PLDc domain contains the HKD motif forming the active site.

Fig.3 Phylogenetic tree of TrPLD1,TrPLD2,TrPLD3 and TrPLD4

Fig.4 Prediction of transmembrane domains(A—D),signal peptides(E—H),and hydrophobicity predictions(I—L) of TrPLD1,TrPLD2,TrPLD3 and TrPLD4 proteins

Fig.5 TrPLD1,TrPLD2,TrPLD3 and TrPLD4 genes coding products of secondary and tertiary structure

Tab.2 Prediction of TrPLD1,TrPLD2,TrPLD3 and TrPLD4 subcellular localization

蛋白名称 Protein name	细胞核 Nuclear	质膜 Plasma membrane	细胞外 Extracellular	细胞质 Cytoplasmic	线粒体 Mitochondrial	内质网 Endoplasmic reticulum	过氧化酶体 Peroxisomal	溶酶体 Lysosomal	高尔基体 Golgi
TrPLD1	3.08	1.09	2.71	0.66	1.75	0.00	0.37	0.00	0.35
TrPLD2	0.05	5.91	0.00	4.03	0.00	0.00	0.00	0.01	0.01
TrPLD3	2.65	2.02	1.84	0.69	1.09	0.03	0.44	0.00	1.25
TrPLD4	0.82	2.75	1.68	1.55	1.99	0.26	0.40	0.02	0.42

Fig.6 Protein phosphorylation sites analysis of TrPLD1,TrPLD2,TrPLD3 and TrPLD4 proteins

Fig.7 Expression of TrPLD1,TrPLD2,TrPLD3 and TrPLD4 genes infected melon fruit Vertical line indicating standard error(±s); different letters indicating significant differences(P<0.05).

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