| The C2H2-type zinc finger protein transcription factor family is a proteins family containing ZnF-C2H2 conserved domains.The family members,which are widely involved in regulating growth and development,stress response and production of secondary metabolites,widely exist in plant pathogenic fungi,such as Magnaporthe oryzae,Fusarium graminearum and Sclerotinia sclerotiorum.However,it has not been reported in the pathogen of Setosphaeria turcica.Therefore,in the present study,the C2H2 zinc finger protein transcription factor family was identified in the genome of S.turcica.The structural characteristics,physicochemical properties and protein interaction among the family members were analyzed.The expression patterns of C2H2 zinc finger protein family members in pathogen growth and development,and infection were further clarified by RNA-Seq technique.The main results are as follows:1.A total of 27 C2H2-type zinc finger protein transcription factor family members were identified in S.turcica.Each member contained at least one highly conserved ZnF-C2H2 conserved domain.Analysis of gene structure showed that 88.9% of the genes contained more than one intron and most of them were short introns.The number of exons was up to 8,and the most number of introns was 7.The phylogenetic tree of S.turcica,S.cerevisiae and M.oryzae was constructed.According to the affinity relationship,all the C2H2-type zinc finger protein transcription factor genes were divided into four subfamilies,and 77.8% of StZFs and MoZFs were clustered together.We speculated that the StZFs and MoZFs had a close evolutionary relationship.Conservative motif analysis revealed that family members had six classes of conserved motifs,in which motifs 1,2 and 3 were the core sequences in the protein family.2.Based on the protein interaction network of S.cerevisiae,the protein interaction network of members of the StZFs family was constructed by homology mapping.In order to verify the reliability of the protein interaction network,a node protein(StZF8)and two protein(StZF7 and StZF16)with strong interaction in the network were selected to further identify by yeast two-hybrid test.The results showed that StZF7 and StZF16,StZF7 and StZF8,StZF8 and StZF16 could interact directly in yeast,however,the interaction between StZF8 and StZF16 was weak.The results further confirmed that the accuracy of the above-mentioned protein interaction network.3.By analyzing the RNA-Seq data from 5 key growth and development stages including hypha,conidium,germ tube,appressorium and penetration peg.12 of the 27 family members expressed at five different developmental stages and the expression level of StZF1/5/7/11/16/22 were relatively high,suggesting that these genes may be involved in regulating the growth and development of the pathogen.In addition,five of them(StZF1/5/7/19/22)were randomly selected to do qRT-PCR analysis.The results showed that their expression trends were consistent with the transcriptome data obtained above.4.The RNA-Seq data of resistance(B73Ht)and susceptibility(B73)maize varieties infected by S.turcica for 24 hours were analyzed.It was found that 23 family members were expressed and the expression level of StZF2/7/8/11/12/16/22 was enhanced after infection of resistant and susceptible varieties.In addition,with conidium suspension as control,8 family members in all expressed family members were found in resistant varieties(B73).The expression level was significantly up-regulated,and no genes with significantly down-regulated was found;in the susceptible variety(B73Ht),11 family members were significantly up-regulated,and 3 members were significantly down-regulated.At the same time,8 genes(StZF4/11/12/14/17/18/23/25)were randomly selected for qRT-PCR analysis.It was found that the expression levels of 8 genes were up-regulated compared with the conidia stage,and the expression trends consistent with the results of RNA-Seq.Above-mentioned results indicated that members of the StZFs family might play an important role in the infection process. |
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