| A St STE12 gene, encoding transcription factor St Ste12 of Setosphaeria turcica, which located downstream in Fus3/Kss1-homolog pathway has been cloned in our previous research. We obtained the St STE12 silencing transformants by using the RNAi technique. In the preliminary analysis of the gene RNAi transformants, we found that St STE12 gene has an important function in appressorium development and infection process of S. turcica, but the target genes regulated by St Ste12 is not known. It is reported that STE12 gene in Saccharomyces cerevisiae regulated the expression of Homeobox Gene Family, which regulated the expression of more target genes in order to regulating the growth and development of Saccharomyces cerevisiae. Based on this above research, we are trying to identification, classification, evolutionary expansion of Homeobox Gene Family in Setosphaeria turcica genome by bioinformatics analysis and to study the Homeobox genes expression in wild-type and St STE12 silencing transformants by RT-q PCR technique in order to identify the genes probably regulated by St Ste12. The main results are as follows:1. It is shown that there are 8 Homeobox genes according to the bioinformatics analysis in S. turcica, and they are named as St HTF1, St HTF2, St HTF3, St HTF4, St HTF5, St HTF6, St HTF7 and St HTF8.2. The Homeobox transcription family are identified and their characteristics are further analyzed. The members scattered in the genome of S. turcica, and all genes contain introns, the number of introns which are contained in all genes ranged from 1 to 3; These eight genes encode proteins which all contain the conserved homeobox domain(the homeodomain), and the homeodomain located in different positions in each protein.3. From the phylogenetic tree, it can be analyzed that the homeodomain protein in S. turcica can be clustered with other fungal species, suggesting that at the amino acid level the homologous gene in different fungal species show a high degree of similarity. Based on the conserved domain analysis, they can be further subdivided into four subgroups.4. Through the senior of the homeodomain protein structure prediction, shows that the protein of the family has the spatial structure(HTH), and it is coincided with conserved motifs analysis results.5. The expression profiling of Homeobox genes in the wild-type were analyzed during in conidia germination, appressorium formation, penetration peg formation and penetration hyphae formation period(0 h, 3 h, 6 h, 12 h and 24 h) after conidia induced respectively) by RT-q PCR using 18 S r RNA as the reference gene. The expression levels were significantly down-regulated in conidia germination, appressorium formation, penetration peg formation and penetration hyphae formation period(p<0.05). This shows that the Homeobox genes expression is bad for the conidia germination, appressorium formation and infection process.6. Homeobox genes expression level in the wild-type and St STE12 silent transformants was detected using RT-q PCR. Compared with WT, the expression levels of 8 Homeobox genes were significantly upregulated in St STE12 silent transformants. The result indicated that St STE12 silencing relieved the prohibition to Homeobox genes expression.7. The expression vectors of St STE12 gene are constructed using p BARKS1-GFP as plasmid vectors. The PEG-mediated method is used to transform the gene expression vectors into wild-type and the overexpression transformants are obtained by glufosinate screening, PCR identified and RT-PCR analysis. This result provides a basis for subsequent Ch IP test. |
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