Mining The Downstream Regulatory Genes Of Transcription Factor PhMYC2a In Petunia Hybrida Anthers

Petunia hybrida is not only a widely used flower in flower beds around the world,but also a model plants for studying flower development.Studying the regulatory network of petunia stamen development and creating male sterile line materials can shorten the cycle of hybrid breeding and reduce production costs,which has important scientific and practical significance.Our previous studies showed that the transcription factor PhMYC2 a plays an important role in jasmonic acid mediated anther development of Petunia hybrida.In order to further verify the effect of jasmonic acid on anthers development through PhMYC2 a and identify its downstream regulatory genes in Petunia hybrida,this study observed the anther tissue structure of the PhMYC2 a overexpression lines OE-F-16 and the floral organ phenotype of plants treated with methyl jasmonate.Furthermore,the downstream candidate genes of PhMYC2 a were screened through anther RNA-Seq,and their bioinformatics analysis and yeast single hybrid verification were performed.The research results are as follows:(1)Compared with the wild-type Petunia hybrida,the OE-F-16 lines exhibited abnormal anthers,no pollen grain production,and delayed degradation of the tapetum;The expression level of PhMYC2 a gradually increased in the anthers from 0.3 cm to 1.5 cm,and then significantly decreased.Methyl jasmonate treatment led to the reduction of petunia anther size,the significant reduction of pollen germination rate and the number of germinable pollen,and the significant increase of PhMYC2 a expression in anthers.(2)The number of differentially expressed genes in each RNA-Seq differential group ranges from 1514 to 8308,and they were mainly enriched in RNA modification,jasmonic acid mediated signal pathway regulation,heat response,plant hormone signal transduction,and α-linolenic acid metabolism;33 shared differentially expressed genes were obtained in8 differential groups of different strains(OE-F-16 and wild-type)and different treatments(MeJA treated and control group),,including 8 sHSP genes.The q PCR validation results showed that the expression changes of 12 genes between different samples were consistent with the RNA-Seq data;Some genes related to jasmonic acid biosynthesis responded to the stimulation of methyl jasmonate,and the expression of some genes related to jasmonic acid signal transduction in the anthers of OE-F-16 was significantly higher than that of the wild type.(3)The prediction results of cis-regulatory element showed that there were MYB binding elements,MYC binding elements,G-box elements and MeJA response elements in the promoter of 8 sHSP genes;The analysis of gene expression patterns showed that different sHSP genes had different expression trends during the anther development of Petunia hybrida.The results of Y1 H showed that PhMYC2 a showed negative binding to the promoters of HSP21.9,HSP26.9,HSP16.1,HSP40.8,and HSP70 in yeast.Although the relevant research of plant MYC2 and jasmonic acid has been relatively in-depth,but it was mostly related to resistance,and research in ornamental plants was still insufficient.This study used exogenous MeJA treated and transcriptome sequencing to explore the function of PhMYC2 a and jasmonic acid in petunia anther development,tap the downstream regulatory genes of PhMYC2 a,and improve PhMYC2 a centered anther development regulation network of Petunia hybrida.The research results indicate that,PhMYC2 a and jasmonic acid were involved in the regulation of Petunia hybrida anther tapetal degradation and pollen grain development;The biosynthesis of jasmonic acid in Petunia hybrida involves both positive feedback regulation and negative feedback regulation mediated by PhMYC2a;PhMYC2 a may regulate the expression of the above sHSP genes by interacting with other proteins or directly binding to the exon sequence.