Screening And Functional Analysis Of Hsf Genes Regulating Benzene/Phenylpropanoid Synthesis In Petunia

Petunia hybrida is one of the most widely used herbaceous flowers in gardens around the world.Volatile benzene/phenylpropane compounds are the most important class of floral aroma compounds in petunia cultivars with flower fragrance.Heat shock transcription factor(HSF)is a major regulator involved in the heat stress response,and it has also been found to regulate the biosynthesis of benzenes/phenylpropanes and other secondary metabolites.This study systematically analyzed the bioinformatics characteristics of the petunia HSF gene family,detected the expression of candidate genes,and selected the petunia key gene PhHSF19 to study its interaction with target genes involved in floral aroma regulation.The main findings of this study are as follows:1.Bioinformatics analysis of petunia HSF gene family: 34 HSF gene family members were obtained in petunia,and most of the petunia HSF genes contained two exons and one intron.Phylogenetic analysis showed that 23 petunia HSFs belonged to the largest HSFA subfamily,eight petunia HSFs belonged to the HSFB subfamily,and there were only two petunia HSFs in the HSFC subfamily.DBD and NLS domains were present in almost all petunia HSFs,some conserved motifs were present in specific HSF subfamily,and AHA motif and NES motif were only present in HSFA subfamily.The HSF binding elements HSE and/or STRE were present in the promoters of many petunia HSF promoters and most petunia HSF promoters contain STRE motif,which was also the largest number of cis-acting elements.2.PhHSF expression analysis and selection of PhHSF19: Based on the reported transcriptome sequencing data of the day 0(bud)or day 2(post-anthesis)corolla tissue of P.hybrida ‘Mitchell’,six candidate genes for PhHSFs were screened.q RT-PCR analysis of the candidate genes in different tissues and developmental stages showed that PhHSF19 was highly expressed in petal tubes,followed by stems and petal limbs.During different flower development stages,the expression level of PhHSF19 was significantly higher at early flowering stage than that at bud stage.3.Interaction verification between PhHSF19 and Ph PAL2 promoter: PAL was the first key gene in the phenylpropanoid pathway,the expression level of Ph PAL2 was significantly higher at early flower opening stage than that at bud stage.The expression pattern of PhHSF19 was positively related with that of Ph PAL2.Three STRE elements are present in the promoter of Ph PAL2.The interaction between PhHSF19 and Ph PAL2 promoter was proved by yeast one-hybrid assays.Based on dual luciferase assay,PhHSF19 could positively regulate the expression of Ph PAL2.