Analysis Of Promoter Characteristics And Downstream Gene Mining Of PhTFL1 In Petunia

Petunia(Petunia hybrida)is a widely used perennial and biennial flower.With the acquisition of petunia genome sequencing,petunia is often used in plant molecular evolutionary biology,flower development and other related research.The TFL1 gene belongs to the FT/TFL1 gene family.In most studies,the function of FT gene is to promote floral transformation,while the function of TFL1 gene focuses on inhibiting flowering and maintaining vegetative growth to promote branch development.In our previous research,we found that Ph FT1 in the FT subfamily of petunia has undergone a functional transformation,showing a flowering-inhibiting phenotype,which replaces the flowering suppressive function exercised by the TFL1 gene;The function of PhTFL1 gene is also different from that of other species.PhTFL1 gene has the highest expression level in the roots,and its function is to affect the development of petunia roots.Overexpression of PhTFL1 gene in petunia will seriously inhibit the root development of petunia plants,while RNAi PhTFL1 transgenic petunia plants show increased lateral roots.In order to further elucidate the molecular mechanism of the petunia PhTFL1 gene affecting plant root development,we analyzed the PhTFL1 gene promoter;We sampled PhTFL1 transgenic lines at seedling stage and performed digital gene expression profiling(RNA-SEQ)to analyze the differentially expressed genes;Finally,we cloned PhTFL1 gene interaction gene Ph VDAC2,preliminarily analyzed the sequence and structure of VDACs gene,and made quantitative expression analysis of different parts.The main findings are as follows:(1)In this study,we analyzed the PhTFL1 gene promoter sequence and found some basic elements such as CAAT-box and TATA-box,we also find some elements related to environmental factors,hormones,and metabolism.These analysis results indicate that the TFL1-like gene promoter may be mainly regulated by hormones,and it is also regulated by many abiotic stresses.The results of our GUS staining experiments also prove that the PhTFL1 gene has specific expression in the roots of plants,which is consistent with the previous quantitative analysis of PhTFL1 gene.(2)We find that overexpression of PhTFL1 will seriously inhibit the root development of petunia,while the number of developed lateral branches of RNAi PhTFL1 transgenic petunia increased.Considering that the root development of PhTFL1 transgenic plants was affected,We sampled at the seedling stage where PhTFL1transgenic lines showed the most significant phenotypic differences and performed transcriptome sequencing.Through the analysis of differentially expressed genes in the transcriptome,we screened for differentially expressed genes in growth-related,hormone-related,lateral branch growth,flower development(flowering induction),enzyme activity,transcription factors,transmembrane transport,etc.We also performed relative quantitative analysis of partial differential gene QRT-PCR,which proved the reliability of transcriptome data.Many genes related to root development were screened,which reduced the range of candidate genes downstream of PhTFL1 gene to a certain extent.(3)VDACs have 4 homologous genes in the petunia genome.Using homologous cloning method,4 VDACs homologous genes were cloned in petunia W115,named Ph VDAC1,Ph VDAC2,Ph VDAC3,PhVDAC4.Through genetic structure analysis,we find that the g DNA of the four VDAC subfamily genes have the same structure: all contain 6 exons and 5 introns(Ph VDAC3 has an extra 12 bp exon),and their c DNA encodes about 277 highly similar amino acids.VDAC1 and VDAC4 were not significantly expressed in different parts of petunia,and the expression levels of VDAC2 and VDAC3 genes were relatively high,of which VDAC2 was the highest in petunia roots.