TRV- based virus induced gene silencing (VIGS) of MADS box gene(s) in Petunia hybrida

Abstract

The Petunia hybrida, an ornamental plant is a member of the Solanaceae family and considered as research model plants in molecular biology. In the present study, publically available RNA seq data of flower bud, flower stage1 and flower stage2 of Petunia downloaded from European Nucleotide Archive. The finding analysed in-silico revealed total number of 3634, 3349, 2955 DEGs in flower bud_vs_stage1, flower bud_vs_flower stage2 and flower stage1_vs_ flower satge2 were observed respectively. The comparison of DEGs in flower bud_vs_stage1, flower bud_vs_flower stage2 and flower stage1_vs_ flower satge2 revealed 1919, 2175, 1805 up-regulation of genes while 1715, 1179, 1150 gene were down-regulated. The transcriptome analysis revealed 38 M-type and 28 MIKC type MADS box gene expression in three different flower stages and leaf tissue. In present study, the work was focused on molecular and functional characterization of PhFBP20, PhFBP21 and PhFBP28 MIKC type MADS box genes in P. hybrida, to enhance our knowledge about their role in regulation of flower development and reproductive transitions. For the molecular characterization, amplified the CDS of 651bp, 657bp and 648bp genes from the cDNA of leaf of P. hybrida encoding 216, 218 and 215 amino acid in PhFBP20, PhFBP21 and PhFBP28 respectively. Further, the protein multiple sequence alignment and conserved motif analysis of PhFBP20, PhFBP21 and PhFBP28 revealed the presence of highly conserved MADS-box and a K-box domain respectively. The expression pattern of PhFBP20, PhFBP21 and PhFBP28 genes from three different tissue (leaf, sepal and petal) and three different flower stages (initial bud: S1, mature bud: S2 and open flower without petal: S3) of Petunia hybrida suggested their requirement in floral developmental processes. The 3-D proteins structure modeling of Petunia hybrida (PhFBP20, PhFBP21 and PhFBP28) MADS box proteins were predicted by PHYRE2 showed α-helix region 58%, 61% and 57% while, β-sheets 4%, 5% and 5% respectively. The in-silico functional analysis of modelled structure of PhFBP20, PhFBP21 and PhFBP28 proteins revealed the high conservance, good alignment with other reported template and significant protein pocket in active site. For VIGS study, we developed and validated TRV constructs (TRV2:PD, TRV2:CHS, TRV2:CHS:PhFBP20 and TRV2:CHS:PhFBP21, TRV2:CHS:PhFBP28) in pTRV2 vector backbone using PhFBP20, PhFBP21 and PhFBP28 genes along with CHS as scorable marker for efficient symptomatic (phenotypic) validation. During experimental observations the silencing of CHS & PDS genes in terms of their corresponding visible phenotypic symptoms in agro-inoculated plants were observed. The phenotypes were observed in TRV2:CHS:PhFBP28 agro-inoculated plants which suggest delayed flowering time in Petunia.