Study On Over-expression And CREST Silencing FBP29 Gene Affect Morphological Development In Petunia

MADS-box gene encoded protein which was a kind of important transcription regulatory factors, and it widely existed in eukaryotic organisms. Plant MADS-box gene had vital roles in the vegetative growth and reproductive development. API/FUL subfamily was one of the MADS-box family, and class A genes controlling sepals and petals was belonged to the family, which was found in he process of researching Arabidopsis thaliana and Antirrhinum mutants in the first. A large number of studies had shown that the subfamily gene not only controlled the development and formation of flower organ, also played important roles in deciding in inflorescence meristem characteristics, flowering time, fruit ripening, leaf development and so on. The studying of Petunia PFG gene in the AP1/FUL family showed that it not only had an effect on from vegetative reproduction turning to reproductive growth, but also was related to keep reproductive growth status; occasional flowers of co-suppression plants had no phenotypic changes, which seemed to have nothing to do with flower development A function in petunia. FBP29 gene was another gene of petunia in the subfamily, therefore, through researching it could further explore function of the subfamily gene, rich related theory,promote its application in the creation of new flower varieties. The main contents and results were as follows:1. The cloning of petunia FBP29 gene and sequence analysisAccording to the logged-in Petunia MADS-box transcription factor FBP29 (GeneBank login number:AF335245.1) cDNA encoding frame sequence, we designed and syntheticed specific primers, and made PCR amplification with Petunia MD cDNA as a template, then obtained purpose fragment after sequencing and comparison. The biggest ORF of FBP29 gene had 738 bp, and encoded 245 amino acids. We got a FBP29 gene 5’end and 3’end non-coding region (UTR) sequence by using RACE technology to make PCR amplification, and got FBP29 gene full length cDNA sequences after splicing together. We also obtained FBP29 coding genome sequences by using PCR amplification, and analyzed introns comparing with the cDNA sequence. Full length cDNA sequence had 1377 bp, ORF had 738 bp,5’UTR had 366 bp, and 3’ UTR had 273 bp; Genome sequence had 5781 bp and contained eight exons and seven introns, one of the largest introns had 2.5 Kb. The initial result of BLAST protein analysis showed that FBP29 gene belonging to plant unique MIKC-type MADS-box gene. Phylogenetic analysis showed that the gene belonging to FUL-like clade in AP1/ FUL subfamily. Multiple sequence alignment showed FBP29 gene has high homology with AP1/FUL genes, and whose C terminal contained FUL-like motif.2. The expression analysis of FBP2’9 gene in PetuniaWe applied RT-PCR to detect FBP29 gene expression in petunia organizations, the results showed that the gene expressed in the vegetative organs, such as stems and leaves, except roots, it also expressed in the bud, and the expression increased in turn; And the each part of flower organs also had expression, the sepals was highest expression, but petals and stamens was weak expression.3. The initial function analysis of FBP29 gene in petuniaWe made FBP29 gene and FBP29::SRDX respectively connected to the plant expression vector to construct plant over-expression vector and CRES-T silencing vector and transform petunia. By basta screening and PCR detection, we respectively received 25 and 32 genetic transformation strains. Phenotype investigation showing that the branch of over-expression TO generation transgenic plants got to reduce, leaf got to smaller, flowers had no phenotypic change, but the flowering earlier; the style did not fall off after seeding. CRES-T silencing TO transgenic plants cylinder color turned to be green, whose periphery appeared 1-5 additional irregular petals; Corolla decreased significantly, whose vascular bundle got to green, and the tip of it was similar to sepal flake. CRES-T silencing T1 generation transgenic plants dwarfed significantly, stems internode was shorter; leaves became smaller significantly, whose color was more green and appeared asymmetric phenomenon, epidermal cells were bigger than wild type; the flowering time of middle phenotype was similar to wild type, but the strong phenotype delayed flowering.