Molecular Mechanism Of GhFPF1 Participating In Plant Shape And Root Development In Gossypium Hirsutum L.

As the main textile fiber and oil seed crop,cotton is one of the most important economic crops.Because of the lack of land resources,the cotton planting area is decreased dramatically.One of the most important way to solve this problem is to breeding short season cotton varieties and achieving double cropping of wheat and cotton.Flowering is a important changing process for plants from vegetative growth to reproductive growth,and is closely related to crop prematurity.Understand the rule of flowering and control this process is meaningful for enhance the earliness of cotton.FLOWERING PROMOTING FACTOR 1(FPF1)gene was originally cloned from Arabidopsis and understood on account of its role in flowering.FPF1 was expressed in apical meristems immediately after the photoperiodic induction of flowering in long-day plants that could flower in response to long days.Up till now,homologous genes of FPF1 have been characterized to regulate flowering positively tobacco(Nicotiana tabacum)but the molecular mechanism was still unclear.We cloned the GhFPF1 to study its function.We performed the bioinformatics analysis,tissue expression analysis,transgenic verification and so on.According to our laboratory findings,we detected the function of GhFPF1 in cotton,we have simply known it's role,the results are as follows:1.Twelve FPF1 homologs were identified from the diploid cotton genomic databases of G.raimondii L.and G.arboretum L with the coding region of Arabidopsis FPF1 gene as the reference sequence.Orthologous sequences from the two cotton species were compared with each other,suggesting that nucleic acid sequences of the six pairs of orthologus were distinct(similarities,84%-99%),though two pairs of orthologous genes possessed the same deduced protein sequence as a result of codon degeneracy.High constraints of genetic divergence might occur during speciation for five genes had higher synonymous changes between the two species.2.Six genes were identified from CCRI36 and named as GhFPF1,GhFLP-1,GhFLP-2,GhFLP-3,GhFLP-4,and GhFLP-5.Until now,as were characterized in several species,members of this gene family were short in length as well as lacking in intron of their genomic sequences.GhFPF1 gene family displayed tissue-specific expression because abundant transcripts of the six genes were found in roots and floral apices,but were barely detectable in leaves or fibers.More importantly,we focused on the contrastive analysis of gene expression in floral apices of CCRI 36(a short-season cotton variety)and TM-1(a genetic standard line).Results uncovered that GhFPF1 had more than four-fold transcript levels in CCRI 36 than in TM-1.Higher expression of GhFPF1 in the short-season cotton suggested that it was the most possible FPF1 orthologous gene as AtFPF1 involved in the promotion of flowering.3.The fusion expression vector p BI121-GFP-GhFPF1 was introduced into onion epidermal cells by the particle bombardment method.The onion epidermal cells carrying recombinant plasmid emitted fluorescence throughout the entire cytoplasm and the nucleus.A 5'-and 3'-RACE strategy was performed to gain transcription initiation and termination sites of GhFPF1.A full-length cDNA of 701 bp composed of 56 bp 5'-UTR,315 bp 3'-UTR and 330 bp ORF was isolated.Cis-acting regulatory elements involved in light,defense and stress responsiveness were found in the promoter of GhFPF1.The hormone treatment assay indicated that GhFPF1 could respond to SA and JA,suggesting that GhFPF1 might be involved in the defense responses of plants.4.After transforming GhFPF1 into Arabidopsis and cotton,seven and nine transgenic lines were obtained,respectively.Results indicated that transgenic Arabidopsis flowered 5.4 days earlier with fewer rosette and cauline leaves than the wild-type.Contrast analysis of Arabidopsis thaliana endogenous genes related to flowering time in wild-type and transgenic lines suggested that the early flowering conferred by GhFPF1 over-expression in Arabidopsis was possibly mediated through AtAP1 and AtFLC.5.On the basis of elements analyses,salicylic acid(SA),abscisic acid(ABA)and jasmonic acid(JA)were selected to spray the leaves in two-true-leaf stage.After 0 h,6 h,12 h,24 h,36 h and 48 h of the treatment,samples were collected to study the expression of GhFPF1.