Cloning, Expression And Functional Analysis Of An Upland-cotton Gene GhFPF1Defined As Flowering Promoting Factor1

Cotton (Gossypium spp.), one of the most important natural fiber crops occupies animportant position in national economy and social development in China. Because of thecontradiction between grain and cotton, more people and less land, the earliness of cotton hasbecome a vital subject for plant breeders to solve the problem and ensure the food security.FLOWERING PROMOTING FACTOR1(FPF1) gene was originally cloned fromArabidopsis and understood on account of its role in flowering. FPF1was expressed in apicalmeristems immediately after the photoperiodic induction of flowering in long-day plants thatcould flower in response to long days. Up till now, homologous genes of FPF1have beencharacterized to regulate flowering positively tobacco (Nicotiana tabacum) but the molecularmechanism was still unclear.Databases of G. raimondii L. genome and G. arboreum L. genome were screened toidentify and compare homologs of AtFPF1from the two diploid cotton species. The geneswere cloned and characterized from G. hirsutum L. as one of the major cultivated species. Toselect excellent candidate gene, their expression pattern in cotton were compared. In addition,more deep research work was conducted on its function. The specific results were as follows:1. Twelve FPF1homologs were identified from the diploid cotton genomic databases ofG. raimondii L. and G. arboretum L with the coding region of Arabidopsis FPF1gene as thereference sequence. Orthologous sequences from the two cotton species were compared witheach other, suggesting that nucleic acid sequences of the six pairs of orthologs were distinct(similarities,84%-99%), though two pairs of orthologous genes possessed the same deducedprotein sequence as a result of codon degeneracy. High constraints of genetic divergencemight occur during speciation for five genes had higher synonymous changes between thetwo species.2. Six genes were identified from CCRI36and 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 genomicsequences. GhFPF1gene family displayed tissue-specific expression because abundant transcripts of the six genes were found in roots and floral apices, but were barely detectable inleaves or fibers. More importantly, we focused on the contrastive analysis of gene expressionin floral apices of CCRI36(a short-season cotton variety) and TM-1(a genetic standard line).Results uncovered that GhFPF1had more than four-fold transcript levels in CCRI36than inTM-1. Higher expression of GhFPF1in the short-season cotton suggested that it was the mostpossible FPF1orthologous gene as AtFPF1involved in the promotion of flowering.3. The fusion expression vector pBI121-GFP-GhFPF1was introduced into onionepidermal cells by the particle bombardment method. The onion epidermal cells carryingrecombinant plasmid emitted fluorescence throughout the entire cytoplasm and the nucleus. A5’-and3’-RACE strategy was performed to gain transcription initiation and termination sitesof GhFPF1. A full-length cDNA of701bp composed of56bp5’-UTR,315bp3’-UTR and330bp ORF was isolated. Cis-acting regulatory elements involved in light, defense and stressresponsiveness were found in the promoter of GhFPF1. The hormone treatment assayindicated that GhFPF1could respond to SA and JA, suggesting that GhFPF1might beinvolved in the defense responses of plants.4. After transforming GhFPF1into Arabidopsis and cotton, seven and nine transgeniclines were obtained, respectively. Results indicated that transgenic Arabidopsis flowered5.4days earlier with fewer rosette and cauline leaves than the wild-type. Contrast analysis ofArabidopsis thaliana endogenous genes related to flowering time in wild-type and transgeniclines suggested that the early flowering conferred by GhFPF1over-expression in Arabidopsiswas possibly mediated through AtAP1and AtFLC.5. Except accelerated flowering and fewer leaves, compared with wild-type, transgenicArabidopsis generated longer hypocotyl and petioles, lower content of chlorophyll. Moreover,for the first time we found that expression of PHYB in transgenic plants was decreased bymore than fifty percent. Taken together, transgenic plants were recognized as so-calledshadow-avoidance syndrome (SAS) which was also a new discovery for GhFPF1involved inthe regulation of plant growth and development.6. In addition, GhFPF1was introduced into cotton through Agrobacterium tumefaciensmediated transformation, nine transgenic lines were obtained after checking the DNA andmRNA. T2generation of genetically modified cotton was in the stage of seedling. To uncoverthe signaling pathways of GhFPF1involved in regulation of plant growth and developmentprocess, further work still needs to be done.