Cloning And Expression Analysis Of Floral Related Genes In Strawberry

Flowering is very important for the production of strawberry. The flowering time and thefloral quality directfly influence the fruit forming time and the yield of strawberry. Most ofthe present studies are focusing on the physiological or cultivated mechnisms that how theexternal factors affect the flowering of strawberry, while its molecular mechanism is poorlyunderstood now. The deepgoing studies in the model plant, such as Arabidopsis andsnapdragon, revealed that a series of important genes envolved in the regulation of theirflowering. Among the hundreds of such genes, the AP1, LFY, TFL1, FT and CO homologousgenes play important roles in the regulation of flowering in many different plants. In thisstudy, we isolated homologous genes of the five genes mentioned above from strawberry bythe motheds of RACE and PCR amplification. Also we studied the expression patterns of thefive genes in different tissues, organs and different growth stages in flowering of strawberryby Real time RT-PCR. The results of our study will be helpful for the understanding of themolecular mechanism of the strawberry. Main results of this study are as follows.1. Fragments of AP1, LFY and TFL1homologous genes in strawberry were isolated withPCR using degenerate primer. The lengths of these fragments are464bp、986bp and1025bprespectively. Their nucleotide identities compared with other AP1, LFY, TFL1homologousgenes form different plants are highly conserved.2. The full-length cDNA sequences of AP1, LFY, TFL1, FT and CO homologous genesin strawberry were isolated with the methods of RACE or specific RT-PCR. Such genes werenamed FaAP1, FaLFY, FaTFL1, FaFT and FaCO-2respectively. The full-length CDS ofFaAP1is735bp and it codes a protein of245amino acids which contains the typicaldomains of MADS-box gene family, it shows a highest homology with the homologousprotein in Rosa, up to90.90%; The full-length CDS of FaLFY is1236bp and it codes aprotein of412amino acids and the protein containes two conserved domains just as otherLFY-like proteins at the N-teminal and C-teminal, it shows the highest similarity (88%) withthe homologous protein of Rosa; The full-length CDS of FaTFL1is516bp and it codes aprotein of172amino acids and the protein containes DPPE domains which shows it belongsto the member of PEBP family, the nucleotide identity is highest when compared withFragaria Vesca, up to98%; The full-length CDS of FaFT is528bp and it codes a protein of176amino acids and the protein aslo containes DPPE domains, the nucleotide identitycompared with Fragaria Vesca is the highest, up to98%; The full-length CDS of FaCO-2is1146bp and it codes a protein of382amino acids and the proetin contained the typicalstructural features of CO-like family.3. We further got the full length DNA sequences of the five genes with PCR using thespecific primers according to their cDNA sequences. The length of DNA sequences of FaAP1,FaLFY, FaTFL1, FaFT and FaCO-2are2911bp,2250bp,1138bp,1138bp,2104bp,1182bp, respectively. FaAP1has eight exons and seven introns, FaLFY has three exons andtwo introns, FaTFL1has four exons and three introns, FaFT has three exons and two intronsand FaCO-2has two exons and one introns. 4. Real time RT-PCR was used to analyze the expression patterns of the five genes indifferent tissues, organs and different growth stages of flowering in strawberry. FaAP1mainly expresses in litter flower, speal and petal, and no transcription was detected in thevegetative tissues; FaLFY mainly transcripts in the reproductive tissues, especially in flowerbuds, and no transcription was detected in vegetative tissues and floral organs; FaTFL1expresses mainly in the bud of vegetative growth and the floral shoot apex, flower and youngfruit and its highest transcription level was detected in stolon stem, no expression wasdetected in floral organs; FaFT mainly transcripts in reproductive tissues with the highestexpression level in mature flower, no transcription was detected in vegetative tissues such asleaf; FaC0-2was detected its transcription in all vegetative tissues and some reproductivetissues, its highest transcription leves was detected in expanded leaf, it was also detected itstranscription in the sepal.5. Promoter of FaAP1which is about700bp in length was isolated from strawberry genomeby chromosome walking methods. Projections indicate that the promoter includes the essential functionalelement of TATA-box, the enhancer element of CAAT-box and some plant meristem spcecialregulating element.