Cloning and Expression Analysis of Flower Development Related Genes from Grapevine (Vitis Vinifera×Vitis Labrusca ’ Fujiminori’)
|School||Nanjing Agricultural College|
|Keywords||Grape Promoter FLOWERING LOCUS T FLOWERING LOCUS C APETALLA3 AGAMOUS Cloning Expression|
In order to understand the complicated biological phenomena of plant flowering, scientists have done plenty of work on the flowering mechanism over the past century. The researches on flower development gradually came from physiological and biochemical level to the molecular level. The understanding of plant flower formation molecular mechanism has contributed fundamental breakthroughs in model plants such as Arabidopsis thaliana and Antirrhinum majus. A large number of functional genes have been identified and represent a complex and accurate functional genetic network, in which the homologous genes FLOWERING LOCUS T, FLOWERING LOCUS C, APETALA3 and AGAMOUS are four key genes in the determination of flowering time, differentiation and development of floral meristems. However, there are different extent differences and relatively high conservation in sequence features, expression patterns and molecular functions among these four homologous genes in different species. Study on the flowering molecular mechanism of fruit trees begins not so far and still quite simple. In this article, we cloned the full length of AGAMOUS (AG), APATALLA3 (AP3), FLOWERING LOCUS C (FLC), FLOWERING LOCUS T(FT) homolog cDNA and their promoter regions from Vitis Vinifera×Vitis Labrusca’Fujiminori’, then analyzed structures and functions of these genes through biological software. We studied the expression characterization of these genes in different flowering stages, in order to better understand flowering responses and regulation of fruiting of grapevine. The main results were as follows:1. Using the same primers in cloning FLOWERING LOCUS T, FLOWERING LOCUS C, APETALA3 and AGAMOUS from Vitis Vinifera x Vitis Labrusca’Red Fuji’, we amplified cDNA sequences of these four genes from Vitis Vinifera×Vitis Labrusca ’Fujiminori’inflorescence by RT-PCR and 3’RACE PCR. The result showed that FT gene is 757 bp (GeneBank accession number HM 192810) and contained an open reading frame of 525 bp coding a polypeptide of 174 amino acids. FLC gene is 1038 bp (GeneBank accession number HM192809) and contained an open reading frame of 632 bp coding a polypeptide of 210 amino acids. AP3 gene is 1049 bp (GeneBank accession number HM 192808) and contained an open reading frame of 681 bp coding a polypeptide of 226 amino acids. AG gene is 1123 bp (GeneBank accession number HM192807) and contained an open reading frame of 681 bp coding a polypeptide of 226 amino acids.2. A 1605 bp FLOWERING LOCUS T homolog upstream fragment was amplified from Vitis Vinifera x Vitis Labrusca’Fujiminori’by genome walking method based on thermal asymmetric PCR, include 135 bp gene region sequence and 1470 bp promoter region sequence(GeneBank accession number HM192806). FLOWERING LOCUS C homolog upstream fragment is 1788 bp, include 90 bp gene region sequence and 1698 bp promoter region sequence (GeneBank accession number HM 192805). APETALA3 homolog upstream fragment is 1143 bp, include 82 bp gene region sequence and 1061 bp promoter region sequence (GeneBank accession number HM 192804). AGAMOUS homolog upstream fragment is 1254 bp, include 122 bp gene region sequence and 1132 bp promoter region sequence (GeneBank accession number HM192803). Using PlantCARE to analyze the four promoter sequences showed that they all have TATA-box, CAAT-box and some other transcription factor-binding sites.3. The expression character of FLOWERING LOCUS T, FLOWERING LOCUS C, APETALA3 and AGAMOUS were studied under different flowering stages in Vitis Vinifera x Vitis Labrusca ’Fujiminori’ inflorescence. The results showed that the expression level of FT gene steadily decreased, but sharp raised before flowering and had a higher peak when the flowering day came. The expression of FLC was the other way round FT, showed a quick raise first and then decreased. AP3 kept a low expression level in all the flower development stages, and slowly decreased. AG also showed an evident raise first and then decreased, kept high expression level for several days until flowering.