Cloning And Expression Of Flower Development Related Genes From Grape (Vitis Vinifera×V. Labrusca)

For a long time,with traditional breeding methods,we met with many difficultics such as the long breeding period,leading to the breeding process become slowly.Genetic engineering technique has been becomeing the important potential breeding method.The floral development is the important foundation of fruit yield and qualitiy.The research of model plant flower development in recent years provided an opportunity for fruit trees.Grape is one of the most important fruit trees in the world,its culture area and total yields are advanced in the world.Different from perennial woody fruit crops,grape has advantages of short juvenile period,multiple flowering in a year and so on.Therefore,it is of great importance to research on flower development of grape.According to the research hotspot of the model plant flower development,we studied on several flowering related genes and the promoter from grape(Vitis vinifera×V. labrusca),cloned the full length of AGAMOUS(AG),APATALLA3(AP3),FLOWERING LOCUS C(FLC),FLOWERING LOCUS T(FT) homolog and the promoter of LEAFY gene. We studied the expression characterization of these genes,we expect that it will be used in the genetic engineering breeding or regulate gene expression.The main results are as follows:1.As a kind of transcription factors,MADS-box genes play an important role in various plant development processes,especially in the development of floral organs and fruit.In order to isolate new MADS-box genes and find out the function of these genes in the development of grape floral organs and fruit,we designed degenerate primers according to the conservative area sequences of MADS-box family gene from various plants, obtained 34 MADS- box gene cDNA fragments amplified by RT-PCR from the inflorescence of Vitis vinifera×V.labrusca 'Red Fuji'.Sequence analysis indicated the size of these fragments is among 138-152 bp,including the initiation codon of genes,deduced amino acid sequences have a higher identify of more than 83%with MADS-box genes of grapevine and other species.By analysis of phylogeny construction,all of these gene fragments have been divided into the Arabidopsis MADS-box gene subfamily.These results showed that there are many MADS-box genes in grape,these gene fragments cover all type floral development genes of ABCDE model.2.In order to study the rule of grape LEAFY gene expression and regulation,we amplified a 1.8kb fragment from Vitis Vinifera×V.Labrusca 'Fujiminori' by PCR.The fragment is 1833bp,deduced 402 amino acids,has two introns,which is 99%identity with VFL gene.Nine hundred and twenty five bp promoter region sequence was cloned by genome walking method.The gene and promoter are 2692bp in length(GenBank accession is EF222286).Promoter sequence analyzed by PLACE and PlantCARE showed that it has TATA-box,CAAT-box and some cis-acting element such as MYB binding site,cis-acting regulatory element involved in ABA response,some light-induced responsive elements and other transcription factor-binding sites.Compared the promoter of grape LEAFY homologue with 4 plants,by FootPrinter analysis we found that these promoter has conservatism and diversity and the distributing of transcription factor-binding sites has similarity and difference.It implied the accuracy and diversity of LEAFY gene expression and regulation.3.As a kind of transcription factors,MADS-box genes play an important role in plant various development processes,especially in the development of floral organs.According to a MADS fragment sequence obtained by degenerate primer RT-PCR,an AG-like MADS-box gene was cloned from inflorescene by the method of 3'RACE PCR,named VvAG(GeneBank accession number EF209334),This gene is 978 bp and contained an open reading frame of 681bp coding a polypeptide of 226 amino acids.Homology analysis showed that the deduced VvAG protein was highly homologous to AG,SHP1/SHP2 of Arabidopsis thaliana,the identity is 77.9%,71.2%and 69.9%,respectively.Phylogenetic analysis also indicated that VvAG belongs to the euAG lineage of AG subfamily in MADS-box gene family.RT-PCR analysis showed that VvAG expresses at high levels in leaves,tendrils,inflorescenes,young fruits and mature fruits,and also expressed in all of 4 wheel floral organ.By blast of VvAG with a shotgun genome sequence,the result indicated that VvAG genome is consist of 7 exons and 6 introns.We constructed a overexpression vector of VvAG and transferred it into tobacco,obtained 8 hyg positive tobacco plants.PCR analysis showed that these lines are all PCR positive.Analysis of 5 PCR positive lines by RT-PCR revealed VvAG expressed in all these lines.4.An APETALA3(AP3) homologue was isolated from grape(Vitis vinifera×V. labrusca 'Red Fuji') inflorescence by bioformatic analysis and RT-PCR.It is 681 bp, coding a polypeptide of 226 amino acids,named as VvAP3(GenBaak accession number EF211123).Homology analysis showed that the deduced VvAP3 protein was highly homologous to the MADS-box genes,PMADS1,NTDEF and AP3,the identity are 71.2%, 70.4%and 59.7%,respectively.Phylogenetic analysis also indicated that VvAP3 belongs to the euAP3 lineage of AP3 subfamily in MADS-box gene family.RT-PCR analysis showed that VvAP3 expressed in inflorescenes,young and mature fruits,and in petal and stamen, but not expressed in sepal and carpel.Blast of VvAP3 with a shotgun genome sequence indicated that VvAP3 genome is consist of 7 exons and 6 introns,the number and length of exon is the same as AP3.We constructed a overexpression vector of VvAP3 and transferred it into tobacco,obtained 7 hyg positive tobacco plants.PCR analysis showed that these lines are all PCR positive.Analysis of 5 PCR positive lines by RT-PCR revealed VvAP3 expressed in all these lines.5.An putative FLC homologue full length cDNA was isolated from grape(Vitis vinifera×V.labrusca 'Red Fuji') inflorescence by bioformatic analysis and RT-PCR.It is 948bp in length,coding a polypeptide of 210 amino acids,named as VvFLC(GenBank accession number is EF520739).The predicted protein sequence shows homology to FLC (43%identity) and to BvFLC1-1 and BvFLC1-4(49%and 49%identity,respectively). Phylogenetic analysis also indicated that VvFLC belonged to the FLC subfamily of MADS-box gene family.VvFLC expresses in leaves,tendrils,inflorescenes,young and mature fruits.Blast of VvFLC with a genome shotgun sequence indicated that VvFLC genome is consist of 7 exons and 6 introns.We constructed a overexpression vector of VvFLC and transferred it into tobacco,obtained 7 hyg positive tobacco plants.PCR analysis showed that these lines are all PCR positive.Analysis of 4 PCR positive lines by RT-PCR revealed VvFLC expressed in all these lines.6.An Flowering Locus T(FT) homologue was isolated from grape(Vitis vinifera×V. labrusca 'Red Fuji') inflorescence by bioformatic analysis and RT-PCR.It is 525bp in length,coding a polypeptide of 174 amino acids,named as VvFT(GenBank accession is EF203918).The deduced amino acid sequence shows a high degree of homology to PnFT1a(89%identity) and to FT and MdFT(75%and 74%identity respectively).VvFT protein has a typical RKIP domain.Phylogenetic analysis showed VvFT belongs to FT homologues of the FT/TFLl gene family.Cloning VvFT genome sequence by PCR(GenBank accession is EF203919),blast of it with VvFT mRNA showed the structure of VvFT gene is consist of 4 exons and 3 introns,the structure is the same as Arabidopsis and poplar.There has some sequence repeat in the intron of VvFT gene,such as(TCC)₁₀ and(CA)₁₀.VvFT is expressed in the developing inflorescene,young and mature berry showed VvFT is related with flower and fruit development.We constructed a overexpression vector of VvFT and transferred it into tobacco,obtained 8 hyg positive tobacco plants.PCR analysis showed that 7 of them are PCR positive.Analysis of 4 PCR positive lines by RT-PCR revealed VvFT expressed in all these lines.Overexpression of VvFT in tobacco hastened flowering showed that VvFT function as promoters of flowering when ectopically expressed in a heterologous plant,and the flower number of transgene plant is more than untransgene plant.A transgene plant showed a dwarfism phenotype,and no change in flower organ shape.