Study On Flowering Regulation Of 3 COL Genes In Onion

Onion(Allium cepa L.)belongs to Alliaceae Allium,is one of the main vegetables in the world,and is one of the main export vegetables in our country.Onion is a typical biennial plants in suitable light cycle,the bulb was formed in the light,after the winter storage of vernalization,the flower into fruit under suitable light cycle in second year,so as to completed the life cycle.In this process,photoperiod is a decisive factor,which not only determines the formation of product organs,but also induces the transition from vegetative growth to reproductive growth.The transition from vegetative to reproductive growth of flowering is an important turning point in the life cycle of plants,CO(CONSTANS)gene is an important element to measure the duration of light between a biological clock and flowering time genes play an important role in the photoperiod pathway.According to the results of transcriptome sequencing,the Co-like gene was cloned and studied.The experimental results are as follows:(1)According to the results of transcriptome sequencing,PCR primers were designed,AcCOL1 gene was cloned by using onion leaves of vegetative growth stage cDNA as the template,the full-length is 1185 bp,accession number : KX553865.AcCOL3 gene,full-length 954 bp,accession number : KX553867;AcCOL7 gene,full-length 1101 bp,accession number :KX553871.(2)The results of structural analysis showed that the three genes of AcCOL family had same Box and CCT domains structure,however,the results of homologous sequence alignment showed that the similarity between three genes were very low,and the similarity was 27.84,and the similarity of any two genes was 24.21-29.13.(3)Phylogenetic analysis showed that the three genes were not in the same branch,and the AcCOL1 gene was in the same branch with radish,tall fescue,barley and tomato,and the closest relationship with radish;The AcCOL3 gene with Arabidopsis and barley in same branch,and most closely related to Arabidopsis COL15 gene,AcCOL7 gene with Arabidopsis,barley,beans,dates and palm oil in the same branch.(4)The transcriptive double wave of AcCOL3 in onion was detected by qRT-PCR.(5)The relative expression of AcCOL3 in different parts of onion was detected by fluorescence quantitative PCR,and the expression of AcCOL3 gene and the expression time were confirmed.The expression of AcCO gene was highest in young leaves,lowest expression in inflorescences.(6)The over expression vector AcCOL1-1250,AcCOL3-1250,AcCOL7-1250 of 3 genes was constructed,and then the recombinant plasmid was transferred into Agrobacterium tumefaciensLBA4404 by freeze-thaw method.The recombinant plasmid AcCOL1-1250,AcCOL3-1250 and AcCOL7-1250 were infected into Columbia wild type Arabidopsis by means of inflorescence impregnation,5 mg/L PPT-containing MS medium was used to screen the resistance of plants transferred overexpression vector,11 AcCOL transgenic plants,14 AcCOL3 transgenic plants,and8 AcCOL7 transgenic plants were obtained.(7)The over expression vector was transferred into co mutant(SALK-096361C),and 5 mg/L PPT-containing MS medium was used to screen the resistance of plants transferred overexpression vector,6 AcCOL1 transgenic plants,8 AcCOL3 transgenic plants,and 5 AcCOL7 transgenic plants were obtained.(8)The wild type Arabidopsis thaliana,which had been transferred into three onions COL gene overexpressing vector,did not change significantly in the time of bolting and flowering and plant phenotype compared with wild-type Arabidopsis thaliana;The co mutant Arabidopsis thaliana,which had been transferred into three onions COL gene overexpressing vector,the AcCOL1 gene was about 9 days ahead of the co mutant,the AcCOL3 gene was about 12 days ahead of the co mutant,and the AcCOL7 gene was about 7 days ahead of the co mutant,and the transgenic mutant plant grew rapidly after bolting.