Constrution Of Expression Vector And Function Analysis Of AcLFY

Onion, a member of the Liliaceae allium, it has functions of anti-atherosclerosis, prevention of high blood pressure, cancer, cataract, coronary heart disease,myocardial infarction, So it has been widely used in the medical, health care and food fields. Onion is identified as strict biennial plants which have flowers must be induced by vernalization and photoperiod. The information on model crop indicate that flowering is a process in which plants integrated all kinds of signals to the inflorescence and floral meristem, so the inflorescence identity gene is considered as the key to connect upstream singals and start downstream flowering. In the early research, we cloned the AcLFY and found its high expression at the beginning of bolting in inflorenscence meristem with also expressing in the other tissues. In this test, the constructed expression vector about onion inflorescence meristem identity gene AcLFY sense, antisense and RNA interference were imported into Agrobacterium tumefaciens EHA105by freeze-thawing method and transferred into Arabidopsis thaliana. By observing the change in the phenotype of transgenic plants and analyzing the function of AcLFY during flowering, it provided information possible to achieve the gene regulation on bolting and flowering of onion.The results are following:(1Construction of the sense plant expression vector pCAM3301-p35S-AcLFY-Tnos, antisense plant expression vector pCAM3301-CaMV35S-AcLFY反-Tnos and RNAi plant expression vector pTCK303-CAcLFY正-CAcLFY反.(2) etermination of the selection pressure in Arabidopsis transformation:the selection pressure of PPT is3.0mg/L; The selection pressure of Hyg B is30mg/L.(3)Recombinant plasmid pCAM3301-p35S-AcLFY-Tnos, pCAM3301-CaMV35S-AcLFY反-Tnos and pTCK303-CAcLpY正-CAcLFy反were introduced into Agrobacterium tumefaciens EHA105by freeze-thawing method and transferred to Arabidopsis by floral-dip method, respectively. We obtained78resistant plant including32sense gene transformed resistant plants,19antisense gene transformed resistant plants and27interference gene transformed resistant plants after resistance screening.(4)The results of PCR identification for the resistance of the three resistant plants showed that there were about69positive strains containing27sense gene transformed positive strains,18antisense gene transformed positive strains and24interference gene transformed positive strains, positive rate was88.5%.(5)The expression test of endogenous AaLFY gene in arabidopsis thaliana by RT-PCR proved that there were high quantity of AaLFY gene expression in sense transformed plants, low expression in antisense transformed plants and no gene expression in RNAi transformed plant.(6) The phenotypic analysis on transgenic plants declared that the transgenic arabidopsis with AcLFY over-expression flowered earlier about5-8days, both number and growth potential of leaves were reduced and lateral inflorescence transformed into solitary flower with the structure of the flower not change much. On the contrary, the transgenic arabidopsis with antisense gene expression flowered later about10days, part of the inflorescence did not form a flower, growth potential was weak, and the number of rosette leaves reduced with branches increasing. The transgenic arabidopsis with interference gene expression did not flower with weak growth potential, increased branches,reduced rosette leaves and the plant height was shorter than antisense AcLFY gene transgenic plants.