Construction Of Plant Over Expression Vector With The Wheat TaGAPC1 And Transgenic Arabidopsis Thaliana

As one of the world's major food crops,wheat is supplied to people in all aspects of life.Therefore,its quality and yield have become the focus to researchers.Gap C gene play an important role in the process of anti-stress in plants,inorder to know the resistance mechanism and the function of wheat TaGAPC1 gene,In this experiment,the target gene used Jinmai 47 cDNA as template gaining cloned gene was connected to pMD19-t T vector,and the sequence was carried out the bioinformatics analysis of the sequence.plant overexpression vector CaMV35S::TaGAPC1 was constructed by homologous recombination method,and transformed Agrobacterium containing overexpression vector into Arabidopsis by Agrobacterium-mediated.screening of T2 seeds with the optimal concentration of hygromycin.The results are as follows:1.According to the full length TaGAPC1 of wheat CDS sequence,the target gene was amplificated with the primers designed by DNAMAN and Premier 5.0 software.it was connected with pMD19-T which named T-1,and successfully sequenced.Sequence analysis indicated that TaGAPC1 was located on the 7D chromosome at 67.28 Mb.It's ORF had the length of 1014 bp,There are two transmembrane domains correspond to amino acid positions respectively(165-185)and(200-219).2.The plant expression vector CaMV35S:: TaGAPC1 was constructed successfully with the method of homologous recombination,which was introduced into E.coli and was sequenced successfully.3.Successfully worked out a suitable to being transformed into Arabidopsis thaliana planting conditions: for the short sunshine 8/16 h it produce nutritional growth,that is the rosette growth in initial,for the long sunshine 16/8 h it produce reproductive growth,that is the bolting and flowering,in the early transplanting period to promote root growth continued watered with nutrient solution.When the length of inflorescence was 3-5 cm,we removed the top inflorescence,and the length of inflorescence and diameter of flower were about 10 cm and 1.5 mm,respectively,a lots of pods on the main stem,Having a large number of unopened inflorescence,the secondary inflorescence was in flowering state.4.The over expression vector CaMV35S:: TaGAPC1 was transferred into Arabidopsis thaliana with agrobacterium mediated floral dip method,the optimum conditions of infection: when the agrobacterium solution containing expression vector OD600 1.6-2.0,the resuspend of agrobacterium was OD600 about 0.8,the best time of infectionfor in 10s or so,in the dark cultured for 24 h,a week after repeat the infection steps.5.25 mg/ml concentration was determined as the optimal hygromycin concentration,and 13 strains of T1 Arabidopsis thaliana were successfully obtained by screening,and the T2 generation seeds were harvested.