Prokaryotic Expression Of TaCYP78A5 Gene And Identification Of Transgenic Wheat Progeny

Wheat is one of the most important food crops in the world,and the increase of its production plays an important role in solving the problem of food shortage.Cytochrome P450 is one of the largest protein families in plants,and CYP78 A is one of its most important members.Many members of the CYP78 A subfamily have been identified to play an important role in the growth and development of seeds.The TaCYP78A3 and TaCYP78A5 gene were cloned by homology cloning method in our research Group,and the results of overexpressing these genes in Arabidopsis thaliana and the BSMV-VIGS(gene silencing induced by barley stripe mosaic virus)in wheat showed that these two genes were related to grain size.In order to further study the function of TaCYP78A5 gene,a series of studies were carried out as follow.(1)Prokaryotic expression and purification of protein TaCYP78A5Firstly,the basic physical and chemical properties and the structure of of TaCYP78A5 were analyzed by bioinformatics.The results showed that the protein had no signal peptide but had a transmembrane structure,and it was a hydrophilic protein.The molecular weight of TaCYP78A5 protein was 55.805 kD,the theoretical pI value was about 8.32,and it belonged to unstable protein.The molecular weight of truncated TaCYP78A5 protein and its fusion protein GST-TaCYP78A5 were 51.973 kD and 80.385 kD respectively,and the theoretical pI values were 8.71 and 7.00 respectively.Then the target TaCYP78A5 gene without N terminal transmembrane structure was amplified from the plant expression vector pCAMBIA3301-TaCYP78A5.The recombinant plasmid pGEX-6p1-A5 was constructed and transformed into the expression strain E.coli BL21(DE3)pLysS for induced expression.The results showed that the GST-TaCYP78A5 fusion protein is expressed in the form of inclusion bodies.After purification and renaturation of the inclusion body protein,the GST tag was removed by Prescission Protease,and high purity target protein was obtained by gel recovery.But it did not produce antibodies after immunized rabbits.(2)Genetic transformation of wheat with TaCYP78A5 gene and genetic and function analysis of transgenic wheat overexpressing TaCYP78A5 geneThe plant expression vector pCAMBIA3301-35S-TaCYP78A5 was transformed into wheat Mianyang 19 by Agrobacterium tumefaciens EHA105.The one positive plant were obtained by PPT screening and PCR identification,but it could not survive after transplanting to the greenhouse.The T0 transgenic wheat JW1 with the pINO::TaCYP78A5 gene overexpressing were provided by Ji'nan Bondi biological science and Technology Co Ltd.After identified by Bar strip test and PCR detection,the copy number of target exogenous gene in T0 transgenic plant was tested.The optimal concentration of Basta for screening transgenic plants by smearing leaf with Basta solution was explored,and the separation of the exogenous target gene in the T1 lines and the variation of the grain size of the positive transgenic lines were statistically analyzed.The results showed that there were 14 positive plants out of 21 regenerated seedlings,among which 6 plants had single copy target exogenous gene,5 plants had double copies,2 plants had 3 copies and 1 plants had 7 copies.The identification of 6 T1 single copy transgenic lines showed that only 3 lines meet the separation ratio of 3:1.200 mg/L of Basta solution smearing the leaf can effectively identify positive plant from transgenic lines.Compared with the wild type JW1,the grain width,grain thickness of 7 progeny plants of T1 positive lines increased in varying degrees,2 positive plants of grain length decreased,but all positive plants of grain weight increased significantly,reaching 10~17%.