Construction Of Wheat ZCCT2 Gene Expression Vector And Agrobacterium Transformation

Wheat is the most widely grown food crop in the world,and about 40% of the population feeds on wheat.Wheat provides 20 percent of the total energy and about 25 percent of the total protein for the world's population.Vernalization gene(VRN)and photoperiod gene(PPD)play an important role in regulating the reproductive growth of wheat.The allelic diversity of these two genes determines the broad adaptability of wheat to the environment.The main flowering inhibitor of wheat is considered to be the VRN2 gene,which can prevent winter wheat from flowering in winter.However,low temperature can reduce the expression of this gene and eliminate the inhibition of this gene,so that winter wheat can bloom in the following spring.The VRN2 gene locus includes two tandem ZCCT1 gene and ZCCT2 gene.Although the current research suggests that ZCCT1 gene is related to vernalization of wheat and ZCCT2 gene is genetically related to flowering,there is no evidence that it is a vernalization-related gene.In this study,single-target knockout vectors,multiple-target knockout vectors,overexpression vectors,and silencing vectors for the ZCCT1 gene and ZCCT2 gene on A,B,and D chromosomes were constructed.Agrobacterium-mediated transgenic technology was used to initially verify the function of ZCCT2 gene in wheat,and to clarify the relationship between ZCCT2 gene and wheat winter characteristics.It will help to evaluate varieties suitable for the target environment and marker-assisted breeding.The main results are as follows:1.Construction of single-target knockout vector: six targets with high scores and good specificity of ZCCT1 gene and ZCCT2 gene were designed by using the online target design website,and six single-target knockout vectors were constructed,which were transferred into the sensing state of agrobacterium EHA105 by heat shock method.2.Construction of multi-target knockout vector: multiple target sequences of ZCCT1 gene and ZCCT2 gene with high scores and good specificity were designed using the online target design website.Six multi-target knockout vectors were constructed and transferred into the sensing state of agrobacterium EHA105 by heat shock method.3.Construction of overexpression vector and silencing vector: six overexpression vectors and six silencing vectors were constructed respectively by using high-fidelity enzyme to amplify the gene sequence and insert the expression vector.4.Protoplast validation: multi-target expression vectors ZCCT1-A1-U6,ZCCT1-D1-U6,ZCCT2-A2-U6,ZCCT2-B2-U6,and ZCCT2-D2-U6 were introduced into the extracted wheat protoplast.The extraction and counting of protoplast met the requirements,and the green fluorescence could be observed under the fluorescence microscope,indicating that the plasmid was successfully transformed into the protoplast,but the conversion efficiency was relatively low.5.Agrobacterium-mediated transgene technology was used to transform Kenong 199,and a total of 30 single-target knockout vector transgenic seedlings were obtained.As a winter variety,Kenong 199 should undergo vernalization in order to switch from vegetative growth to reproductive growth.Among the 30 transgenic wheat seedlings obtained,9 transgenic plants were found to have entered the reproductive growth without vernalization after the transgenic event,and seeds were successfully harvested.21 plants remained in vegetative state without vernalization until yellowing was inactive or capable of heading but no seed.