Preliminary Functional Verification Of The TaGAPC5 Gene In Transgenic Wheat

Glyceraldehyde-3-phosphate dehydrogenase(GAPDH)is widely exists in various organisms,and is a key enzyme in the glycolysis and gluconeogenesis process.GAPDH can be involved in stress tolerance response in plants,the study of the expression and regulation of GAPDH gene has very important significance in improving wheat stress resistance and yield.GAPDH can be classified into four distinct groups: GAPC,GAPA/B,GAPCp and NP-GAPDH.As an important member of GAPDH gene family,GAPC plays an important role in the plants life activities.In this experiment,we use the green fluorescent protein(GFP)subcellular localization technology research the localization of TaGAPC5 in onion epidermal cells,then lay a foundation for GAPDH drought-resistant regulation pathway.And in the previous study,our group had cloned the full-length sequence of GAPDH gene,and on this basis,by using gene gun mediated method,the over-expression vector of TaGAPC5 gene was transferred into water sensitive wheat cultivar Zhengyin 1,and the RNAi vector was transferred into drought resistant wheat cultivar Changwu 134,then obtained T1 generation transgenic lines that the target gene is stably expressing.In order to determine the stability of transgenic plants furtherly,the main purpose of this study is obtaining T2 generation seeds on the basis of the T1 generation and carring on the positive verification,continue to cultivate to get T2 generation transgenic plants,and determine the related physiological and molecular biological indexes to detection the gene expression levels,so as to verify the function of the gene.The results are as follows:1.Establishing transgenic wheat cultivation system after planting and cultivating the transgenic and wild type seeds of T1 generation.The agronomic characters of the transgenic and wild type plants were detected,the results showed that the agronomic characters of the plants were influenced by the target gene.The positive T2 generation plants were successfully obtained by the PCR detection,the positive plants included 4 strains of Zhengyin 1,2 strains of Changwu 134.2.Preliminary validated the function of TaGAPC5 gene in transgenic wheat.The results showed that in the transgenic plants of T2 generation of Changwu 134,TaGAPC5 gene expression was 18% of the control,indicating that RNAi vector was successfully transferred into and interfered the gene expression.For T2 transgenic plants of Zhengyin 1,TaGAPC5 gene expression was 4% of the control,indicating that the target gene did not cause significant changes about expression,results of related physiological indexes under drought stress showed that TaGAPC5 gene expression changes with the time of drought stress,and the changes of gene expression may have a certain impact on drought resistance of plants.3.Construct green fluorescent protein fusion expression vector,then using gene gun bombard the onion epidermal cells,and observing subcellular localization of the target after training in laser confocal microscope.The results show that the green fluorescent scattered throughout the whole cell after transferred the 16318-GFP empty vector into onion epidermal cells,while after transferred the 16318-TaGAPC5-GFP vector into onion epidermal cells,the cell membrane,cytoplasm membrane and cell nuclear membrane three parts are observed the green fluorescence,indicating TaGAPC5 gene is located in the entire biofilm system.