Identification And Analysis Of TaCWI-B1-transgenic Wheat Lines And Cloning And Functional Characterization Of TaWTG1 In Wheat(Triticum Aestivum L.)

Wheat（Triticum aestivum L.）is a widely grown food crop in the world.The grain size and grain weight of wheat are the important factors influencing its yield.Therefore,discovery and functional analysis of the genes that control grain size in wheat have important theoretical significance for molecular breeding and improvement of wheat.Cell wall invertase（CWI）irreversibly catalyzes the decomposition of photosynthetic product sucrose into glucose and fructose,regulates sucrose unloading,promotes grain filling,and thus affects rice and maize yield.However,the biological function of the wheat Ta CWI gene is still unclear.The rice WIDE AND THICK GRAIN 1（WTG1/Os OTUB1）gene encodes an OTU-related protease with deubiquitinating enzyme activity,which is related to rice grain size and shape.However,the role of the WTG1 homolog gene Ta WTG1 in grain growth and development is still unknown.In this study,in order to reveal the function of Ta CWI gene in wheat growth and development,the T₂ transgenic wheat lines overexpressing Ta CWI-B1 driven by 35S promoter were identified and their glycometabolism related physiological and biochemical indexes were analyzed.In addition,the Ta WTG1 gene was cloned and its molecular characteristics were analyzed.The prokaryotic expression,purification and deubiquitinase activity of Ta WTG1 protein were verified in vitro.The transgenic overexpression of arabidopsis was used to study the biological functions of Ta WTG1 gene.The main results obtained are as follows:1.Using PCR and detection of hygromycin resistance gene screening,94 positive plants were identified from 195 T₂ plants.The coincidence rate of the two test results reached 98%;q RT-PCR analysis showed that the expression level of target gene Ta CWI-B1 in four wheat transgenic lines was significantly higher than that in non-transgenic control JW1.Western blot analysis showed that overexpression of Ta CWI-B1 gene in four wheat transgenic lines resulted in significantly higher expression of target protein than control JW1.2.The cell wall invertase activity,sucrose,glucose and fructose contents of T₂ generation Ta CWI-B1 wheat transgenic lines were measured at the seedling stage,and the sucrose and starch contents of the grain samples at 15 d and 25 d after flowering were measured.Compared with JW1,the enzymatic activity,glucose and fructose contents in the leaves of transgenic wheat lines increased extremely significantly,and the sucrose content increased significantly.The sucrose content in the grain samples decreased significantly and the starch content increased significantly in the seed of 15 d and 25 d after flowering.Overexpression of Ta CWI-B1 can promote the metabolism of sucrose at the seedling stage and the accumulation of starch at the grain filling stage in the grain of wheat.3.Three homoeologous genes Ta WTG1-A,Ta WTG1-B and Ta WTG1-D of Ta WTG1 gene were obtained from wheat cv.Xiaoyan 6 by homologous cloning strategy,and their sequence similarity was 99%.Ta WTG1 encodes an OTU-related protease containing a conserved amino acid of the OTU-related protease family and a conserved domain of otubain;genomic sequence analysis revealed that three copies of the Ta WTG1 gene all contain 8 exons and 7introns.4.The analysis of spatiotemporal expression pattern indicated that Ta WTG1 gene was highly expressed in the late stage of grain development.The subcellular localization results showed that Ta WTG1 protein was mainly located in the nucleus.5.The prokaryotic expression vector of Ta WTG1 was constructed and the optimal induction conditions of fusion protein were determined:IPTG concentration was 0.4 mmol/L,and induced at 28°C for 6 h.The recombinant protein was isolated and purified by Ni-NTA Sefinose TM Resin column and the high-purity fusion protein His-Ta WTG1 was obtained.In vitro deubiquitinase activity assay results showed that Ta WTG1 has deubiquitinating enzyme activity and can cleave K48-and K63-linked tetrameric ubiquitin chains.6.The Ta WTG1 gene overexpression transgenic arabidopsis was obtained by agrobacterium inflorescence infection.Compared with the wild type,the transgenic plants showed a significant decrease in the number of branches and siliques,and a significant increase of the 100-grain weight.In conclusion,this study used overexpression Ta CWI-B1 transgenic wheat as experimental material.Studies have shown that the overexpression of Ta CWI-B1 gene affects the content of glucose,fructose and sucrose in wheat seedling stage,and promotes the accumulation of starch in grain during grain filling.In addition,the wheat Ta WTG1 gene was cloned,which proved that Ta WTG1 has deubiquitination function,and overexpression of Ta WTG1 in Arabidopsis initially confirmed that Ta WTG1 affects grain size and grain weight.