Cloning And Functional Analysis Of The TaXTH-7A Gene In Common Wheat(Triticum Aestivum,L)

Wheat is often subjected to stresses such as drought and high temperatures,which poses a threat to its production.The use of molecular biology tools and genetic engineering provides an effective method for improving wheat resistance breeding.Xyloglucan endoglycosyltransferase/hydrolase belongs to the glycosyl hydrolase 16 family,which helps to improve the drought tolerance of plants and is considered as a candidate gene for plant drought tolerance genes.In this study,the Ta XTH-7A gene was cloned and its structure,expression and function were studied to provide candidate gene for wheat resistance breeding.The main findings are as follows:1.The Ta XTH-7A gene was cloned by PCR.We found that it contains 3 exons and 2introns,of which CDS is 870 bp in length and encodes 290 amino acids.Sequence analysis of the encoded protein revealed that the protein has a conserved site unique to XET;the protein also contains a signal peptide and a transmembrane domain,possibly belonging to a secreted protein.2.The Ta XTH-7A was constructed on p Cold^TMTF vector,and the recombinant protein was induced by IPTG under low temperature conditions.The recombinant protein was successfully expressed in prokaryotic cells by SDS-PAGE electrophoresis and Western blot analysis.XET enzyme activity was determined by colorimetry,and it was found that Ta XTH-7A did have the effect of transferring the xyloglucan glycosyl group,and has maximum activity at p H5.5.This proves that Ta XTH-7A is indeed a xyloglucan endoglycosyltransferase.3.The expression product of Ta XTH-7A was subcellular localization by Agrobacterium injection.It was found that Ta XTH-7A protein mainly exists in cytoplasm,cell membrane and apoplast.The presence of Ta XTH-7A protein in apoplast indicates that the protein is indeed secreted protein.The expression pattern of Ta XTH-7A was studied under different stress conditions.The results showed that the gene was up-regulated under drought,high salt（Na Cl）,ABA,SA conditions,and down-regulated under high temperature and low temperature conditions.Arabidopsis thaliana was transformed with the promoter of Ta XTH-7A,and GUS staining revealed that the gene was mainly expressed in roots and anthers.4.The 35S:Ta XTH-7A-p Ca MBIA1302 vector was constructed,and the Ta XTH-7A gene was driven by 35S,and Arabidopsis thaliana was infected by Agrobacterium tumefaciens to obtain the transgenic Arabidopsis T₃ generation.The functional analysis of Ta XTH-7A was carried out by comparison of transgenic Arabidopsis and wild type.The results showed that:Under normal growth conditions,the germination rate of transgenic Arabidopsis increased,the main roots were longer,and the average diameter of roots increased.Further analysis revealed that the endogenous hormone content of transgenic Arabidopsis seeds changed,and the root cell volume increased.These may be responsible for the increased rate of germination and root growth of transgenic Arabidopsis.Under the conditions of simulated drought treatment by PEG6000 and Mannitol,transgenic Arabidopsis thaliana’s germination rate was faster and the number of lateral roots increased significantly.Under drought treatment conditions,the drought tolerance of transgenic Arabidopsis thaliana was significantly higher than that of wild type;The determination of the index found that the Ta XTH-7A gene increased the antioxidant capacity of transgenic Arabidopsis root’s cells,reduced the degree of membrane peroxidation,and maintained the integrity and normal function of the cell membrane.This may be the physiological cause of the drought tolerance of transgenic Arabidopsis.In addition,the root length of wheat was positively correlated with the expression of Ta XTH-7A gene,and the increase of wheat root length was beneficial to drought tolerance.The results of the above studies in Arabidopsis and wheat consistently indicate that the Ta XTH-7A gene contributes to the improvement of plant drought tolerance.