Cloning And Functional Analysis Of EuGT3, A Glycosyltransferase Protein-encoding Gene Of Eucommia Ulmoides

Eucommia ulmoides Oliv.is a national secondary protected plant with a wide range of uses.The medicinal ingredients of Eucommia ulmoides mainly include phenylpropanoids,lignans,flavonoids and polysaccharides,etc.,which can be used to treat diseases such as hypertension,diabetes,and rheumatoid arthritis.Glycosyltransferase is a class of enzymes responsible for catalyzing the glycosylation reaction of small molecule compounds.It transfers active sugar groups from nucleoside sugars to hormones,secondary metabolites,pathogenic bacteria infectants,and a series of toxic substances inside and outside plants.Plant small molecule compound receptors,thereby affecting various metabolic activities of the plant.In recent years,a large number of scholars have cloned glycosyltransferase-related genes in plants and analyzed their functions.Glycosyltransferases mainly affect compound glycosylation,flavonoids,lignin synthesis,and participation in plants.A variety of biological approaches such as stress resistance.In order to further enrich the members of the glycosyltransferase family in plants and to better understand the biological functions of glycosyltransferases in different plants,this study cloned the Eucommia glycosyltransferase encoding gene EuGT3 and performed bioinformatics analysis on it.And to analyze its expression characteristics in Eucommia ulmoides tissues and organs,and construct the plant overexpression vector p CAMBIA1300-35S-EuGT3,genetically transform Arabidopsis thaliana by Agrobacterium inflorescence dip method,and successfully obtain transgenic Arabidopsis plants.The obtained transgenic Arabidopsis thaliana was used to verify and analyze the function of EuGT3 gene.The following results were obtained:1 Cloning and bioinformatics analysis of EuGT3 geneA gene encoding glycosyltransferase protein was successfully cloned from Eucommia ulmoides by RACE(rapid amplification of c DNA ends)technology,and a CDS sequence with a full length of 1441 bp was obtained by splicing,encoding a total of 481 amino acids.Analysis of the protein domain encoded by the gene revealed that the sequence contained a conserved sequence PSPG box structure,which was identified as a glycosyltransferase family and named EuGT3.Further bioinformatics analysis of EuGT3 showed that EuGT3 has a molecular weight of 64 KDa,a theoretical isoelectric point of p I:4.97,no signal peptide,no transmembrane structure,and a hydrophobic protein with multiple glycosylation sites.2 EuGT3 gene subcellular localization and tissue expression analysisThe results of bioinformatics analysis predict that the protein is present in the inner membrane structure of the cell.The fusion expression vector p CAMBIA1300-35S-EuGT3-GFP was constructed,tobacco was used as the experimental material,and EuGT3 was subcellularly localized through the transient expression experiment of tobacco leaf epidermis.The results showed that EuGT3-GFP fusion protein was expressed in the cytoplasm.The q RT-PCR method was used to detect the expression of EuGT3 gene in different parts of Eucommia ulmoides seedlings.The result analysis showed that EuGT3 was expressed in roots,stems and leaves,but there was no significant difference in the expression amount in roots,stems and leaves.Therefore,the expression of this gene has no tissue specificity.3 Construction of EuGT3 Gene Plant Expression Vector and Genetic Transformation of Arabidopsis thalianaUsing pCAMBIA1300-35S-GUS as the basic vector,construct the plant expression vector p CAMBIA1300-GUS with target genes EuGT3 and 35 S as promoters,GUS as reporter gene,and resistance screening as kanamycin(Kan)and hygromycin(Hyg).35S-EuGT3.Using the Agrobacterium transformation system,the p CAMBIA1300-35S-EuGT3 plasmid was genetically transformed into Arabidopsis thaliana by the Agrobacterium inflorescence infection method.According to GUS histochemical staining and PCR identification,a total of 21 transgenic Arabidopsis thaliana lines were obtained.The q RT-PCR results showed that among the 21 transgenic Arabidopsis lines,the EuGT3 gene expression levels of TP-2,TP-9,TP-12 and TP-21 were higher in four lines,so the progeny of these four lines were repeatedly screened to obtain T3 generation Arabidopsis thaliana plants for follow-up experiments.4 The effect of overexpression of EuGT3 gene on the phenotype of Arabidopsis thaliana plantsThe phenotypes of EuGT3 gene transgenic plants,wild-type and empty vector plants were observed under the same culture conditions.During the entire growth process of Arabidopsis plants,the growth process of transgenic Arabidopsis was faster than that of the same age wild-type and empty vector Arabidopsis Mustard plants,and transgenic Arabidopsis plants bolting and flowering earlier than wild-type and empty vectors.The EuGT3 gene,wild-type and empty vector Arabidopsis plants transplanted to the nutrient soil were used for the statistics of leaf length,leaf width,leaf number,lotus diameter,plant height and branch number.The results showed that the average leaf length of transgenic Arabidopsis was 4.21 ± 0.32 cm,leaf width was 1.49 ± 0.42 cm,leaf number was 19.41 ± 0.48,lotus diameter was 8.52 ± 0.41,plant height was25.23 ± 0.43 cm,and branch number was 9.31 ± 0.24;wild type The average leaf length of Arabidopsis is 4.04±0.12 cm,the leaf width is 1.43±0.37 cm,the number of leaves is 16.02±0.35,the diameter of the lotus is 8.21±0.52,the height of the plant is21.92±0.26 cm,the number of branches is 6.05±0.30;The average leaf length is3.97±0.28 cm,leaf width is 1.44±0.12 cm,leaf number is 16.35±0.02,lotus diameter is 8.34±0.09,plant height is 21.35±0.18 cm,and branch number is 6.18±0.23.Compared with the wild-type and the empty vector Arabidopsis,transgenic Arabidopsis has significant differences in leaf number,plant height and branch number,but the differences in leaf length,leaf width and lotus diameter are not significant.It is speculated that the glycosyltransferase EuGT3 may be involved in the regulation of plant hormones such as cytokinin and auxin in Arabidopsis thaliana transfected with EuGT3 gene,thereby affecting the number of leaves,plant height and branch number of Arabidopsis plants.But its specific regulation mechanism needs further study.5 Detection of flavonoids in Arabidopsis plants overexpressing EuGT3 geneThe total flavonoids of Arabidopsis plants were extracted with organic solvents.According to the absorption spectrum characteristics of the extract,the absorbance was measured at a specific wavelength of 325 nm with a microplate reader,and the standard curve was used as a reference to analyze and calculate the transgenic EuGT3 gene and wild-type A.thaliana.The total flavonoid content of mustard.The results showed that the total flavonoid content of EuGT3-2,EuGT3-9,EuGT3-12 and EuGT3-21 were all higher than that of the wild type,which were 2.25,2,2.4 and 2.53 times of the total flavonoid content of wild-type Arabidopsis,respectively.,Have reached a very significant level.It shows that EuGT3 participates in the metabolism of flavonoids in Arabidopsis thaliana,and overexpression can significantly increase the total flavonoids content in Arabidopsis.The newly harvested T3 generation seeds of the transgenic EuGT3 gene homozygous Arabidopsis,the empty vector and the wild-type plants were inoculated on the same medium and germinated.The results showed that the germination rate of T3 seeds transduced with EuGT3 gene,empty vector and wild-type plants on the normal medium was not different,and the germination rate could reach 100%.Then the Arabidopsis seeds were subjected to stress treatment with different concentrations of Na Cl and mannitol to observe the germination rate of Arabidopsis seeds under salt stress and drought stress.The results showed that the seed germination rate of transgenic Arabidopsis with EuGT3 gene,empty vector and wild-type Arabidopsis was different under stress treatment,that is,the germination rate of transgenic Arabidopsis was higher than that of wild-type and empty vector,and with increasing stress intensity Increase,this phenomenon is more obvious.It is speculated that the glycosyltransferase EuGT3 may be involved in the regulation of hormones in plants and the synthesis of anti-stress active ingredients in the stress environment,thereby improving the ability of plants to resist stress environments.