Cloning, Expression And Transformation Of A MsUGT73B2 Gene From Alfalfa (Medicago Sativa. L)

Alfalfa(Medicago sativa L.),known as the "king of forage",is a leguminous herbage which has the capable of nitrogen fixation with nitrogen-fixing nodules.It is an excellent pasture and has the advantages of high yield,good quality,good adaptability,and so on.Alfalfa encounters various adversities(such as drought,high temperature,low temperature,salinity,etc.)during the growth period,affecting its normal development and nodule activity.It is very feasible to use molecular methods to cultivate good varieties.In recent years,alfalfa gene cloning,functional verification and genetic transformation have become increasingly mature.Glycosylation is one of the most important metabolic reactions in plants.Glycosylation in plants is catalyzed by glycosyltransferases and affects various metabolic activities of plants.In this study,the gene was selected from the cDNA microarray and the alfalfa MsUGT73B2 gene was cloned by RACE-PCR.The bioinformatics analysis of the MsUGT73B2 gene was carried out.Based on this,the expression pattern of MsUGT73B2 gene in different tissues under different treatments such as drought,ABA and salt was analyzed by qRT-PCR.The plant overexpression vector pCAMBIA1300-35S-MsUGT73B2-EGFP was constructed.Introduced the vector into Arabidopsis thaliana,MsUGT73B2 transgenic Arabidopsis was successfully obtained.Now all the findings are summarized as follows:1.Using RACE-PCR,the full length cDNA of MsUGT73B2 gene was successfully obtained.Sequence analysis showed that the full-length cDNA of this gene was 1512 bp in length and encoded 503 amino acid residues,which was highly similar to GT99 D gene of Medicago truncatula.2.qRT-PCR analysis of MsUGT73B2 gene expression pattern showed that the leaves expression was significantly higher than the root;in drought and ABA treatments,the gene expressions showed upward trends from 0 to 2 hours and decreased after 2 hours.When the gene was treated by NaCl,the gene expression was found to increase at 0-4 h,reached a maximum at 4 h,and then decreased.3.Overexpression vector pCAMBIA1300-35S-MsUGT73B2-EGFP was successfully constructed and injected into tobacco for subcellular localization.The Arabidopsis thaliana was used to transfer MsUGT73B2 gene by dip-flower method,and transgenic Arabidopsis of MsUGT73B2 gene was successfully obtained through antibiotic screening and PCR assay.