Isolation And Characterization Of A UDP-Glucose Dehydrogenase Genc From Broussonetia Kazinoki×broussonetia Papyifera

Paper mulberry(Broussonetia kazinoki×Broussonetia papyifera)is widely used in the paper manufacturing,animal feed,medicine industries and in other industries as well,and plays an important role in poverty alleviation industries,environmental protection,and ecological restoration.Uridine diphosphate glucose dehydrogenase(UGDH)is a key enzyme in the hemicellulose and pectin biosynthesis pathway that catalyzes the conversion of uridine diphosphate glucose(UDP-Glc)to UDP-glucuronate(UDP-GlcA)and participates in the regulation of growth and development in plants.Therefore,studying the function and expression characteristics of the UGDH gene of paper mulberry is of great significance for the development and utilization of this multifunctional and common tree species.In this study,we isolated a UGDH ortholog,referred as to BpUGDH,from paper mulberry and analyzed its function and expression characteristics.At the same time,selected the paper mulberry tissue culture seedlings which were with low temperature stress treatment and under non-stress conditions for transcriptome analysis,and then functional clustering analysis the obtained differentially expressed genes and also predicted the protein-protein interaction network,and selected the key proteins.Through research,the purpose is to provide a good candidate gene for improving cold resistance,accelerating fiber cell development,and enhancing plantvegetative growth.The main results are as follows:(1)The orthologs of UGDH were isolated from paper mulberry by RT-PCR and RACE,referred to as BpUGDH,which contains 1443bp open reading frame,encodes a protein of 480 amino acids with a predicted molecular weight of 53.04kDa and isoelectric point of 5.41;The relationship between BpUGDH and UGDH of Boehmeria nivea were close in genetic relationships and the BpUGDH protein was expressed in the chloroplast.(2)To investigate the expression of BpUGDH by Real time Q-PCR,the result shows that BpUGDH were expressed in roots,stems and leaves of paper mulberry with highest expression in stems.(3)The BpUGDH 1324bp promoter sequence was isolated using a genome walking method,and a GUS reporter gene was used to analyze the pattern of BpUGDH expression.A Pro.BpUGDH::GUS gene construct was highly expressed in transgenic Arabidopsis thaliana seedlings and its expression was induced by low temperature,methyl jasmonate,gibberellin,ethylene and auxin.While its expression decreased in mature organs and no GUS staining was observed in seeds.(4)To further analyze the features and functions of BpUGDH,we constructed the pBIl01-BpUGDH plasmid containing the BpUGDH cDNA sequence with expression driven by the CaMV35S promoter and introduced it into A.thaliana.The results showed that BpUGDH overexpression increased the soluble sugar content,promoted the accumulation of hemicellulose and pectin,enhanced the vegetative growth of transgenic plants and four transgenic lines showed stronger cold tolerance.(5)A total of 59056 unigenes were obtained from the transcriptome sequencing of paper mulberry tissue culture seedlings.Comparing with the commonly used 6 large databases,the results were annotated to 31358 unigenes in the NR database and the most similar to M.notabilis,reaching 60.40%.There were 27426,23431,7957,18183 and 15574 unigenes in Swans-Prot,Pfam,COG,GO and KEGG databases have the annotated results,respectively.(6)A total of 7320 differentially expressed unigenes were obtained during cold stress response in the paper mulberry.Among them,3219 were up-regulated and 4101 were down-regulated.The analysis of GO enrichment results showed that more unigenes involved in catalytic activity,metabolic processes,membrane,binding,cellular processes,single-organism process and membrane fractions;The results of KEGG pathway analysis showed that 1552 differential genes covered 124 pathways and mainly concentrated in the metabolic part.(7)The protein PPIs of differentially expressed genes were predicted by using the arabidopsis protein interaction network data of String 11.0 database as reference,revealed that PCNA2,THY-1,DUT1 and CDC2 the four key proteins.In the same way,a total of 47 genes were screened from the three gene sets of functional enrichment of BpUGDH and then predicted for protein PPIs,obtained 9 proteins with close interaction with BpUGDH and combined with the results of RNA-Seq,the mechanism of BpUGDH to improve the cold tolerance of plants was preliminarily investigated.