Cloning,Expression And Functional Verificationof Ramie Hemicellulose Synthesis Related Gene

Ramie（Boehmeria nivea L.）is a perennial fiber crops,belong to Urticaceae,which is mainly represented by harvested bast fibers and is a typical representative of perennial fiber plants.At present there are related literatures found that ramie hemicellulose plays an important role in the cadmium tolerance of ramie.Therefore,the study of hemicellulose-related genes in ramie has provided a theoretical basis for improving the accumulation of cadmium in ramie and its tolerance to cadmium and reducing the cadmium transformation in forage crops.Based on the results of transcriptome sequencing and the results of ramie root response to heavy metal cadmium,a total of 14 hemicellulose-related genes responding to cadmium stress were screened（3 β-mannanase genes（MAN）and 11 woods.Dextran glycosyltransferase/hydrolase gene（XTH）.There are related literatures showing that the hemicellulose of ramie cell wall has outstanding contribution to cadmium tolerance,so the full-length cDNAs of 6 hemicellulose related genes（1 MAN and 5 XTH）were cloned,and their tissue expressions and pairs were studied.The response to stress;the construction of overexpression vectors and transgenic Arabidopsis gene function studies,as well as revealing the cadmium-tolerant molecule mechanism of cadmium,the specific results are as follows:1）The ramie BnMAN1 gene was cloned and encoded 375 amino acids.The predicted molecular weight was 42.47 kD and the theoretical isoelectric point was 9.45.The similarity comparison revealed.Analysis of the Protein Domains Encoded Thereof Finds that BnMAN1 has a β-mannanase domain（COG3934）and a glycosyl hydrolase domain（Cellulase）.The promoter sequence contained several cis-acting elements,such as TATA-box,CAAT-box,AE-box,Box 4,I-box,GAG-motif,GATA-motif,Skn-1_motif,ARE,AuxRR-core,MBS.Comprehensive analysis found that the gene may respond to ramie stress resistance by participating in hormone signal transduction.2)Five XTH genes,named as BnXTH1,BnXTH2,BnXTH3,BnXTH4,BnXTH5 was isolated.The analysis revealed that all five BnXTHs contained conserved motifs that callde GH16_XET and Glyco_hydro₁6.And the encoded amino acid sequences contained the conserved catalytic motif DE（L/I）DFEFLGN.The cluster analysis of five BnXTH-encoding amino acid sequences revealed that BnXTH1,BnXTH2,and BnXTH3 proteins are closely related,and BnXTH4 and BnXTH5 proteins are closely related.3)The results of real-time PCR suggested that the BnMAN1 gene expressed in roots,stems,leaves,shoot tips and flowers,with the high expression levels in female flowers and male flowers,among which female flowers had the highest expression levels;Five BnXTHs genes were found in roots,stems and leaves.Expression was found in stem tip and flower,and it was mainly expressed in female flower and male flower.Analysis of abiotic stress showed that all five genes were induced by abiotic stresses such as drought,cadmium,and high salt.4)The BnMAN1,BnXTH1,BnXTH2,BnXTH3,BnXTH4 and BnXTH5 overexpression vectors were constructed,and import it into EHA105,and obtained the transgenic Arabidopsis thaliana.The function of BnXTH1 gene was preliminary verified.Overexpression BnXTH1,which can improve transgenic arabidopsis had higher root length and biomass yield than the wild type under cadmium stress,indicating that it was related to heavy metal cadmium tolerance.