Cloning And Expression Of Endoglucanase Gene EG146 From Rhizoctonia Solani,the Pathogen Of Rice Sheat Blight

As one of the three most devastating diseases of rice,sheath blight occurs in all rice producing areas of the world.The pathogen causing the disease is Rhizoctonia solani Kiihn,which has strong saprophytic and mainly destroys the host cell wall by secreting cell wall degrading enzymes(CWDE)and toxins,so as to kill the host cell and obtain the nutrients needed for its own growth and development,and to facilitate its expansion in the host.However,because the genetic transformation system of R.solani has not been established,the pathogenetic mechanism of the pathogen has not been studied well yet.In order to understand the role of CWDE in the pathogenic progress of R.solani,the endoglucanase EG146 gene was cloned and the pathogenicity and function of its coding product were studied.The results are as follows:An endoglucanase gene EG146 from the 16th family of glycoside hydrolase was cloned from the strain YN-7.Sequencing results showed that the gene has a total length of 1565 bp,including 9 exons and 8 introns,and the complete ORF size is 1125 bp,encoding 374 amino acids.The encoding product has a signal peptide of 19 amino acids while the amino acids from 23 to 70 are chitin binding domain,and from 92 to 305 are catalytic domain.According to bioinformatics prediction,EG146 protein is about 39 kDa in size,contain transmembrane domain,and mainly is secreted from endocrinic to extracellular.The tertiary structure of this protein is sandwich structure,which is the special structural characteristics of endoglucanaseEG146 gene was constructed into prokaryotic expression vector pET-28 and the recombined vector was transformed into Rosetta’s receptive state cell.No target protein was found in the supernatant of the culture medium after IPTG induction,but it mainly existed in the supernatant of the broken cell and had no endoglucan activity.The gene was transferred to Pichia pastoris GS115 and KM71.When the engineering strain was induced by methanol,a protein consistent with the predicted size was detected in the supernatant of broken cell,but no target protein had been detected in the culture supernatant.Enzyme activity assay showed that EG146 gene could be expressed in E.coli,but its expression product could not be folded correctly.After transient expression of EG146 gene in Nicotiana tabacun,Nicotiana benthamiana,tomato and pepper,we found that a large area of cell necrosis could be found in the leave of N.tabacun but other plant could not.DAB staining showed that there were a large number of brown particles burst along the leaf vein in the tobacco leaves injected with Agrobacterium containing EG146 gene,but only a small number of particles were found in the leaves untreated or injected with Agrobacterium without EG146 gene.This means that EG146 gene can cause the HR response in N.tabacun.Conductivity test showed that EG 146 could cause cell damage and electrolyte leakage of the N.tabacun leaves which conductivity value was 2.5 times that of the control group.When the total proteins of treatment leaves were analyzed by SDS-PAGE,we found that comparing with the control a protein band about 55 kDa was significantly thinner.MS detection showed that this protein is Rubisco enzyme,a very important enzyme involved in photosynthesis in chloroplast of plant cells.As to the reason of EG146 gene causing the decrease of Rubisco enzyme level in tobacco cells,the receptor of EG146 in plant cells and the specific role of EG 146 in the pathogenic process of the pathogen need further study.