| The plant soil borne disease is caused by many kinds of pathogens. Because of the variety of pathogen species and the complicated factors, it has been one of the most important issues in agricultural production. The use of effective microorganisms from plant rhizosphere to control soil borne disease has an unique advantage. Diversity of microbial community exist in plant rhizosphere, they play an important role in control of plant diseases and promoting plant growth.The most common plant rhizosphere bacteria including Paenibacillus spp., Bacillus spp., Pseudomanas spp. and Burkholderia spp. and other types, are collectively called plant growth promoting rhizobacterium(PGPR). The important part of the research is to screen beneficial microorganisms. This study obtained strain X2-3 from the screening strains of PGPR population which was more antibacterial. We analyzed its antibacterial activity and effect to seed germination. And on this basis, we studied cloning, expression and activity of encoding ?1,3-glucanase genes, to provide the foundation for the further understanding of the biological control mechanism. The concrete results are as follows:1 The screening, identification and biological characteristics of strain X2-3We collected the rhizosphere soil from Shouguang, Shandong Province and other places, using plate dilution method to obtain bacterial isolates with antibacterial effect. The strain X2-3 was screened out. By morphology observation and analysis of 16S rDNA sequence of the strain X2-3, it was identified as Lysobacter capsici. The strain formed a clear and transparent circle on brewer's yeast (Saccharomyces cerevisiae) cell wall, laminarin and skim milk medium. Which showed that the bacteria could produce ?-1,3-glucanase and protease.2 Analysis of L.capsici X2-3 against other pathogensThe antimicrobial effect of the strain X2-3 on was analyzed by plate confrontation method. The results showed that the strain X2-3 has different degrees of inhibition of Fusarium graminearum, Rhizoctonia cerealis, Thielaviopsis basicola, Bipolaris sorokiniana, Phytophthora parasitica Var. nicotianae, Pythium ultimum.3 Effects of L. capsici X2-3 on germination of wheat seedsLUMAI 21 and QINGMAI H03 were used by soaking wheat seeds method. We found L. capsici X2-3 could significantly promote the seed germination, the length of taproots was increased by 36.9% and 9.7%, the length of lateral roots increased by 53.8% and 14.8% respectively. The pot experiments showed that X2-3 could reduce the occurrence of wheat root rot, the disease index of the control reduced 7.3%.4 Cloning and expression of ?-1,3-glucanase genes from L. capsici strain X2-3We designed and synthesized primers, and amplified three (3-1,3-glucanase genes from L. capsici X2-3 genome DNA by PCR, based on homologous sequences, according to reported ?-1,3-glucanase genes of strain Lysobacter enzymogenes C3, OH 11 and N4-7. The length of three ?-1,3-glucanase genes gluA, gluB and gluC is respectively 765bp,1200bp and 1149bp.According to the amino acid sequence deduced from the nucleotide sequence. Analyzed by DNAMAN, the similarity of gluA, gluB and gluC amino acid sequence of L. capsici X2-3 and those of L. enzymogenes C3, OH11 and N4-7 were 96.36%,94.99%,95.89% respectively. The amino acid sequence length of gluA of L. capsici X2-3 were same with L. enzymogenes C3, OH11, N4-7. The amino acid sequence of gluB of L. capsici X2-3 were longer than that of L.enzymogenes C3, OH11 N4-7 4 amino acids, in the position of seventeenth amino acid and twentieth amino acid of GLUB protein, the sequence is GVAG. The amino acid sequence of gluC was less than that of C3, OH11, N4-71 amino acids (glycine), in the position of the 258th amino acid of GLUC protein.The gluA?gluB?glnC gene and plasrmid pEHISTEV digested by BamHI and NotI were transformed to E. coli (Rossetta) formed prokaryotic expression strain pE-GluB, pE-GluA and pE-GluC. After 1PTG induction, the target protein was expressed in large quantities and detected by SDS-PAGE, there was a distinct band at 44.3 KDa and 29KDa respectively in pE-GluB and pE-GluA. Protein molecular weight was consistent with the predicted molecular weight of the amino acid sequence deduced from the gene. The empty plasmid pEHISTEV transformed into E.coli (Rossetta) did not has the corresponding protein.The GLUA and GLUB protein were purified by Ni-NTA His.Bind Resin, and the activity of the protease was determined by DNS colorimetry method. The activities of GLUA and GLUB were 27.06U/ml and 60.46U/ml respectively. |
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