Screening Of Rare Antagonistic Strains From Special Eco-environment And The Cloning And Analysis Of Biocontrol Related Genes

The special eco-environment microorganisms could generate new bioactive substances and lead compounds such as enzymes,antibiotics,polysaccharides and lipids,etc.For their unique genetic types,specific physiological and metabolic mechanisms,they are a potential repository for exploiting new anti-factors,and are also a new hotspot of microorganisms resources development.This study is to screen antagonistic strains from special eco-environment that have the capability of antagonizing Fusarium oxysporum on cabbages and Botrytis cinerea on tomatoes,which is a severe disease at production in recent years,laying a foundation for product development and application of the strain.The test screened out 728 special eco-environment microorganisms from Mohe permafrost,snow-covered plateau of Tibet,mangroves and other special eco-environment,and obtained 11 actinomycete strains,40 bacteria strains.Identification of strains by 16 S r DNA gene sequencing showed that Bacillus sp.was a dominant flora.Collimonas sp.ZL261 was isolated from the alpine meadow soil at Sejila Mountain in Tibet of China,of which the altitude is 4530 meters,had special colony morphology and cultural characteristics.The bacterium was a kind of new microorganism in China.Therefore,this research was focusing on studying and analysizing Collimonas sp.ZL261 for determining its antibacterial activity,taxonomic status and function-related gene cloning.The results were shown as follows:The results from antibacterial spectrum test showed that the strain ZL261 had strong specialization on antagonizing pathogens.Among the tested pathogens including bacteria,yeast,and pathogenic fungi,only Monilinia fructicola was greatly inhibited by ZL261.The efficacy of the strain fermentation broth inhibiting peach brown rot caused by M.fructicola in vitro was up to 86.21%.The strain had capability of producing siderophore,protease,chitinase,phosphate enzymes and other biocontrol related substances.By morphological,cultural,biochemical and physiological characterizations,16 S r DNA sequencing and DNA-DNA hybridization analysis,the strain was identified as C.pratensis,which was a new record of the genus in China.In order to clarify the category and the function characteristics of the protease,the protease gene(named as capro)was cloned by constructing the gene library with restriction endonuclease method,and successfully expressed in Escherichia coli DH5α.An open reading frame of 1092 bp encoding a 363-amino acid(aa)protein with a calculated molecular weight(Mr)of 38.0 k Da and a p I of 6.65,submitted to Gen Bank and obtained an accession numbers KF992845.Protein BLAST and phylogenetic tree analysis of the deduced amino acid sequence from capro showed that the encoded protein had 83% homology to a protease of C.fungivorans Ter331(NC015856).The analysis of primary,secondary and three-dimensional structures showed that the amino acid sequece of Capro include zinc-binding conservative regions.One recombinant protein with the size of 38 k Da was obtained by expressing the gene capro in E.coli DH5α.The protease Capro was sensitive to EDTA which was an inhibitor of metalloproteases.The optimum p H and optimum temperature of Capro activities were p H 7 and 30 °C,respectively.When exposed to 10 °C,the enzyme relative activity still remained more than 70%.Summarizing the results above,we reached the conclusion that the protease Capro was a cold-active proteases belonging to matrix metalloproteinases M35.Based on the reported extracellular chitinase gene chi I(EU599185.1)of C.fungivorans Ter331,primers were designed to amplify the chitinase gene from the strain ZL261.The results showed that the chitinase gene(named as chi IQ),was successfully cloned,and the gene had an open reading frame of 1338 bp(Gen Bank: KJ704781)and encoded a protein composed of 445 amino acids.The molecular weight of Chi IQ was 46.04 k D and the predicted isoelectric point was 5.67.Sequence alignment and homology analysis revealed that the chi IQ gene had a high similarity(92%)to the chi I gene(ACF93782.1)of the strain Ter331.The amino acid sequence of Chi IQ contained a highly conserved sequences SXGG and GXDXDXE of chitinase GH18 family.Therefore,Chi IQ chitinase was a member of GH 18 family.The chitinase gene prokaryotic expression vector was constructed using restriction endonucleases(Bam H I and Hind III)digestion method and expressed in E.coli BL21.This work was essential for further studying the role of chitinase secreted by the strain ZL261 in the activity against peach brown rot.