Determination Of Infectivity Of Meloidogyne Enterolobii And Screening Of Nematicidal Activity Clones From Soil Metagenome Library

In recent years, Meloidogyne enterolobii has broken out in tropical and subtropical regions of guava orchard,causing devastating damage and financial loss to guava,has been gradually rising as the most important specie tropical and subtropical regions is one of the root knot nematode species. With the development of modern and efficient agriculture, the increasing greenhouse and facilities to improve the ground temperature, the intrusion and proliferation of Meloidogyne enterolobii is possible, which will pose a threat to the country's crop production. Therefore,make clear its host range and infectivity,and find out efficient control methods is the key to prevent the spread of the disease.Through the identification of host plant root-knot nematodes in Hainan Island, found that the population has been further proliferation,replacing Meloidogyne incognita is showing, and in tropical and subtropical regions of china's most important trend of root-knot nematodes in this study. In order to avoid chemical nematicides fertilization to cause chemical pollution to the environment, the researches apply the metagenomic technology,directly from the occurrence and prevention and control of nematodes and most closely related to the rhizosphere microbial screening of Nematicidal protease gene resources, and make the preliminary research on the activity of enzyme, aim at finding out a new way to biological control of Meloidogyne enterolobii. The specific research results are as follows:1) The determination of infectivity of Meloidogyne enterolobii.To explore the minimum inoculation quantity of Meloidogyne enterolobii and evaluate the inoculation quantity effects on the growth and different infectivity of seven kinds of varieties of potted tomatoes by planting tomatoes in the greenhouse.The results show that the minimum inoculation quantity reached the second-stage juveniles ?J2? of M.enterolobii is40in every500cm3.In seven tomato varieties,"Eminem Stan F1"is the middle resistant variety,"zhong za No9"?"li sheng No1"?xi fen No1"?da an ji ri"are the resistant varieties,"USA903" and "jin sheng"are susceptible varieties to disease.2) The construction and screening of Metagenomic library. From a seriously infected field by Meloidogyne enterolobii in guava orchard, healthy guava rhizosphere sample, total DNA extraction, purification, then repair, connection, such as packaging and transfection process, to construct metagenomic Fosmid expression library. The results show that characteristic analysis:The library containes31104clones,the average insertion fragment is36.5kb, empty loading rate is1.9%,contains more than1Gbp microbial genomic information.the skim milk as substrate, the transparent circle phenotypic choice screening positive clones containing protease one hundred and eleven strains, nine strains have a toxic effect to Meloidogyne enterolobii,including21fa6?5ba8?5fa8?20aa2?60ha9?14ba10?83fa2?83ha8and178fa8.Especially21fa6clone poison works best.3) Study on the containing active protease gene clone about21fa6.As21fa6clone for the research object, rotation speed150r/min,culture temperature35?is the best activation conditions;shaking speed200r/min,culture temperature was35?,is a clone of bacteria optimum enzyme production conditions;adding inducer of skim milk,the induction of FAS dual role,when48h,fermentation liquid protein absorption valuereached the maximum value.Preliminary study on21fa6protease clone, is a protease genes subcloned, sequenced, separation and purification of the protease activity of the foundation.4) Further research was done on21fa6., the subclone library was constructed and the subclone sprol14a5,which could degraded the protein was screened. After analysis of gene structure, sprol14a5is a secreted extracellular protease and a database search for homologies revealed it possessed45%identities with peptidase S15from Maricaulis maris MCS10?accession no. YP₇56822at NCBI?.It is a novel serine protease. Besides these, it has the serine protease-conserved catalytic triad residues, Asp469, His541and the catalytic nucleophile Ser348. Indor biological nematicidal showed that cloning broth could kill the nematodes without inducer and the promoter which existed in the clone plasmids. So this protease gene had its own promoter. At the same time,this protease is a secreted extracellular serine protease.