| Bacillus subtilis is an aerobic,thermophilic,non-pathogenic and environment friendly(GRAS,generally recognized as safe)Gram-positive bacteria.Its simple structured cell wall secretes high aplication value proteins.Escherichia coli,Gram-negative bacteria,is the first host to express recombinant protein.Compared to other gene expression systems,its ability to highly express the exogenous genes and expression of the protein far exceeds the amount of total protein of bacteria and used as most widely system in recent years.In this study,c DNA libraries of Pinella ternata contructed in B.subtilis and E.coli expression systems were screened against phages,Nematodes and pathogenic bacteria in order to find the key resistance genes in P.ternate libraries,responsible for the effective control of plant diseases.In this study,93 autolysis genes were found after repeated screening of P.ternata c DNA library.From these 93 autolysis genes,plasmids of 28 autolysis genes were extracted and transformed into B.subtilis,among them 25 autolysis genes showed positive antimicrobial results by plate streaking method.Crude protein from autolytic strain was tested against different bacteria and empty vector was taken as a negatrive control.Five genes(280,857,946,1259,1366)showed obvious bacteriostatic effect against the B.cereus 905 and Ralstonia solanacearum R21-5.Tricine-SDS-PAGE gel electrophoresis results confirmed that the molecular weight of the protein is between10-17 KD.In order to verify whether these genes have broad spectrum resistance,we screened anti-phage genes from P.ternata library by using E.coli phage JMPW1.Finally 7suspected resistant genes were selected for further experiment.To prevent the genetic mutation,extracted plasmids were transformed and streaked again on the plate for confirmation.We used B.subtilis WB800 phage Nf for screening of anti-phage gene from P.ternata library by using B.subtilis.We initially screened resistant genes and then for their stability the screening was repeated for the same genes.In this study,different screening methods were used for different expression systemsto find resistant genes against nematode.Extracellular metabolites of 93 autolysis genes from P.ternata c DNA library constructed in B.subtilis expression system were used to perform food preference assay and progeny assay of C.elegans.The Results showed that a total of 22 genes can influence C.elegans food preference but have no effect on C.elegans progency.Genes were randomly selected from P.ternata library constructed in E.coli expression system and induced by IPTG,which have significant effect on food preference for C.elegans. |
