| Streptomyces lavendulae gCLA4 was a plant endophytic actinomycetes,isolated from the cucumber leaves.Indoor and field experiments have shown that it could inhibit the growth of a variety of pathogens,had a good biological control function.In order to explore its bioactive substances,reveal the biogenesis mechanism of gCLA4,and lay the foundation for the creation of biological pesticides.In this study,the gCLA4 gen function were predicted,and the total 102 proteins-94 unknown function secreted proteins which amino acid numbers greater than 100 and less than 1000 in gCLA4 and 8 functional proteins,which may inhabit the biocontrol activity such as chitosanase(278),chitinase(3253,4025,4203,5554),chitin-binding protein(1879),ricin b lectin(2380),necrosis-inducing protein(4955)were taken to expressed in E.coli BL21(DE3).The antimicrobial activity of the crude protein solution were detected by confrontation in the dish,and the tobacco allergies were detected.The results were as follows:(1)The gCLA4 genome size was about 7.59 Mb.The GC content was 72.36%.There were 6906 coding genes in the whole genome,and the length of the coding region was 86.83% of the total length of the genome.The gCLA4 gene sequence was compared with GO,COG,KEGG and NR database,then 978,2363,2852 and 6634 genes were annotated.gCLA4 genome sequence was analyzed by antiSMASH,then 24 secondary metabolytic biosynthetic gene cluster was obtained.Its 6 custers were 100% similar to the known biosynthetic gene cluster,and one was 87%.It may synthesize Streptothricin,Coelichelin,which with antibacterial activity.A total of 271 secreted proteins were found in gCLA4,accounting for 3.9% of the total genome.Of which 104 proteins had functional descriptions.(2)In this research,the signal peptide of 102 proteins was delected and ligated to pET-28 a and pCold TF to construct the recombinant vectors and expressed in E.coli BL21(DE3).Then total 86 soluble proteins were obtained,12 soluble proteins were obtained by pET-28 a,74 were obtained by pCold TF.The 86 proteins had no antibacterial activity against target bacteria such as Escherichia coli,Staphylococcus aureus,Bacillus subtilis,Saccharomyces cerevisiae,Pseudomonas syringae pv.Actinidiae and Valsa mali 03-8.Necrosis-inducing protein 4955 could cause tobacco allergy,the others were inactive.(3)The basic properties of necrosis-inducing protein 4955 was analyzed by Protarsam and the three-dimensional structure was simulated by SWISS-MODLE.The stability of protein 4955 was explored.The activities of CAT,SOD,POD,PAL and the expression of PR1-a,PR1-b,NPR1,LOX and PAL in tobacco were detected after the treatment of protein 4955.The results showed that protein 4955 consists of 225 amino acids,molecular weight was 24491.12,PI was 5.96,contained conserved domain NPP1.Toleranted temperature up to 40°C.Tolerated pH from 6 to 10.The 5 times dilution of the original protein was active,while 10 times dilution was weak.The activity of CAT,SOD and PAL increased at 2 days after the treatment of protein 4955,and the expression of PR1-b and LOX genes were up-regulated at the 1st,3rd and 5th day,and the PAL gene was up-regulated on the 4th day.The results indicated that protein 4955 can induce tobacco resistance.(4)Chitosanase 278,chitinase 3253,4025,4203,5554,chitin-binding protein 1879 composed of 255,533,576,754,574,172 amino acids,respectively.Molecular weight were 27540.71,53830.43,60100.10,80762.58,59326.23,18571.79,PI were 5.86,5.57,5.80,6.00,5.90,8.72.Chitosanase 278 contained Glyco-hydro-46 domain;Chitinase 3253,4025,5554 were 18-family chitinase containing both the GH18 domain,CMB domain and FN III domain;chitinase 4203 contained both GH18-chitinase domain,a 19 family ChiC domain and a ChiC-BD domain;chitin binding protein 1879 contained LPMO-10 domain.About 24.5% chitin binding protein could bind to chitin when reacted for 24 h at 40°C.Chitosanase 278,chitinase 3253,4025,5554 had no degradation activity against chitosan and chitin. |
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