| Plant virus disease is seriously harming agricultural production and had made great losses to world economic because of common occurrence and difficult control. Microbe is an important resource for research and development of antiviral agents. It is a research focus that isolation and screening of antiviral compounds from microbial metabolites for scholars around the world. Streptomyces kanasensis ZX01 was isolated from soil round Kanas lakeside of Xinjiang by Research & Development Center of Biorational Pesticide, Northwest A&F University. Previous tests showed that metabolites of strain ZX01 have great antiviral activity. So, this study included isolation and identification of antiviral compound, sequencing and analysis of genome, construction of conjugation system, construction of mutant with nsdA disruption, and so on. Main results are as follows:(1) Glycoprotein GP-1 with anti-TMV activity was isolated from S. kanasensis ZX01 by using extraction, adsorption chromatography, ion-exchange chromatography, et al. Molecular weight of GP-1 is 8479 Da, including 40.23% saccharide and 54.36% protein. GP-1 contains 15 kinds of amino acids. High amount of Asp (15.15%) and Glu (12.63%), low level Arg (0.62%) and Lys (4.88%). GP-1 comprises D-mannose, D-xylose, D-arabinose, D-glucose, D-glucosamine and D-galactose in a molar ratio of 0.38:0.14:0.40:2.62:0.10:1.00. GP-1 not only contains N-glycan but also O-glycan. The secondary structure content of GP-1 was estimated to be 20.10% of β-sheet,21.70% of β-turn and 58.30% of unordered structure. Heat affects anti-TMV activity of GP-1 but secondary structure. The anti-TMV activiy of GP-1 is stable under 60℃ condition, but it will lose activity when temperature is higher than 80℃. The results of MALDI-TOF-MS/MS showed that GP-1 was a novel glycoprotein. A method of rapid purification and detection was established by using ultrafiltration, affinity chromatography and HPLC.(2) The results of anti-TMV activity tests in vivo and in vitro showed that GP-1 with great protective effect prevented host plant from TMV infection. The results of inactivating tests and scanning electron microscope showed TMV particles were disrupted by GP-1, then lost infective ability. Moreover, GP-1 inhibited capsid protein accumulation of TMV in host plant. Induction tests showed GP-1 has ability of induced host resistance which increased at first and decreased subsequently with the induction time. The activities of SOD, PPO and PAL increased sharply in plant after treating with GP-1, but the MDA content decreased.(3) High-throughput sequencing technologies were applied to sequence genome of strain ZX01. The draft genome of 7,026,279 bp was distributed in 225 contigs with G+C content of 73.88%. This draft genome consisted of one linear chromosome with 7 rRNA operons,65 tRNA genes, and 6245 coding sequences (CDSs). For the CDSs,3996 proteins could be assigned to COG families, and 2249 CDSs encode proteins with no match to any known proteins in the public databases.(4) Twenty-one biosynthetic gene clusters for secondary metabolites (including terpenes, polyketide synthases, phosphonate, bacteriocin, et al; distributing in 19 contigs) were identified by antiSMASH v3.0.0. Cluster 1,9,16,18 and 20 were PKS, NRPS or hybrid PKS/NRPS. In normal condition, cluster 1,9 and 20 were expressed, but cluster 18 wasn’t expressed and cluster 16 was expressed partly. In γ-butyrolactone inductive condition, cluster 1,9,16,18 and 20 were all expressed, cluster 9 and 16 were overexpressed. Moreover, the results of genic analysis of glycosylation and global regulation by multiple methods showed that plenty of glycosyltransferase and global regulators which may be involved in glycosylation and metabolic regulation of GP-1 in S. kanasensis ZX01 genome.(5) The conjugation system of strain ZX01 was developed and optimized. The spores were used as the receptor,50℃ heat shock 10 min,37℃ incubated 2-3 h, donator:receptor=10:1, in Gause I medium containing 10～30 mM MgCl2 to conjugate, covering antibiotics after 16～18 h incubation.(6) The recombinant vector pRV5455 (pKC1139::nsdAUD::KanR) was developed by using Overlap PCR method. nsdA in strain ZX01 was disrupted succesfully and mutant of ZX01 without nsdA was obtained. The nsdA deficiency made colonial morphology changed, spores and hypha production increased, and then GP-1 production enhanced.In conclusion, S. kanasensis ZX01 has a wide application for great secondary metabolism ability. The sequencing of S. kanasensis ZX01 genome and establishment of conjugation system provide plenty of data for researches on molecular genetics, metabolic regulation and construction of engineering S. kanasensis ZX01. This study will lay the foundation for application of S. kanasensis ZX01 on crop protection。... |
PDF Full Text Request