GabT Regulated GABA Metabolism Pathway Of Nematicidal Streptomyces Snea 253 Strain

Root-knot nematode(RKN)is one of the important pathogens for economic crops,with a wide range of hosts and distribution.Using biocontrol bacteria can effectively control the population of root knot nematodes and reduce the damage they caused.Streptomyces venezuelae Snea253 strain belongs to actinomycetes with nematicidal activity to plant-parasitic nematode which preserved in our laboratory previously.Bioinformatics analysis showed that γ-aminobutyrate transaminase gene(gabT)is one of the important genes involved in carbon metabolism in Snea253 strain.The purpose of this study is to clarify that the gabT gene affects the activity of the strain by regulating the γ-aminobutyric acid(GABA)metabolic pathway of Snea253.The attenuated strain(Snea253-R)obtained by UV mutation was used as experienmental strain,Meloidogyne incognita as the target;The content of GABA and the downstream metabolite succinic acid were detected by enzyme-linked immunoassay(ELISA)and high performance liquid chromatography in attenuated strain overexpressed the gabT gene,nematicidal activity was detected too.Meanwhile,the gabT gene expression level,product content and nematicidal activity of wild-type strain under different carbon sources conditions were measured.The results as follows:1.Construction of overexpression vector: DNA of the wild-type strains was used as templates,pIB139 as vector to construct overexpression vectors.Then,the recombinant plasmid pGEM-gabT was transformed and was extracted,the plasmid pGEM-gabT and pIB139 were double digested by Xba I and Nde I and ligated,the double enzyme digestion of recombinant plasmid pIB139-gabT showed that overexpression vector pIB139-gabT was constructed successfully.2.Analysis of GABA metabolic pathway regulated by overexpression gabT gene in Snea253 strain: the overexpression vector pIB139-gabT and protoplasts of Streptomyces venezuelae attenuated strain(Snea253-R)were mixed by PEG-mediated method,Snea253 R-pIB139 stain with pIB139-gabT plasmid was on cultured by R2 YE regeneration medium and GA medium.By using real-time quantitative PCR,the relative expression level of gabT gene in strain R-pIB139 was 3.69 times than the attenuated strain Snea253-R.GABA and succinic acid content detection: GABA and succinic acid content in the fermentation broth of the strain was measured,R-pIB139 strain had a significant lower GABA content,but higher succinic acid than the attenuated strain Snea253-R.After 24 h,nematicidal activity of the overexpression strain R-pIB139 was increased by 39% than the attenuated strain Snea253-R.The nematicidal activity of succinic acid was measured,the higher concentration,and the higher corrected mortality to Meloidogyne incognita J2.After 48 h,the corrected mortality of J2 was 98.75% with 2000 μg/m L concentration.3.Effects of carbon source regulation on the GABA metabolism of Snea253: The eight carbon sources had different effects on the GABA metabolism of Snea253 strain.Real-time quantitative PCR was used to detect the relative expression of gabT gene in wild-type strains.The relative expression of gabT gene in corn starch and soluble starch as culture medium was higher,in potato starch and glucose carbon was lower.The content of GABA was lower when corn starch and soluble starch were used as carbon sources,the nematicidal activity of culture medium with corn starch and soluble starch as carbon source was the highest.The gabT gene produces more downstream products such as succinic acid by regulating the GABA pathway,and the nematicidal activity is stronger.Also,the expression of gabT gene is influenced by the carbon source of the culture medium.Those results can provide a new theoretical basis for altering the biocontrol activity and effect by regulating genes related to carbon metabolism,and laid a theoretical foundation for industrial production of biocontrol bacteria to against plant nematodes.