Cloning And Bioinformatics Analysis Of RpsL And RpoB Genes From X.nematophila YL002Resistant Mutations

Entomopathogenic nematodes symbiotic bacteria can produce a variety of biologicalactive substance, it has great application potential in agriculture, medicine and other fields.Screened X. nematophila YL002from shaanxi yangling of entomopathogenic nematodesisolated strains have stronger antibacterial activity of entomopathogenic nematodessymbiotic, under the minimum inhibition concentration of4kinds of antibiotics selectionpressures，12strains of gentamicin and streptomycin resistant and stable genetic mutationstrains were screened that YL002wild strains after uv-acridine orange compoundmutagenesis from previous studies, mutant strain and wild strains have certain differences inantibacterial activity, metabolic characteristics, cultivation characteristics and DNA level,etc. And microbes resistant to streptomycin or gentamicin may be due to the small subunitencoding ribosomal rpsL gene mutation or coding RNA polymerase beta subunits of therpoB gene point mutation. To preliminary explicit the mechanism that YL002strainsresistant to gentamicin and streptomycin, we cloned YL002wild strains and resistantmutant strains of rpsL and rpoB gene, conducts a preliminary analysis about bioinformatics,From gene level analysis of drug resistance mutations cause of mutant strain, get thefollowing results:(1) Through in vitro and in vivo measurement, we determine the inhibitory effect onbotrytis cinerea pers of wild strain and12mutant strains. Results show that most of theactivity of mutant strains is better than that of wild strain, including that L0501-2strain hasobvious inhibitory effect on spore germination of botrytis cinerea, the inhibition rate offermented liquid which was diluented2times was87.99%at the time of18h,3.52timesthat of wild strain; Q025-2strain has the best inhibitory effect on mycelial growth, theinhibition rate was75.25%, it is1.3times that of the wild strains; Q1220-3strain has goodtreatment effect on botrytis cinerea pers, and the relative control effect was50.43%, it is1.65times that of wild strain; Q025-1, Q025-2, Q0104-2and L0420-3strains have goodprotection effect, the relative control effect were96.0%,89.17%,83.76%and82.62%, respectively, is1.65,1.53,1.44, and1.42times of a wild strain.(2) Using of PCR cloning technology,we measured the whole rpsL gene sequences ofYL002strain and9strains. Through comparison we found that rpsL gene sequences of8mutant strains are the same as wild strains. Through bioinformatics analysis, we found thatrpsL encodes126amino acids, without functional domains, and a low complex area;compared with wild strain, rpsL of Q025-7strain of Gentamicin resistance has changed,30th lysine mutate into asparagine, changed from alkaline mutate into neutral,94th and118th of glycine mutate into arginine, changed from neutral amino acid to alkaline aminoacid.(3) Using of PCR cloning technology，we measured rpoB gene sequences of YL002wild strain and8resistant mutant strains, through bioinformatics analysis, we found thatrpoB gene encodes1342amino acids, which contains six important structure domains,respectively，they are ATP binding sit, GTP binding sit, DNA binding sit, mRNA protection,protein binding sit and spiral enzyme activity; By8strains of resistant mutant strainscompared with YL002wild strains of rpoB amino acid sequences, we found4of mutantstrains and wild strain sequences are the same, rpoB gene of the4mutant strains (L0420-3,and Q1220Q025-1-3and L0501-2) had occurred point mutations, L0420-3strain are16lys-Thr,356Thr-Ala and639Try-Asn，structure of the GTP binding sit and protein bindingsit are changed; L0501strain230Phe-Pro; Q025-1strain413Asp-Glu,414Val-Trp,1041Asp-Glu, structure of the DNA binding sit and GTP binding sit were changed;Q1220-3strains743Ala-Pro,1125Asp-Val and1263Ala-Ser, changed the DNA bindingstructure.