| Iron is essential to the growth of virtually all organisms. Despite its abundance in nature, iron is often a growth limiting nutrient because of the low solubility of ferric iron under aerobic conditions at neutral pH. Consequently, many bacteria produce ferric iron chelating compounds known as siderophores to gain access to various iron sources. Siderophore producing microorganisms can promote plant growth by increasing the bioavailability of iron via secreting siderophore and control plant diseases by antagonizing against plant pathogens via competing iron nutrient with them. In response to iron deprivation, B.subtilis secretes the catecholic siderophore bacillibactin (BB) to acquire iron.In this study, we isolated a series of siderophore producing bacteria from the rhizosphere soil of the rubber trees in Hainan Island by CAS agar plate assay, and CAS15, which produced distinct orange halo at CAS agar plate and had strong antagonism to common plant pathogens, was selected for the following studies. We amplified the siderophore producing gene dhbC and expressed it in E.coli, and then verified the function of dhbC by delecting it and retransforming it into to the dhbC- mutants via the homologous recombination method. The results of this study will provide the basis for the exploitation and utilization of the siderophore producing microorganisms and the study of the mechanism of their biological control, and provide a new way for the biological control of the plant diseases. The main results and conclusions are as follows.According to the characteristics of morphological, cultural, physiological and biochemical, CAS15 was identified as Bacilllus primarily. 16S rDNA sequence analysis result showed that the 16S rDNA sequence of CAS15 shared 99.4% identity with that of Bacillus subtilis. The genetic distance were closest between the 16S rDNA of CAS15 and that of Bacillus subtilis (accession No. DQ207730 and EU047884) and Bacillus sp.(accession No. AB188212), they are located at the same branch of the constructed phylogenetic tree, thus, CAS15 was identified as Bacillus subtilis.The type of siderophore produced by CAS15 and the siderophore productivity were analyzed by ESI-MS assay and DHB(G) assay respectively. The results showed that the siderophore produced by CAS15 was catecholic siderophore 2,3-dihydroxybenzoate-glycine- threonine trimeric ester bacillibactin, with the calculated mass of 881.25, and the siderophore productivity was significantly inhibited by iron. The inhibition was stronger with the higher concentration of iron. When there was no FeCl3, the OD510/OD600 value is 0.10～0.39, when there was 5μmol/L FeCl3 in the medium, the OD510/OD600 value is 0.005～0.04, while when there was 50μmol/L FeCl3 in the medium, the OD510/OD600 value is 0.001～0.01.The influencing factors for the siderophore production of CAS15 were studied, the results showed that modified minimal medium (MM) was the optimal medium for CAS15 siderophore production, and when cultured at pH7.2 and 37℃with glucose as the carbon source and tryptophan as the exogenous nitrogen source, a high yield of siderophore was obtained from the culture broth of CAS15, which indicated that the up-mentioned culture condition was benefit for CAS15 siderophore production.The antagonism of CAS15 against the common plant fungal pathogens was studied by the duel-culture test, the result showed that CAS15 had strong antagonism against the 15 selected fungal pathogens, with the inhibition width of 6～10 mm. Then the inhibiton of the cell-free supernatant of CAS15 was studied, the result showed that the cell-free supernatant of CAS15 significantly inhibited the growth of the 15 pathogens significantly, with the inhibiting rate of 20.18%～94.07%, which indicated that CAS15 had wide range of antagonistivity against plant fungal pathogens.Then we study the promotion of CAS15 cell-free supernatant on the seed germination of cucumber, the result showed that 100×and 1000×dilution promoted the growth of cucumber seedling significantly, and 100×dilution had the best effect. The result of inoculation in vitro showed that the cultured broth of CAS15 inhibited the mango anthracnose effectively by decreasing 53.34% of the disease index than the control, and the prevention rate was 78.95%, which indicated that CAS15 had great potential in plant growth promotion and biological control.A pair of specific primers were designed based on the reported genomic sequence of Bacillus subtilis, and then a 1197 bp fragment of CAS15 dhbC was amplified by PCR. CAS15 dhbC was predicted to encode a 43.8 kDa polypeptide with 398 amino acid residues. The BLASTn result showed that the nucleotide acids of dhbC gene (accession No.FJ194456) of CAS15 shared 99.74% and 99.58% identity with that of dhbC gene of B.subtilis subsp. subtilis str.168 (accession No. Z99120.2) and Bacillus subtilis subsp. subtilis str. NCIB 3610 (accession No. NZ_ABQL01000005.1) repectively. There were only 4- and 5-base difference in nucleotide sequence and 2- and 3-residue difference in amino acid sequence with that of Bacillus subtilis strain 168 and NCIB 3610 respectively.The structure analysis and prediction of DhbC encoded by CAS15 dhbC was studied by network (http://www.expasy.org), the results showed that the protein encoded by CAS15 dhbC was a acidic polypeptide, with the theoretical PI of 5.30. The instability index of DhbC was computed to be 53.00, which classified the protein as unstable. The Grand average of hydropathicity (GRAVY) is -0.303. The total number of negatively charged residues (Asp + Glu) and positively charged residues (Arg + Lys) of DhbC was 57 and 39 respectively, and its formula was C1910H3039N545O592S12, with a total number of 6098 atoms. DhbC contains 5 protein kinase C phosphorylation sites, 6 casein kinaseⅡphosphoralylation sites, 1 tyrosine kinase phosphorylation site and 2 N-myristorylation sites. According to its unstability, DhbC could form 3 3D structures by 14-394, 122-397 and 69-393 amino acids.The gene fragment of CAS15 dhbC was ligated to expression vector pET-30a(+) and then transformed into E.coli BL21(DE3) by calcium chloride transformation method, and the recombinant E.coli BL21(DE3)/pET-30a-dhbC was obtained. The recombinant DhbC of 48.8 kDa was exressed mainly in soluble form via 1 mmol/L IPTG induction in E.coli, and the amount reached highest at 30℃for 4 h. According to the N-terminal fusion 6 His-tag, the recombinant protein was purified by Ni2+ metal affinity chromatography and then identified by western blot, the result showed that the recombinant DhbC had the antigenicity to rabbit anti-his-tag polyclonal antibody.Finally, CAS15 dhbC gene was deleted by gene knockout and dhbC gene-delected mutant (CAS15 dhbC-del) was obtained. Then the dhbC gene was retransformed into the CAS15 dhbC-del via homologus recombination, and dhbC gene-comlemented mutant (CAS15 dhbC-com) was obtained. The function of siderophore production was tested at CAS agar plate, the result showed that CAS15 dhbC-com could produce the distinct orange halo at CAS agar plate as CAS15, while CAS15 dhbC-del could not, which indicated that dhbC gene was directly related to the siderophore production and it was confirmed to play an important role in the siderophore biosynthesis of CAS15.
|
PDF Full Text Request