The Conditions For Siderophore Producing Of CAS19 And Cloning And Analysis Of The Siderophore Synthesis-Related Gene CepR

In this study, we selected siderphore producing bacterium Burkholderia cepacia CAS 19 as the starting strain, to study the influencing factors of siderophore production, comparison of two siderophore detection mediums, the antagonistic spectrum, the promoting effect on tomato and cucumber, cloning and bioinformatic analysis of the siderophore synthesis-related gene cepR.The main results are as followings:1. Absorption spectrometry was used to study the influencing factors of siderophore production from Burkholderia cepacia CAS 19. The results showed that MSA was the optimum medium for CAS 19 to produce siderophore, and there was a specific absorption peak at 320 nm wavelength in the culture filter; When the iron concentration in the culture medium is higher than 1μmol/L, the production of siderophore was almost completely inhibited. When using sucrose as carbon source, proline as nitrogen source, with the C/N ratio of 10:1 and medium pH of 7.5, inoculated at 30℃with media amount of 20/250 (mL), the siderophore producing of CAS 19 strain reached the peak value.2. By comparing the halo size of CAS 19 and CAS 17 on these two siderophore detection mediums, We find that halos of two Burkholderia cepacia strains on the MSA-CAS agar plate are bigger than that on the CAS agar plate, which indicated that the MSA-CAS agar plate is more suitable for detecting the siderophore of Burkholderia cepacia than CAS agar plate.3. The antagonism of isolate CAS 19 against 20 fungal phytopathogens were studied by using duel-culture test. The results showed that isolate CAS 19 could significantly inhibit the mycelium growth of 13 of the fungi tested, which indicated that CAS 19 had broad antagonistic spectrum against common plant pathogens.4.100×dilution of CAS 19 fermentation solution was used to irrigate the cucumber seedlings, the result showed that plant height,stem circumference and fresh weight of the irrigating treatments were higher than water and MSA control treatments, which indicated that the growing of cucumber seedlings was significantly promoted by 100×dilution of the CAS19 fermentation solution.5. The germination experiment results showed that the germination of tomato seeds was significantly promoted by siderophore producing bacteria CAS 19,CAS5,CAS 12,CAS 17 and CAS15.100x dilution is best for CAS 19,CAS5,CAS12,CAS17 fermentation solution to promote the germination of tomato seeds while 100×dilution for CAS15, probably because MSA medium is more suitable for siderophore production from Burkholderia, but not for Bacillus subtilis CAS15. There was just a little difference between the water and liquid MSA control treatments.6. The effect of fermentation, supernatant and bacterial cell suspension of CAS 19, cAS5, CAS 12, CAS 17 and CAS 15 on the growth of tomato was studied, the results showed that there was little effect on the growth of tomato by bacterial cell suspension, while the effect of fermentation and supernatant were obviously distinct, so we can infer that it is the secretions not the bacterium itself that promote the growth of tomato plants. We can infer siderphore producing bacteria can promoting plant growth. The data of plant heignt, root length, stem perimeter and fruit weight showed that among the 5 bacteria strains, CAS 19 and CAS 17 promoted the growth of tomato most significantly.7. The full-length cDNA of siderophore synthesis-related gene cepR was cloned by PCR, and the structure and function of the cepR gene encoding protein was analysis and forecaste by the biological information. The informatic analysis results showed that the nucleotide sequence of cepR have 99%identity with that of Burkholderia cepacia LMG1222.The length of cepR gene was 768 bp containing a complete open reading frame that encodes 239 amino acids with a LuxR-type HTH domain, several Casein kinaseⅡphosphorylation site, protein kinase C phosphorylation site, Tyrosine kinase phosphorylation site, N-myristoylation site, N-glycosylation site and a transmembrane helices. The results of this study will be addicted to Burkholderia cepacia siderophore synthesis of research related to regulation of genetic information and reference, and for laying the foundation for further development and use.