Cloning And Functional Analysis Of PurL Gene Involved In Siderophore Biosynthesis In Pseudomonas Mediterranea G-229-21T

Pseudomonas mediterranea G-229-21T was a strain which conserved in our lab. It was screened from tobacco rhizosphere by dual culture with P. parasitica var. nicotianae (Breda de Hann) Tucker on low iron Sucrose-L-Asparagine (SA) plate. The strain produced high-affinity siderophore under low iron conditions. Tested by the plate of nutrition agar with antibiotics, G-229-21T was sensitive to kanamycin, but could tolerate 20μg/mL chloromycetin. The Tn5-1063a was used to mutagenize G-229-21T. One siderophore-deletion mutant named G-229-21TA was obtained.The specific-fragment of the mutant G-229-21TA was amplified with primers PC5/1 and PC5/2. The amplification result approved that G-229-21TA was a mutant of G-229-21T. Designing primers according to luxA sequences from Tn5-1063a, the DNA of the wild strain, pRL1063a and the mutant as the templates, the sequences of luxA was obtained by PCR. The luxA of the mutant is identical to that of Tn5-1063a at 100% level, it indicates that Tn5-1063a was inserted directly into the genome of G-229-21T.Three specific primers were designed according to Tn5-1063a, and seven arbitrary degenerate primers were designed at the same time. By TAIL-PCR (thermal asymmetric interlaced PCR) the gene purL involved in siderophore synthesis was obtained from the mutant G-229-21TA.The whole sequences of purL gene were obtained by using degenerate primer and TAIL-PCR methods. According to the result of BLAST analysis, the gene was identical to purL of Pseudomonas fluorescens Pf-5 at the level of 90 %. PurL gene encoding a 5-phosphoribosylformylglycinamidine synthase among the biosynthesis pathway of purine, is responsible for the forth step to catalyze the conversion of 5-phosphoribosyl-N-formylglycinamide to 5-phosphoribosyl-N-formylglycinamidine.The research proved that the blocking of the purine pathway would result in a number of abnormal processes which depend on the ATP, and some enzymes which with purine as their essential group would be inactive. So purine biosynthesis pathway has multiple complicated effects. On the other hand, the synthesis and transport of microbial siderophore was a very complex process. The metabolize pathway in microbial cell also has effects on the synthesis and transportation of siderophore. purL gene has relationship with siderophore biosynthesis. The amplification of promoter regions and functional analysis of purL gene were done.