Cloning And Functional Expression Of Acc Deaminase Gene From Klebsiella Oxytoca Strain Rs-5

Secondary salinization has largely occurred in the northwest of china and became a major factor limiting crop yields of Xinjiang province. In a previous study, Klebsiella oxytoca Rs-5(Genebank Accession NO. EF551363)and Pseudomonas putida Rs-198 (Genbank Accession NO. FJ788425), two bactearial strains which could increase the germination rate, shoot lengths, and dry weights of potted cotton plants during salt stress conditions, were isolated from salinized cotton fields. The present study focuses on ACC Deaminase (ACDS) gene isolation from K. oxytoca, expression vestor construction, genetic transformation of P. putida, and functional analysis of cotton plants treated with genetically engineered P. putida.The ACDS gene was cloned from K. oxytoca Rs-5 by Polymerase Chain Reaction(PCR),using degenerated oligonucleotide primers matching publically available ACDS sequences. The obtained PCR product was sequenced and shown to include an open reading frame (ORF) of 1017 bp encoding a protein of 338 amino acids. This putative protein shared high similarity to ACDS from Enterobacterc loacae UW4 and Cal2 (Genebank Accession NO. AF047710 and AF047840),having amino acid identities of 95.56% and 96.75%, respectively. Thus, the gene was named Koacds..The prokaryotic expression vector pDSK519-Koacds was constructed by inserting the cloned Koacds gene fragment into a shuttle plasmid pDSK519, and transformed into E. coli Bl21. SDS-PAGE and enzyme activity analyses indicated that the specific activity of the recombinant enzyme was up to 0.302±0.042 U/mg under optimum condition [0.4 mM IPTG ( at 25℃)].To determine whether the Koacds gene played an important role in relieving salt stress, the shuttle expression vector pDSK519-Koacds was transformed into P. putida Rs-198, a strain lacking ACDS activity. P.putida strain Rs-198 was isolated by our own lab and shown to relieve salt stress and to promote plant growth. Optimum conditions including growth stage of the strain, concentration of competent cells, electroshock voltage, and the medium were optimized for electroporation of pDSK519-Koacds into P. putida Rs-198. The transformation efficiency was up to 1.3×10⁷ CFU/μg DNA under optimium conditions, using 4.6×10¹²/ml competent cells that were collected at logarithmic growth phase (OD₆₀₀ = 0.5) and electroporation of pDSK519 plasmid DNA at 13 kV/cm. Compared with untreated control, the germinating rates of cotton seeds treated with strains Rs-5,Rs-198, and 198--Koacds, respectively, were enhanced 15.9%,16.8%, and 16.7%; the average stem length of cotton increased 30.6%, 31.0%, and 32.1%; and the average dry weight of cotton increased 10.8%, 13.6%, and 12.2%.In an effort to find the reason of the above mentioned phenomenon, the stability of the plasmid in LB culture and soil was studyed. It was previously reported that the stability of plasmid in recombinant strains pDSK519 and pDSK519-Koacd cultivated in LB were obviously higher then that in soil. After Continuously cultivating for 96 hours, no plasmid in soil were left, while about 65% and 68% of plasmid in LB media were left.