Isolation, cloning and sequence analysis of 1-aminocyclopropane-1-carboxylate deaminase gene from native sinorhizobium meliloti

Abstract

Background: Many plant growth-promoting bacteria including Rhizobia contain the 1-aminocyclopropane-1-carboxylate (ACC) deaminase enzyme that can leave ACC, and thereby lower the level of ethylene in stressed plants. Drought and salinity are the most common environmental stress factors for plants in Iran. Objectives: The main aim of this research was development of bio-fertilizers containing ACC deaminase enzyme which is very important in conditions of stressed drought and salinity. Materials and Methods: In this research 168 isolates of native Sinorhizobium meliloti were evaluated for ACC deaminase activity. These isolates were classified in four groups based on growth rate on ACC containing medium and enzyme activity. One isolate from each group was selected for molecular characterization. The nucleotide sequence of 16S rRNA gene of the selected isolates were determined. The ACC deaminase genes (acdS) on total and chromosomal DNA of S. meliloti KYA40, and KYA71 strains were isolated and cloned in pTZ57R/T vector and the obtained recombinant plasmids were used for sequence analysis. Results: The sequence of acdS genes from strains KYA71 and KYA40 and corresponding proteins were analyzed with respect to available sequences in NCBI database. The 16S rRNA gene sequences of S. meliloti strains submitted to the GeneBank/NCBI database. The acdS gene of KYA71 may be located on chromosomal DNA and in KYA40 it is located on one of the mega plasmids. These two genes have 99% similarity with three nucleotide differences which only lead to a change in one amino acid 48, threonine in KYA40 acdS gene and methionine in KYA71. Conclusions: The comparison of amino acid sequences of KYA40 and KYA71 with other sequences in the database showed that the amino acids 37 to 58 in almost all strains were similar. Therefore, it was concluded that it was a conserved region in this location of acdS genes and any changes in this region may cause change in ACC deaminase activity.