Expression of nodulation and nitrogen fixation genes in 6 strains of bradyrhizobium japonicum under different temperatures

Abstract

At present 15% of domestic soybean consumption are grown locally. The rest 58% of soybean consumed are imported. Rhizobial biofertilizers have been known to increase soybean productivity. Packages of soybean biofertilizers need to be kept in cool places or in refrigerators. Therefore, if a high nitrogen-fixing, thermotolerant soybean rhizobial strain (s) is developed, it will save cost and energy. In this research, six soybean rhizobial strain of Bradyrhizobium japonicum SK3, SK4, SK26, SK28, S76 and S162 were grown in yeast extract mannitol broth and in yeast extract glycerol broth under different temperatures from 25 ํC-45 ํC. Viable plate counts on both medium at 30 ํC and 40 ํC were also carried out. Results showed all the six B. japonicum strains were thermotolerant. RAPD-PCR fingerprints using either RPOI or CRL-7 were different for the six strains of B. japonicum, hence, the six B. japonicum constituted distinct strains. Results of RT-PCR with each set of primers specific for nodD[subscript1], nodA, nodB, nifH, nifD showed differential expression of nodD1 when B. japonicum SK4, SK26 or SK28 was grown under 25 ํC, 30 ํC, 35 ํC and 40 ํC. nodD1 expression increased when SK26 was grown at high temperatures ( 35 ํC, 40 ํC). On the other hand, expression of nodD1 of SK4 and SK28 was found to decrease when growth temperatures were 35 ํC and 40 ํC. Differential nodB expression was observed when B. japonicum S76 was grown at 25 ํC, 30 ํC, 35 ํC, 40 ํC with high expression at 35 ํC and 40 ํC with high expression at 35 ํC and 40 ํC. No differential gene expression was observed for nodD[subscript1] of B. japonicum SK3, S76 and S162 and for nodA, nodB, nifH and nifD of every B. japonicum strain under all experimental conditions. In addition, RT-PCR products were obtained when total RNA from each B. japonicum strain grown at 45 ํC for 5 h was isolated and used for RT-PCR