Identification of nifH and nodC genes from Rhizobium aegyptiacum THE 14TH NATIONAL CONFERENCE ON BIOCHEMISTRY AND MOLECULAR BIOLOGY 6th of October City, Cairo; 29th – 30th March 2017 Identification of nifH and nodC genes from Rhizobium aegyptiacum Ahmed G. Abo-Elwafa1, Ali M. El-Refy1, Fawzy A. El-Feky1 1Biotechnology Department, Faculty of Agriculture, University of Al-Azhar, Cairo. Egypt. ABSTRACT Rhizobium is nitrogen-fixing bacteria for legumes. The nifH and nodC are genes marker for identification of Rhizobium Species. In the present study, eleven Rhizobium isolates which had been collected from nine Governorates were positive in nodulation of Trifolium alexandrinum. Molecular biology studies of nifH and nodC genes of G3 isolate, they have been identified based on PCR tool using universal primers. These two genes partially sequenced and submitted into Genbank in NCBI databank under accession numbers (KY404231) and (KY404232), respectively. Also, the result of phylogenetic analysis based on Neighbor Joining method showed that these two genes are closely related to Rhizobium aegyptiacum. The aim of this research is: Isolation and classification of Rhizobium sp. that nodulate Egyptian Winter Berseem Clover (EWBC) based on identification of nifH and nodC gene using molecular biology studies and bioinformatics tools . RESULTS AND DISCUSSION Figure . 1 Gram negative rod shape of R. aegyptiacum EG1 strain on the plates and it growth in YEMA media containing Congo red 1.0kb 900bp 800bp 700bp 600bp 500bp Figure 5: Phylogenetic based on nifH sequences, shown the relationships between members of the genus Rhizobium. M 1 2 3 Figure .2 PCR amplified fragments using nifH and nodC primers, M = 3kb DNA ladder. Lane1 primer (nifH1): amplified fragment 660 bp. Lane2 primer (nodC): amplified fragment 940 bp. Lane3 Primer (nifH): amplified fragment 780 bp. METHODOLOGY Plants and microorganisms Elevens rhizobial isolate were obtained from root nodules of Trifolium alexandrinum L. were collected from nine governorates. The isolates purity was achieved by adding Congo red to YEMA media (0.25g/100 ml of EtOH; 10 ml stock/liter of YEMA), Gram stain test, and their ability to nodulate Berseem Clover. DNA manipulation and quantification Total genomic DNA of rhizobium isolates was isolation by Promiga DNA isolation kit. DNA concentration was detected by Quantiflour ds DNA system kit with Quantus fluorimeter, then diluted to 50 ng and stored at -20 C. Amplification of nifH and nodC genes nifH flat files of different trifolii strains were retrieved from NCBI database were used to design degenerative nifH1 primer to amplify 660 bp of nifH gene, in addition to two universal primers were used to amplify 780 bp of nifH and 940 bp of nodC genes by PCR with annealing temperatures 57˚C and 55˚C respectively. The obtained fragments from nifH and nodC amplification were purified by using Gel and PCR Clean-Up System (promega). Amplified fragment Phylogenetic analysis The purified fragments were sequenced, and the sequence similarity achieved with NCBI BLASTN online tool. Phylogenetic trees for nifH and nodC genes were constructed based Neighbor Joining method. Figure 6: Phylogenetic based on nodC sequences, shown the relationships between members of the genus Rhizobium. The results were showed all isolates are Gram negative, rod shape, white colored on YEMA media containing Congo red stain, and have the ability to form nodules on EWBC roots. Our primers amplified nifH (704 bp) and nodC (890 bp) genes of G3 isolate successfully. The two isolated genes were submitted to the NCBI under accession numbers (KY404231) and (KY404232) respectively. Alignment based on NCBI BLASTN online tool, and the result of phylogenetic analysis based on Neighbor Joining method showed that these two genes are closely related to Rhizobium aegyptiacum . Recommendation: The amplification of nifH and nodC genes is new strategy for identify nitrogen-fixing Bacteria in legume, and for other nitrogen fixing free living bacteria only nifH gene can be used to identify it. Figure 3: nifH gene flat file Acknowledgement We would like to thank the Academy of Scientific Research & Technology (ASRT) for it's financial support Figure 4: nodC gene flat file