Molecular cloning, phylogenetic analysis and structure prediction Of amt1 from azolla-anabaena azollae symbiotic system

Azolla anabaena azollae is a symbiotic association between the aquatic fern Azolla and nitrogen fixing cyanobacteria anabaena azollae, by the exchange of carbon and nitrogen between them. The nitrogen fixed by the cyanobacterium is converted into ammonium and is transported via ammonium transporters. AMT1family of ammonium transporters are high affinity transporters hence are quick to respond under very low levels of nitrogen supply.AMT1 will be the best AMT to be studied under nitrogen limiting growth conditions. Putative AMT1 sequence was cloned from the symbiotic Azolla-Anabaena using reverse transcription PCR and phylogenetic analysis was conducted with the nearly similar sequences using BLAST. The amplified putativeAMT1sequence from the cDNA of Azolla anabaena azollae was sequenced and the sequence was submitted to NCBI. Multiple sequence alignment was performed using ClustalW and the Maximum Likelihood and Neighbour Joining tree was developed using Tamura-Nei model of MEGA 6.0.The reliability of each branch was tested by bootstrap analysis with 1,000 replications. In the phylogenetic analysis, AMT1of Azolla anabaena azollae showed a close similarity with Azolla caroliniana AMT1.The transmembrane AMT1 protein structure was predicted with Raptorx structure prediction program.