Functional Research Of 3',5'-Bisphosphate Nucleotidase Gene ArHAL In Arthrospira Platensis

Given the limitation of genetic improvement measures, the investigation on the halotolerance mechanisms of Arthrospira sp. is still insufficient, although its adaptability to high salt stress environment was recognized early. Based on the accomplishment of whole genome sequencing of Arthrospira sp., elucidating halotolerance mechanisms of Arthrospira sp. in molecular level will be viable, which could also be a complement to the theory on the plant.In yeast, HAL2 encodes the 3',5'-bisphosphate nucleotidase, a salt sensitive enzyme whose overexpression probably counteracts the decrease in activity produced by toxic levels of Na+ or Li+. The bioinformatics methods were utilized to analyze the sequence of hypothetical 3',5'-bisphosphate nucleotidase gene ArHAL from Arthrospira platensis. Given the high sequence homology between the ArHAL gene product of Arthrospira platensis and HAL2, the ArHAL gene was cloned and expressed in Escherichia coli BL21(DE3) and Anabaena sp. PCC7120. Furthermore, the enzymology of ArHAL and its capacity of salt tolerance were determined in vitro and in vivo respectively.1. The analysis of sequence homologyIt was found that the deduced amino acid sequence of ArHAL gene from Arthrospira platensis shows significant homology with the putative products of the gene HAL2 of yeast. The coding region of the ArHAL gene holds 975 base pairs, coding a 319 amino acid protein. Sequence analysis revealed that these homologic sequences belong to an ancient conserved protein family, named inositol phosphatase.2. The functional research of ArHAL geneThe coding region of the ArHAL gene was reconstructed to contain a Ndel site at the start codon and a EcoKl site right after the stop codon by standard PCR method. Then, the entire open reading frame was inserted into the expression vector pET28a(+) to generate a reconstructed plasmid pET28a- ArHAL. After the purification of the ArHAL gene product from E. coli, the enzyme characteristics of ArHAL were investigated.The purified ArHAL gene product showed 3',5'-bisphosphate nucleotidase activity, which was confirmed by the kinetic parameters. The Km values of substrates PAP, PAPS, 5'-ADP are 3.67, 0.52, 41.53mmol/L, and the Vmax are 1111, 124, 263.16umol/L.min-1.mg-1, respectively. Its substrate specificity , Mg2+ requirement, and the ions inhibition were studied and compared to those of other 3',5'-bisphosphate nucleotidase. The lack of inhibition by the low concentrations of Li+ is the most pronounced differences between the ArHAL gene product and its homologic proteins fromother sources. The possible causes of these differences are under discussed, based on the active site sequence alignment between Arthrospira platensis and yeast 3',5'-bisphosphate nucleotidase.Furtherly, ArHAL gene was cloned into the shuttle vector pRL25C, then conjugated and overexpressed in Anabaena sp. PCC7120. ArHAL gene was detected in Anabaena by PCR and RT-PCR in the transcriptional level. But the phenotype difference between transgenetic Anabaena and the negative control under salt stress was not distinctive.