Molecular cloning and characterization of an inorganic phosphate transporter protein encoding gene in Polyporus umbellatus / 中草药

ABSTRACT

Objective To clone an inorganic phosphate transporter (PiT) gene from Polyporus umbellatus and perform the bioinformatics and expression mode analysis. Methods Using RT-PCR.to clone the full-length cDNA of PiT. The characteristics of physiochemical properties, conserved domains, signal peptide and transmembrane domain of the predicted PiT protein were determined by using bioinformatic tools. Results A inorganic phosphate transporter (PiT) gene (NCBI KU179154), designated as PuPiT, was cloned from Polyporus umbellatus sclerotia by RT-PCR. The full open reading frame cDNA sequence of PuPiT was 1 590 bp, encoding a putative PiT protein with 530 amino acids with a molecular weight of 57 552, and a theoretical pI of 6.82. The amino acids possess 12 membrane-spanning domains. Phylogenetic tree analysis indicated that PuPiT had the highest similarity with PiT from Moniliophthora rorer, and had high similarity with Moniliophthora roreri, Laccaria bicolor, and Heterobasidion irregulare. Quantitative real-time PCR showed that PuPiT expressed in both the symbiotic part and non-symbiotic part. Meanwhile, the expression of PuPiT in the symbiotic part was significantly up-regulated, about 12 times more than that in the non-symbiotic part. This result showed that PuPiT might play an important role in the Pi accumulating. Conclusion Molecular cloning and characterization of the novel PuPiT gene will be useful for further functional determination of the gene involving in phosphorus translocation regulation and symbiotic process.