Cloning And Functional Analysis Of High-affinity Potassium Transporter Gene PaHAK1 Of Phytolacca Acinosa Roxb

Phytolacca acinosa Roxb is one of the plants which contain more potassium by far. Phytolacca acinosa Roxb could normally grow and develop in soil containing very low K+ concentration (μM level), and High-affinity K+ transporter's higher expression or more efficient transporting of potassium may be the resons. To study the mechanism of high-affinity potassium absorption in some high-K plants, the PaHAK1 cDNA was cloned from Phytolacca acinosa Roxb by homology cloning and RACE technology. Moreover, PaHAK1 was overexpressed in rice and the yeast mutant W△6 which is defective in K+ uptake. The function of PaHAK1 was preliminarily determined by analysis of gene expression, K+ uptake dynamics and bioinformatics. The results are as follows:(1) The K+ uptake dynamics of Phytolacca acinosa Roxb and rice were tested. The Km, Cmin and Imax was obvious difference, but Phytolacca acinosa Roxb had smaller Km and Cmin, higher Imax when the concentration of potassium was low, and the difference decreased with the concentration of potassium increasing, this suggested Phytolacca acinosa Roxb had stronger K+ uptake abillity and better resistance to the low potassium than rice.(2) The full-length cDNA sequences of PaHAK1 was 2337 bp which was cloned. The putative protein was 771 amino acids with molecular weight 86.66 KDa and isoelectric point 7.54. Hydrophobicity plot and topology prediction results indicated the protein had 12-13 putative transmembrane regions. Blastn results showed it was 88% homology to HAK1 of Mesembryanthemum crystallinum L.(3) The PaHAK1 expression were analized by Real-time PCR. The results showed that PaHAK1 mainly expressed in plant roots and could highly express with inducing by low potassium or free potassium. The relative expression quantities of gene gradually reduced with the increasing of the K+ concentration outside. So PaHAK1 was a putative High-affinity potassium transporter related with K+ uptaking in roots.(4) Plant over-expression vectors of PaHAK1 were constructed, and then PaHAK1 over-expression vector was transferred into the rice via Agrobacterium-mediated rapid transformation.25 positive lines of rice were obtained by PCR.(5) The over-expression vector basis of pYPGE15 was constructed and transferred into yeast mutant W△6 with defect in K+ uptake by electro-transformation. This will provide a convinience for the deeper functional study of PaHAKl.