Cloning And Fuctional Characterization Of Potassium Channel Gene GhAKT1and Potassium Transporter Gene GhKT2from Cotton(Gossypium Hirsutum L.)

Potassium (K) is the most important and abundant cation in living plant cells and plays crucial roles in plant growth and development. Under low-K+conditions, most crops do not grow well and show K+-deficient symptoms. Cotton(Gossypium hirsutum L.) is more sensitive to low K+availability than most other major field crops, and often shows signs of K deficiency on soils not considered deficient in K+. In this study, we identified and characterizednovel members of K+transporter and channel genes, from the root of cotton cv. Liaomianl7. The results would be beneficial for elucidating how cotton acquires K+and developing K+efficient cotton genotypes by using biotechnological approaches.Compared with nodel species such as Arabidopsis and rice, little is known about the molecular mechanism in response to K+-deficient stress of Gossypium hirsutum. Hence, K+transporter and channel genes were chosen for further expression and functional study. The main results were as follows:1. The cotton homologue of the Arabidopsis Shaker-like K+channel, GhAKTl, was isolated by RT-PCR and RACE method form Liaomianl7. The GhAKT1cDNA sequence contained a130bp5’-UTR and a280bp3’-UTR, as well as an open read frame of2628bp encoding a875amino acids. The deduced transcripts GhAKT1shares a high homology with AtAKT1and other members of group I of the plant Shaker-like K+channel family. The deduced polypeptide exhibited all of the structural features that are shared by plants inwardly rectifying K+channels, such as six transmembrane domains (S1-S6), S5and S6connected by the P-loop, the K+selectivity sequence of TxxTxGYGD in the P-loop, a putative cyclic nucleotide binding domain (cNMP), and five ankyrin repeat sequences that are present only in the AKT1subfamily of plant inwardly rectifying K+channels.2. To define the subcellular localization GhAKT1, we constructed GhAKTl-GFP expression vector, and then transferred into by Arabidopsis. The results of subcellular localization showed that GhAKT1::GFP fusion protein observed at the plasma membrane.3. The results of the Real-Time PCR showed that GhAKTl transcripts occured highest levels in cotton leaves, and they were also detected in shoot apex, roots, and stem. We isolated the2180bp promoter of GhAKT1, histochemical assay revealed that the GUS staining was detected at mesophyll cells and veins. In roots, GhAKTl was expressed in both primary and lateral root tips as well as mature regions. Furthermore, we found that GUS staining was preferentially located in epidermis of mature root; and staining in cortex and endodermis was weaker than in EP.4. The ORF of GhATKl was constructed into plasmid Pbil21and transferred into Arabidopsis via Agrobacterium-madiated method. The transformed plants were verified by PCR and Real-Time PCR analysis. Overexpression of GhAKT1in Arabidopsis enhanced both potassium nutrition and seed germination of the transgenic plants. Noninvasive Micro-test Technique (NMT) demonstrating that the K+uptake mediated by GhAKTl is also Ba2+-sensitive like AtAKTl, and we also suggest that GhAKT1 can mediate K+uptake from very low concentrations, within the range of operation of the high-affinity K+uptake system.5. The cotton homologue of the Arabidopsis potassium transpoter gene, GhKT2, was isolated by RT-PCR and RACE method form Liaomian17. The GhKT2cDNA sequence contained a171bp5’-UTR and a280bp3’-UTR, as well as an open read frame of2379bp encoding a792amino acids. The deduced transcripts GhKT2share a high homology with those KUP/HAK/KT plant potassium transpoters. The deduced polypeptide exhibited GVVYGDLSTSPLY structural features that are shared by plants potassium transpoters, and predicted containing eleven putative hydrophobic regions.6. To define the subcellular localization GhKT2, we constructed GhKT2::GFP expression vector, and then transferred into by Arabidopsis. The results of subcellular localization showed that GhKT2::GFP fusion protein observed at the plasma membrane.7. The results of the Real-Time PCR showed that GhKT2transcripts occured highest levels in cotton leaves, and they were also detected in shoot apex, roots, and stem, the expression of GhKT2was induced by low K+stress. We isolated the2800bp promoter of GhKT2, histochemical assay revealed that the GUS staining was detected at mesophyll cells and veins. In roots, GhKT2was expressed in both primary and lateral root tips as well as mature regions.8. The ORF of GhKT2was constructed into plasmid pBI121and transferred into Arabidopsis via Agrobacterium-madiated method. The transformed plants were verified by PCR and Real-Time PCR analysis. Overexpression of GhKT2in Arabidopsis enhanced potassium nutrition of the transgenic plants. Noninvasive Micro-test Technique (NMT) demonstrating that GhAKT1can mediate K+uptake from very low concentrations.