Molecular Cloning And Sequences Analysis Of Voltage-gated K~+ Channel β Subunit Genes In Wheat And Barley

Based on PCR cloning, four gene sequences encoding voltage-gated K+ channelβsubunit were isolated from wheat (cv Liang mai 4, Chinese spring), wild emmer and barley for the first time. Amino acids sequences variations ofβsubunit in Arabidopsis, rice, wheat and barley were detected. Bioinformatics analysis was also conducted. The results were outlined as following.1. The voltage-gated K+ channelβsubunit sequences from Liang mai 4, Chinese spring, wild emmer and barley were 987bp, encoding 328 amino acids. The nucleotide sequences of KTB and KHB shared 92.08% and 95.08% sequence similarity with those from Arabidopsis and rice K+ channelβsubunits(KAB1 and KOB1), respectively. The genes had complete open reading frames, start codon ATG, and stop codon TAA. The amino acids sequences shared 96.04% and 95.37% sequence identity with KAB1 and KOB1. These clones, the first K+ channel from wheat and barley, were designated as 'KTB1' and 'KHB1'.2. The region of the KOB1 sequence (amino acids 79-115) shared little homology with KAB1,34 amino acids were mutated including a deletion. However, KTB1 and KHB1 had a high degree of sequence conservation in this region for each other; only 2 mutations, which were Pro→Gln and Thr→Leu, were found. In Chinese Spring, the mutation of Tyr115→Cys115 in this region resulted in a deletion of tyrosine kinase phosphorylation site. KOB1 lacked 3 protein kinase C phosphorylation sites (STK, SRK, SLK) and 1 tyrosine kinase phosphorylation site but had an additional casein kinaseⅡphosphorylation site (SSWE) in this region. Two out of 4 protein kinase C phosphorylation sites which both plant and mammalian shared are present here (S/TTK, SRK). 3. Bioinformatics analysis indicated that KTB1 and KHB1 were hydrophilic polypeptides without signal peptides or membrane spanning regions. Secondary structure prediction showed that they had more coil (46.65%) andα-helix (39.02%) thanβ-strand(14.33%). Subcellular location prediction demonstrated that KTB1 and KHB1 mainly existed in cytoplasm (60.9%), followed by chondriosome (26.1%) and nuclear (8.7%), the rest (4.3%) were located in cytoskeletal.