人工改造甜瓜钾离子通道基因ΔAT2/MIRK转拟南芥的生理特性分析
Physiological Characteristics of Transgenic Arabidopsis thailand into Artificial Modified K+ Channel Gene ΔAT2/MIRKin Melon

K+通道是介导植物K+吸收转运的主要途径之一。甜瓜K+通道MIRK对外源Na+敏感,生物信息学分析其敏感位点可能位于孔区外(即S5-P-S6)。为了验证MIRK受Na+抑制位点,本研究将拟南芥中不受Na+抑制的同源K+通道基因KAT2孔道区替换到MIRK序列上,生成人工改造甜瓜K+通道基因ΔAT2/MIRK。电生理实验结果显示ΔAT2/MIRK仍保持了MIRK介导K+的属性,但其内向电流则不受外源Na+的抑制。将MIRK和ΔAT2/MIRK分别转化到拟南芥K+通道KAT1突变体kat1中,抗性筛选和PCR检测结果表明成功获得p35S::MIRK-kat1和p35S::ΔAT2/MIRK-kat1转基因植株。分别用50、100 mmol/L的NaCl溶液处理转基因拟南芥和kat1突变体,发现MIRK及ΔAT2/MIRK在kat1中的过表达可以介导K+内流,并参与植株对盐胁迫的响应。
Potassium channel is one of the main ways for K+ absorption and transport.Melon potassium channel MIRK was found to be inhibited by external Na+.The putative sensitive sites may be located in the pore area(S5-P-S6)by bioinformatics analysis.To explore the molecular base of blockage site(s),the segment S5-P-S6 of MIRK was replaced by the same segment of KAT2 which was not inhibited by Na+,then the artificial modified melon potassium channel gene ΔAT2/MIRK was obtained.The electrophysiological results indicated that ΔAT2/MIRK still can mediate K+ uptake,but the inward current was not suppressed by Na+.MIRK and ΔAT2/MIRK were transferred into Arabidopsis thaliana (kat1) by Floral Dip method.Resistance testing and PCR results showed that MIRK and ΔAT2/MIRK have been integrated into the genome of Arabidopsis(the p35S::MIRK-kat1 and p35S::ΔAT2/MIRK-kat).Transgenic plants(T3-generation)were treated with 50,0 mmol/L NaCl solution and the ratio of potassium to sodium of plants was measured.The results indicated that melon K+ channel gene ΔAT2/MIRK transformation could mediate K+ influx and participate in plants’ response to salt stress.