In silico 예측과 전기생리학적 방법을 이용한 벼의shaker type K+ 채널 유전자의 선별
Screening of a Rice Gene Encoding Shaker Type K+ Channel by Combining the In Silico Prediction and the Electrophysiological Measurement
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본 연구는 식물에 내염성을 줄 수 있는 유전자인 shaker type K+ 채널을 인코딩하는 유전자를 벼의 유전자로부터 선별하기 위해서 수행하였다. 먼저, 전압의존성 K+ 채널의 공통적인 특징인 6개의 막 관통부위와 K+ 이온선택필터 보존서열 (TXXTXGYG)을 보유하는지 여부에 따라 30,778개의 벼 게놈 유전자로부터 K+ 채널을 지정할 가능성이 가장 높은 5개의 유전자를 TMHMM과 CLUSTALW를 사용하여 in silico 방식을 통해 선별하였다. 선별된 5개의 cDNA를 pSP64T Xenopus 발현 벡터에 서브클로닝한 후 in vitro 전사로 cRNA를 제작하였다. 합성된 cRNA를 개구리 난모세포에 미세 주입시킨 후 발현된 K+ 채널의 이온투과도를 two electrode voltage clamping (TEVC) 방법을 이용하여 측정하였다. 그 결과, 클론 J023118D04가 shaker type K+ 채널의생물리적 특성을 갖는 것으로 확인하였다. 수많은 벼 유전자로부터 shaker type K+ 채널을 인코딩하는 유전자를선별하는데 in silico 예측 및 개구리 난모세포 이형발현과 전기생리학적 측정 방법의 결합이 매우 유용함을 확인하였다.
Abstract
Five rice cDNAs that are most likely to encode putative voltage-gated K+ channels were selected by the in silico sequence homology and membrane topology analyses of transmembrane domains (TMs) and of a wellpreserved K+ selectivity filter (TXXTXGYG) in reference to Arabidopsis shaker type K+ channel (KAT1). The five candidate cDNAs werefurther subcloned into pSP64T vector, a Xenopus expression vector, and then in vitrotranscribed to generate cRNAs. Each cRNAs were microinjected into Xenopus oocytes, and their K+ channel conductance was measured elecrophysiologically by using two electrode voltage clamping (TEVC). Among them, one of the rice cDNAs (clone J023118D04) gave rise to K+ current with biophysical characteristics similar to those of the shaker type K+ channels. Collectively, cloning and functional characterization of plant ion channels could be greatly facilitated by combining the in silico analysis and heterologous expression in Xenopus oocytes.
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