CAJ | 학술논문

CEL I内切酶特异识别并切断错配碱基，是建立TILLING实验技术平台的关键之一。本文从贵州省贵阳市当地种植的西芹中获得CEL I酶粗提物，利用一对具有已知单碱基差异的质粒作为底物验证所提CEL I酶粗提物的酶切活性，通过琼脂糖电泳和毛细管电泳对酶切产物进行检测，表明所获芹菜粗提物具有CEL I活性。本实验建立了适宜烟草TILLING实验的CEL I粗提物酶切体系：酶切温度42℃，酶切时间60 min，酶切反应总体积15μL，包括8μL PCR产物、4.5μL ddH2O、1.5μL酶切缓冲液(pH 7.5,500 mmol/ L KCL,100 mmol / L Tris-Cl,15 mmol / L MgCl2)和1μL CEL I酶（稀释为原始粗提液0.05倍）。
CEL I내절매특이식별병절단착배감기，시건립TILLING실험기술평태적관건지일。본문종귀주성귀양시당지충식적서근중획득CEL I매조제물，이용일대구유이지단감기차이적질립작위저물험증소제CEL I매조제물적매절활성，통과경지당전영화모세관전영대매절산물진행검측，표명소획근채조제물구유CEL I활성。본실험건립료괄의연초TILLING실험적CEL I조제물매절체계：매절온도42℃，매절시간60 min，매절반응총체적15μL，포괄8μL PCR산물、4.5μL ddH2O、1.5μL매절완충액(pH 7.5,500 mmol/ L KCL,100 mmol / L Tris-Cl,15 mmol / L MgCl2)화1μL CEL I매（희석위원시조제액0.05배）。
cellI enzyme that specifically cleaves mismatch in DNA double strands is one of the most important components of TILLING experiment platform. Crude extract of cellI enzyme was obtained from celery growing in Guiyang of Guizhou province. A pair plasmids with a known single base difference were used as substrate to verify cellI restriction enzyme activity of crude extracts, and cleavage products were detected by agarose gel electrophoresis and capillary electrophoresis, which confirmed cellI activity in crude extracts. An effective cellI digestion system was thus established: cellI enzyme effectively cut mismatch DNA in 15 μL digestion reaction solution including 8 μL PCR product, 4.5 μL ddH2O, 1.5 μL 10×cleavage buffer (pH 7.5, 500 mmol/ L KCL, 100 mmol / L Tris-Cl, 15 mmol / L MgCl2 ) and 1μL the 20 times dilution of cellI crude extraction after 60-min incubation under 42℃.