CAJ | 학술논문

以蓖麻种子为研究对象,优化其cDNA-AFLP反应体系,为研究不同蓖麻材料或不同发育阶段的蓖麻种子内脂肪酸基因表达差异奠定理论基础。试验结果表明：提取蓖麻种子总RNA；将RNA逆转录为cDNA,用高频限制性内切酶EcoR Ⅰ和Mse Ⅰ进行酶切,并与EcoR Ⅰ、Mse Ⅰ接头连接,得到连接产物；连接产物稀释40倍作为预扩增模板,进行预扩增；优化选择性扩增反应体系,得到最佳反应体系为：10× PCR Buffer 2.0μL、dNTPs(10 mmol/L)1.4μL、Mg2+(25 mmol/L)1.4μL、模板稀释80倍1.0μL、Ex扩增引物(50 pmol/μL)1.2μL、My扩增引物(50 pmol/μL)1.2μL、LA Taq(5 U/μL)0.2μL、ddH2 O 11.6μL。采用优化后的体系,利用聚丙烯酰胺凝胶电泳对256对选择性引物进行筛选,得到适合蓖麻种子cDNA-AFLP分析的选择性扩增引物10对。
이비마충자위연구대상,우화기cDNA-AFLP반응체계,위연구불동비마재료혹불동발육계단적비마충자내지방산기인표체차이전정이론기출。시험결과표명：제취비마충자총RNA；장RNA역전록위cDNA,용고빈한제성내절매EcoR Ⅰ화Mse Ⅰ진행매절,병여EcoR Ⅰ、Mse Ⅰ접두련접,득도련접산물；련접산물희석40배작위예확증모판,진행예확증；우화선택성확증반응체계,득도최가반응체계위：10× PCR Buffer 2.0μL、dNTPs(10 mmol/L)1.4μL、Mg2+(25 mmol/L)1.4μL、모판희석80배1.0μL、Ex확증인물(50 pmol/μL)1.2μL、My확증인물(50 pmol/μL)1.2μL、LA Taq(5 U/μL)0.2μL、ddH2 O 11.6μL。채용우화후적체계,이용취병희선알응효전영대256대선택성인물진행사선,득도괄합비마충자cDNA-AFLP분석적선택성확증인물10대。
Castor is one of the most important oil crops with high economic value. In this work,the cDNA-AFLP system of castor seed was established and optimized to lay a foundation of research fatty acid gene expression in dif-ferent castor materials or different developmental stages. The main results were as follows:total RNA was extracted and then reversed transcription into cDNA. cDNA digested with EcoR Ⅰ and Mse Ⅰ was joined with EcoR Ⅰ and MseⅠjoint. The jointing products diluted by ddH2 O according to the ratio of 1∶40 were used in pre-amplification. And the optimized selective amplification system was performed with 10 × PCR Buffer 2. 0 μL、dNTPs(10 mmol/L) 1. 4 μL、Mg2+(25 mmol/L) 1. 4 μL,jointing product 1. 0 μL,Ex primer(50 pmol/μL) 1. 2 μL,My primer(50 pmol/μL) 1. 2 μL,LA Taq(5 U/μL) 0. 2 μL、ddH2 O 11. 6 μL. With the optimized PCR reaction system,we esti-mated 256 primer combinations with SDS-PAGE and gained 10 combinations which were suitable for cDNA-AFLP analysis of castor seed.