毕业设计（论文）《ACC氧化酶基因PCR扩增体系优化研究》.doc

ACC氧化酶基因PCR 扩增体系优化研究 河南科技大学本科生毕业论文 PAGE 20 PAGE 19 ACC氧化酶基因PCR 扩增体系优化研究 摘　要 乙烯作为植物的五大类激素之一，在植物的生长发育过程中发挥着重要作用，而乙烯在植物体内的生物合成主要是由ACC氧化酶和ACC合成酶这2个限速酶来控制的，因而通过调节这两种酶的表达能有效地控制乙烯的合成量，从而获得所需要的植物新种质。本实验采用DP103质粒小提试剂盒（离心柱型）提取质粒的方法，从大肠杆菌中提取得到含有ACC氧化酶基因的重组质粒（PGEM-T），并采用琼脂糖凝胶电泳的方法检测其目的基因的获得；同时对目的基因进行PCR扩增，并通过对参与PCR基因扩增反应程序中退火温度和循环次数不同的梯度设置，即：退火温度分别设置为30℃、40℃、50℃、60℃、70℃，循环次数分别设置为：10次、20次、30次、40次、50次，进行单因子检测，观测各因子在不同条件下多扩增结果的影响，并利用琼脂糖凝胶电泳的方法筛选出最优扩增条件。实验结果表明重组质粒上700 bp ACC氧化酶基因在PCR基因扩增退火温度50℃，循环次数30次时电泳条带清晰无拖尾现象；重组质粒上600 bp ACC氧化酶基因在PCR基因扩增退火温度20℃、30℃、40℃，循环次数30次、40次其电泳条带清晰均无拖尾现象，且均无明显差别。 关键词：ACC氧化酶，质粒提取，PCR，退火温度，循环次数 ACC Oxidase Gene PCR Amplification System at The Experimental Study of Optimize ABSTRACT As one of five plant hormones, ethylene plays an important role in the process of plant growth and development. The biosynthesis of plant ethylene is mainly controled by ACC oxidase and ACC synthase 2 rate-limiting enzymes. So it is effective in controlling the synthesis of ethylene by regulating the expression of these two enzymes, which is necessary for new plant germplasm. In this experiment recombinant plasmid which included ACC oxidase gene was extracted from E. coli with the method of Kit. and the two kinds of target genius were evaluated with the method of agarose gel electrophoresis. At the same time, the target gene was carried out PCR amplification at the different annealing temperature and cycle numberthe set, that was the annealing temperature set to 30℃, 40℃, 50℃, 60℃, 70℃, and the cycle number was 10, 20, 30, 40 and 50 times, respectively. Then we observated the effect of one factor on the results of multi-amplified in different conditions. The optimal amplification conditions were screened with the method of agarose condensate gel electrophoresis. The experimental results showed that electrophoretic bands of the recombinant plasmid of 700 bp ACC oxidase gene were clear without tailing phenomenon, which was in the PCR gene amplification under the conditions of a