摘要植酸酶作为一种由微生物发酵生产的酶制剂，可以分解动物饲料中的天然有机磷。在饲料中添加植酸酶可以分解饲料中的天然磷，使得动物能够加以吸收利用，从而代替部分磷酸氢钙。大多数鱼类的胃肠呈中性偏碱性，在水产养殖业中，用碱性植酸酶作饲料添加剂，更加有效。然而，相对于酸性植酸酶，碱性植酸酶的酶活较低。本实验拟通过克隆技术获得能高效表达碱性植酸酶的质粒。84084
采用基于氯仿一步法原理(Trizol)进行改良的RNA plant plus试剂盒从百合花粉中获取总RNA，根据NCBI中已报道的碱性植酸酶基因(phyC)序列(AF029053)，以该序列的两端为基础，设计并合成一对引物，以总RNA为模板，采用逆转录方法获取cDNA，用获取cDNA全长为模板，使用经优化PBO polymerase作为DNA连接酶，运用PCR技术扩增出一段长度为1200bp左右的片段，经凝胶检测选择片段扩增成功的产物，用切胶回收工艺回收并纯化产物。对载体进行双酶切处理，将产物与载体经DNA重组技术在22℃恒温条件下连接，转化至大肠杆菌特殊感受态中，于37℃恒温培养，经阳性克隆筛选，获取阳性克隆菌，过夜培养，采用基于SDS碱裂解法改进的AxypRep质粒DNA小量试剂盒提取出克隆质粒phyCI，分光光度计测定质粒浓度，双酶切克隆质粒提取成功，测定其碱基序列，与phyC基因（AF029053）比对，同源性为92%。
毕业论文关键词：植酸酶；百合花粉；基因；克隆
Abstract As a kind of phytase enzyme production by microbial fermentation, natural organic phosphorus in animal feed。 In the feed in the addition of phytase to feed in natural phosphate decomposition, allowing the animals to can be absorbed by, to replace part of dicalcium phosphate。 Most fish gastrointestinal was neutral and alkaline, in the aquaculture industry, with an alkaline phytase as feed additive and more effective 。 However, compared with the acidic phytase, alkaline phytase activity is relatively low。 This study is to obtain high expression plasmid alkaline phytase by cloning technology。
Using the modified RNA plant plus kit based on of the chloroform one-step principle (Trizol) to obtain total RNA from lily pollen。According to the reported in the NCBI alkaline phytase gene (PHYC) sequence AF029053, at both ends of the sequence based, a pair of primers were designed and synthesized。 Using total RNA as template, using PCR method to amplify the cDNA, for obtaining full-length cDNA as template, using optimized PbO polymerase as a DNA ligase, the use of PCR amplified with a length of about 1200bp fragment, by in gel detection of selected fragment was successfully amplified product, using recycled plastic recycling process for cutting and purified product。 Double enzyme digestion treatment。 Will product and vector by DNA recombination technique in 22 DEG C under the condition of constant temperature connection, was transformed into E。 coli special feeling state, in a constant temperature of 37 DEG C for culture, the positive clone screening, to obtain positive clones were, overnight culture, based on the improved SDS alkaline lysis method AxypRep plasmid DNA small reagent box extracted plasmid phyCI, photometric meter were used to measure the concentration of plasmid and double enzyme cutting cloning plasmid was successfully extracted by using, its nucleotide sequence was determined, and PHYC gene (AF029053) ratio on the homology of 92%。
Keywords:Phytase; Lily pollen; Gene; Cloning
目 录
第一章 文献综述 1
1。1 植酸酶及其分类 1
1。2 植酸酶的应用 1
1。2。1 动物饲料工业 1
1。2。2 水产养殖业 百合花粉碱性植酸酶基因的克隆及其表达载体的构建:http://www.youerw.com/shengwu/lunwen_99519.html