【作者】 林峰；

【导师】 张英汉； 樊俊华；

【作者基本信息】 西北农林科技大学 ， 动物遗传育种与繁殖， 2003， 博士

【摘要】 利用基因工程技术生产生物素是国际上高技术竞争的又一热点。由于生物素能够提高家畜胴体质量和改善肉质，提高家畜繁殖力，是家畜品种性能正常发挥所必需的物质。目前，生物素只能通过化学合成方法获得，且均从国外进口，价格昂贵，制约了我国畜牧科技和产业的发展。随着基因工程技术的兴起，人们开始转向生物素生物合成的研究。生物合成方法可以进行生物素的专一性合成，克服了化学合成法收率低，不能专一性合成，需经高成本化学拆分过程才能获得d-生物素的弊端，可使生物素的生产成本降低，从而提高畜牧业的经济效益。我国尚无这方面的研究，因此，开展本研究具有重要的理论意义和经济价值。 本研究以枯草杆菌ASl.1094菌株为研究材料，克隆了生物素操纵子基因，并对基因序列进行改造。而后对改造后的生物素操纵子基因进行了功能表达检测及bioW与bioB基因的诱导表达。旨在探索如何提高生物素操纵子基因的表达效率，为该操纵子基因的高效表达提供可行的研究路线和理论依据。并构建了可商业化操作的研究素材，为以后的研究提供探索性经验。本论文主要作了以下几方面的研究： 1．大片段基因组DNA的提取 为了获得用于PCR扩增(长距离PCR扩增和分段PCR扩增)的高纯度、大片段(至少为PCR产物长度的4倍)的DNA模板，应用三种方法：低熔点琼脂糖包埋法，SDS-蛋白酶K-酚氯仿抽提法和细菌基因组DNA提取试剂盒法，分别提取获得了枯草杆菌基因组DNA，并对3种方法的操作程序进行了不同程度的改进，结果表明：低熔点琼脂糖包埋法提取的基因组DNA片段明显大于后两种方法，采用0.5％琼脂糖凝胶电泳3h，仍然跑不出加样孔。将该方法提取的基因组DNA稀释100倍作为模板，采用长距离PCR方法，获得了枯草杆菌生物素操纵子基因全长。说明以低熔点琼脂糖包埋法提取枯草杆菌基因组DNA，并获得较大的DNA片段，是完全可行的。 2．枯草杆菌生物素操纵子基因的克隆 将枯草杆菌基因组DNA稀释后，通过PCR反应条件的优化，分别扩增得到了生物素操纵子基因的长距离PCR产物(10.3kb)和3个分段PCR产物。而后采用TA克隆方法，经PCR产物的纯化、两端加A、pGEM-T载体连接、转化大肠杆菌、阳性克隆的酶切鉴定及重组质粒测序，分别获得了生物素操纵子基因3个片段(bio-1、bio-2、bio-3)的克隆质粒。应用分子生物学软件对测序结果进行了分析，并与Genebank中枯草杆菌168菌株的生物素操纵子基因序列进行了对比分析：共有12个碱基差异，且7个碱基导致了氨基酸变异，但未出现移码突变现象。 利用长距离PCR扩增引物为测序引物，利用PCR产物直接测序法，在长距离PCR产物两侧各测了1个反应，并与Genebank中168菌株序列进行了对比分析：两个菌株的序列基本吻合，只有1个碱基的差异。 通过2种方法对3个基因片段的转化效率比较和质粒浓度的测定(重复多次)，结果表明:biow基因作为大肠杆菌的外源基因，插入高拷贝的pGEM一T载体中，重组后的质粒具有不稳定性。 3.枯草杆菌部分基因组文库的构建及筛选 首先，通过对枯草杆菌生物素操纵子基因序列和基因组序列的分析，选择刀。脚Hl和KPnl位点。用这2个内切酶分别将基因组DNA和BluescriPt SKM13载体完全酶切消化，分别纯化回收(酶切的基因组DNA回收4.3一7kb间的片段)，并用几DNALigase连接，转化大肠杆菌DHS。。 应用PCR快检法对构建的枯草杆菌部分基因组文库进行了筛选，共检测了250个菌落，得到了3个阳性克隆子，经酶切鉴定和测序，获得了生物素操纵子5.Ikb的BamHI一KPnl亚克隆片段。测序结果表明:ASI .1 094菌株与168菌株bioAFDB基因序列完全一致，9个差异碱基是由于PCR扩增导致的。 枯草杆菌生物素操纵子基因4个亚克隆序列的测序结果证实，ASI .1 094菌株和168菌株生物素操纵子基因序列是完全一致的(见附件1)。 4.枯草杆菌生物素操纵子基因序列的改造 基因序列改造的关键是删除bioB与biof基因间的终止子，在Bowe:等(1 996)[’97]序列研究分析的基础上，设计一对PcR引物，以bi。一2片段克隆质粒为模板，采用Skip PCR方法，得到了约4.3kb产物，以TA克隆方式，插入pGEM一T载体中。将经鉴定的阳性克隆质粒进行测序，结果表明:bioB与biol基因间的5 lbp的终止子序列已被删除。在基因序列分析的基础上，采用特征酶酶切的方式，还删除了生物素操纵子基因上、下游与生物合成无关的序列。并将改造后的枯草杆菌生物素操纵子基因的3个片段依次插入pGEM一T载体中。并经binw、bioB及biol基因3’端的引物进行PCR扩增和重组质粒酶切鉴定而得到证实。 5.改造后的生物素操纵子基因的功能表达检测及biow和 bioB基因的诱导表达 为了检测改造后的生物素操纵子基因是否能够正常表达，我们利用生物素是细菌生长的必需物质这一特点，用4种大肠杆菌生物素缺陷株(R874、R875、R877、R879)经遗传互补分析表明:改造后的生物素操纵子中的bioA、binF、bioD、bioB4个基因均能够表达。因此，bioB与biof基因间终止子序列的删除，不影响基因的表达。 同时也证实，虽然所克隆的枯草?更多还原

【Abstract】 It was one of hot spots of high-technologic competition that biotin was produced by using genetically engineered technology. It could improve carcass quality meat quality, reproductive ability of livestock, and was indispensible matter for normal elaboration of livestock’s varietal performance.Up till now, biotin could only be gained by the method of chemical synthesis and only imported from the foreign countries,and its price was very expensive and seriously restricted the development of our country’s animal husbandry. Alongside rising of genetically engineered technology, the people begined to research biotin biosynthesis. The biosynthetic method could carry on the specific synthesis of biotin and overcome the low yield, nonspecific synthesis and nessary use of the chemical separation for chemically synthetic method.This method could reduce manufacturing cost of biotin, and improved economic benefits of animal husbandry. The use of this method had importantly theoretical meaning and economic value.Used As 1.1094 strain of Bacillus subtilis as researched material, the structural genes of biotin operon(bio operon) were cloned, and its sequence were engineered. Then the engineered genes of bio operon were carried on testing of the functional expression, and bioW & bioB gene were induced to express. This experiment probed how to improve the expression efficiency of the structural genes of bio operon, and provided the feasibly researched route and scientifically theoretical foundation. For highly efficient expression of the structural genes of bio operon, a lot of researched materials were constructed which could commercially operated in this experiment and these materials provided some explored experience and reference materials for future research. The study mainly carried on several research followed: 1. Extraction of large-fragment genomic DNAIn order to gain DNA template of PCR amplification (Long PCR amplification and salvage PCR amplification) which was high purity and large fragment, three methods were used to extract genomic DNA of Bacillus subtilis, i.e. low melting-point agarose embedding method, SDS-Proteinase K-Phenol Chloroform extraction method and bacterial genomic DNA extraction kit method. The genomic DNA of Bacillus subtilis were gainedby these methods, and the operated programs of the methods were improved. The results showed that the genomic DNA extracted by low melting-point agarose embedding method were obviously biggest than that of another two methods. Used the genomic DNA extracted by low melting-point agarose embedding method as PCR template, the full length of structural genes of Bacillus subtilis bio operon were gained by long PCR method. The results also explained that it was feasible that the genomic DNA of Bacillus subtilis were extracted and gained large DNA fragments by low melting-point agarose embedding method.2. Cloning of structural genes of Bacillus subtilis bio operonDiluted the genomic DNA of Bacillus subtilis as the template, long PCR product (10.3kb) and three salvage PCR products were separately gained by optimization of reaction conditions of PCR. Then used TA cloning method, The plasmids of three fragments( bio-1 bio-2 bio-3) of structural genes of Bacillus subtilis bio operon were gained by the purity of PCR products adding A in the two side of the products ligation with pGEM-T vector transformed Escherichia col identification and sequencing of recombinant plasmid. Contrasting analysis with the genes sequence of Bacillus subtilis 168 strain’s bio operon, the sequence of bio operon of As 1.1094 strain had 12 bases difference with that of 168 train,and 7 bases caused variation of amino acid.Used long PCR primers as sequencing primer, the two sides of sequence of the long PCR product were separately sequenced one reaction by the direct sequencing method of PCR products, and the sequencing sequence were carried on constrasted analysis with sequence of 168 strain in Genebank. The results showed that the sequence of two strains basically coincided but更多还原