Gene targeting by homologous recombination can realized targeted integration . at the same time we can knock - out some gene to know its genetic function and create specific mutation to study some kinds of metabolizing mechanism 第一部分：立碗藓原生质体分离及培养的研究基因打靶技术通过利用同源重组可以在一定程度上实现外源基因的定向整合；同时还可通过对某基因的剔除研究基因的功能或通过创造定点突变来研究某种代谢机理。
Attachment to host tissue is essential for colonization by most mucosal pathogens , such as mycoplasma hyopneumoniae . according to the reported sequence of p97 gene in strain 168 , a pair of primers was designed and the rl region of p97 gene in strain 168f485 was amplicated by pcr 本研究根据已报道的序列为参考，设计一对带有ecor和hind酶切位点的引物，并通过引物的定点突变，利用pcr方法从猪肺炎支原体168f485株扩增到其黏附因子p97基因的抗原决定簇r1区。
After absorption , the all - frans - retinal isomerizes to a 13 - c / s configuration and br undergoes a photocycle : br570 k590 l550 m410 n520 o640 br570 bacteriohodopsin is a promising biological photoelectric material . we intent to conduct a more thermostable br by site - directed mutagensis . a point ( no . 274 t , no . 274 a turn to c g ) mutation was introduced to br gene by successive pcr technique , and cloned into puc - 19 vector 本论文利用连续pcr的方法定点突变了br基因的一个氨基酸，突变位点选择了br基因第273的t和274位的a ，把它们变为cg ( tyr替换为arg ) ，然后把突变的br基因克隆入嗜盐菌表达载体pnov - r ，经测序鉴定的表达载体转化br缺陷的嗜盐菌，构建了tyr79 argbr突变体。
At the same time , vp4 gene was mutated in a certain point by pcr . the plant expression vector : pbi121 was constructed and then transformed to agrobacteriutn tumefaciens eha105 directly . a . tumefaciens eha105 harboring pbi121 / vp4 was used to transform the boechmeria nivea l . guad according to the leaf disc procedure 为便于基因操作，对外壳抗原蛋白vp4基因进行适当的修饰和改造：通过引物设计，利用pcr反应，在基因的起始编码前引入有助于真核生物表达的kozak序列和限制性内切酶位点；使用套叠pcr对vp4基因进行定点突变，以便于将改造后的基因插入pbi121构建植物表达载体，通过直接转化法，把pbi121 vp4转入农杆菌eha105 ，构建了农杆菌工程菌。
In escherichia coli , arog gene encodes phenylalanine - sensitive 3 - deoxy - d - arabino - heptulosonate - 7 - phosphate synthase isoenzyme arog that catalyzes the first committed step of shikimate pathway . here we study the essential amino acid residues involved in the formation of feedback inhibition site of arog , and the effects of n - terminus on feedback inhibition and its quaternary structure , and the importance of the structural " d2 " symmetry to allosteric inhibition 本博士论文工作以大肠杆菌k - 12来源的arog为研究对象，通过定点突变、反馈抑制实验和酶学动力学参数的测定，深入地研究了arog的反馈抑制位点的特性，并对arog的n -末端在反馈抑制机理和维持稳定四级结构中的作用，以及蛋白质结构的“ d2 ”对称性对酶功能的重要性等进行了具体的研究。
We successfully construct the eukaryotic expression vector of gfp - eif - 5a and its mutational vector using genetic engineering techniques . we found that eef - 5 a localized in nucleus as well as in cytoplasm just for a short time after its transient expression , then distributed only in cytoplasm Eif - 5a的hypusine修饰是其活性和功能发挥所必需的，我们通过pcr方法实现了hypusine位点的定点突变，并进一步构建了含gfp标签的eif - 5a及其hypusine位点突变的真核表达载体。
Furthermore , by inserting " anther box " element to the mutated area of two site - mutation promoters , another two promoters , ipmas and ipmal , were created . in order to study the chemical - inducible capacity of wild and modified pr - la promoters , a coding sequence of gus ( | 3 - glucuronidase ) gene was fused to their downstre am , and the chimeric genes were cloned into pbin ! 9 - based plant expression vector 为了检测得到的启动子驱动效率及诱导活性，将所得到的启动子、定点突变启动子和插入花药盒的启动子与gus基因连接，构建了6个植物表达载体，同时分别构建包含ipl barnase 、 ipml barnase 、 ipmal barnase嵌合基因的植物表达载体。
Several amino acid residues of sed have been proved to be important in the interaction between sed and mhc , but the mode of sed binding to mhc is not yet clear . this study is designed to explore the above issues . the main content and results of this study are as follows : firstly , the prokaryotic expression system of sed was constructed 基于以上问题，我们对sed可能的活性位点进行预测，构建了一系列超抗原sed免疫识别机制的研究sed定点突变体，检测这些突变体的mhc11结合活性和tcrvfi特异性，寻找与tcrv6结合的关键位点，进一步探讨sed与mhc和tcr的相互作用方式。
