【作者】 侯桂琴；

【导师】 薛乐勋；

【作者基本信息】 郑州大学 ， 药理学， 2004， 硕士

【摘要】 近些年来，人们在转基因植物方面进行了大量的研究。与微生物发酵、动物细胞和转基因动物等生产系统相比，转基因植物不需要昂贵的设备和严格的培养条件，具有光合自养、成本较低，植物病毒不感染人类等优点，已成为一类比较廉价和安全的生物反应器。然而高等植物培养周期过长，生长受季节性限制，许多研究兴趣集中到生长迅速，培养简单的单细胞绿藻上。近几年来，莱茵衣藻的遗传背景已逐步研究清楚，有多种外源性蛋白质在衣藻中表达成功。遗憾的是，与衣藻比较，更具有优点的盐藻作为生物反应器的研究至今国内外尚缺乏。盐藻属绿藻门团藻目，是一种原生质裸露的嗜盐性浮游单细胞真核藻，长约6～15μm，呈椭圆形或梨形，无细胞壁只有细胞膜，且有双鞭毛，能在水中游动。有一个大型杯状叶绿体约占细胞体积的48％左右，可以进行光和自养，叶绿体的基部有一个淀粉核。盐藻是一种没有细胞壁的单细胞真核藻类，也是真核植物中抗逆性最强的一种藻类。该藻的突出优点在于培养条件极其简单，光合自养，抗盐性极佳，可在0.05-5M的盐水中生长，最佳生长繁殖盐度为2-3M。其强大的渗透调节能力，被认为可能是耐盐机理研究的模式植物。同时它又是单细胞光合自养生物，适于研究光合作用机理。 以细胞核为外源基因受体的传统植物基因工程虽已发展趋于成熟并得到广泛应用，但由于细胞核基因组大，背景复杂，外源基因的整合位点和整合的拷贝数难以人为控制，造成外源基因表达效率低，容易出现基因失活、基因沉默、位置效应等现象；同时转入多个基因时操作步骤过于复杂，所表达的原核基因必须经过修饰改造，环境郑州大学2以吟年硕士学位论文杜氏盐藻(D“nal曰lasa如“)叶绿体atPA基因片段的克隆分析及杂交安全难以保证等。叶绿体转化系统的出现为克服这些困难带来了希望。与核基因转化系统相比，叶绿体基因组小，遗传操作简单，外源基因是通过同源重组机制定点整合进叶绿体基因组。这样有利于人为控制外源基因的插入位点，可以将目的基因定位在适于表达的位点，能较好地解决“顺式失活”、“位置效应”等类的基因沉默问题。由 于叶绿体中DNA分子有多个拷贝，同时叶绿体对表达产物的积累有较强的承受能力，保障了外源基因在叶绿体中的高效表达。另外，由于叶绿体基因组的原核性质，对来自原核生物的外源基因无需改造就可以在叶绿体中高效表达，而且可以将多个外源基因采取“多顺反子”的原核表达形式同时弓!入，并由共同的启动子控制，既方便操作又可避免由于存在多个相同的启动子所带来的“共沉默”。这是核基因转化无法做到的。 要建立杜氏盐藻叶绿体转化体系，最关键的是构建一个稳定的叶绿体表达载体。因此选择理想的启动子是至关重要的。研究表明，在莱因衣藻中atPA基因(编码叶绿体ATP合成酶u亚基)簇(包括 atpA，psbl，CemA和atpH四个基因)共用一个启动子，这个强启动子位于。tpA基因的仁游，它可以榨为叶绿体载体表达系统的必要元件。本研究室曾采用莱因衣藻叶绿体atpA基因启动子作为盐藻叶绿体转化系统的启动子，虽然也有转录起始活性，但表达效率不高。若能把盐藻叶绿体自身的atPA基因强启动子克隆出来并用于其叶绿体表达载体无疑是最理想的。故本研究根据近缘藻类atpA基因的氨基酸高度保守序列，设计简并引物，用PCR方法扩增得到了盐藻叶绿体atpA部分核昔酸序列(GenBank ACceSSion AY435096)，旨在为下一步启动子的克隆和盐藻高效叶绿体表达载体的构建打下基础。同时还通过设计简并引物，用PCR方法直接从叶绿体基因组中克隆了atpA基因片段，这为从叶绿体中克隆某些基因提供了一种快速而简便的途径。 方法 从GenBank卜搜索五种藻类莱因衣藻(ch/二厂成咖，nas二盯助a刀t，’l’)、普通小球藻(乙为了。厂。11a。u1朋r1’s)、肠sost，’那失2叮厂j陇】、卵形肾藻(从岁为二s曰。1’Lvo1，’二cea)和〔F叔nfdl’osc汽口on、rojae叶绿体的atpA全基因所编码的氨基酸序列进行比对，找出两段比较保守的序列，设计简并引物，以提取的CtDNA为模板，用PC尺方法扩增atPA基因片段。并把其与pMD一18T一veCt()r(全长2.69kb)载体连接进行体外重组和转郑州大学20汉年硕士学位论文杜氏盐藻(D期。肠llasa枷。)叶绿体at衅基因片段的克隆分析及杂交化，用含Amp、X一gal和工PTG的LB平板筛选，挑取阳性菌落提质粒，后用EcoRI和HindIH进行双酶切鉴定，将含有插入片段的重组质粒送上海基康公司测序，序列结果与GenBank七其他藻类的atPA基因片段进行分析较。最后利用克隆的片段为探针分别与盐藻叶绿体基因组和核基因组进行southern杂交，证实其是否来自盐藻叶绿体，为下一步启动子的定位打下基础。 结果 1.pcR扩增产物 经PCR扩增后，电泳检测在约4oobp处有一明显亮带，与预期扩增的片段大小一致。 2.盐藻叶绿体atpA基因的克隆及重组质粒的鉴定 将PCR扩增后获得的片段经琼脂糖凝胶回收后与PMD一18T一vector相连进行体外重组和转化，用含Amp、X一gal和IPTG的LB平板筛选，挑取14个阳性菌落进行快速DNA鉴定，用Ec0RI和Hin更多还原

【Abstract】 Dunaliella salina belongs to chlorophyta, Chlorophycease, Volvocales and its shape and structure are very similar to Chlamydomonas reinhardtii except for lacking of cell wall. It is a kind of unicellular alga, can swim through its diflagella and has a large cup-shaped chloroplast covering about 48% of the cell volume.D.salina can grow in extreme environment such as in a variety of salt concentrations ranging from 0.05 to 5M, and the optimum salt cencentration for growth is 2 to 3M. Because its growth condition is very simple, D.salina is a favorable host for producing pharmaceutical proteins.Nucleic transformation in plants has many problems such as the complicated manipulations and low efficiency expression of the foreign genes, but the chloroplast transformation may resolve the problems mentioned above. The chloroplast genome is very small and genetical manipulation in it is easier, so the foreign gene is sited-direct integrated into the chloroplast genome through homologous recombination, which is helpful to locating the foreign gene introduced at a certain site where it can be expressed efficiently. Furthermore, because the chloroplast DNA has many copies, it is very possible to express high the foreign genes in the chloroplast.In order to find a chloroplast transformation system of D. salina, we constructed a high efficiency expression vector, and the key to it is that wemust find out a powerful promoter for the vector. It has been shown that the atpA gene cluster in the C. reinhardtii chloroplast genome comprises the aptA, Psbl, cemA and atpH genes, which encode a a -subunit of the coupling-factor-I (CFi) ATP synthase, a small photo system II polypeptide, a chloroplast envelope membrance protein, and subunit III of the CFo ATP synthase, respectively. The four genes have a common functional promoter that lies immediately the upstream of psbl and the atpAgene.D. salina has high homologys to C.reinhardtti, so the powerful promoter may be evolved either in D. slina or in C. reinhartii. For finding this powerful promoter, we designed a pair of degenerate primers and amplified the atpA gene fragment from the chloroplast of D. salina by PCR according to conserved motifs of the homologous amino acid sequences of five kinds of algae. In this study, the strong promoter was cloned to a construct high efficiency expression vector of the chloroplast of D.salina. On the other hand, the fragment of the atpA gene directly from the chloroplast genome was cloned by PCR technique through designing degenerate primer, which provides a fast and simple method for cloning some genes from the chloroplast.Methods:To find the conserved motifs, known amino acid sequences were compared. According to the conserved motifs of the homologous amino acid sequences of about ten kinds of algae, we designed a pair of degenerate primer and amplified the atpA gene fragment from the chloroplast of D. salina by PCR technique. The resulting PCR products were inserted into pMD-18 T-vector, and then transformed into E.coli JM109. Positive colonies were selected to determine their sequences. Homologous analysis of the deduced amino acid sequences was performed by BLAST and subsequently compared with GenBank data. The cloned fragment was used as a probe, which was hybridized with the chloroplast DNA of D. salina by SouthernResults and discussion:1. The products of PCRThe products amplified by PCR were analyzed by electrophorisis and a bright brand of about 400bp appeared.2. Cloning of the atpA gene and identification of the recombinant plasmid.The resulting PCR products were inserted into PMD-18 T-Vector, and then transformed into E.coli JM109. Positive colones were selected to determine their sequences. By the methods of enzyme digestion and electrophorisis, there were the expected fragments in four colones, respectively.3. analysis the result of sequencing.Homologous analysis of the deduced amino acid sequence were performed by BLAST and subsequently compared with GenBank data. Finally, we got a 405bp nucleotide seque更多还原