【作者】 张贵星；

【导师】 薛乐勋；

【作者基本信息】 郑州大学 ， 病理学与病理生理学， 2003， 博士

【摘要】 杜氏藻包括盐生杜氏藻(Dunaliella salina)及Dunaliella bardawil等，属于自养的耐盐单细胞真核绿藻，细胞形状呈梨形，具双鞭毛，胞内有一个大的杯状叶绿体。盐藻可在0.05～5.0 mol／L(饱和度)的氯化钠溶液中生长繁殖，盐藻细胞无细胞壁，其营养体细胞属于单倍体细胞。由于这些生物学特性，盐藻是一个非常理想的生物反应器候选宿主。此外，盐藻是光合自养生物，所以它的培养只需简单的无机培养基，而模式生物如大肠杆菌、酵母等则需要富含有机物的培养基；盐藻耐高浓度的氯化钠，故盐藻培养过程中不容易污染其它微生物；盐藻无细胞壁，载体DNA等容易导入细胞内，故其转化等遗传操作较普通的植物细胞容易；盐藻是真核生物，细胞内存在蛋白质的糖基化等翻译后加工、修饰过程，故利用盐藻生产的基因工程产物如蛋白质或多肽药物等，较之大肠杆菌等生产的基因工程产物更接近真核蛋白。另外，从转基因的角度考虑，由于盐藻营养体细胞的基因组是单倍体，不存在双倍体或多倍体细胞的基因型到表型的显性、隐性等遗传关系，所以其基因型能直接体现在表型上，非常有利于遗传突变株的筛选。目前国内外报道较多的对盐藻的研究主要仍集中在胡萝卜素的生产和利用以及盐藻的耐盐机理上，但对盐藻进行转基因研究，在国内外尚未见到报道。 盐藻Dunaliella bardawil属于杜氏藻的一种，它较之盐生杜氏藻郑州大学医学院2003年博士论文光诱导表达载体的构建及其在盐藻除草剂抗性转化中的应用(Dunaliella salina)具有更强的胡萝卜素合成能力，胡萝卜素含量占其干重可达10%I8]。Dunaliellaba炸lawil中的cbr基因是一种与胡萝卜素生物合成相关的基因，cb:的表达与高等植物的早期光诱导基因(e arlylight一induced genes，e一ip)的表达密切相关[81，eBR蛋白后被认为是一种玉米黄质结合蛋白，后者与CBR蛋白结合后形成的复合物对盐藻的光合作用系统起一种保护作用I9]，cBR基因是一种诱导表达的基因，诱导条件包括强光、硫酸盐剥夺以及norflurazon(一种除草剂)处理等[0]。 业已证明，野生盐藻对许多抗生素具有抗性[10，1‘];虽然盐藻对氯 霉素敏感，但根据我们实验室过去研究经验，用氯霉素抗性来筛选转 化的盐藻，现象不明显，筛选周期长;而后来我们自己的研究表明，野生的盐藻，无论是Dunalzella:alina[‘2]还是Dunaliella ba凡lawil(数据未显)，均对除草剂草T磷(phosphinothricin，即T)敏感。最低3 .omg几 剂量的草丁嶙就能够完全抑制盐藻的生长。草丁嶙系谷氨酸的结构类似物，进入细胞后可以抑制谷氨酞胺合成酶从而导致细胞内积累高浓度的氨而使细胞发生氨中毒【‘’〕。Bar基因是除草剂抗性基因，其编码产物草丁嶙乙酞转移酶可以使PPT乙酞化而失去毒性[l4，”]。 本实验室实践证明，对盐藻使用除草剂草丁嶙筛选体系，效果明 显。 为对盐藻进行转基因研究，我们设计并克隆了一种光诱导的盐藻表达载体，用于盐藻的瞬时转化和稳定转化。在该载体中我们将外源的bar基因插入到Dunal心la ba尹’c lawil的CBR基因的启动子(强光诱导表达)之下，并在bar基因下游接上CBR基因的终止子(3，一UTR，3‘一tintranslated region)，构成一个bar基因的表达盒。 作者在盐藻的培养等生物学特性的研究基础上，建立了两种盐藻转化方法，即电激转染和磷酸钙共沉淀转染。利用自己构建的盐藻光诱导载体，分别通过电激转染和磷酸钙共沉淀转染，进行盐藻的除草剂抗性郑州大学医学院2003年博士论文光诱导表达载体的构建及其在盐藻除草剂抗性转化中的应用转化，从细胞生物学和分子生物学的角度对两种转化方法进行了比较和分析，为转基因盐藻生物反应器研究初步建立了两种比较稳定的转化方法。 本研究论文分两部分，第一部分阐述盐藻光诱导表达载体pPB3RZa的分子克隆过程;第二部分阐述对盐藻基本生物学特性的研究和用载体pPB3RZa转化野生盐藻Dunaliella bardawil赋予其除草剂抗性以及对Dunaliella.Bardawil转基因方面的研究工作，包括转基因的检测等。1.方法盐藻光诱导表达载体pPB3RZa的构建盐藻Dunaliella ba程lawil的。br基因启动子区片段的PCR扩增 首先提取盐藻Dunal招lla baz农匕wil的基因组DNA，根据cbr基因序列，通过计算机引物设计程序Primer Premier，设计巢式PCR两对引物，外侧上游弓}物cbrsws:s‘GGeGGeGGAGAAAGGGAoAAGAA以G3‘，外侧下游引物:cbrswx，5‘AGAG“GeGGGAeGA鱿灯TGAIGGA3’;一对内侧上游引物的5’端具限制序列及保护序列，上游引物为cbrsns，5‘CGGG工皿工丛昼鑫CCCCATTCGTCeT以TTeTGGeTeT3‘(Xbal)，下游引物为cbrsnx，5‘月A旦燮里里G门℃戌rc汀CC月厌汀GCAfCGU哎汀卫岌孙‘(EeoRx)。PCR扩增反应分两步进行，第一轮反应应用上述1对外侧引物和自己研究的一种改良的降落PCR程序，以提取的Dunaliella ba矛农云wil基因组DNA为模板，进行扩增反应。巢式PCR第二轮反应以第一轮反应的产物为模板，使用上述一对内侧引物，通过普通PCR程序进行更多还原

【Abstract】 The alga Dunaliella, such as Dunaliella salina and Dunaliella bardawil, is an autotrophic unicellular eukaryotic green alga which looks much like another much related unicellular eukaryotic green alga, Chlamydomonas reiharditti, in morphology. But the varieties of Dunaliella species differ from C. reiharditti in two aspects of biological characteristics; they are halotolerant and lack a rigid cell wall. Dunaliella algae potentially are ideal candidate hosts for bioreactor, which can be used to generate many transgenic products including many pharmaceutical proteins, such as the interferins, interleukin-2 (IL-2), and tumor necrosis factor (TNF), etc. Dunaliella, as a bioreactor host, has a great deal of advantages over other model organisms. Since Dunaliella belongs to autotrophic organism, the culture of Dunaliella needs only light, water, and some inorganic salts without the use of costly organic medium as in the culture of E.coli and yeast, that is, the cost of Dunaliella is relatively very low. Dunaliella cells are much halotolerant, they can propagate in medium of 0.05-5.0 mol/L sodium chloride, and the medium most often used in laboratory contains 1.0-2.0 mol/L sodium chloride which prevents propagation of other microorganisms, implying that the laboratorial culture of Dunaliella is easier and could efficiently avoid the contamination of other microbes. Since Dunaliella cell has no cell wall, it is convenient to make geneticmanipulation on it, i.e. it is facile to introduce foreign DNA into Dunaliella cells, and even, many procedures of transfection of mammalian cells, as electroporation, phosphate calcium coprecipation, and liposome-mediated transfection, can be assumed to transform Dunaliella. Besides, on transgenic consideration, since the genome of vegetative Dunaliella cell is haploid, the expression of transgene is direct and manifest without the interference of recessive effect as in diploid cells, and this makes the screening of Dunaliella mutant facilitated. Finally, because Dunaliella cells are eukaryotes, the proteins inside them can be processed after translation, accordingly, the protein product by Dunaliella genetic engineering behaves more like the mammalian protein than that of E.coli gene engineering, namely, protein product from Dunaliella genetic engineering, in industry, requires less posttranslational procession than that from prokaryotic organisms. Another advantage for protein manufactured from Dunaliella is that we can use Dunaliella to produce edible vaccine for Dunaliella itself is safe to human being. In comparison of tansgenic higher plants, Dunaliella cells propagate rapidly and independent of seasons guaranteeing the production of protein at any moment in a year.But most studies on Dunaliella, so far, have been focusing on the mechanism of its halotolerance and on production of carotene, and few transgenic studies are performed on Dunaliella. The purpose of this study is to construct an expression vector and to find some methods that can be used to transform Dunaliella bardawil, and finally to establish a bioreactor on the ground of Dunaliella.To begin the transgenic research of Dunaliella, the author designed and cloned a light inducible expression vector which is applicable totransiently and/or stably transform Duiialiella bardawil. In the vector mentioned above, the exogenous herbicide-resistant gene bar (Basta resistant) was put under control of the promoter of gene cbr (carotene biosynthesis related) from Dunaliella bardawil, and at the downstream of bar gene lay the 3-UTR (3’-untranslated region) of cbr acting as a terminator of gene expression. These three fragments comprised an expression cassette of bar which was expected to confer Dunaliella bardawil a resistance of phosphinothricin (the major ingredient of Basta). Meanwhile, the two fragments flanking bar may act as homologous fragments and make the bar gene integrated into the genome of Dunaliella bardawil by double cross-over. The homologous integration of bar into genome of Dunaliella bardawil would更多还原