绿色巴夫藻的纯化培养及cdna文库的构建-微生物学专业毕业论文.docxVIP

山东大学硕士学位论文 山东大学硕士学位论文 绿色巴夫藻的纯化培养及eDNA文库的构建 行了扩增实验，检测到文库中的插入片段长度在500bp-I．2kb之间，符合构建 cDNA文库的要求。 对已报道的A9．脂肪酸去饱和酶基因序列进行同源性分析，发现不同来源的 脂肪酸去饱和酶具有高度保守性，即含有三个富含组氨酸的保守区。根据第一个 保守序列HXXXXH和第三个保守序列HXXHH，设计简并引物，以绿色巴夫藻 染色体DNA为模板，进行PCR扩增，得到一段长约600bp的片段(His box)。 将扩增产物与pGEM．TEasy载体连接，成功构建了重组质粒pGEM-T Easy- His box，经过双酶切及PCR分析，证明克隆到pGEM-T Easy载体上的片段为扩 增后产物。进行核苷酸序列测定表明，该His box片段由589个碱基组成：经 DNATOOLS分析，该序列不含内含子，编码由196个氨基酸残基组成的多肽； 通过BLAST X在GenBank中进行同源性比较，该多肽与Pirellula sp．的△9．脂肪 酸去饱和酶同源性达到69％。该多肽含有三个组氨酸保守序列，第一个与第二 个之间的距离为31个氨基酸残基，第二个与第三个之间的距离149个氨基酸残 基：利用TMHMM 2．0对该多肽进行分析，发现该片段有两个跨膜区域，均与已 报道的A9．脂肪酸去饱和酶保守区相同。 关键词：绿色巴夫藻，A9．脂肪酸去饱和酶，纯化，cDNA文库，SMART技术， 基因克隆，序列分析 IV 山东大学硕士学位论文 山东大学硕士学位论文 绿色巴夫藻的纯化培养及cDNA文库的构建 Pavlova viridis，a marine microalga,is rich in the very long chain polyun— saturated fatty acids(vLCPUFAs)such豁eicosapentaenoic(20：5n-3)and docosahexaenoic(22：6n-3)acids．It has been considered to be a new powerful source for∞一3 fatty acid desaturase genes．Selection ofPavlova viridis from the mixed culture was made by antibiotic for purifying strain．The growth condition was optimized．Total RNA from Pavlova viridis was extracted and eDNA library of Pavlova vwidis was construeted． Because of no suitable method for Pavlova viridis storage，the method of inoculating frequently was carried out，which resulted in the contamination with bacteria and fungi occasionally．Based on the different sensitivity to antibiotic for cell wall synthesis between bacteria and microalgae，it’s possible to add antibiotic to the mixed microalgae culture for eliminating bacteria contamination．The results indicated that purified Pavlova viridis stain was obtained by adding penicillin、 gentamycin and amikacin to the culture one by one． Pavlova viridis is autotrophic organism谢m low cell density．To harvest a large quantity of microalgae cells，cultural condition was optimized．The results showed that Pavlova viridis grew best in seawater F／2 medium by adding 5960 stock solution III and illuminated all day． Pavlova vf，idi