高质量毕赤酵母基因组DNA提取方法比较

旨在比较5种毕赤酵母基因组DNA的提取法,以便获得简便高效的提取高质量酵母基因组DNA的优化方法。分别使用蜗牛酶破壁法,超声波破碎法,液氮研磨法,Lyticase破壁法,试剂盒法提取毕赤酵母基因组DNA,然后进行DNA电泳检测以及紫外分光光度计测定DNA浓度和纯度。结果显示,5种方法均能提取出酵母基因组DNA,而酶法所提取的酵母基因组DNA质量最好。由此证实,蜗牛酶法成本低、效果好,是理想的提取高质量酵母基因组DNA的方法,完全满足后续试验要求。     

利用毕赤酵母系统高效表达灵芝中的免疫调节因子LZ-8蛋白

根据Murasugi等发表的LZ-8基因的DNA序列,利用PCR方法从灵芝菌丝体的基因组DNA中扩增到lz-8基因.将该基因构建到毕赤酵母表达载体上,电激转化毕赤酵母.对转化子先后进行PCR和PCR-Southem鉴定,表明lz-8基因已转入毕赤酵母.转化子经发酵培养后用SDS-PAGE电泳方法检测发酵液,证明毕赤酵母...     

一种简便的适用于酵母双杂交系统的酵母质粒提取方法

目的:建立一种适用于酵母双杂交系统的简便快捷的酵母质粒提取方法。方法:以酿酒酵母为供试材料,用玻璃珠振荡法破除酵母细胞壁,提取酵母总DNA,最后通过电转化大肠杆菌DH10B获得目的质粒。结果:粗提得到的质粒可直接转化DH10B,作为模板用于PCR分析及酵母双杂交后续的序列分析等,大大降低了工作量。结论:该方法简便快捷,经济实用,降低了成本,提高了效率,可以作为一种实验室酵母质粒提取方法。     

荧光定量PCR检测重组新蛭素中毕赤酵母基因组DNA的残留量

目的:建立real-time PCR定量检测毕赤酵母基因组DNA残留量的方法,提高重组新蛭素的产品安全性。方法:选择拷贝数高且分布广泛的毕赤酵母5S rRNA基因为靶标基因设计扩增引物,提取酵母基因组DNA,稀释后作为扩增模板。以罗氏荧光定量PCR_LightCycler480平台为基础,建立基于SYBR GreenⅠ荧光染料的real-timePCR的检测方法,并考察用该方法检测重组新蛭素中毕赤酵母基因组残留量的灵敏度、精密度和回收率。结果:该法检测宿主DNA残留量灵敏度高,DNA浓度为0.1~1000 pg/μL范围内呈现良好的线性关系,其标准曲线的误差值小于0.2;用该法对5批注射用重组新蛭素(酵母)产品中宿主基因组DNA残留量进行了测定,结果分别为0.03、2.3、0.2、0.6、0.2 pg/mg。结论:该方法具有操作简便、灵敏度高等优点,可用于重组产品中酵母基因组残留DNA的定量测定。     

一种改进的丝状真菌DNA提取方法

以丝状真菌雅致枝霉(Thamnidium elegans)和深黄伞形霉(Umbelopsis isabellina)为材料, 使用优化的CTAB法提取基因组DNA。改进后的方法使用液氮冻融以及玻璃珠振荡的方法代替了传统的液氮研磨, 所需菌体量少, 而得到的基因组DNA比用传统的CTAB法得到的基因组DNA产率高、纯度好、且步骤简单, 适用于一次微量提取多个样品的基因组DNA。这种方法得到的基因组DNA可用于大部分分子生物学基本实验如PCR和DNA的酶切等。     

一种改进的丝状真菌DNA提取方法

以丝状真菌雅致枝霉(Thamnidium elegans)和深黄伞形霉(Umbelopsis isabellina)为材料,使用优化的CTAB法提取基因组DNA.改进后的方法使用液氮冻融以及玻璃珠振荡的方法代替了传统的液氮研磨,所需茵体量少,而得到的基因组DNA比用传统的CTAB法得到的基因组DNA产率高,纯度好、且步骤简单,适用于一次微量提取多个样品的基因组DNA.这种方法得到的基因组DNA可用于大部分分子生物学基本实验如PCR和DNA的酶切等.     

适于涡虫基因组DNA快速制备的改良的CTAB法

提取得到高质量的DNA样品是进行分子生物学研究的必要前提。为了找到一种适用于提取涡虫基因组DNA的常规方法,我们以东亚三角头涡虫为材料,分别用改良的CTAB法、SDS法、SDS-蛋白酶K法对涡虫的基因组DNA进行了制备,并对3种方法制备的涡虫基因组DNA进行了检测与比较。根据比较结果,我们认为改良的CTAB法最适合于涡虫基因组DNA的快速制备,为涡虫的分子生物学研究打下了基础。     

抗菌肽牛乳铁蛋白肽衍生肽在毕赤酵母中的表达及活性鉴定

利用巴斯德毕赤酵母系统表达抗菌肽牛乳铁蛋白肽衍生肽简称LfcinBD,获得的表达产物具有较强的抗菌活性.将人工设计的用化学合成法合成的以酵母偏爱密码子编码的LfcinBD基因片段克隆到巴斯德毕赤酵母分泌型表达载体pPIC9K中,获得的重组质粒pPIC9K-LfcinBD通过限制性内切酶Sac Ⅰ酶切线性化,电击法转化毕赤酵母GS115宿主菌,G418抗性筛选,得到高拷贝转化子.经PCR检测,LfcinBD基因与毕赤酵母染色体稳定整合.阳性克隆经甲醇诱导表达LfcinBD,诱导表达5 d,每24 h取上清1 mL,进行抑菌试验.结果表明,抗菌肽牛乳铁多肽衍生肽基因已整合到酵母细胞基因组中并获得表达,经0.5%甲醇在30℃诱导48 h可产生较强抗菌活性的抗菌肽,而且对氨苄青霉素抗性的大肠杆菌亦有较强的抑菌作用.     

金针菇免疫调节蛋白基因fip-fve在毕赤酵母GS115中诱导型和组成型表达

为获得稳定来源并且具有生物学活性的重组金针菇免疫调节蛋白(Fip-fve),将fip-fve基因转至毕赤酵母GS115中进行诱导型和组成型表达。用PCR方法从金针菇子实体基因组DNA中扩增fip-fve基因,连接至pPIC9构建诱导型表达载体pPIC9-FIP-fve,从毕赤酵母基因组DNA中扩增三磷酸甘油醛脱氢酶启动子(pgap),替换pPIC9-FIP-fve上乙醇氧化酶启动子(paox1)构建组成型表达载体pPIC9-PGAP-FIP-fve。将线性化的两种表达载体用PEG法转化毕赤酵母GS115,经组氨酸缺失培养基筛选和酵母菌落PCR鉴定后进行表达。结果表明,重组Fip-fve在以甲醇(1%,V/V)为碳源进行诱导型表达4 d达到最高,粗蛋白表达量为158.2 mg/L,在以葡萄糖(10%)和甘油(1%,V/V)为碳源进行组成型分别在表达第4天和第5天达到最高,粗蛋白分别为46.3 mg/L和29.5 mg/L。SDS-PAGE及Western blotting证明重组Fip-fve已正确表达,血细胞凝集活性检测初步证明重组Fip-fve具有良好生物学活性。     

莱茵衣藻ω-3脂肪酸脱氢酶基因在毕赤酵母中的表达与活性分析

采用RT-PCR方法克隆到莱茵衣藻ω-3脂肪酸脱氢酶基因lyd(l)5,与毕赤酵母表达载体pPIC3.5K连接,电击法转化毕赤酵母GS115.转化子经高浓度G418筛选出高抗性重组子,经PCR鉴定目的基因已整合入毕赤酵母基因组中.甲醇诱导表达,RT-PCR检测表明莱茵表藻ω-3脂肪酸脱氢酶基因在毕赤酵母中得到了表达;毕赤酵母总脂肪酸甲酯经气相色谱(GC)分析结果显示亚油酸的含量明显降低,而α-亚麻酸的含量有所提高.     

Preparation of megabase-size DNA from plant nuclei

A novel technique has been developed for the preparation of high molecular weight (HMW) DNA from plant nuclei. This technique involves physical homogenization of plant tissues, nuclei isolation, embedding of the nuclei in low-melting-point agarose microbeads or plugs, and DNA purification in situ . This technique is simple, rapid, and economical, and the majority of the DNA prepared is over 5.7 Mb in size. The genomic DNA content of the HMW DNA prepared by this technique is enriched by at least threefold and the chloroplast DNA content is reduced by over twofold relative to that prepared from plant protoplasts by existing methods. The DNA is readily digestible with different restriction enzymes and partial digestions of the DNA could be reproducibly performed. This method has been successfully used for the preparation of HMW DNA from a wide range of plant taxa, including grasses, legumes, vegetables, and trees. These results demonstrate that the DNA prepared by this technique is suitable for plant genome analysis by pulsed-field gel electrophoresis and for the construction of yeast and bacterial artificial chromosomes.     

Isolation of circular yeast artificial chromosomes for synthetic biology and functional genomics studies

Circular yeast artificial chromosomes (YACs) provide significant advantages for cloning and manipulating large segments of genomic DNA in Saccharomyces cerevisiae. However, it has been difficult to exploit these advantages, because circular YACs are difficult to isolate and purify. Here we describe a method for purification of large circular YACs that is more reliable compared with previously described protocols. This method has been used to purify YACs up to 600 kb in size. The purified YAC DNA is suitable for restriction enzyme digestion, DNA sequencing and functional studies. For example, YACs carrying full-size genes can be purified from yeast and used for transfection into mammalian cells or for the construction of a synthetic genome that can be used to produce a synthetic cell. This method for isolating high-quality YAC DNA in microgram quantities should be valuable for functional and synthetic genomic studies. The entire protocol takes ～3 d to complete.     

有效提取病毒诱导基因沉默植株中DNA的方法

根据PCR反应的要求,用改良的CTAB法,以番茄植株为材料,实现了微量番茄叶片基因组DNA的快速提取。提取基因组DNA所用的组织量少,所得的DNA经过电泳检测虽有降解,但足以用于PCR检测,以其作模板扩增中国番茄黄化曲叶病毒诱导的硫黄素酶（Su）基因沉默植株中病毒组分中的DNAmβ和1.7 A,片段大小分别为1 300、500 bp。测序结果证明是相应基因的部分片段。该方法的材料不需要使用液氮,可以单人大批量提取,并在基因沉默的番茄植株中能稳定而准确的规模化PCR检测。     

Physical Map of the Saccharomyces Cerevisiae Genome at 110-Kilobase Resolution

A physical map of the Saccharomyces cerevisiae genome is presented. It was derived by mapping the sites for two restriction endonucleases, SfiI and NotI, each of which recognizes an 8-bp sequence. DNA-DNA hybridization probes for genetically mapped genes and probes that span particular SfiI and NotI sites were used to construct a map that contains 131 physical landmarks--32 chromosome ends, 61 SfiI sites and 38 NotI sites. These landmarks are distributed throughout the non-rDNA component of the yeast genome, which comprises 12.5 Mbp of DNA. The physical map suggests that those genes that can be detected and mapped by standard genetic methods are distributed rather uniformly over the full physical extent of the yeast genome. The map has immediate applications to the mapping of genes for which single-copy DNA-DNA hybridization probes are available.     

Development of large DNA methods for plants: molecular cloning of large segments of Arabidopsis and carrot DNA into yeast.

Procedures for the preparation, analysis and cloning of large DNA molecules from two different plant species are described. Arabidopsis and carrot protoplasts were used for the preparation of large DNA molecules in agarose "plugs" or in solution. Pulsed-field gel electrophoresis (PFGE) analysis of large plant DNA preparations using a contour-clamped homogeneous field (CHEF) apparatus indicated that the size of the DNA was at least 12 Mb. Large DNA preparations were shown to be useful for restriction enzyme analysis of the Arabidopsis genome using both frequent and infrequent cutting enzymes and for the molecular cloning of large segments of DNA into yeast using artificial chromosome (YAC) vectors. PFGE and blot hybridization analysis of Arabidopsis and carrot DNA-containing YACs indicated that both unique and highly repeated DNA sequences were represented in these libraries.     

An improved method for photofootprinting yeast genes in vivo using Taq polymerase.

We have developed an improved method for photofootprinting in vivo which utilizes the thermostable DNA polymerase from T. aquaticus (Taq) in a primer extension assay. UV light is used to introduce photoproducts into the genomic DNA of intact yeast cells. The photoproducts are then detected and mapped at the nucleotide level by multiple rounds of annealing and extension using Taq polymerase, which is blocked by photoproducts in the template DNA. The method is more rapid, sensitive, and reproducible than the previously described chemical photofootprinting procedure developed in this laboratory (Nature 325. 173-177), and detects photoproducts with a specificity which is similar, but not identical to that of the previously described procedure. Binding of GAL4 protein to its binding sites within the GAL1-10 upstream activating sequence is demonstrated using the primer extension photofootprinting method. The primer extension assay can also be used to map DNA strand breakage generated by other footprinting methods, and to determine DNA sequence directly from the yeast genome.     

Homologous gene targeting in Caenorhabditis elegans by biolistic transformation

Targeted homologous recombination is a powerful approach for genome manipulation that is widely used for gene alteration and knockouts in mouse and yeast. In Caenorhabditis elegans, several methods of target-selected mutagenesis have been implemented but none of them provides the opportunity of introducing exact predefined changes into the genome. Although anecdotal cases of homologous gene targeting in C.elegans have been reported, no practical technique of gene targeting has been developed so far. In this work we demonstrate that transformation of C.elegans by microparticle bombardment (biolistic transformation) can result in homologous recombination between introduced DNA and the chromosomal locus. We describe a scaled up version of biolistic transformation that can be used as a method for homologous gene targeting in the worm.     

Replication origins in yeast chromosomes

DNA replication initiates at many sites in eukaryotic chromosomes. It has been difficult to isolate such replication origins, but a family of sequences from the yeast genome have properties which suggest that they may serve this function. The identification of these sequences together with sophisticated methods of genetic analysis, make yeast a useful organism for the study of eukaryotic DNA replication.