绒毛状烟草BAC文库的构建

绒毛状烟草是重要的野生种烟草之一。本研究以CopyControl^TM pCC1BAC^TM(Hind Ⅲ Cloning-Ready)Vector为载体,构建了绒毛状烟草的BAC文库。分析结果表明,BAC文库DNA插入片段为50~200 kb,平均110 kb,空载率<2%。随机挑选15个克隆进行稳定性继代试验,100代后插入片段仍然稳定存在。该绒毛状烟草BAC文库的建成,为进一步克隆重要功能基因提供了必备的物质平台。     

新疆野生油菜BAC文库的构建

本实验以新疆野生油菜18号的基因组DNA为材料构建了其BAC（bacterial artificial chromosome）文库。该文库共有14592个克隆,插入片段在50-300kb之间,平均插入片段105kb,空载率低于5%,覆盖了新疆野生油菜基因组约4.0倍。新疆野生油菜基因组BAC文库的构建,为进一步研究其基因组结构及功能基因的利用等奠定了基础。     

南昆山毛叶茶cDNA文库构建

本研究以天然低咖啡碱茶树品种-南昆山毛叶茶春季嫩稍为材料,采用Stratagene公司ZAP系列试剂盒成功构建了该品种全长cDNA文库.原始文库滴度为6.0×105pfu/ml,重组率为97.3%,1.0～2.0kb的插入片段占整个文库的70%左右,达到一个高质量cDNA文库的要求,文库经扩增后滴度为1.9×109pfu/ml,重组率达到99.1%.     

红曲霉HMW-DNA的制备

用低熔点琼脂糖包埋浓度为1×109～1×1010个/mL红曲霉原生质体液制备得到DNA胶块。经裂解和PMFS纯化后脉冲场电泳检测,胶块中的DNA片段长度在800kb以上,每个胶块DNA含量为8～13μg。DNA胶块经脉冲场电泳去除小于600kb的非目的小片段后用限制性内切酶EcoRI、BamHI、HindIII作用,结果大部分能被酶解。可以用于BAC文库等大片段基因组文库的构建,为红曲霉功能基因组的研究奠定了基础。     

树干毕赤酵母基因组文库的构建及纤维二糖酶基因的筛选

首次构建了树干毕赤酵母DNA基因组文库,该文库包含3 000个克隆,插入片段长度为0.5～8.0 kb。树干毕赤酵母染色体基因为15 441 179 bp,该文库覆盖1.08倍树干毕赤酵母染色体基因,筛选出任一基因或序列的概率为97%。提取文库中的质粒醋酸锂法转化酿酒酵母W303-1A,涂布于纤维二糖为唯一碳源的平板,通过纤维二糖酶对底物纤维二糖的底物特异性筛选文库,首次获得树干毕赤酵母来源的一个1.82 kb的纤维二糖酶基因。     

甘蔗UGPase DNA及其5’侧翼序列的克隆与序列分析

以甘蔗（FN95-1702）为材料,首次克隆得到甘蔗UGPaseDNA片段,测序结果表明该基因全长约5-3kb,包含21个外显子,20个内含子。开放读码框（ORF）共编码476个氨基酸。通过接头连接PCR方法克隆得到约0．8kb的该基因5’侧翼序列,并对得到的序列进行基础启动子区及调控元件分析,预测在该序列上存在TAT...     

蓖麻成熟期胚抑制消减cDNA文库构建及其差异表达基因分析

利用抑制性消减杂交（suppression subtractive hybridization,SSH）技术,以高含油量蓖麻品种油蓖5号开花后第15d胚和36d胚为实验材料构建36d成熟期胚消减cDNA文库。文库的插入片段平均长度约400bp,随机挑取700个克隆进行PCR筛选,获得596个阳性克隆,经过点杂交筛选后选择了521个阳性克隆进行测序。经过同源性比对归并后得到96个差异表达基因,其中包括31个未知基因。已知基因涉及油脂合成、糖的分解、蛋白质贮藏与降解等多个方面。本研究还通过RT—PCR检测了部分可能与蓖麻油脂合成相关基因的差异表达水平,对其可能的功能进行了简要的分析。     

乳链球菌乳糖酶基因的分子克隆

以LacDNA片段为探针，筛选了pUC19为载体构建的乳链球菌（S.lactis)6030菌株的质粒DNA文库，得到插入片段约5.0kb的阳性克隆，并对插入片段进行限制性酶切分析，用ONPG法测定了重组质粒在大肠杆菌细胞中乳糖酶的表达水平。     

烟草优质品种叶片全长cDNA文库的构建和质量分析

为研究优质烟叶的分子基础,以翠碧一号不同生长时期的叶片为材料,CTAB法提取总RNA,利用SMART技术合成全长cDNA,之后按经修改的Clontech技术成功构建了烟草叶片全长cDNA文库.经鉴定,初级文库库容为3.85×106个克隆,重组率为100％,重组子插入片段集中在750-2000 bp之间.随机挑取25个克隆测序后,经生物信息学分析表明,烟草上已经报道的基因占10条,11条为全长基因序列;20条序列有功能注释.综合分析表明,所构建的文库质量较高,达到了基因的分离、筛选和克隆的建库目的.     

噬菌体随机肽库淘选桔霉素模拟表位的研究

目的:从噬菌体随机肽库中淘选模拟桔霉素表位的噬菌体粒子。方法:以抗桔霉素的单克隆抗体为配基,分别免疫亲和淘选以融合蛋白形式表达在丝状噬菌体M13外壳蛋白Ⅲ上的随机7肽库和12肽库,以ELISA方法鉴定阳性克隆,同时进行DNA测序以分析插入的7肽和12肽的氨基酸序列。结果:经过3轮淘选,在7肽库中淘选到20株能与该抗体特异性结合的阳性克隆,在12肽库中淘选到33株能与该抗体特异性结合的阳性克隆,且该结合均能被桔霉素阻断,模拟表位的共有序列为X-组氨酸-赖氨酸-X-X-X-X,X为任意氨基酸。以7肽库中亲和力最强的克隆(P10)建立了竞争ELISA检测方法,线性范围为10～325ng/ml,检测下限为10ng/ml;以12肽库中亲和力最强的克隆(P1)建立了竞争ELISA检测方法,线性范围为10～439ng/ml,检测下限为10ng/ml。结论:噬菌体展示技术可成功淘选到桔霉素模拟表位,高度保守的His和Lys的存在,提示His和Lys在CIT与其配体的结合中可能起重要作用。     

Identification and analysis of specific chromosomal region adjacent to exogenous Dhfr-amplified region in Chinese hamster ovary cell genome

Chinese hamster ovary (CHO) cells are widely used for the stable production of recombinant proteins. Gene amplification techniques are frequently used to improve of protein production, and the dihydrofolate reductase (DHFR) gene amplification system is most widely used in the CHO cell line. We previously constructed a CHO genomic bacterial artificial chromosome (BAC) library from a mouse Dhfr-amplified CHO DR1000L-4N cell line and one BAC clone (Cg0031N14) containing the CHO genomic DNA sequence adjacent to Dhfr was selected. To identify the specific chromosomal region adjacent to the exogenous Dhfr-amplified region in the CHO cell genome, we performed further screening of BAC clones to obtain other Dhfr-amplified regions in the CHO genome. From the screening by high-density replica filter hybridization using a digoxigenin-labeled pSV2-dhfr/hGM-CSF probe, we obtained 8 new BAC clones containing a Dhfr-amplified region. To define the structures of the 8 BAC clones, Southern blot analysis, BAC end sequencing and fluorescence in situ hybridization (FISH) were performed. These results revealed that all the selected BAC clones contained a large palindrome structure with a small inverted repeat in the junction region. This suggests that the obtained amplicon structure in the Dhfr-amplified region in the CHO genome plays an important role in exogenous gene amplification.     

High-Resolution cosmid mapping of the left arm of Saccharomyces cerevisiae chromosome XII; A first step towards an ordered sequencing approach

For the sequencing of the left arm of chromosome XII of Saccharomyces cerevisiae, we fine-mapped the entire 450 kb fragment between the ribosomal DNA (rDNA) and the left telomere. Total yeast DNA in agarose blocks was digested with I-PpoI, which exclusively cuts once in each repeat unit of the rDNA. The resulting fragment was isolated from pulsed-field gels, together with the equally sized chromosome IX. A cosmid library of some 30-fold chromosome coverage was generated from this material, with the cloning efficiency being around 20 000 clones per microgram genomic DNA. The chromosome XII and IX specific clones were identified by complementary hybridizations with the respective chromosomes. For the left arm of chromosome XII, a contiguous cosmid array (contig) with an average map resolution better than 9 kb was generated by clone hybridization procedures. The ordered library serves as a tool for the physical mapping of genetic markers. Also, a minimal set of 15 clones was selected that covers the entire fragment. This subset forms the basis for the generation of a template map of much higher resolution for a directed sequencing of the left arm of chromosome XII.     

Comparative genomics uncovers large tandem chromosomal duplications in Mycobacterium bovis BCG Pasteur

On direct comparison of minimal sets of ordered clones from bacterial artificial chromosome (BAC) libraries representing the complete genomes of Mycobacterium tuberculosis H37Rv and the vaccine strain, Mycobacterium bovis BCG Pasteur, two major rearrangements were identified in the genome of M. bovis BCG Pasteur. These were shown to correspond to two tandem duplications, DU1 and DU2, of 29 668 bp and 36 161 bp, respectively. While DU1 resulted from a single duplication event, DU2 apparently arose from duplication of a 100 kb genomic segment that subsequently incurred an internal deletion of 64 kb. Several lines of evidence suggest that DU2 may continue to expand, since two copies were detected in a subpopulation of BCG Pasteur cells. BCG strains harbouring DU1 and DU2 are diploid for at least 58 genes and contain two copies of oriC, the chromosomal origin of replication. These findings indicate that these genomic regions of the BCG genome are still dynamic. Although the role of DU1 and DU2 in the attenuation and/or altered immunogenicity of BCG is yet unknown, knowledge of their existence will facilitate quality control of BCG vaccine lots and may help in monitoring the efficacy of the world's most widely used vaccine.     

低耗水管道化制浆技术对豆浆品质及稳定性的影响

通过开展低耗水管道化制浆技术对豆浆品质及稳定性进行研究,对比分析不同制浆工艺所得豆浆的基本组分、粒径、离心沉淀率、稳定性、色差和感官评价等指标的差异。结果表明：新型低耗水浸泡技术结合胶体磨制浆得到的豆浆蛋白质浸提效果（3.89 g/100 g）最佳;平均粒径（2.55 μm）相对较小,粒径峰值较高、峰宽较窄,分布均一;离心沉淀率（1.81%）及稳定性指数斜率最低,体系稳定性显著高于传统技术制备的豆浆（P＜0.05）;豆浆颜色亮白、口感细腻,感官评分为96.20 分,感官品质显著优于其他处理组（P＜0.05）。低耗水浸泡技术结合胶体磨制浆可有效提高大豆蛋白提取率,减小豆浆粒径,改善豆浆稳定性和感官品质,为豆制品管道化加工提供参考。     

蒸汽爆破辅助提取高温豆粕中的蛋白质

研究了蒸汽爆破对高温豆粕中蛋白质溶解性的影响和后续蛋白质提取工艺。结果表明:当蒸汽压力较低时,高温豆粕的氮溶解指数随着蒸汽压力的升高而提高,但当超过2.1 MPa时,压力对爆破处理的效果的影响不明显;豆粕的粒度对蒸汽爆破效果有一定的影响,当豆粕颗粒的平均小于100目时效果不佳,在20～80目时效果较好。当高温豆粕颗粒大小为20～80目,爆破条件为1.8 MPa,180 s时,蒸汽爆破处理使高温豆粕的氮溶解指数提高了1.1倍。对经蒸汽爆破处理的高温豆粕中蛋白质的提取条件研究表明,pH值和温度对蛋白质的提取率有较大影响。在优化条件下,经蒸汽爆破处理的高温粕的蛋白质提取率达到76.04%。     

免培养技术对浓香型白酒大曲中细菌多样性的影响

采用免培养(culture independent)技术直接从浓香型白酒大曲中提取细菌微生物基因组DNA,利用细菌16S rDNA通用引物扩增大曲混合微生物的16S rDNA,采用PCR扩增技术、分子克隆技术以及序列同源性分析等方法测定大曲中细菌的16S rDNA基因全序列,通过与基因数据库中相似菌群序列同源性的比较,构建系统发育树。结果显示,成熟的大曲中细菌共分为Lactobacillus,Pantoea,Enterobacter,Klebsiella,Leuconostoc,Erwinias,Pseudomonas,Bacillus licheniformis几大类群,表现出高度的细菌多样性。     

Preparation of low-fat uptake frying batter composite by dry particle coating of microparticulated soybean hull

This study examined the influence of soybean hull-coated frying batter composite on fat uptake during deep fat frying. Soybean hull was microparticulated by impact mill at impact mill speed (IMS) of 6000, 10,000 and 14,000 rpm, and air-classified into coarse and fine fractions at Air Classifying Wheel Speed (ACWS) of 4000, 8000 and 12,000 rpm, respectively. Each soybean hull was dry-coated into wheat flour by dry particle coating system. As the difference in particle size between wheat flour and soybean hull got larger, the coating process became more effective, which indicates the size difference between wheat flour and soybean hull was important for coating effectiveness. When the ratios of wheat flour to soybean hull were 99:1 and 95:5, there were about 3.3 (g/100 g) and 24.4 (g/100 g) of fat content reduction, respectively. Inner crust structures showed slight reduction in cell size and improved cellular integrity with shrinkage in the cell membrane, with increase in soybean hull content. This means soybean hull can form a protective layer and can be applied to the food industry as a frying batter composite to reduce fat uptake.