克隆

Molecular cloning and characterization of the novel lection gene. Primers were designed based on conserved sequences of other lectin genes from Amaryllidaceae. A novel lectin gene was isolated from Zephyrathes grandiflora. The full-length cDNA of Zephyrathes grandiflora lectin was 955bp, containing an open reading frame (576bp) encoding a peptide of 191 amino acids. The gene was named as zga (Zephyrathes grandiflora agglutinin), with its encoding protein showing 59% similarity to GNA.

基因克隆及序列分析：根据石蒜科植物凝集素的保守区段，设计引物，采用RACE技术从韭莲中克隆出长度为952bp的新凝集素基因，包含了完整的编码序列(576bp)，它编码一个全长191个氨基酸的蛋白，与雪花莲外源凝集的相似性达59％，被命名为ZGA(Zephyrathes grandiflora agglutinin)。

The stable clones are further identified by RT-PCR and Western blot; 6 MTT assay is used to investigate the effect of ZNRD1 on the cell growth of cells (AGS, SGC7901, MKN28, NIH3T3, GES-1); 7 Soft agar assay is used to investigate the effect of ZNRD1 on the clonality of cells (AGS, MKN28); 8 Nude mice assay is used to investigate the effect of ZNRD1 on the cell growth of gastric cancer cells (AGS, MKN28); 9 Flow cytometry is used to investigate the effect of ZNRD1 on the cell cycle distribution of cells (AGS, MKN28, NIH3T3, GES-1); 10 Flow cytometry is used to investigate the effect of ZNRD1 on the cell apoptosis of cells (AGS, MKN28, NIH3T3); 11 MTT assay is used to investigate the effect of ZNRD1 on the drug sensitivity of cancer cells (SGC7901, SGC7901/VCR, HL-60, HL-60/VCR) in vitro; 12 SRCA is used to investigate the effect of ZNRD1 on the drug sensitivity of gastric cancer cells (SGC7901, SGC7901/VCR) in vivo; 13 Flow cytometry is used to investigate the effect of ZNRD1 on adriamycin accumulation of cancer cells (SGC7901, SGC7901/VCR, HL-60, HL-60/VCR); 14 Transmission electron microscope is used to investigate the effect of ZNRD1 on the sensitivity of SGC7901 cells towards drug-induced apoptosis; 15 Flow cytometry and DNA ladder assay are used to investigate the effect of ZNRD1 on the sensitivity of cells (SGC7901, SGC7901/VCR, HL-60/VCR) towards drug-induced apoptosis; 16 Microarray is used to investigate the profiling of ZNRD1-responsive genes in gastric cancer cells (AGS, MKN28, SGC7901, SGC7901/VCR); 17 RT-PCR and Western blot are used to identify the results of microarray; 18 Reporter gene assay is used to investigate the effect of ZNRD1 on the transcriptional activity of cyclin D1; 19 Reporter gene assay is used to investigate the effect of ZNRD1 on the transcriptional activity of MDR1; 20 Kinase assay is used to investigate the effect of ZNRD1 on the activity of cyclin E-CDK2 kinase; 21 The antisensenucleic acids of p21 is used to inhibit the expression of p21, and flow cytometry is used to investigate the effect of p21 on ZNRD1-induced cell cycle arrest in gastric cancer cells; 22 The antisensenucleic acids of p27 is used to inhibit the expression of p27, and flow cytometry is used to investigate the effect of p27 on ZNRD1-induced cell cycle arrest in gastric cancer cells; 23 Liposome is used to up-regulate the expression of Skp2, and flow cytometry is used to investigate the effect of Skp2 on ZNRD1-induced cell cycle arrest in gastric cancer cells; 24 Western blot is used to investigate the effect of ZNRD1 on the stability of Skp2 and p27 in gastric cancer cells; 25 MVD assay is used to investigate the effect of ZNRD1 on the angiopoietic activity of gastric cancer cells; 26 ELISA is used to investigate the effect of ZNRD1 on the expression of VEGF165 in gastric cancer cells; 27 The roles of DARPP-32 in MDR of gastric cancer cells are investigated using gene transfection, MTT assay, SRCA, flow cytometry and DNA ladder assay.

应用杂交瘤技术制备ZNRD1的首个单克隆抗体；2）利用RT-PCR、Western blot和免疫组化检测ZNRD1在胃癌组织、胃炎组织、正常胃上皮组织、胃癌细胞和正常胃组织上皮细胞中的表达；3）构建ZNRD1的小干扰RNA载体，并测序鉴定；4）利用脂质体将ZNRD1的真核表达载体及其空载体转染胃癌细胞（AGS、SGC7901、MKN28）和小鼠成纤维细胞（NIH3T3），G418筛选后进行鉴定；5）利用脂质体将ZNRD1的小干扰RNA载体及其空载体转染药敏胃癌细胞（SGC7901）、正常胃组织上皮细胞（GES-1）、对长春新碱耐药的胃癌细胞（SGC7901/VCR）、药敏白血病细胞（HL-60）、对长春新碱耐药的白血病细胞（HL-60/VCR），G418筛选后进行鉴定；6）利用MTT实验检测ZNRD1高/低表达对细胞（AGS、SGC7901、MKN28、NIH3T3、GES-1）生长的影响；7）通过软琼脂克隆形成实验检测上调ZNRD1对AGS、MKN28细胞克隆形成能力的影响；8）通过裸鼠成瘤实验检测上调ZNRD1对AGS、MKN28细胞体内成瘤性的影响；9）通过流式细胞仪分析ZNRD1高/低表达对细胞（AGS、MKN28、NIH3T3、GES-1）的细胞周期的影响；10）通过流式细胞仪分析上调ZNRD1对细胞（AGS、MKN28、NIH3T3）的凋亡的影响；11）通过MTT实验检测ZNRD1高/低表达对细胞（SGC7901、SGC7901/VCR、HL-60、HL-60/VCR）体外药物敏感性的影响；12）通过肾包膜下移植法检测ZNRD1高/低表达对细胞（SGC7901、SGC7901/VCR）体内药物敏感性的影响；13）通过流式细胞仪分析ZNRD1高/低表达对细胞（SGC7901、SGC7901/VCR、HL-60、HL-60/VCR）内阿霉素蓄积和泵出的影响；14）通过透射电镜检测上调ZNRD1对SGC7901细胞凋亡敏感性的影响；15）通过流式细胞仪和DNA梯度试验检测ZNRD1高/低表达对细胞（SGC7901、SGC7901/VCR、HL-60）凋亡敏感性的影响；16）通过基因芯片检测ZNRD1高/低表达对胃癌细胞内基因表达谱的影响；17）利用RT-PCR、Western blot对基因芯片的结果进行鉴定；18）利用报告基因实验检测ZNRD1对cyclin D1的启动子活性的调节作用；19）利用报告基因实验检测ZNRD1高/低表达对MDR1的启动子活性的调节作用；20）利用激酶试验检测ZNRD1对cyclin E-CDK2 激酶活力的影响；21）利用反义核酸技术抑制p21的表达；通过流式细胞仪检测抑制p21对ZNRD1介导的细胞周期阻滞的影响；22）利用反义核酸技术抑制p27的表达；通过流式细胞仪检测抑制p27对ZNRD1介导的细胞周期阻滞的影响；23）利用脂质体转染法上调Skp2的表达；通过流式细胞仪检测上调Skp2对ZNRD1介导的细胞周期阻滞的影响；24）利用Western blot检测ZNRD1对p27和Skp2的蛋白稳定性的影响；25）利用微血管密度实验检测ZNRD1对AGS、MKN28细胞裸鼠移植瘤微血管形成的影响；26）利用ELISA检测ZNRD1对AGS、MKN28细胞培养上清和移植瘤匀浆中VEGF165含量的影响；27）利用脂质体转染法、MTT实验、肾包膜下移植法、流式细胞仪和DNA梯度试验检测新耐药相关分子DARPP-32对细胞（SGC7901、SGC7901/VCR、对阿霉素耐药的胃癌细胞SGC7901/ADR）多药耐药表型的影响；利用脂质体转染法和MTT实验检测下调ZNRD1对DARPP-32介导的胃癌多药耐药的调控作用。

The cDNA document of ACC oxidase gene of Citrus aurantium L. was cloned. By using the program of BLAST on NCBI GenBank database, the sequence presented a very high match with the ACC oxidase genes from other plants. The base sequence was analysed by using biology programe of DNAStar 5.0. 278 amino acids were coded by the base sequence and the base sequence had the same conserve region of ACC oxidase gene of many kinds of other plants. The base sequences comparability was more than 72% compared with those of many kinds of other plants. The amino acid sequences comparability was more than 70% compared with those of a lot of other plants.

克隆了酸橙1-氨基环丙烷－1-羟酸氧化酶基因cDNA片段，将片段序列在NCBI网站上进行同源性搜索，显示的皆为不同植物的ACC氧化酶基因，因而认为所克隆的片段就是酸橙ACC氧化酶基因；并运用DNAStar5.0软件进行序列分析，推导的氨基酸序列为278个残基；具有所有植物ACC氧化酶基因共有的保守区域；与多种植物的ACC氧化酶基因的核苷酸和氨基酸序列的同源性都在72%和70%以上。

Pneumoniae FH strain was cloned and the sequence was analysed by M13 DNA sequencing method. Comparing the PCR product sequcence with MP M-129 strain P1 gene, we found that there are 4 bases different. This may result from the different MP DNA templates. The maximum homology is 98.8%. The result confirmed the fidelity and specificity of the amplified target DNA segment by PCP, and suggested that two categories of MP P1 gene still exist a few differences even in the conservation region. The cloning MP DNA segment was labelled by random hexanucleotide priming, after hybridization, the probe detection was completed using an anti-digoxigenin antibody alkaline phosphatase conjugate, and the substrates 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium. This hybridization system is much superior to the radioactive probe hybridization, because it is safe, easy to handle and has no limitation of decay time. The time required for colormetic detection is also much less than the corresponding autoradiographic exposure time needed to achieve similar detection limits with 32P-labelled probes. The Dig-probes could be used repeatedly, and this made them not only much convenient to use, but also lower the cost, and worthwhile to be used popularly.

将PCR产物进行重组，并将阳性重组质粒，应用M13测序系统对产物进行DNA序列分析，并与MPM-129株P1基因核苷酸进行同源性比较，发现有4个位置的碱基发生了变化，其同源性为98.8％，证实了PCR所扩增DNA片段的准确性和特异性，同时也证实了不同MP组型的P1基因即使在保守区也存在着一定的差异，将克隆的目的DNA片段用异羟基洋地黄毒苷配基用随机引物法标记制备MP DNA探针，杂交后用碱性磷酸酶标记的抗Dig多克隆抗体与杂交体反应，再用BCIP和NBT呈色，制备MP DNA探针，鉴定所扩增片段的特异性，与同位素探针比较，Dig探针不受半衰期限制，可反复使用，而且价格低廉，值得推广使用。

More than fourty avirulence genes have been cloned from plant path...

目前，已经从植物病原物中克隆了40多个无毒基因，其中只有6个无毒基因是从植物病原真菌中克隆到的。

More than fourty avirulence genes have been cloned from plant pathogens.

目前，已经从植物病原物中克隆了40多个无毒基因，其中只有6个无毒基因是从植物病原真菌中克隆到的。

In order to clone AVR-Pia, the flanking sequence of MS220K4 was used as a probe to screening the TAC library and 17 positive clones were obtained. Further restriction enzyme mapping and Southern blot analysis on 10 of the 17 positives clones narrowed the avirulence gene to a region of 10 kb.

为克隆AVR-Pia基因，以MS220K4的侧端序列为探针筛选稻瘟菌基因组TAC文库，得到17个阳性克隆，通过对其中10个进行的限制性内切酶分析并结合Southern杂交确定了目的基因所在区域。

The composition diversity of microbial community shows that 13 genuses of microorganisms were identified by the technologies of clone labrary and isalation of pure strain and the dominant genuses were Hydrogenophaga,Pseudomonas,Bacteroides and Clostridium taking up 78% in one hundred of positive clones.

复合系组成多样性的研究表明，通过克隆文库构建和单菌株分离共确定出13个菌属的微生物，优势菌群有Hydrogenophaga、Pseudomonas、Bacteroides和Clostridium，占100个阳性克隆子的78%。

An infectious clone of the sat-RNA of BBSV-X was also constructed by placing the cDNA under the control of the 35S promoter. This plasmid could infect Chenopodium amaranticolor with the help of in vitro transcripts of BBSV-X.

构建了35S启动子控制下的BBSV卫星RNA全长侵染性克隆，侵染性检测表明，所获得的3个克隆均有较好的侵染性。

The impact of the FDA's decision will be felt beyond US borders. Chikara Kubota, an animal-cloning researcher at Kagoshima University in Japan, says he was relieved to hear of the FDA's decision and he expects the announcement to affect the status of moratoriums in other countries, including Japan.

FDA此项决定的影响范围将不仅仅限于美国本土，日本鹿儿岛大学研究动物克隆的Chikara Kubota研究员对此非常欣慰，他认为这项声明会影响包括日本在内的其它国家对克隆食品暂缓入市的态度。

It has been put forward that there exists single Ball point and double Ball points on the symmetrical connecting-rod curves of equilateral mechanisms.

从鲍尔点的形成原理出发，分析对称连杆曲线上鲍尔点的产生条件，提出等边机构的对称连杆曲线上有单鲍尔点和双鲍尔点。

The factory affiliated to the Group primarily manufactures multiple-purpose pincers, baking kits, knives, scissors, kitchenware, gardening tools and beauty care kits as well as other hardware tools, the annual production value of which reaches US$ 30 million dollars.

集团所属工厂主要生产多用钳、烤具、刀具、剪刀、厨具、花园工具、美容套等五金产品，年生产总值3000万美元，产品价廉物美、选料上乘、质量保证，深受国内外客户的青睐