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To study the applicable prospect of suicide gene on tumor therapy in clinic,we cloned the gene of D-amino acid oxidase and the gene of yeast cytosine deaminase.We hope to establish the transgenic mices of DAAO gene and YCD gene,and the two transgenic strain mices are valuable animal system for studying the biological characteristic of DAAO gene and YCD gene ,for studing the killing activity of DAAO/D-Ala and YCD/5-FC gene therapy systems on tumor.The D-amino acid oxidase gene derived from R.gracilis and it could oxidize D-amino acids. Hydrogen peroxide(H2O2) is a reactive oxygen species generated in the deamination of D-Ala catalyzed by DAAO.

为了探讨自杀基因在临床肿瘤治疗中的应用前景,建立自杀基因肿瘤治疗评价的动物模型,我们克隆了D-氨基酸氧化酶(D-amino acid oxidase,DAAO)基因和酵母菌胞嘧啶脱氨酶(yeast cytosine deaminase,YCD)基因,希望建立DAAO基因及YCD基因转基因小鼠,为研究DAAO及YCD基因的生物学特性、开发和评价DAAO/D-Ala及YCD/5-FC自杀基因系统进行肿瘤治疗建立良好的实验动物模型。

METHODS: Tum5 gene was amplified from plasmid pSPORT1Sfi by PCR technique and subcloned into the expression plasmid of lentiviral vector, pGCFU, to generate the lentiviral expression vector, pGCFUTum5. The correct Tum5 gene was confirmed by endoenzyme digestion and sequencing. Recombinant lentiviruses were produced by 293T cells following the cotransfection of pGCFU Tum5 and packaging plasmids-pHelper1.0and pHelper2.0. The resulting recombinant lentiviruses (GCFUTum5) which carried Tum5 and EGFPgene were then used to infect human umbilical vein endothelial cells.

采用PCR技术从含有tumstatin基因的质粒克隆模板 pSPORT1Sfi钓取Tum5基因,并将基因克隆到慢病毒载体表达质粒pGCFU中,构建慢病毒载体表达质粒pGCFUTum5,通过酶切、测序验证Tum5基因后,将pGCFUTum5质粒和包装质粒pHelper1.0,pHelper2.0共同转染人胚胎肾上皮细胞系293T细胞,获得携带Tum5基因和EGFP基因的重组慢病毒GCFUTum5,并转染靶细胞人脐静脉血管内皮细胞。

Results The expression levels of γ-IFN and IGTP in allograft were higher compared with that in isograft, and expression of IGTP was even more significant than γ-IFN in allgraft. In islgraft the expression levels of the both were very low or beyond the capacity to be detected.

结果γ-干扰素和γ-干扰素诱导基因在异基因移植物中显著表达,且γ-干扰素诱导基因的表达水平高于γ-干扰素,在同基因移植物中2个基因表达水平很低,甚至难以检测出。

PCR-RFLP in FSHR gene 5'–flanking region with exon 1 digested by restriction endonuclease TaqⅠdemonstrated that the frequencies of allele A (0.5500) and genotype AA (0.5000) in twinning cows were higher than that of allele A (0.3500) and genotype AA (0.3000) in monovular cows for Qinchuan cattle, while The frequencies of allele B (0.5937) and genotype BB(0.6875) in twinning cows were higher than that of allele B (0.5416) and genotype BB (0.4167) in monovular cows for Holstein cattle. Key words: Bovine; Twinning; FSHR gene; PCR-RFLP

FSHR基因5'端(包括侧翼序列和第一外显子)的限制性内切酶TaqⅠ的PCR-RFLP标记研究发现,在秦川牛中双胎母牛的A基因频率(0.5500)和AA基因型频率(0.5000)分别高于单胎母牛的A基因频率(0.3500)和AA基因型频率(0.3000);在黑白花奶牛中双胎母牛的B基因频率(0.5937)和BB基因型频率(0.6875)均高于单胎母牛的B基因频率(0.5416)和BB基因型频率(0.4167),也就是FSHR基因的5'端的PCR-RFLP标记在一个品种内单胎母牛和双胎母牛之间有一定趋势,但两个品种的趋势不同。

The interaction fashions between avirulence genes and resistant genes were pilot studied.

本文通过无毒基因的克隆和分析,对基因-基因学说中病原菌无毒基因和抗病基因互作方式进行了初步探讨。

In our study, 3 genotypies have been respectively demonstrated in UL139 and UL149 for the first time, and the corelations between certain genotypical structure and certain diseases were also proposed; The existence of the 3 genotypes of UL144 was foremost verified in isolates from congenital infants; Furthermore, the UL140, UL141 and UL145 genes were observed to be greatly discrepant to those had been described previously: comparing with Toledo, 231 nt are inserted in UL140, 2 more ORFs are obtained in UL141, and the UL145 ORF moves upstream by 90 nt. Except UL144, the sequences of 18 else genes in clinical isolates were submitted for the first time, and 479 sequences were assigned by GeneBank in total. Relevantly, 9 papers were published.

我们在学术界首次证实了HCMV UL139、UL149两个基因在临床分离株中分别存在三种基因型,发现了其中某个基因型的基因结构与特定来源的分离株存在一定的对应关系;首次在先天感染分离株中验证了UL144三种基因型的存在;首次证实了UL140、UL141和UL145等基因与原先认识的基因结构不同,其中UL140基因较Toledo株增加了231个碱基、UL141产生2个新的基因编码区、UL145 ORF较Toledo株前移90个碱基;首次或最先提交了除UL144基因外其余18个基因的临床分离株序列,本项目组共有479个HCMV相关基因序列被GenBank收录;发表研究论文9篇。

A trivalent plant expression vector containing Bt cry1Ah gene, cry1Ie gene and glyphosate-tolerant 2mG2-epsps gene was constructed, glyphosate isopropylamine salt as a screening agent, 2mG2-epsps gene as a selectable maker gene. The vector was transfer into maize immature embryonic calli by microprojectile bombardment, and 69 T0 generation plants were obtained. PCR analysis showed that 17 plants had the integration of insect-resistant cry1Ah, cry1Ie gene and glyphosate-tolerance 2mG2-epsps gene.

同时构建了含有Bt cry1Ah和cry1Ie基因和耐草甘膦2mG2-epsps基因的三价植物表达载体,通过基因枪轰击法转化玉米愈伤组织,以2mG2-epsps为筛选标记基因,以草甘膦异丙胺盐作为筛选剂,获得T0代转化植株69株,PCR检测结果表明:有17株已完整的整合有抗虫基因cry1Ah、cry1Ie和耐草甘膦基因2mG2-epsps的3个目的基因,RT-PCR分析外源基因可以正确转录,已获得结实转基因植株。

We initiated a genetic screen for Drosophila olfactory genes by means of SG18.1-Gal4/UAS system: The result validates our screen as an rapid, effective approach for recovering genes controlling glomerular map patterning; From a misexpression screen of 1,515 P{GS} lines, we identified 23 genes that, when forcibly expressed in the olfactory receptor neurons, disrupted the stereotyped anatomy of the Drosophila antennal lobes; These genes, which have not been shown previously to control olfactory map development, encode novel proteins as well as proteins with known roles in axonal outgrowth and cytoskeletal remodeling.

本文首次利用SG18.1-Gal4/UAS基因筛选系统对果蝇嗅觉基因进行了筛选和鉴定:结果表明,所采用的方法可以快速、有效地分离到果蝇嗅觉相关基因;从1515株果蝇P{GS}品系分离到86株突变体,其中40株有明显表型,从中鉴定了23个基因,这些基因在ORN强迫表达时可以损坏果蝇嗅叶的固有结构;在筛选中发现的一些调节嗅觉图谱发育的基因可以编码新蛋白或编码参与轴突生长和细胞骨架重塑的蛋白;为了便于研究,根据基因的功能和所编码蛋白的特点,我们对分离到的基因进行了分类,即参与轴突引导和突触产生、调节转录、调节细胞骨架和功能未确定或未知的基因;并通过对嗅觉感觉器的检测发现,除了调节转录的基因外,其他基因对触觉器的数量和分布影响不明显。

The association of microsatellites in rice orphan and non-orphan genes was computed and censused. The result showed: rice has 28532 orphan genes, which accounts for 50.9%, and 27524 non-orphan genes, which accounts for 49. 1%; the microsatellite content in orphan genes is significantly higher than in non-orphan genes; in constitution, tri-nucleotide microsatellite content in ether orphan genes or non-orphan genes exceeds 50%, while the total tri-nucleotide microsatellite content in orphan genes accounts for 68% and that in non-orphan genes accounts for 58%, of total microsatellites.

对微卫星在水稻孤儿基因与非孤儿基因之间的关系进行了计算和统计分析,结果表明:水稻孤儿基因总数为28532条序列,占50.9%,而非孤儿基因为27524条,占49.1%;孤儿基因中微卫星含量明显高于非孤儿基因;在组成上,不论在孤儿基因还是在非孤儿基因中,三核苷酸微卫星的含量都超过了50%,孤儿基因中的含量为68%,明显高于非孤儿基因的58%。

An expression construct specified in mammary gland with double cistrons of human G-CSF gene and IRES-EGFP gene under control of ovine beta-lactoglobulin gene flanking sequence,has been constructed in two parts (named fragment I and Fragemnt II)that share an overlapping region of 2.2 kb sequence.Two sites of loxp and lox2272 for homologous recombination were inserted into both flanking regions of G-CSF.The lengths of fragment I and Fragment II are 5.9kb and 5.6 kb,respectively.The whole length of the expression vector (β-LG-hG-CSF-IRES-EGFP) is 9.3 kb.

以绵羊β乳球蛋白基因(β-Lactoglobulin,β-LG)为转基因表达框架,将人G-CSF(Human Granulocyte Colony Stimulating Factor, hG-CSF)基因与报告基因-增强型绿色荧光蛋白(Enhanced Green Fluorescenct Protein, EGFP)基因作为双表达单元拼接到β-LG基因的第一外显子处,并在G-CSF基因两侧引入同源重组位点loxP、lox2272,将打靶基因表达构件β-LG-hG-CSF-IRES-EGFP(总长9.3 kb)分为两段进行构建,片段Ⅰ(长:5.9kb)与片段Ⅱ(长5.6 kb)两段重叠部分为2.2 kb。

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