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Results as bellows: AtSIRT1 was located in Mitochondrial as hSIRT4 of human, and maybe take part in respiration and electron transformation chain, AtSIRT2 was located in nucleolus as hSIRT6 of human, maybe play important role in extend lifespan;mutation in AtSIRT1 leaded to cotyledon of plant turn to yellow and caused short life span. Mutation in AtSIRT2 could make the color of leaf turn to purple and accumulate a lot of anthocyanin;Sirtinol, a inhibitor of SIRT which did not cause the same model of the mutation of AtSIRT1 and AtSIRT2 indicated that the mechanism of Sirtinol was different from other organism;the structure of AtSIRT1 and AtSIRT2 were similar to other known Sir2, which indicated that they maybe have the same function;AtSIRT2 was overexpressed and its activity was detected.

结果表明,1,拟南芥AtSIRT1与人的同源蛋白hSIRT4相同,定位于线粒体,可能参与呼吸作用和电子传递,SIRT2与人的同源蛋白hSIRT6相同,定位于细胞核,可能同它的功能类似,在延缓衰老及调节细胞寿命方面起作用。2,AtSIRT1突变,可引起幼苗和植株的子叶变黄和早衰;AtSIRT2突变,可引起叶片发紫,沉积大量花青素。3,SIRT蛋白的抑制剂Sirtinol不能表型模写AtSIRT1和AtSIRT2突变体,说明Sirtinol在拟南芥中的作用机制不同于其他生物。4,AtSIRT1和AtSIRT2蛋白质结构预测表明与已知的Sir2蛋白相似,揭示其功能的相似性。5,在大肠杆菌中过量表达了其中一个基因(AtSIRT2),可体外检测其酶学活性,进一步证明其功能。

A clone from each genotype was randomly selected as representative for sequencing. The obtained 16S rDNA gene sequences had a similarity of 87%-100% with those in the GenBank (www. ncbi. nlm. nih. gov), and more than half of them had a similarity lower than 97%, being of new species. Based on phylogenetic analysis, the bacteria in the two soils were classified into 10 groups, with 5 groups in common. The dominant bacterial groups in the two soils differed obviously. In primeval forest soil, the dominant group was Proteobacteria, which had 39 genotypes, occupying 58.0% of all the clones; while in the soil of degraded ecosystem the dominant groups were Acidobacteria and Proteobacteria, which had 19 and 15 genotypes occupying 32.5% and 30.5% of all the clones, respectively. In the soil of degraded ecosystem, Proteobacteria group decreased while Acidobacteria group increased markedly, compared with those in primeval forest soil.

从每种基因型中随机选择一个克隆子作为代表进行测序分析,所有序列与GenBank数据库中序列的同源性为87%~100%,且两样地中均有超过一半的基因型序列与数据库中已知序列同源性低于97%,属于分类在"种"地位上的新发现细菌;通过系统发育研究将两样地的细菌分为10大类群,两样地共同拥有5大类群,但两样地的细菌优势类群明显不同,原生土壤为Proteobacteria,含39种基因型,占总克隆子数的58.0%,退化生态系统土壤为Acidobacteria和Proteobacteria,分别含19种和15种基因型,占总克隆子数的32.5%和30.5%;与原生土壤细菌类群相比,退化生态系统土壤Proteobacteria类群明显减少,Acidobacteria类群明显增加。

In this experiment, we screen the major protective antigen gene-SOD gene of M. paratuberculosisin order to study the sensitive, specific diagnostic reagent and prophylaxis preparation, especially theDNA vaccine. The SOD gene was amplified from Mycobacterium paratuberculosis C-2 chromosomalDNA by using the PCR technique and cloned into pMD18-T Vector System. We gained a SOD gene of624bp.The recombinant clone was identified byα-complementarity, enzyme digestion and PCRidentification. The result indicated that the recombinant plasmid pMD18-T-SOD was successfullyconstructed. Moreover, through sequential determination and DNASTAR analysis between the clonedSOD gene of M. paratuberculosis C-2 and that of the M.paratuberculosis K-10 strain, the sequentialhomogeneity reached 99%, and the amino acid homogeneity reached 99.5%. The preceding analysisindicated that the SOD gene was very conservative in M. paratuberculosis.

为了研制副结核病敏感、特异的诊断试剂和新型、高效的预防制剂,尤其是DNA疫苗,本研究筛选了M.paratuberculosis主要保护性抗原SOD基因,以M.paratuberculosis C-2染色体DNA为模板,以SOD基因的特异性引物进行PCR扩增,获得了624bp的SOD基因,通过T-A克隆技术,将PCR产物克隆至pMD18-T Vector中,以质粒大小、酶切分析、PCR扩增及序列分析鉴定重组克隆,成功地构建出克隆质粒pMD18-T-SOD,序列测定及DNASTAR分析表明,所获得的M.paratuberculosis C-2 SOD基因与Gen Bank中M.paratuberculosis K-10 SOD基因的大小完全一致,两者核苷酸序列的同源性为99%,氨基酸序列的同源性为99.5%,表明该基因在副结核分枝杆菌中是高度保守的。

It also can be used as alternative antigen of newgeneration vaccine.In this experiment,we screen the major protective antigen hsp65 gene of MAP in order to developnew vaccine especially the DNA vaccine for the prevention of paratuberculosis disease.The hsp65 genewas amplified from MAP C-2 chromosomal DNA by using the PCR technique.We gained a hsp65 gene of 1 626bp.Then PCR product was cloned into pGEM-T vector by T-A clone technique and therecombinant clone was identified by plasmid size,enzyme digestion and PCR identification.The cloneplasmid of pGEM-T- hsp65 was successfully constructed.The nucleotide sequence and deduced aminoacid sequence ofclone gene was analyzed by DNASTAR software.The result indicated that the size ofhsp65 gene consist with M.paratuberculosis K-10 strain in GenBank and the sequential homogeneityreached 99.1%,the amino acid homogeneity reached 99.3%.The preceding analysis indicated that thehsp65 gene was very conservative in M.paratuberculosis.

为了研发预防副结核病的新型疫苗尤其是DNA疫苗及相关蛋白功能,本研究选择了MAP的主要保护性抗原Hsp65蛋白,以副结核分枝杆菌C-2株染色体DNA为模板,以hsp65基因的特异性引物进行PCR扩增,获得了1 626bp的hsp65基因,通过T-A克隆技术,将PCR产物克隆至pGEM-T Vector中,以质粒大小、酶切分析、PCR扩增及序列分析鉴定重组克隆,成功地构建出克隆质粒pGEM-T-hsp65,以DNASTAR软件分析了所克隆基因的核苷酸序列和推导的氨基酸序列,结果表明,所获得的hsp65基因与GenBank中MAPK-10株该基因核苷酸大小完全一致,两者核苷酸序列的同源性为99.1%,氨基酸序列的同源性为99.3%,表明该基因在副结核分枝杆菌中高度保守。

In this study, the cDNA fragments of G-L intergenic region and polymerase activity module in L gene of two street rabies virus were cloned and sequenced. The nucleotides sequence of these cDNA fragments mentioned above and their deduced amino acid sequence were compared with that of rabies virus strains, rabies-related virus strains and other Rliabdoviridae published previously. The result show that the homology of nucleotide sequence of G-L IGR among MRV, DRV is 83.3%, compared with other rabies-related virus strains, the homology of G-L IGR is between 58.3%-91.7%. the length of G-L IGR of rabies virus is different in Rhabdoviridae.

本实验对狂犬病病毒野毒株MRV、DRV的G-L间隔区以及L基因聚合酶活性部位进行了克隆和测序,将所测定的结果和推定的氨基酸序列与国内外公开发表的狂犬病病毒其它固定毒株、野毒株和同科不同属的病毒的相应部分进行了分析比较,结果表明MRV、DRV G-L IGR核苷酸序列的同源性为83.3%,与其它毒株的同源性在58.3%~91.7%之间;狂犬病毒G-L间隔区序列的长度在弹状病毒科不同病毒属之间有所不同。

The cloned BBE gene sequences were 94.84% identified with the reported BBE genes in GenBank previously. Based on the cDNA sequences of COR and BBE ,two fragments about 400~500 bp from each gene with lower identity among them were cloned. The fusion gene BC (744 bp) is fused by the PCR technique. Then the promoter CaMV 35S driven, containing'forward BC fusion fragments-reverse pdk intron-reverse BC fusion fragments', plant siRNA expression vector were constructed based on the vectors pHANNIBAL and pART27.The work will lay the foundation for breeding a low morphine and high thebaine poppy.

利用blast及分子生物学软件DNAStar对COR和BBE基因的cDNA序列同源性进行分析比较,分别从各基因中筛选和克隆了一段同源性极低、约400~500 bp的片段;并应用重叠PCR法将其拼接成744 bp的融合基因BC,以中间载体pHANNIBAL和植物表达载体pART27为基础,构建了以CaMV 35S启动子驱动的含有"正向BC融合片段- pdk内含子-反向BC融合片段"的ihRNAi植物表达载体,通过转化野生罂粟,初步研究了以COR和BBE基因为靶标的RNAi对内源吗啡合成的抑制效果,为进一步培育低吗啡高蒂巴因的罂粟种质提供了依据。

Molecular homology trees were constructed based on the amino acid sequences of the RdRp,CP and MP,and the results showed that TVDV shares much more homologous with the specieses of the genus Polerovirus than other genuses of family Luteoviridae,which indicated that TVDV should be a definitive member of the genus Polerovirus in the family Luteoviridae.

根据三个ORF编码的氨基酸序列构建的分子同源树的分析,也都显示了TVDV与黄症病毒科马铃薯卷叶病毒属病毒的同源性最高,进一步证实了TVDV应该是黄症病毒科马铃薯卷叶病毒属的确定成员。

Researches on proteid reveal that VSV N gene encoding nuclear protein is a polypeptide which is made up of 422 amino acid and has a strong hydrophility. Comparing its homogeneity with that of VSV wild strains, it shows that the nuclear protein is highly conservative due to the high homogeneity of sequences which is above 98. 8%. It provides a basis for establishing clinical VSV diagnostic method.

蛋白质分析发现,VSV N基因编码的核蛋白是由422个氨基酸组成的多肽,具有较强的亲水性,同VSV野生型病毒株的核蛋白同源性比较,其氨基酸序列的同源性高达98.8%以上,证明VSV核蛋白具有很好的保守性,为临床上建立VSV诊断方法奠定了基础。

In this study, to define the functional roles and mode of the upstream regulatory elements ofα-globin gene cluster,αMⅡwas obtained, in which the 31.7kb sequence including HS-33, HS-10、HS-8 and HS-4 was deleted by defective prophage mediated by the homologous recombination;αMⅢwas also constructed, in which the 35.4kb sequence including HS-40、HS-33、HS-10、HS-8 and HS-4 was deleted by the method of temperature sensitive shuttle vector mediated homologous recombination.

在此基础上,为了定义α-珠蛋白基因簇上游各调控元件的调控功能,并探讨其作用机制,本研究利用缺陷型原噬菌体介导的同源重组法修饰α-BAC,删除包括HS-33、HS-10、HS-8和HS-4在内长达31.7kb的序列得到BAC突变体αMⅡ;又利用温度敏感型的穿梭载体介导的同源重组法删除包括HS-40、HS-33、HS-10、HS-8和HS-4在内达35.4kb的序列,得到BAC突变体αMⅢ。

The phylogenetic tree was constructed using maximum evolution approach in Mega. The negative selection sites were analyzed by using Datamonkey. There is an apparent 30 bp nucleotides deletion mutation in homonids FKN comparing to that of Old World Monkeys besides other point mutations. Homology of nucleotide sequence between human and chimpanzee, gorilla, orangutan, gibbon, macaque and leaf monkey is 99.2%、98.4%、98.1%、96.5%、95.9% and 93.8% respectively. Homology of amino acid sequence of them is 98.5%、98.0%、97.7%、94.7%、93.7% and 90.5% respectively.

序列分析发现人猿超科较旧大陆猴FKN基因除了有散在的点突变外,还有一明显的30bp的核苷酸缺失突变;人FKN基因序列与黑猩猩、大猩猩、红毛猩猩、长臂猿、猕猴和叶猴的同源性分别是99.2%、98.4%、98.1%、96.5%、95.9%和93.8%,由此推导的氨基酸序列同源性分别是98.5%、98.0%、97.7%、94.7%、93.7%和90.5%;FKN基因进化树表明人与黑猩猩关系更近,FKN基因进化和通常认为的物种进化一致;Datamonkey分析结果显示FKN存在3个负选择位点53Q、84D、239N。

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