基因型

To better understand the roles of LsrA protein in nodulation, a series of analyses of nodules using scanning electron microscopy, genetic and biochemical approaches have been carried out. Our analyses suggest that the lsrA1 nodules contain bacteroids in the invasion and establishing zone only. The lsrA gene expression is active early in the invasion zone and activated later than bacA genes. The lost of LsrA protein dramatically reduced the expression of nifA and fixK, and completely blocked the expression of the nifH gene for nitrogenase. LsrA protein functions early in the bacteroid development and it is essential for the development nitrogen fixing bacteroids.

前期的工作中，苜蓿中华根瘤菌Rm1021中90个候选LysR基因已经被定向插入突变，并筛选在自生生长时期、共生生长时期的表型，以期寻找更多在自生状态或共生固氮中有功能的LysR转录因子。1 针对前期鉴定出的共生固氮必需的lsrA基因，我们应用了一系列扫描电子显微镜技术、生物化学、分子遗传学等方法，发现lsrA基因主要在根瘤侵染区开始表达，表达时序也在侵染阶段左右，但晚于bacA基因表达；LsrA蛋白缺失后根瘤固氮区中缺乏具有固氮能力的类菌体，nifA和fixK基因的转录水平降低，nifH基因的转录被完全阻断，因此LsrA蛋白为根瘤发育所必需，是新的根瘤发育信号传导途径成员。2 通过表型筛选我们鉴定了苜蓿中华根瘤菌的oxyR基因，并研究了它的调节特性。oxyR突变后，苜蓿中华根瘤菌对过氧化氢敏感性提高，适应性降低。

In 42 ACME-arcA-positive isolates, 8 isolates harbored SCCmec V, 8 isolates harbored class C1 mec complex and ccrAB_, 22 isolates harboring class C1 mec complex and 4 isolates harboring class C2 mec complex were negative for all known ccr allotypes.

本研究在ACME-arcA基因阳性菌株中发现整合酶基因ccr的一种新亚型ccrAB_(SHP.42株ACME-arcA基因阳性菌株，8株携带V型SCCmec，8株携带C1型mec基因复合体和ccrAB_，剩余26株携带C型mec基因复合体和未分型ccr。

After thecomplete genome extraction of the strain was performed, the genomic DNA was partiallydigested by restriction enzyme Sau3AⅠ, the DNA fragments from 1 to 5Kb was clonedinto prokaryote expression vector pET-28a-c, and transformed host bacteria. The resultsshowed that we succeeded in constructing the gene expression library of haemophilusparasuis serovar 5, which is fundamental for the study of advanced gene screening. Inaddition, primer design was performed based on haemophilus influenzae in this study. In addition, PCR was performed by using genomic DNA of haemophilus parasuisserovar 5 as the template. The results demonstrated that we obtained two neo-gene:23SrRNA gene(conserved gene belonging to the large-subunit of ribosome) and adenylatecyclase gene(encodes adenylate cyclase and participates in converting adenyl nucleosidetriphosphate to cyclic adenosine3",5"-monophosphate). Furthermore, the phylogeneticanalyses between the species was performed, and neighbor-joining tree was constructedbased on comparison of 23S rRNA gene sequences, so it was illuminated betweenHaemophilus parasuis and other species in molecular evolution relationship.

选择我国流行优势菌株副猪嗜血杆菌血清5型地方株为研究对象，提取细菌基因组DNA，用限制性内切酶Sau3AⅠ对基因组DNA进行部分酶切，回收大小为1～5Kb的DNA片段，将其连接入原核表达载体pET-28a-c，最后转化宿主菌，结果成功地构建了基因表达文库，为后续的基因筛选工作奠定基础；另外，本研究选择嗜血杆菌属的流感嗜血杆菌为参考对象进行引物的设计，以副猪嗜血杆菌血清5型地方菌株的基因组DNA为模板，进行PCR扩增反应，结果表明成功地获得两个新基因：23S rRNA基因(存在于核糖体大亚基中的保守性基因)和腺苷酸环化酶基因(负责将腺嘌呤核苷三磷酸转变为环腺苷酸)，并进一步做了不同物种之间的分子系统发育分析，构建了基于23S rRNA基因的邻接法系统发育树，阐明了副猪嗜血杆菌与其它菌种的分子进化关系。

Methods The 26th exon, the 30th exon and the 21st intron of gene GP Ⅱb in 110 individuals were amplified by polymorphism and sequenced to investigate whether there was linkage among the polymorphisms of the gene. Human platelet antigen-3 (HPA-3) gene frequency was detected by Fok 1 enzyme in 147 patients with hematologic diseases, and was compared with that in 110 normal individuals. Forty-four patients who received apheresis platelet transfusion repeatedly were randomly divided into the HPA-3 homotype group and the control group. The antibodies of the platelet were detected after 3 times of platelet transfusion.

聚合酶链反应－单链构型多态性分析检测110例正常人GP Ⅱb基因第26和30外显子(Exon 26， Exon 30)及21内含子(Intron21)基因多态性，进行基因序分析，研究这些基因多态性是否存在连锁关系；应用Fok1酶切法对147例血液病人人类血小板抗原－3(human platelet antigen-3， HPA-3)基因进行分型，并与110例正常人进行比较；将接受单采血小板输注的44例血液病人随机分为HPA-3同型输注组和对照组，血小板输注3次以后检则血小板抗体。

Five floral organ identity genes of Mussaenda pubescens were cloned and their expression patterns among different organs were detected by RT-PCR. The AP1 homologue, MupAP1, is expressed in enlarged and small sepals, petals and stamens, carpels and fruits; the SEP3 homologue, MupSEP is strongly expressed in all floral organs and weak in leaves as well. Two of the B class gene, AP3 homologue MupDEF and TM6 homologue MupTM6, were detected expressed in all floral organs, however, the other B class gene, MupGLO, homologous to PI, were detected expressed in small sepals, petals and stamens, carpels and fruits, but not in enlarged sepals.

本论文选殖出五个毛玉叶金花花器同源基因，其中A群基因AP1的同源基因MupAP1在两型花萼、花瓣及雄蕊、心皮、果实中有表现，E群基因SEP3的同源基因MupSEP则在所测部位均有表现，B群基因中，AP3的同源基因MupDEF及TM6的同源基因MupTM6在两型花萼、花瓣及雄蕊、心皮及果实中均有表现，另一个B群基因，PI的同源基因MupGLO则在花瓣及雄蕊、心皮、果实及小花萼中有表现，但在膨大花萼中并没有侦测到表现。

The results showed that in evaluation of the method by detecting 50 RHD 1227A positive and 50 RHD 1227A negative individuals, the genotyping method displayed a sensitivity of 100% and a specificity of 100%; in evaluation of the method by detecting 33 DEL positive and 89 DEL negative individuals, the sensitivity was 100%, however, there were two serologically negative samples which were confirmed as positive using genotyping method.

结果表明：在50例RHD 1227A阳性和50例RHD 1227A阴性的Rh阴性样本中基因分型方法的灵敏度和特异性都是100％；在33例DEL阳性样本和89例DEL阴性的样本中，基因分型方法的灵敏度为100％，有2例样本血清学结果为阴性而基因分型结果为阳性，重新用血清学方法和序列分析方法复核这2例样本，发现2例都是血清学漏检，因而基因分型方法的特异性是100％。

The results showed that in evaluation of the method by detecting 50 RHD 1227A positive and 50 RHD 1227A negative individuals, the genotyping method displayed a sensitivity of 100% and a specificity of 100%; in evaluation of the method by detecting 33 DEL positive and 89 DEL negative individuals, the sensitivity was 100%, however, there were two serologically negative samples which were confirmed as positive using genotyping method. After re-testing these two samples with serological method and sequence analysis, it was found that original serological method gave false negative results and genotyping method still showed 100% specificity. The minimal target DNA concentration of this genotyping method is 8.13 ng/μl.

结果表明：在50例RHD 1227A阳性和50例RHD 1227A阴性的Rh阴性样本中基因分型方法的灵敏度和特异性都是100％；在33例DEL阳性样本和89例DEL阴性的样本中，基因分型方法的灵敏度为100％，有2例样本血清学结果为阴性而基因分型结果为阳性，重新用血清学方法和序列分析方法复核这2例样本，发现2例都是血清学漏检，因而基因分型方法的特异性是100％。

By use of site mutation strategy and PCR technology, we obtained the gene P12X3C that includes full length P1, 2A, 3C and a part of 2B and 3B and the gene P12X3C3D that includes full length P1, 2A, 3C, 3D and a part of 2B and 3B. After being digested by restriction enzyme respectively, the gene P12X3C and the gene P12X3C3D were cloned into the pcDNA3. 1 and pTARGET expression vector that were digested by the same enzyme. Recombinant plasmids were checked by restriction enzyme analysis and nucleic acid sequencing. Further more, recombinant plasmids were transfected into BHK-21 cells by using lipoid. The proteins of foot-and-mouth disease virus , which were expressed in BHK-21 cells, were confirmed by sandwich-ELISA and fluoroscopy, and the capsid of FMDV was tested by electron microscope. In order to evaluate enhanced immune response of guinea pigs against FMDV, DNA vaccines which were designed to produce viral capsids lacking infectious viral nucleic acid and contained the gene P12X3C and the gene P12X3C3D were injected respectively with FMDV 3D protein which was expressed in Pichia Pastoris Secreted expression System and purified or with pcDNA3. 1/IFN which includes the gene IFN-α of cattle. Subsequently, Recombinant plasmids were injected to cattles with or without pcDNA3. 1/IFN. Anti-FMDV antibodies were detected by ELISA, and the T lymphocyte proliferation response was tested by MTT assay, neutralization antibodies titers were analyzed by micro-neutralization assay.

为研制带有O型口蹄疫病毒(Foot-and-Mouth Disease Virus，FMDV)China99株结构蛋白基因及多个非结构蛋白基因的DNA疫苗，本研究通过定点突变方法和PCR扩增方法，获得包含有FMDV China99株结构蛋白P1、非结构蛋白2A、3C以及部分2B、3B编码基因的片段P12X3C和包含有FMDV China99株结构蛋白P1、非结构蛋白2A、3C、3D以及部分2B、3B编码基因的片段P12X3C3D，将获得的基因片段直接/酶切后与同样处理的真核表达质粒连接，分别得到重组质粒pcDNA3.1/P12X3C和pcDNA3.1/P12X3C3D、pTARGET/P12X3C3D；对重组质粒进行序列测定、分析，并将重组质粒分别转染BHK-21细胞，通过双抗体夹心ELISA方法和间接免疫荧光标记方法检测细胞中FMDV抗原的表达，用电子显微镜观察病毒空衣壳的组装；为评价重组质粒作为DNA疫苗对实验动物及本动物的免疫效果，将重组质粒经肌肉注射方法接种豚鼠，并与酵母表达的纯化FMDV China99株3D蛋白及带有牛α干扰素的真核表达质粒pcDNA3.1/IFN分别/同时免疫，第二次免疫后第三周豚鼠攻以1OOID〓或1000ID〓的O型FMDV China99株；随后将质粒pcDNA3.1/P12X3C、pcDNA3.1/P12X3C3D与带有牛α干扰素的真核表达质粒pcDNA3.1/IFN同时免疫牛，三周后经牛舌皮攻以10〓ID〓的O型FMDV China99株。

By use of site mutation strategy and PCR technology, we obtained the gene P12X3C that includes full length PI, 2A, 3C and a part of 2B and 3B and the gene P12X3C3D that includes full length PI, 2A, 3C, 3D and a part of 2B and 3B. After being digested by restriction enzyme respectively, the gene P12X3C and the gene P12X3C3D were cloned into the pcDNA3.1 and pTARGET expression vector that were digested by the same enzyme. Recombinant plasmids were checked by restriction enzyme analysis and nucleic acid sequencing. Further more, recombinant plasmids were transfected into BHK-21 cells by using lipoid. The proteins of foot-and-mouth disease virus, which were expressed in BHK-21 cells, were confirmed by sandwich-ELlSA and fluoroscopy, and the capsid of FMDV was tested by electron microscope. In order to evaluate enhanced immune response of guinea pigs against FMDV, DNA vaccines which were designed to produce viral capsids lacking infectious viral nucleic acid and contained the gene P12X3C and the gene P 12X3C3D were injected respectively with FMDV 3D protein which was expressed in Pichia Pastoris Secreted expression System and purified or with pcDNA3.1/lFN which includes the gene IFN-a of cattle. Subsequently, Recombinant plasmids were injected to catties with or without pcDNA3.1/IFN. Anti-FMDV antibodies were detected by ELISA, and the T lymphocyte proliferation response was tested by MTT assay, neutralization antibodies liters were analyzed by micro-neutralization assay.

为研制带有O型口蹄疫病毒(Foot-and-Mouth Disease Virus，FMDV)China99株结构蛋白基因及多个非结构蛋白基因的DNA疫曲，本研究通过定点突变方法和PCR扩增方法，获得包含有FMDV China99株结构蛋白P1、非结构蛋白2A、3C以及部分2B、3B编码基因的片段P12X3C和包含有FMDV China99株结构蛋白P1、非结构蛋白2A、3C、3D以及部分2B、3B编码基因的片段P12X3C3D，将获得的基因片段直接／酶切后与同样处理的真核表达质粒连接，分别得到重组质粒pcDNA3.1／P12X3C和pcDNA3.1／P12X3C3D、pTARGET／P12X3C3D；对重组质粒进行序列测定、分析，并将重组质粒分别转染BHK-21细胞，通过双抗体夹心ELISA方法和间接免疫荧光标记方法检测细胞中FMDV抗原的表达，用电子显微镜观察病毒空衣壳的组装；为评价重组质粒作为DNA疫苗对实验动物及本动物的免疫效果，将重组质粒经肌肉注射方法接种豚鼠，并与酵母表达的纯化FMDV China99株3D蛋白及带有牛α干扰素的真核表达质粒pcDNA3.1／IFN分别／同时免疫，第二次免疫后第三周豚鼠攻以100ID_(50)或1000ID_(50)的O型FMDV China99株：随后将质粒pcDNA3.1／P12X3C、pcDNA3.1／P12X3C3D与带有牛α干扰素的真核表达质粒pcDNA3.1／IFN同时免疫牛，三周后经牛舌皮攻以10~4ID_(50)的O型FMDV China99株。

Total RNAs from KAx-3 cells and AK127 cells(developed for 14h) were isolated. After the reverse transcription and PCR reaction, two distinct differential fragments were acquired., fragment A was from KAx-3 cells and fragment C was from AK127 cells. After retriving and reamplifying the differentially expressed fragments, white-blue plaqueselection, the fragments were purified. Northern blot proved that fragment A was from KAx-3 cells and fragment C was from AK127 cells. The results of sequencing and researching for NCBI database have been showed: part sequence of fragment A shows 91% similarity to the gene encoding DhkA, 92% similarity to the gene encoding DhkF, 91% similarity to the gene encoding STATc, 97% similarity to the homoeobox gene encoding protein. These genes play important part in controlling cell differentiation and cell proportion in Dictyostelium discoideum.

本研究通过提取盘基网柄菌发育14小时的野生型KAx-3细胞和突变型AK127细胞的总RNA，运用mRNA差异显示法分离出了两条明显的差异表达片段，其中片段A来自野生型KAX-3细胞，片段C来自突变型Ak127细胞；并通过凝胶回收差异片段、对差异片段进行再次PCR、蓝白斑筛选克隆、提取质粒、酶切电泳纯化差异片段；接着进行Northern杂交的结果表明，片段A只与野生型KAx-3细胞的总RNA有杂交信号，片段C只与突变型AK127细胞的总RNA有杂交信号，这就排除了差异片段假阳性的可能；最后通过测序，搜索NCBI BLAST数据库发现：片段A的小部分序列与编码组氨酸激酶DhkA基因中一段序列的相似性高达91%，与编码组氨酸激酶DhkF基因中的一段序列相似性高达92%，与编码STATc蛋白基因的一段序列相似性达91%，以及与编码同源框蛋白的基因中的一段序列相似性达97%，这些基因在盘基网柄菌细胞分化和细胞比例调控过程中起着相当重要的作用，这些数据进一步说明了突变细胞不能完成发育的原因。

This one mode pays close attention to network credence foundation of the businessman very much.

这一模式非常关注商人的网络信用基础。

Cell morphology of bacterial ghost of Pasteurella multocida was observed by scanning electron microscopy and inactivation ratio was estimated by CFU analysi.

扫描电镜观察多杀性巴氏杆菌细菌幽灵和菌落形成单位评价遗传灭活率。