黄山梅的组织培养和快速繁殖

1植物名称黄山梅(Kirengeshoma palmata Yatabe.). 2材料类别成熟种子. 3培养条件(1)种子萌发培养基:MS 6-BA 1.0mg·L-1(单位下同) IAA 0.5 活性炭(AC)0.1%;(2)增殖培养基:MS 6-BA 2.0 IAA 0.2;(3)壮苗培养基:MS 6-BA 0.5 IAA 0.5;(4)生根培养基:1/2MS 6-BA 0.1 NAA 1.0.各种培养基均附加3%蔗糖和0.6%琼脂,pH 6.0.培养温度为(25±2)℃,光强为30 μmol·m-2·s-1,光照时间12 h·d-1.     

胡萝卜与川西獐牙菜不对称体细胞杂种的核/质基因组特征

胡萝卜(Daucus carota var.sativa)原生质体与经260μW/cm2紫外线照射的川西獐牙菜(Swertia mussotii)原生质体用PEG法诱导融合.对融合再生的克隆的5SrDNA间隔序列和RAPD分析结果得知,各再生克隆均存在双亲的核DNA及重组DNA.杂种的核基因组组成以胡萝卜(受体)为主,供体川西獐牙菜DNA谱带较少;UV照射剂量对体细胞杂种核基因组组成没有明显的影响.进一步对再生克隆叶绿体DNA的SSR分析表明,杂种细胞中双亲叶绿体基因组随机分离并发生重组.     

用8质粒病毒拯救系统产生H9N2/WSN重组A型流行性感冒病毒

把禽流行性感冒(流感)病毒A／Chicken／Shanghai／F／98(H9N2)的血凝素(HA)和神经氨酸酶(NA)基因cDNA克隆至polⅠ-pol Ⅱ双向转录和表达载体pHW2000,用这两种质粒与8质粒病毒拯救系统中流感病毒A／WSN／33(H1N1)6个内部基因cDNA的质粒组合(6 2重排),共转染COS-1细胞,产生了能在鸡胚中高滴度增殖的H9N2／、WSN重组病毒。用A／WSN／33的8个基因cDNA质粒作对照,也产生了转染子病毒。经过EID50测定和MDCK感染实验,新基因型H9N2／WSN病毒感染鸡胚的能力强(EID50为10^-11／0．2m1),而且对鸡胚的毒力弱,在不加胰酶的情况下不使MDCK细胞产牛病变。经电镜观察,两个转染子病毒的形态与野生型流感病毒相似。反向遗传操作技术的建立,为对禽流感病毒基因功能和疫苗构建等方面的研究提供了新的手段。     

Prevalence and evolution of drug resistance HIV-1 variants in Henan, China

To understand the prevalence and evolution of drug resistant HIV strains in Henan China after the implementation of free antiretroviral therapy for AIDS patients. 45 drug naive AIDS patients, 118 AIDS patients who received three months antiretroviral therapy and 124 AIDS patients who received six months antiretroviral treatment were recruited in the southern part of Henan province. Information on general condition, antiretroviral medicines, adherence and clinical syndromes were collected by face to face interview. Meanwhile, 14ml EDTA anticoagulant blood was drawn. CD4/CD8 T cell count, viral load and genotypic drug resistance were tested. The rates of clinical improvement were 55.1% and 50.8% respectively three months and six months after antiretroviral therapy. The mean CD4 cell count after antiretroviral therapy was significantly higher than in drug naive patients. The prevalence rate of drug resistant HIV strains were 13.9%, 45.4% and 62.7% in drug naive patients, three month treatment patients and six month treatment patients, respectively. The number of resistance mutation codons and the frequency of mutations increased significantly with continued antiretroviral therapy. The mutation sites were primarily at the 103, 106 and 215 codons in the three-month treatment group and they increased to 15 codon mutations in the six-month treatment group. From this result, the evolution of drug resistant strains was inferred to begin with the high level NNRTI resistant strain, and then develop low level resistant strains to NRTIs. The HIV strains with high level resistance to NVP and low level resistance to AZT and DDI were highly prevalent because of the AZT＋DDI＋NVP combination therapy. These HIV strains were also cross resistant to DLV, EFV, DDC and D4T. Poor adherence to therapy was believed to be the main reason for the emergence and prevalence of drug resistant HIV strains. The prevalence of drug resistant HIV strains was increased with the continuation of antiretroviral therapy in the southern part of Henan province. Measures, that could promote high level adherence, provide new drugs and change ART regimens in failing patients, should be implemented as soon as possible.     

含三倍体葡萄糖反应元件的质粒的构建及序列分析

用酚氯仿抽提法提取SD大鼠肝脏的基因组DNA,PCR扩增单倍体葡萄糖反应元件（glre）。分别用SacⅠ酶单酶切glre、pUC118载体后,连接、转化,PCR筛选出含三倍体glre的质粒并测序。结果PCR扩增出51bp的目的DNA片段,与文献报道的葡萄糖反应元件glre的大小一致;连接转化后,经PCR筛选出三倍体glre,序列分析显示其基因序列和Genbank数据库中的glre基因一致。以上结果表明构建成功含三倍体glre的质粒,为构建具双向调节的胰岛素分泌调控基因质粒奠定了基础。     

钙荧光探针Fura-2/AM对大肠杆菌细胞的负载行为

Fura-2/AM作为一类高效的钙离子荧光探针,在动物细胞中已得到较成功的应用,但在微生物细胞分析方面鲜见报道。该文系统研究了该探针的荧光光谱特征及其在大肠杆菌细胞内的负载行为,并考察了在大肠杆菌细胞中钙离子与Fura-2的结合情况。为利用其研究大肠杆菌等微生物细胞内钙离子浓度及钙离子跨膜行为奠定了基础。     

扣囊复膜孢酵母β-葡萄糖苷酶基因在毕赤酵母中的克隆与表达

利用PCR技术,从扣囊复膜孢酵母的总DNA中扩增得到β-葡萄糖苷酶（β-Glucosidase）基因（BGL1）,长度为2596 bp,连接到pGEM-T载体上,用限制性内切酶切下目的基因,插入到巴斯德毕赤酵母表达载体pPIC9K中,使之位于α-因子信号肽下游,且与之同框,构建成重组质粒pSHL9K.通过电转化将重组质粒pSHL9K插入到Pichia pastoris GS115菌株染色体中,获得高效表达BGL1基因的毕赤酵母重组工程菌株.重组酶的最适温度为50℃,最适pH为5.4.培养基中β-葡萄糖苷酶活性最高可达47U/mL.     

中国新疆维族人群HLA-B等位基因与HIV-1感染易感性或抗性

通过对中国维吾尔族人群HLA-B等位基因的分布频率的研究,探讨HLA-B等位基因与HIV感染的易感 或抵抗性的相关性。本研究用PCR-SSP的方法对新疆维吾尔族110例无相关的健康对照者(HIV阴性)和128例 HIV阳性感染者进行HLA-B等位基因分型。用POPGEN软件对健康对照者人群进行Hardy-Weinberg平衡检 测,用卡方检验分析HLA-B等位基因在健康对照者和HIV阳性感染者频率分布的差异。在HIV-1阳性感染者 中,B*4901等位基因频率显著性增加(B*4901:P=0．02．OR=3．06,95%CI=1．16～8．10)。而在健埭对照者 中,B*40等位基因顿率增加具有统计意义(B*40:P=0．02．OR=0．39．95％CI=0．07～0．92)。由此可见,B* 4901等位基因可能与HIV-1感染的易感性有关,而B*40等位基因可能与与HIV-1感染的抵抗性有关。     

CCL27的不同剪切机制及其功能差异

CCL27是一种CC型趋化因子,其配体为CCR10。由于mRNA剪切的不同,CCL27形成两种不同的分子,即经典CCL27和其变异体PESKY,前者含分泌肽,对活化CD4＋T细胞有趋化作用,是皮肤炎症反应中扮演主要角色的趋化因子;后者含有核内定位序列,调控细胞骨架结构的重排,这一特性为该趋化因子所独有,可能预示着趋化因子家族的新功能。     

肺炎克氏杆菌GDHt-γ亚基基因克隆与序列分析

目的：克隆编码肺炎克氏杆菌GDHt-γ亚基的基因并对其进行序列分析。方法：用酚氯仿抽提法提取肺炎克氏杆菌基因组DNA,用P1、P2为引物,PCR扩增GDHt-γ亚基基因,并对目的基因进行了测序并应用生物信息学进行序列分析。结果：PCR扩增出约426bp的目的DNA片段,与GenBank中报道的K．pneumoniae GDHt-γ亚基基因序列存在一个同义突变差异（Gin：CAG→CAA）。结论：成功地获得编码GDHt-γ亚基的基因,为进一步研究该亚基的结构与生物学功能奠定了基础。