Production of a truncated human c-myc protein which binds to DNA

Two kinds of truncated human c-myc proteins were produced in Escherichia coli. The human c-myc gene is composed of three exons, exons 2 and 3 having coding capacity for a protein of 439 amino acids. 252 N-terminal amino acids are encoded by exon 2, the C-terminal 187 amino acids being encoded by exon 3. One of the proteins (p42) produced in E. coli corresponds to 342 amino acids from the 98th Gln to the C-terminus, plus 21 amino acids derived from the H-ras gene at the N-terminus. The other (p23) corresponds to 155 amino acids from the 98th Gln to the 252nd Ser, plus five amino acids (Gly-Gly-Thr-Arg-Arg) at the C-terminus, plus 21 amino acids from the H-ras gene at the N-terminus. The p23 protein was produced by using cDNA in which a frame shift occurred at the boundary between exons 2 and 3. We investigated the DNA-binding activity in p42 and p23 proteins. DNA-cellulose column chromatography showed that p42 binds to DNA, whereas p23 does not. This DNA-binding activity of p42 was inhibited by antiserum prepared against p42 but not by antiserum against p23. This indicates that the DNA-binding activity of c-myc protein is localized in the portion encoded by exon 3.     

Oncogenicity of DNA in vivo: Tumor induction with expression plasmids for activated H-ras and c-myc

All vaccines and other biological products contain contaminating residual DNA derived from the production cell substrate. Whether this residual cell-substrate DNA can induce tumors in vaccine recipients and thus represent a risk factor has been debated for over 50 years without resolution. As a first step in resolving this issue, we have generated expression plasmids for the activated human H-ras oncogene and for the murine c-myc proto-oncogene. Their oncogenic activity was confirmed in vitro using the focus-formation transformation assay. Two strains of adult and newborn immune-competent mice were inoculated with different amounts of either plasmid alone or with a combination of the H-ras and c-myc plasmids. Tumors developed only in mice inoculated with both plasmids and only at the highest amount of DNA (12.5 microg of each plasmid). The NIH Swiss mouse was more sensitive than the C57BL/6 mouse, and newborn animals were more sensitive than adults. Cell lines were established from the tumors. PCR and Southern hybridization analyses demonstrated that both inoculated oncogenes were present in all of the tumor-derived cell lines and that the cells in the tumors were clonal. Western analysis demonstrated that both oncoproteins were expressed in these cell lines. These results demonstrate that cellular oncogenes can induce tumors following subcutaneous inoculation. Such information provides a possible way of evaluating and estimating the theoretical oncogenic risk posed by residual cell-substrate DNA in vaccines.     

Induction of Immune Response by Synthetic Fragments of the Bovine Prion Protein and Their Analogues in Mice of Various Lines

Antibodies to the bovine prion protein were produced by immunizing mice of three lines with five synthetic fragments of the protein and six of their analogues. The analogues contained amino acid substitutions that, according to theoretical calculation, should lead to an increase in the immunogenic activity of peptides. All the peptides except for one induced the formation of antibodies. All the sera containing the antipeptide antibodies were tested by an immunohistochemical method. The sera effectively bound to brain preparations from an animal with spongiform encephalopathy were identified; it was shown that they do not interact with preparations of normal brain. Therefore, it was shown that the immunization of mice with synthetic fragments of a prion protein allows one to obtain specific antibodies suitable for the study and diagnostics of prion diseases.     

土壤因子与华重楼生物量和药效成分含量相关性分析

为研究土壤因子对华重楼生长和药效成分积累的影响,该研究利用高效液相色谱技术(HPLC)以及相关性和多元线性回归分析方法,测定了华重楼不同产地土壤成分、样品的生物量和重楼皂苷的含量,分析了土壤因子与华重楼生物量和药效成分的相关性。结果表明:(1)不同产地土壤成分和华重楼产量及其重楼皂苷含量均有差异。(2)相关性分析显示,干重与有机质、全氮、碱解氮显著正相关,重楼皂苷Ⅰ与有机质和速效磷显著正相关,重楼皂苷Ⅱ与速效磷和速效钾显著正相关,而重楼皂苷Ⅶ与土壤各因子的相关性不显著。(3)多元线性回归分析显示,影响干重的主导因子为碱解氮,影响重楼皂苷Ⅰ的主导因子为有机质,影响重楼皂苷Ⅱ的主导因子为速效磷,而重楼皂苷Ⅱ与碱解氮呈线性负相关。综合分析认为,影响华重楼干重的土壤因子主要是碱解氮,而影响华重楼皂苷含量的土壤因子主要为有机质和速效磷,该研究结果为华重楼的人工栽培提供了理论依据。     

我国沿海拟菱形藻属的2新记录种及其产毒特征分析

为澄清我国沿海拟菱形藻属(Pseudo-nitzschia)的物种多样性,并确认中国海域拟菱形藻属是否具有产生多莫酸(Domoicacid)的能力,采用毛细管显微操作技术从我国沿海水体中分离、纯化拟菱形藻细胞,建立了单克隆培养株系,并基于核糖体转录间隔区ITS1-5.8S-ITS2 序列构建了分子系统树。结果表明,结合在光学显微镜和透射电镜下观察的形态学特征和分子系统发育分析数据,鉴定到我国拟菱形藻属的2新记录种:银河拟菱形藻(P. galaxiae Lundholm & Moestrup)和微孔拟菱形藻(P. micropora Priisholm, Moestrup & Lundholm),对其形态学特征进行了详细描述,并与相似种类进行了比较研究。利用高效液相色谱(HPLC)技术对多莫酸特征进行了检测,结果表明培养株系并不产生多莫酸。这些为我国拟菱形藻属物种多样性和产毒特征研究提供了基础数据。     

QTL Analysis in Recombinant Chromosome Substitution Lines and Doubled Haploid Lines Derived from a Cross between Hordeum vulgare ssp. vulgare and Hordeum vulgare ssp. spontaneum

Recombinant chromosome substitution lines (RCSLs) were developed in BC₃ generation to introduce segments of a wild barley strain ‘H602’ (Hordeum vulgare ssp. spontaneum) into a barley cultivar ‘Haruna Nijo’ (H. vulgare ssp. vulgare) genetic background. One hundred thirty four RCSLs were genotyped by 25 SSR and 60 EST markers, which were localized on a linkage map of doubled haploid lines (DHLs) derived from the same cross combination. Graphical genotyping revealed that the observed average substitution ratio of H602 segment (12.9%) agreed with the expected substitution ratio (12.5%), and a minimum set of 19 RCSLs represented the entire H602 genome. Phenotypes of five qualitative and nine quantitative traits were scored in both the RCSLs and DHLs. Five qualitative traits were localized as morphological markers on the linkage map of the DHLs, and these molecular markers were aligned on the respective chromosomal regions in the RCSLs. Simple and composite interval mapping procedures detected a total of 18 and 24 QTLs for nine qualitative traits on the RCSLs and DHLs, respectively. Several QTLs were localized at coincident or very close regions on both linkage maps. In spite of general inferior agronomic performances in wild barley, several H602 QTL alleles showed agronomically positive effects. These RCSLs should contribute to substitution of favorable alleles from wild barley into cultivated barley. These RCSLs are also available as sources of near isogenic lines, with which we can apply advanced genetic analysis methods such as isolation of QTLs and detection of epistatic interactions among QTLs.     

The RustPuccinia abruptavar.partheniicola,a Potential Biocontrol Agent of Parthenium Weed: Environmental Requirements for Disease Progress

The rust fungusPuccinia abruptavar.partheniicola,a potential biological control agent of parthenium weed (Parthenium hysterophorus), was evaluated under controlled environmental conditions. A range of spore germination temperatures as well as dew period durations and temperatures were investigated to determine some of the environmental requirements for disease establishment and disease progress. Plants were inoculated with urediniospores and exposed to dew periods between 3 to 12 h at temperatures of 10, 15, or 20°C. For disease expression, the inoculated plants were then grown in a glasshouse at one of two temperature regimes (30/26°C or 18/13°C; day/night). Urediniospores germinated best at 12 ± 1°C, with lower germination rates at 5°C or above 20°C. No infection occurred when the plants were exposed to dew periods of ≤3 h, regardless of the incubation temperature. The disease progressed most rapidly when plants were inoculated and incubated for a dew period of at least 12 h at a temperature of 15 ± 1°C. The disease progressed most slowly following inoculation at dew periods of 6 h or less. Disease progress was more rapid when the plants were exposed to a cool-temperature regime (18/13°C) than when exposed to a warm-temperature regime (30/26°C). This suggests that good infection of parthenium weed could be obtained when the urediniospores arrive on the plants during the afternoon in the cooler months of the central Queensland autumn when relatively long dew periods are expected.     

青花菜BoSCL3基因的克隆和渍水胁迫下的表达特征分析

为了解SCL3 (scarecrow-like 3)基因的功能,从青花菜(Brassica olreacea var. italica)中克隆得到1个SCL3基因,命名为BoSCL3,其cDNA全长1 355 bp,编码446个氨基酸。BoSCL3分子量为49.96 kD,为疏水性蛋白,与油菜(B. napus)、芜菁(B. rapa)中SCL3蛋白的亲缘关系最近,同科植物的SCL3具有较高的同源性。荧光定量PCR分析结果表明,青花菜BoSCL3基因表达量随渍水胁迫时间延长先下降后上升,推测其可能参与渍水胁迫响应。这为探讨青花菜BoSCL3基因响应渍水胁迫的分子机制提供理论依据。     

香鳞毛蕨DfGNOM基因的克隆及其表达分析

GNOM是一种ADP核糖基化因子(ARF)的鸟嘌呤核苷酸交换因子(GEF),为探索GNOM在香鳞毛蕨(Dryopteris fragrans)中的抗逆功能,该研究克隆了DfGNOM并进行生物信息学分析,采用实时荧光定量PCR(qRT-PCR)方法分析了DfGNOM基因在不同植物激素及逆境胁迫处理下的表达模式,为进一步探索该基因的功能以及香鳞毛蕨的抗逆机制奠定基础。结果表明:(1)成功获得DfGNOM全长4 338 bp,蛋白质多序列比对以及进化树分析表明,DfGNOM与江南卷柏(Selaginella moellendorffii) SmGNOM亲缘较近,motif分析表明该蛋白含有sec7保守结构域。(2) qRT-PCR分析显示,DfGNOM在香鳞毛蕨的根、叶柄和叶中均有表达,但在叶中表达量最高;DfGNOM的相对表达量在生长素(IAA)处理后总体上调,在脱落酸(ABA)处理后总体下调;在NaCl处理下呈"降-升-降"的变化趋势,高温和低温处理下呈"升-降-升"变化趋势;在茉莉酸甲酯(MeJA)、乙烯利(ETH)以及PEG处理下,DfGNOM的相对表达也表现出不同的模式。研究认为,DfGNOM在香鳞毛蕨非生物胁迫响应过程中发挥调控作用。     

云南重楼ITS序列单核苷酸多态性与甾体皂苷特征的相关性分析

为了解云南重楼(Paris polyphylla var. yunnanensis) ITS序列的单核苷酸多态性(SNP)特征与其甾体皂苷构成特征的相关性,并探讨其对药材质量稳定性的影响,利用MegAlign软件对37份云南重楼样本的ITS序列进行对比,根据SNP位点进行分型;采用HPLC法测定7种甾体皂苷成分(重楼皂苷Ⅰ、Ⅱ、Ⅴ、Ⅵ、Ⅶ、H和薯蓣皂苷);用SPSS 25.0软件和SIMCA-P 15.0软件对各基因型甾体皂苷构成特征进行统计分析。结果表明,云南重楼ITS序列存在40个双等位多态性位点,其中碱基转换32个,碱基颠换8个,根据SNP特征可划分为2类基因型YN-I和YN-II。6种甾体皂苷(重楼皂苷Ⅰ、Ⅱ、Ⅵ、Ⅶ、H、薯蓣皂苷)在云南重楼广泛分布,而重楼皂苷Ⅴ稀少。YN-I和YN-II的药典指标成分总含量分别为1.070%和0.93%,样本合格率分别为68.42%和77.78%;YN-I的甾体皂苷总含量为1.65%,YN-II为1.32%。方差分析表明,2类基因型的甾体皂苷特征存在一定程度的区别,但药典指标成分无显著差异,药材应该具有等效性。聚类分析和主成分分析表明,YN-Ⅰ的离散度更高,YN-Ⅱ的聚集度更好,表明YN-II的药材质量更加稳定,植株个体的甾体皂苷合成和累积差异更小,YN-II的SNP特征对云南重楼良种选育的分子遗传标记筛选具有重要意义。     

2种菊苣再生体系及遗传转化效率的比较

以普那菊苣和将军菊苣子叶为材料,通过植物组织培养的方法,探讨了不同激素浓度配比对二者愈伤组织诱导、芽分化以及根再生的影响,并通过农杆菌介导法将编码獐茅液泡膜Na+/H+逆向转运蛋白基因(AlNHX)导入菊苣中,比较普那菊苣和将军菊苣的遗传转化效率。结果表明:不同基因型的菊苣愈伤组织诱导和芽分化条件不同,普那菊苣最佳培养基为MS+1.5mg/L 6-BA+0.2mg/L IBA;将军菊苣最佳培养基为MS+1.0mg/L 6-BA+0.5mg/L NAA;二者最佳生根培养基均为1/2MS+0.1mg/L NAA。获得的抗性芽经PCR检测,初步证实AlNHX已插入到菊苣基因组中,且普那菊苣转化效率为10.0%,将军菊苣转化效率为13.3%。     

Immunogenicity of Ehrlichia ruminantium Grown in Tick Cell Lines

Ehrlichia (previously Cowdria) ruminantium, the pathogen which causes heartwater in domestic and wild ruminants, can now be propagated in cell lines from one vector (Amblyomma variegatum) and five non-vector (Ixodes scapularis, I. ricinus, Boophilus decoloratus, B. microplus and Rhipicephalus appendiculatus) tick species. E. ruminantium isolates from West and South Africa and the Caribbean vary in their cell line preference, growth patterns and immunogenic capability. In laboratory trials, certain combinations of tick cell line and E. ruminantium isolate were highly immunogenic in sheep. These trial vaccines were grown under specific in vitro conditions and administered as a single intravenous dose of freshly harvested whole, live culture. Following immunisation and subsequent exposure to virulent E. ruminantium, protected sheep showed no clinical response and a range of serological responses. This revised version was published online in July 2006 with corrections to the Cover Date.     

勃氏甜龙竹不同部位根的解剖结构比较

该研究选择勃氏甜龙竹7月份(雨季)和11月份(干季)地面以下10 cm处土壤中秆基处生的根,秆下部节内(地上部分3~4节)和秆上部枝条基部在空气中自然生的根,以及7月份枝条高空压条产生的伸长阶段和成熟阶段的根作为实验材料,采用树脂切片和徒手切片法,对不同部位根的根尖和根毛区进行横切,甲苯胺蓝O和苏丹红7B染色,研究不同起源根的形态解剖结构,为竹子根解剖学研究提供新的理论信息。结果显示:(1)不同来源根的皮层宽度占根直径的比例、周缘纤维组织宽度及其细胞层数、内皮层细胞壁厚度均随生长时间延长而增加。(2)所有起源根的外皮层和内皮层细胞壁均有显著的木栓质沉积,但环内皮层木栓化程度存在显著差别。(3)测量不同来源根的后生木质部导管数目和直径发现,7月份采集的根导管数目更多、直径更大。(4)枝条基部自然生根的根尖与其他起源部位的根相比更为钝平,但根冠面积较小;秆下部节内自然生根非常硬,所以未能成功观察其根尖结构特点。研究认为,勃氏甜龙竹4种不同起源根的基本结构一致,但在各自的具体结构上还存在显著差别,可能与其生长的环境以及发育状况密切相关。     

棉花EIN3/EIL家族基因序列分析及GhEIL3基因克隆

该研究以拟南芥AtEIN3基因为探针,从陆地棉TM-1全基因组测序数据库中筛选其同源序列,序列分析得到16条具有EIN3结构域的基因序列。对陆地棉EIN3/EIL家族基因的基因结构、系统进化、序列相似性及结构域分布情况进行分析,发现它们与拟南芥EIN3/EIL家族基因在N端具有较高相似度,其中15条基因序列包含5个保守结构,1条包含4个保守结构。在此基础上,采用RT-PCR方法从抗枯萎病的陆地棉品种‘中棉所12’中克隆得到一个新的EIN3/EIL家族基因,命名为GhEIL3(GenBank登录号为KY072936)。序列分析表明:GhEIL3基因开放阅读框长1 092bp,编码363个氨基酸,含有一个EIN3结构域。实时荧光定量PCR(qRT-PCR)结果显示,GhEIL3基因在枯萎病菌诱导后呈上调表达,诱导后1h其相对表达量达到最大值,推测GhEIL3基因可能参与棉花对枯萎病菌的防御反应。     

天山雪莲LEA14基因克隆及功能分析

从天山雪莲叶片低温诱导的EST文库中获得了1个胚胎发育晚期丰富蛋白基因(LEA)cDNA全长序列。序列分析表明,该基因含有1个468bp编码155个氨基酸的开放阅读框。NCBI保守域预测此蛋白属于LEA_2家族,命名为SiLEA14。系统进化分析表明,该蛋白与北柴胡的LEA-2蛋白亲缘关系最近。荧光定量PCR结果显示,SiLEA14表达量在低温、盐和干旱胁迫条件下迅速升高。亚细胞定位结果表明,SiLEA14蛋白定位于细胞核中。利用农杆菌介导法将该基因导入烟草,测定并分析转基因植株在冷冻和盐胁迫处理下的生理指标,结果表明,SiLEA14基因在烟草中的过量表达提高了烟草的抗冻和耐盐能力。     

三倍体观赏百合与二倍体食用龙牙百合的杂交分析

为探讨异源三倍体百合与龙牙百合(BB)的杂交亲和性,实现观赏百合与食用百合的种质融合与创新,该研究以三倍体百合Triumphator(LLO)作母本,龙牙百合为父本,采用常规授粉与切柱头授粉,利用基因组荧光原位杂交(GISH)技术分析母本及子代的基因组成.结果 显示:(1)通过常规授粉和切柱头授粉共获得17个发育良好的...     

Akkermansia与高原牛肺水肿病相关性研究

【目的】本文通过对高原牛胃肠道菌群结构组成的分析,从微生物学角度探讨Akkermansia与高原牛肺水肿病的关系。【方法】本研究以沈阳地区健康娟姗牛为对照,以引进入拉萨半年的健康娟姗牛、拉萨本地健康黄牛以及引进入拉萨半年患肺水肿病的娟姗牛的粪便作为分析样本,采用Illumina MiSeq高通量测序技术测定样本中微生物16S rRNA基因V3–V4区序列,通过比较4种粪便样本菌群组成及丰度的差异,探讨Akkermansia与高原牛肺水肿病的相关性。【结果】Verrucomicrobia中Akkermansia在拉萨本地健康黄牛的胃肠道中的含量显著高于引进入拉萨半年的健康娟姗牛,在引进入拉萨半年患肺水肿病的娟姗牛胃肠道中的含量显著高于引进入拉萨半年的健康娟姗牛。在属水平上,沈阳地区健康娟姗牛胃肠道菌群中Akkermansia丰度占比为0.07%;引进入拉萨半年的健康娟姗牛胃肠道菌群中Akkermansia丰度占比为0.09%;拉萨本地黄牛胃肠道菌群中Akkermansia丰度占比为6.62%,是优势菌属;引进入拉萨半年的患肺水肿病的娟姗牛胃肠道菌群中Akkermansia丰度占比为11.85%,且是第一优势菌属。【结论】首次从微生物学角度探讨Akkermansia与高原牛肺水肿病的关系,为将Akkermansia丰度作为诊断肺水肿病的监测指标提供参考,但具体丰度值还有待进一步研究。