甘肃省小麦条锈病主要流行小种的RAPD分析及Su11-4小种SCAR标记建立

本研究应用RAPD标记技术对甘肃省小麦条锈菌主要流行的8个生理小种进行多态性标记分析,旨在寻找小麦条锈菌不同生理小种的特异性标记,共选用220条10碱基随机引物进行筛选,其中有147条可得到稳定清晰的扩增条带。研究结果显示,通过使用147条引物对甘肃省流行的8个条锈菌的生理小种进行RAPD分析,发现各致病小种间遗传变异丰富,其中引物S301在条中33号中扩增得到约507bp的特异条带;引物S39在条中32扩增得到长度约183bp的特异条带;引物S36在Hybrid46-8扩增得到约510bp的特异条带;引物S2140在Su11-4扩增得到约317bp的特异条带。另外,本研究还对扩增得到的特异片段进行回收并进行测序分析,其中依据小麦条锈菌生理小种Su11-4特异条带的测序结果设计特异引物,成功将其转化为对Su11-4小种特异的SCAR标记,这对不同条锈菌生理小种的快速准确检测具有重要意义。     

山羊草属植物醇溶蛋白的遗传多样性分析

利用APAGE技术对山羊草属中6个物种30份材料的醇溶蛋白进行了分析,共出现14种醇溶蛋白APAGE谱带类型和67条相对迁移率不同的谱带,其中,每份材料可分离出10~22条带,所有67条谱带均具有多态性。同一物种内的谱带类型为1~4种,种间没有出现相同的带型。种内各材料间谱带类型可完全相同,带型间的最大相似系数为0.941,而种间最大相似系数则为0.606。     

两个SCAR标记与京海Ⅰ号黄鸡体重的相关性研究

通过对两个RAPD条带(1660条带和2326条带)的克隆、测序,根据条带序列设计两对SCAR引物,对京海Ⅰ号黄鸡基因组进行扩增。结果表明:S1(1660条带)与S2(2326条带)标记在京海Ⅰ号黄鸡中出现的频率分别为40%和48%。无S1标记个体12周龄、18周龄和43周龄体重显著高于有S1标记个体,有S2标记个体在4周龄、12周龄、18周龄和43周龄体重显著高于无S2标记个体。     

耐低钾山羊草基因型的筛选与鉴定

为研究小麦近缘属种的耐低钾特性,分析不同山羊草(Aegilops)耐低钾胁迫下各生理指标的差异,筛选和鉴定耐低钾基因型。本试验通过砂培法,以9种不同基因型山羊草为供试材料,采用改进的Hoagland营养液,设低钾(0.02mmol·L~(-1) KCl)和正常供钾(2.0mmol·L~(-1) KCl)两个水平进行试验。对相对根冠比、相对根长、相对地上部干重、相对钾积累量和相对钾利用效率5个指标进行相关分析和主成分分析,筛选出3个综合指标进行山羊草耐低钾基因型的鉴定。结果发现,不同基因型山羊草对低钾胁迫存在显著的差异。地上部干重、钾积累量和钾利用效率可以作为苗期耐低钾种质筛选的重要指标。综合评价结果表明:双角山羊草(Ae.bicornis)、偏凸山羊草(Ae.ventricosa)、粗厚山羊草(Ae.crassa)、拟斯卑尔托山羊草(Ae.speltoides)、尾状山羊草(Ae.caudata)、粘果山羊草(Ae.kotschyi)、易变山羊草(Ae.variabilis)、欧山羊草(Ae.biuncialis)、小伞山羊草(Ae.umbellulata)的综合评价值(D值)分别为0.383,0.898,0.327,0.516,0.007,0.682,0.338,0.346,0.655。由此可知,其中偏凸山羊草耐低钾胁迫能力最强,其次是粘果山羊草,尾状山羊草耐低钾胁迫能力最弱。通过本试验,初步建立了山羊草耐低钾种质的评价指标,为进一步研究山羊草的耐低钾分子机制,以及小麦育种上利用耐低钾山羊草种质提供了材料和理论依据。     

云南圭山山羊及红骨圭山山羊的染色体核型及带型研究

作者采用外周血淋巴细胞培养及染色体分带技术,分析了云南圭山山羊和红骨圭山山羊的染色体核型和C-带带型及G-带带型。结果表明,两种山羊的染色体形态较相似,二倍体染色体众数为2n=60,其中常染色体29对全部为端着丝粒,X染色体相对长度介于1号与2号染色体之间为端着丝粒,Y染色体最小且为唯一的中端着丝粒染色体。两种山羊中都发现一定比例的体细胞染色体多倍体,比例分别为6.8%、9.4%。对2种山羊C-显带及G-显带的带型分析显示没有明显差异。     

粗山羊草抗条锈 (CYR32) 性状的遗传分析

12份粗山羊草材料成株期对中国条锈病新小种CYR32为免疫-高抗,1份为高感.为探讨抗病性遗传规律,组配成抗×感、抗×抗杂交组合.通过对抗×感以及杂交组合的F1、F2群体对CYR32条锈菌抗性表型的分析,结果显示,所有抗源亲本对条锈病抗性呈完全显性遗传, F2群体抗感分离比例为3∶1,条锈病的抗性受1对显性基因控制.抗×抗杂交组合F2均呈抗病性,推测12个抗病粗山羊草材料的抗病基因可能为同一对显性基因.     

利用ACGM和EST-SSR标记对云贵高原野生山蚂蝗属种质的遗传多样性分析

山蚂蝗属野生种质是一个具有巨大经济价值的资源。本研究利用基于基因表达序列数据库开发的２种分子标记ＡＣＧＭ 和ＥＳＴ ＳＳＲ 共８５对引物对山蚂蝗属９个种４６个野生种质资源进行多样性分析。结果显示,ＡＣＧＭ引物中有扩增产物的引物比例为８６．４９％,远高于ＥＳＴ ＳＳＲ 引物的５４．１７％。同时,ＡＣＧＭ 的多态性比率也大于ＥＳＴ-ＳＳＲ,可见ＡＣＧＭ 在山蚂蝗属野生种质中的转移性优于ＥＳＴ-ＳＳＲ。通过ＡＣＧＭ 和ＥＳＴ ＳＳＲ 分析得到的遗传相似性系数为０．５２３～０．９６７,平均相似系数为０．７０３,这表明山蚂蝗属野生种质资源间存在较高的遗传多样性。此外,ＡＣＧＭ 分析能有效区分４６ 个山蚂蝗属种质基因型,而ＥＳＴ-ＳＳＲ 只能区分绝大多数山蚂蝗属基因型。在ＵＰＧＭＡ 聚类图上４６个供试材料被分成９组,与传统分类结果不完全一致。说明基于禾本科和豆科基因表达序列开发的分子标记能用于山蚂蝗属植物的遗传分析,同时这也为其他野生种质资源的遗传多样性研究提供了有益的借鉴。     

Insulin binding is a specific marker of fetal erythrocytes in ruminants

The ability of erythrocytes (RBC) from sheep and cattle of various gestational and postnatal ages to bind insulin specifically was studied. Insulin binding to RBC decreased as gestational and postnatal age advanced and was absent in blood obtained from adult animals. Maximal percentage 125I-insulin bound to RBC (3.6 X 10(9)/ml) was highest in the fetuses of sheep and cattle (7.3 +/- .6 and 7.8 +/- .9, respectively) compared to postnatal animals (2.3 +/- .2 and 2.2 +/- .3, respectively), or adults (no binding) of the same species. The decrease in binding began antenatally, and binding was projected to be insignificant by the end of the second postnatal month. Most of the observed decrease was due to a progressive decrease in the number of receptors on the cell surface. The time course of this phenomenon, as well as the total absence of insulin receptors on the RBC of adult ruminants, provides independent evidence that two distinct populations of RBC in ruminants exist. The gradual appearance of the adult RBC with no insulin binding results in a decrease in observed binding to RBC in a given blood specimen as fetuses and postnatal animals age.     

CD86 molecule is a specific marker for canine monocyte-derived dendritic cells

In this study, canine monocyte-derived dendritic cells (cMo-DC) were produced in presence of canine GM-CSF (cGM-CSF) and canine IL-4 (cIL-4), and they were characterized by their dendritic morphology, MLR functionality and phenotype. We noticed that cMo-DC were labelled with three anti-human CD86 (FUN-1, BU63 and IT2.2 clones), whereas resting and activated lymphocytes or monocytes were not stained. CD86 expression was induced by cIL-4 and was up-regulated during the differentiation of the cMo-DC, with a maximum at day 7. Furthermore, cMo-DC were very potent even in low numbers as stimulator cells in allogeneic MLR, and BU63 mAb was able to completely block the cMo-DC-induced proliferation in MLR. We also observed that cMo-DC highly expressed MHC Class II and CD32, but we failed to determine their maturation state since the lack of commercially available canine markers. Moreover, cMo-DC contained cytoplasmic periodic microstructures, potentially new ultrastructural markers of canine DC recently described. In conclusion, this work demonstrates that the CD86 costimulatory marker is now usable for a better characterization of in vitro canine DC.     

Establishment of a LacZ marker rescue assay to detect infectious RD114 virus

Cats have an infectious endogenous retrovirus, named RD114 virus, and there is a possibility that RD114 virus has contaminated live attenuated vaccines, for which feline cells are used as a substrate. To monitor infectious RD114 virus in vaccines for cats, we developed a LacZ marker rescue assay to detect infectious RD114 virus. Among four human cell lines examined, TE671 cells (human rhabdomyosarcoma) were most susceptible to RD114 virus and supported RD114 replication efficiently. Infection was enhanced approximately 5 times by the addition of polybrene at concentrations of 2 to 8 microg/ml in the medium during viral adsorption. A 4-hr viral adsorption period was sufficient to obtain the maximum titer. By inoculating samples into TE671 cells transduced with the lacZ marker gene, the limiting diluted sample (i.e., less than 10 infectious units) was detected at 12 days post-inoculation by the LacZ marker rescue assay. Based on the results obtained in this study, we propose a standard protocol of the LacZ marker rescue assay to detect infectious RD114 virus.     

Cell-surface lactoferrin as a marker for degranulation of specific granules in bovine neutrophils

OBJECTIVE: To develop a rapid and accurate flow cytometric method for measuring degranulation of specific granules in bovine neutrophils. SAMPLE POPULATION: Blood samples obtained from four 6- to 18-month-old Holstein cattle. PROCEDURE: A monoclonal antibody (BL97) was generated against bovine lactoferrin and tested for applicability in ELISA, immunoprecipitation tests, immunofluorescence microscopy, and flow cytometric analyses. Using this antibody, cell-surface lactoferrin was measured concurrent with amount of secreted lactoferrin from bovine neutrophils activated with phorbol myristate acetate (PMA). Cell-surface lactoferrin also was measured on neutrophils in bovine whole blood stimulated with PMA, platelet-activating factor (PAF), N-formyl-methionyl-leucyl-phenylalanine (fMLF), and interleukin 8 (IL-8). RESULTS: Antibody BL97 recognized bovine lactoferrin in ELISA and western immunoblots and was useful for immunoprecipitation testing, immunofluorescence microscopy, and flow cytometric analyses of bovine leukocytes. Neutrophils activated with PMA had parallel increases in content of secreted lactoferrin (measured by ELISA) and cell-surface lactoferrin (measured by flow cytometry) with increasing PMA concentrations. In addition, fluorescein-conjugated BL97 antibody detected increases in cell-surface lactoferrin on neutrophils in bovine whole blood after activation with PMA, PAF, and IL-8. In contrast, increases in cell-surface lactoferrin were not detected on bovine neutrophils treated with fMLF. CONCLUSION AND CLINICAL RELEVANCE: Measurement of cell-surface lactoferrin on bovine neutrophils by flow cytometry is a valid and rapid method for assessment of release of lactoferrin from specific granules in these cells and represents a means to rapidly measure neutrophil activation. This technique allows for investigation of mechanisms of neutrophil modification in isolated cells as well as in whole blood.     

Alpha-naphthyl acetate esterase activity is not a specific marker for ovine T lymphocytes

Alpha-naphthyl acetate esterase (ANAE) activity was demonstrated in ovine lymphocytes harvested from blood on Ficoll-metrizoate gradients. The enzyme's specificity for T lymphocytes, identified by immunofluorescent staining of T cell-specific antigens, was assessed. Correlation analysis of the results obtained using unfractionated lymphocytes from 12 sheep showed no correlation between ANAE activity and the expression of T cell antigens (r = 0.22). When lymphocytes from 4 sheep were fractionated on nylon wool columns a mean of only 43.2% of the cells in the non-adherent population were ANAE-positive whereas 94.7% of these cells were identified as T lymphocytes. Blood lymphocytes from 5 animals were separated into 3 fractions using Percoll discontinuous density gradients. No significant relationship was seen between ANAE activity and T cells in Fractions 1 and 3 (r = 0.41 and 0.21). Fraction 2 cells, however, did show a significant positive relationship (r = 0.91) between these two features but the biological significance of this relationship is unknown. It was concluded that ANAE activity is not a specific marker for ovine T lymphocytes.     

Establishment and characterization of dengue virus type 2 nonstructural protein 1 specific T cell lines

Immunity against dengue viruses (DENV) infection may include cellular immune responses which involve in the immunopathology of DENV infection hosts. This study was to establish short-term dengue virus type 2 (DENV2) nonstructural protein 1 (NS1) specific T cells from splenocytes from BALB/c mice immunized with DENV2 NS1 in vitro, which may be used to identify immunopathologic mechanism of dengue. Nine DENV2 NS1 specific T cell lines were successfully established by using limiting dilution methods and maintained for 20 weeks by re-stimulated with DENV2 NS1, recombinant mouse IL-2 and antigen presenting cell weekly. Phenotypically, these cells were mainly composed of CD3⁺CD4⁺ T cells. The culture supernatants of these cells contained large amounts of TNF-α and IFN-γ. Vascular tissue pathological change could be found in the mice adoptive transferred with DENV2 NS1 specific T cells. The results indicate that DENV2 NS1 specific T cells could be established and maintained with syngeneic T cell growth factors in vitro. Meanwhile, DENV2 NS1 specific T cells might contribute to the immunopathology of vascular leakage of dengue.     

Microsatellite marker C04107 as a diagnostic marker for copper toxicosis in the Danish population of Bedlington terriers

The linkage phase of marker C04107 was evaluated before implementation of the marker in a diagnostic test. Blood samples from 68 dogs were collected and genotyped by PCR. Two alleles were detected with sizes of 160 bp and 164 bp and allele frequencies of 0.45 and 0.55 respectively. Genotyping revealed that 35 dogs were heterozygous (51.5%), 22 dogs were homozygous for the normal allele (32.3%) and 11 dogs were homozygous for the disease allele (16.2%). Liver biopsies were taken from 14 selected dogs and the copper content was evaluated histologically. Biopsies from 8 dogs homozygous for the disease allele showed many copper granules along with single cell necrosis, haemosiderosis and cellular infiltration. In liver biopsies from 6 dogs genotyped to be heterozygous or homozygous for the normal allele, copper granules were absent or moderate in number and no lesions were present. The survey demonstrates that the linkage phase of marker C04107 in the Danish population of Bedlington terriers is similar to the linkage phase detected in other countries. Thus, the marker can be used in a diagnostic test for copper toxicosis in Denmark.     

高粱EST-SSR标记的建立及其在苏丹草中的应用初探

从NCBI中下载了202 325条高粱Sorghumbicolor EST序列。去除低质量和冗余的序列后,在5 661条高粱EST中共发掘出了6 663个SSR位点,出现频率是20.19%,平均分布频率是1/3.93 kb。在5 197条EST序列中,共有3 446条序列能够设计引物,占总数的66.3%。在高粱EST-SSR中,三核苷酸重复是主要的重复类型,出现最多的重复基元是GGC/GCC。在3 446对高粱EST-SSR引物中,随机选择了20对引物进行了合成。以苏丹草Sorghumsudanense722和高粱Sorghumbicolor TX623A为模板,用这20对引物进行PCR扩增,结果全部可以扩增出条带,可用率为100%,有差异的有4对,多态性比率为20%。研究结果证明了根据高粱EST建立SSR标记是有效、可行的。