鸡大肠杆菌O2/O74混合血清型菌株iss基因的克隆与序列测定

采用PCR方法对1株O2/O74混合血清型鸡大肠杆菌检测其iss基因的存在,并与1株O2血清型鸡大肠杆菌相比较.结果表明:鸡E.coli O2/O74混合血清型菌株携带iss基因,但采用目前通用的PCR方法并不能检测出iss基因,必须经套式PCR扩增才能检测出iss基因.O2/O74混合血清型菌株iss基因序列与已知禽大肠杆菌iss基因序列同源性为99.7%;与人源大肠杆菌iss基因进行比对,同源性为90.6%;与λ噬菌体bor 基因序列进行比对,同源性为88%,显示了此基因的保守性.     

猪瘟病毒巢式RT-PCR检测方法的建立与应用

为建立一种快速、特异、灵敏的检测猪瘟病毒（classical swine fever virus,CSFV）巢式RT-PCR（nested RT-PCR）方法,本研究参照GenBank中公布的CSFV E2基因保守区域序列设计2对特异性引物,以CSFV总RNA为模板,优化反应条件,建立CSFV nested RT-PCR方法,对其进行特异性、敏感性和重复性试验;利用所建立的方法对35份临床疑似CSFV感染样品进行了应用检测,并对阳性PCR产物进行克隆测序鉴定。结果表明,本研究成功建立了CSFV nested RT-PCR检测方法,能够特异性地扩增CSFV,但对ST正常细胞对照和其他8种病原对照未扩增出任何条带;稳定性和重复性好;敏感性高,最低检测病毒含量为1 TCID₅₀;自35份疑似CSFV感染样品中检出19份阳性样品,PCR阳性产物克隆测序结果表明均为CSFV E2基因片段。本研究成功建立了CSFV nested RT-PCR检测方法,可用于CSFV的快速检测,为CSF的早期检测诊断提供了特异、灵敏的方法。     

应用巢式PCR法检测猪单个胚胎中的基因突变

单胚胎基因突变检测技术是以单个胚胎的裂解产物为模板进行DNA或RNA分析的方法,该技术的建立将为在单细胞水平进行基因表达和突变检测提供简便而快捷的方法。本研究设计了2对巢式引物,以显微注射了Cas9 mRNA和猪SS基因2个靶点的gRNA的四细胞期孤雌胚胎为试验材料,通过蛋白酶K对猪单个胚胎进行裂解后直接进行巢式PCR分型,并检测到猪胚胎中SS基因的删除突变。该方法可以最大限度减少假阴性和假阳性结果的出现,在猪基因突变检测中具有重要作用。     

Designing a sampling scheme to reveal correlations between weeds and soil properties at multiple spatial scales

Weeds tend to aggregate in patches within fields, and there is evidence that this is partly owing to variation in soil properties. Because the processes driving soil heterogeneity operate at various scales, the strength of the relations between soil properties and weed density would also be expected to be scale‐dependent. Quantifying these effects of scale on weed patch dynamics is essential to guide the design of discrete sampling protocols for mapping weed distribution. We developed a general method that uses novel within‐field nested sampling and residual maximum‐likelihood (reml ) estimation to explore scale‐dependent relations between weeds and soil properties. We validated the method using a case study of Alopecurus myosuroides in winter wheat. Using reml , we partitioned the variance and covariance into scale‐specific components and estimated the correlations between the weed counts and soil properties at each scale. We used variograms to quantify the spatial structure in the data and to map variables by kriging. Our methodology successfully captured the effect of scale on a number of edaphic drivers of weed patchiness. The overall Pearson correlations between A. myosuroides and soil organic matter and clay content were weak and masked the stronger correlations at >50 m. Knowing how the variance was partitioned across the spatial scales, we optimised the sampling design to focus sampling effort at those scales that contributed most to the total variance. The methods have the potential to guide patch spraying of weeds by identifying areas of the field that are vulnerable to weed establishment.     

Specificity and selectivity of arbuscular mycorrhizal fungal polymerase chain reaction primers in soil samples by clone library analyses

An open question with regard to the community ecology of arbuscular mycorrhizal fungi (AMF) concerns how to best amplify AMF in the soil, which contains a large proportion of DNA from AM extra-radical mycelium and spores. However, to date, a direct comparison of AMF primers for soil samples, which would systematically assess their amplification efficiency, is still missing. In our present study, we compared and characterized four widely used primer sets targeting AMF 18S rDNA or SSU-ITS-LSU rDNA from three soil samples as follows: (1) SSUmAf/LSUmAr?+?SSUmCf/LSUmBr, (2) GeoA2/Geo11?+?NS31/AM1, (3) AML1/AML2?+?NS31/AM1 and (4) AMV4.5NF/AMDGR. These primer sets were compared in terms of the proportion of Glomeromycota detected, AMF diversity and community composition. Our data revealed that the newly combined primer set 3 was the most suitable one for amplifying AMF from soil samples. It yielded the highest AMF alpha diversity, and was very specific to Glomeromycota. Primer set 2 was unable to amplify Claroideoglomus from soil 1, which was the dominant AMF clade as proved by other three primer sets. Primer set 4 demonstrated its instability among different soil samples, since the proportion of AMF in total sequences varied from 5% to 83%. Although primer set 1 showed the highest proportion of AMF (95–100%) in the soil samples, it captured the lowest AMF diversity, and the operational taxonomic units obtained by this primer set were only 36.4% of that by primer set 4. Taken together, our data suggested that AMF diversity in soil samples could be underestimated by primer set 1, 2 and 4. Our result confirmed the important role of the choice of AMF primers for analyzing AMF communities in soil and explored the most suitable one for amplifying AMF from soil samples.     

Functional mechanisms of drought tolerance in maize through phenotyping and genotyping under well watered and water stressed conditions

Developing tolerant genotypes is crucial for stabilizing maize productivity under drought stress conditions as it is one of the most important abiotic stresses affecting crop yields. Twenty seven genotypes of maize (Zea mays L.) were evaluated for drought tolerance for three seasons under well watered and water stressed conditions to identify interactions amongst various tolerance traits and grain yield as well as their association with SSR markers. The study revealed considerable genetic diversity and significant variations for genotypes, environment and genotype × environment interactions for all the traits. The ranking of genotypes based on drought susceptibility index for morpho-physiological traits was similar to that based on grain yield and principal component analysis. Analysis of trait – trait and trait – yield associations indicated significant positive correlations amongst the water relations traits of relative water content (RWC), leaf water potential and osmotic potential as well as of RWC with grain yield under water stressed condition. Molecular analysis using 40 SSRs revealed 32 as polymorphic and 62 unique alleles were detected across 27 genotypes. Cluster analysis resulted in categorization of the genotypes into five distinct groups which was similar to that using principal component analysis. Based on overall performance across seasons tolerant and susceptible genotypes were identified for eventual utilization in breeding programs as well as for QTL identification. The marker-trait association analysis revealed significant associations between few SSR markers with water relations as well as yield contributing traits under water stressed conditions. These associations highlight the importance of functional mechanisms of intrinsic tolerance and cumulative traits for drought tolerance in maize.     

H3亚型禽流感病毒巢式PCR检测方法的建立

为建立一种H3亚型禽流感病毒(AIV)的检测方法,本研究针对H3亚型AIV HA基因保守序列,设计并筛选出2对特异性引物,通过优化反应条件,建立了H3亚型AIV巢式PCR检测方法。对该法进行特异性和敏感性检验,并用该法对96份临床样品进行检测。特异性试验结果表明该法只能检测到H3亚型AIV,对其他常见禽病病原体不扩增;敏感性试验结果表明该巢式PCR对H3亚型AIV检测下限为1×10³拷贝/μL,灵敏度比常规PCR高100倍;96份临床样品检测结果与病毒分离结果一致。本研究所建立的巢式PCR为H3亚型AIV的诊断提供了一种准确、有效的检测方法。     

基于PCR和巢式PCR技术的玉米南方锈病早期检测

【目的】建立巢式PCR方法,快速检测处于病害潜育期的玉米叶片内的多堆柄锈菌(Puccinia polysora),为玉米南方锈病(southern corn rust)的预测和防治提供技术支持。【方法】根据多堆柄锈菌ITS区序列,在其变异区设计3条多堆柄锈菌特异性检测引物NX471-F、NX255-F和NX255-R,建立以NX471-F与真菌ITS区通用引物ITS4为外侧引物,NX255-F/NX255-R为内侧引物的巢式PCR。采用20μL扩增体系：Ex Taq DNA聚合酶(5 U·μL^-1)0.15μL、10×Ex Taq Buffer(Mg²⁺plus)2μL,d NTP Mixture(各2.5 mmol·L^-1)1.6μL,上下游引物(10μmol·L^-1)各0.3μL,DNA 1μL,14.65μL ddH₂O。扩增程序：利用外侧引物NX471-F/ITS4进行第一步PCR扩增,95℃预变性7 min;95℃变性30 s,55℃退火30 s,72℃延伸45 s,...     

小陇山林区天然林森林调查最小取样面积研究

【目的】分析森林结构参数与取样面积之间的关系,确定评价天然林状态特征的最小取样面积,为指导天然林生态监测与经营提供理论依据。【方法】以小陇山林区天然林作为研究对象,选取3块面积为1hm2的天然林固定监测样地,采用巢式取样法,按照10m×10m,20m×20m,…,90m×90m的嵌套方式设置取样面积,分析树种数、树种多样性(Shannon-Wiener指数)、林木株数、胸高断面积、树种隔离程度(混交度)、林木分布格局(角尺度)与取样面积之间的关系,采用对数模型、幂函数模型和逻辑斯蒂模型,分别对样地树种-面积、Shannon-Wiener指数-面积进行拟合。【结果】树种数、树种多样性与取样面积的曲线特征均为初期急剧上升,而后逐渐趋于平缓,曲线拐点出现在取样面积1 600和2 500m2处。树种-面积模型和Shannon-Wiener指数-面积模型均以逻辑斯蒂模型为最优。不同取样面积下每公顷林木株数和胸高断面积的估算值与实测值比较结果表明,当3块样地取样面积增大到1 600m2时,每公顷林木株数估算值与实测值的误差均降至10%以下;1、2号样地取样面积增大到1 600m2、3号样地取样面积增大到2 500m2时,每公顷胸高断面积估算值与实测值的误差均降至10%以下。混交度分析表明,当取样面积较小时,混交度各频率分布表现为无规律,3块样地平均混交度趋于稳定的取样面积依次为1 600,2 500和900m2。角尺度分析表明,1号样地中各取样面积的平均角尺度均表明林地中林木为聚集分布,但当取样面积2 500m2时平均角尺度上下波动较大;2号样地中当取样面积≥900m2时平均角尺度趋于稳定,林地中林木表现为聚集分布;3号样地中当取样面积≥2 500m2时平均角尺度趋于稳定,林地中林木表现为随机分布。【结论】当取样面积达到2 500m2时,各参数所表现的规律均趋于稳定,因此,在小陇山林区研究天然林结构特征的最小取样面积应不小于2 500m2。     

核桃枝枯病小新壳梭孢巢式PCR检测方法的建立

近年来,小新壳梭孢Neofusicoccum parvum导致的核桃枝枯病屡有报道,该病防治困难,发病率高,是核桃的一种灾难性病害。为建立N.parvum的快速、灵敏的分子检测体系,根据N.parvum的几丁质合酶1基因(CHS1)及其前后100bp序列,分别设计了两对特异性引物CT-WK3-S/CT-WK3-A和CT-N-S/CT-N-A,建立了N.parvum的巢式PCR检测方法。结果表明,建立的体系可以从不同来源的5个N.parvum菌株中特异性地扩增出97bp的目的条带,灵敏度达30fg/μL,是常规PCR的10倍,而从其近缘种葡萄座腔菌属Botryosphaeriasp.的病原菌及其他供试菌株均未扩增出任何条带。以感染N.parvum的核桃组织总DNA为模板进行检测,可以在发病早期检测出N.parvum的存在。本研究建立的巢式PCR体系可以为核桃枝枯病的田间检测提供思路。