美人蕉黄斑驳病毒巢式PCR检测方法的建立

本文以我国台湾进境美人蕉病株为材料,根据已报道的美人蕉黄斑驳病毒Canna yellow mottle virus(CaYMV)基因序列设计2对特异性引物(外侧引物1对、内侧引物1对),建立了巢式PCR快速检测CaYMV的方法,并对进境的50份美人蕉样品进行了检测。结果显示,该方法特异性强,且灵敏度高于常规PCR,是常规PCR的1 000倍,表明该方法能够实现对CaYMV的快速、准确、灵敏检测,适用于口岸快速检测CaYMV。     

Size distribution approaches for monitoring and conservation of coastal Cymodocea habitats

• 1. Cymodocea nodosa's leaf length distribution was studied as an easily measurable indicator to monitor and conserve Macedonian, North Aegean, Greek coastal habitats.
• 2. Three Cymodocea meadows off the eastern Kavala Gulf coast (Nea Karvali, Erateino, Agiasma), with that of Nea Karvali close to an industrial area being the most degraded, were sampled during the seagrass main growing season in July 2004. Two further meadows, one pristine to less degraded (Brasidas, Gulf of Kavala) and one degraded (Biamyl, Inner Thessaloniki Gulf), were sampled as benchmarks in July 2005. The results were evaluated using Gaussian fit curves, and non‐parametric and nested parametric ANOVA on a hierarchy of spatial scales: area (tens of metres), site (hundreds of metres) and meadow (kilometres).
• 3. Frequency (%) distribution of leaf length values and CymoSkew index variation were best associated with anthropogenic stress. Frequency (%) distribution of adult and intermediate photosynthetic leaf length values revealed a unimodal distribution possible to be fitted, at least at pristine to less degraded meadows, by normal distribution (R²>0.5).
• 4. Statistically significant variation was estimated for CymoSkew index, a quantitative expression of leaf length asymmetry, on the meadow scale (P<0.001). Biamyl (3.82) and Nea Karvali (3.64) were indicated as heavily degraded meadows, Erateino (2.93) as a degraded meadow, Agiasma (2.18) as a meadow with the first signs of degradation, and Brasidas (1.68) as a pristine to less degraded meadow. These results in combination with other meadow specific biotic parameters were used to suggest a preliminary angiosperm ‘Ecological Status Classes’ classification scheme useful for the implementation of WFD in the north Aegean Sea.
• 5. The CymoSkew index seems to respond to lower levels of stress than is needed for other more conservative plant modules and therefore, could be regarded as an early warning indicator of Cymodocea habitat degradation. Copyright © 2009 John Wiley & Sons, Ltd.

     

柑橘黄龙病菌亚洲种分子检测引物评价及绝对定量PCR体系优化

柑橘黄龙病是对柑橘产业最具毁灭性的病害, 目前没有可用的有效药剂和抗病品种, 分子检测对黄龙病有效防控至关重要。本研究对国内外常用的常规PCR和巢式PCR检测引物进行评价, 针对多拷贝的nrdB和16S rDNA基因, 构建质粒标准品并筛选适用于绝对定量PCR的最佳质粒。结果表明, 使用Es Taq MasterMix对感染 Candidatus Liberibacter asiaticus (CLas)的柑橘样品进行常规PCR检测时, 在评价的16对引物中, OI1/OI2c、Las606/LSS和HLBF468/R877灵敏度最高, 推荐同时使用检测黄龙病菌含量低的样品;各组巢式PCR检测引物有其适用扩增体系, 部分引物用Es Taq MasterMix扩增时出现非特异性扩增, F1/B1→F3/B3则适用Es Taq MasterMix体系, 且最高可稳定特异检出105倍稀释感染CLas柑橘总DNA样品（2×10-3 ng/μL）, 是灵敏度最高的引物组, OI1/OI2c→S3/S4在Es Taq MasterMix和Ex Taq DNA聚合酶体系中均可稳定特异检出104倍稀释感染CLas柑橘总DNA样品（2×10-2 ng/μL）, 是适用扩增体系最广的引物组;构建的5个绝对定量PCR质粒标准品中, pnrdB83扩增效率最接近100%, 且在2次重复试验中波动最小, 稳定性最强, 并且作为标准品对黄龙病待测样品进行绝对定量时, 各样品在2次重复试验中的拷贝数差值最小, 是本研究筛选的最佳质粒。本研究的结果将为柑橘黄龙病菌的定性和定量分子检测提供参考。     

压砂地土壤盐分演替特征研究

针对西北旱区特有的压砂地,分析了不同种植年限压砂地土壤盐分演替特征,结果表明,裸地及新、中、老压砂地土壤盐分平均相对偏差介于-15.43%~28.14%之间,标准差介于4.44%~31.02%之间,变化范围较小,Spearman秩相关系数较大且显著相关,通过时间稳定性可初步确定研究区土壤盐分均值的代表性测点。盐分均值与代表测点值相关系数的变化范围为0.762~0.952,标准误差及平均偏差均较小,代表测点的土壤盐分与区域平均值相关性较高,差异性较小。对新砂地表层土壤盐分进行时空模拟,各时段土壤盐分累计分布曲线变化规律相似,模拟值与实测值略有差异,其空间分布基本趋势与实测数据相符。各时段土壤盐分均属于中等弱变异性,盐渍化程度表现为裸地老砂地新砂地中砂地,压砂地土壤盐分时刻都在发生着演替,中砂地土壤盐渍化程度最小,但伴随着种植时间的延长和覆盖砂石土砂比的增加,老砂地土壤盐渍化呈加剧趋势。     

不同覆盖措施对旱区农田土壤酶活性及西瓜产量的影响

基于大田试验,研究了砂石+地膜覆盖,砂石覆盖,地膜覆盖3种旱区农田地表不同覆盖措施下的土壤酶活性及西瓜产量,以农田不覆盖（CK）为对照。结果表明：砂石+地膜覆盖在西瓜不同生育时期和不同土壤层次由于土壤水分条件的改善,可显著提高土壤酶活性,其中土壤脲酶活性提高了34.5%,碱性磷酸酶提高了13.0%,过氧化氢酶提高了29.5%,砂石覆盖次之（分别提高了31.4%,11.5%, 25.5%）,地膜覆膜处理虽然也有提高土壤三种酶活性的效果,但作用甚微（仅0.9%,1.4%,2.9%）。对西瓜产量及商品率的影响,砂石+地膜覆盖、砂石覆盖处理与对照相比增产效果显著,分别增产50倍和30倍,地膜覆盖与对照相比增产7倍,但是商品率较低（2.16%）。表明在当地旱区农业生产中,砂石+地膜覆盖或砂石覆盖是抗旱、增产增收的有效措施。     

巢式PCR和SYBR Green Ⅰ实时PCR检测转基因大豆方法的研究

为了建立转基因大豆检测技术,采用巢式PCR和SYBR Green Ⅰ实时PCR技术,检测转基因大豆外源基因(CaMV35S、CP4EPSPS)。结果表明,利用巢式PCR可检测出1 ng/μL转基因含量1%的大豆中的CaMV35S基因,而第一轮PCR的检测限为100 ng/μL;利用SYBR Green Ⅰ染料能结合双链DNA的特点,应用实时PCR技术可检测到CaMV35S、CP4EPSPS基因扩增所产生的信号,通过扩增产物的熔解曲线能有效地区分特异性产物,CaMV35S基因的检测限为0.1 ng/μL。同时利用该方法对黄豆、炒黄豆、豆干等样品进行检测,样品中未检出CaMV35S基因成分。巢式PCR方法明显提高了PCR的检测限,SYBR Green Ⅰ实时荧光PCR方法能有效、快速检测CaMV35S转基因成分。     

Methods for detecting the cassava bacterial blight pathogen: A practical approach for managing the disease

Cassava bacterial blight (CBB), caused by Xanthomonas axonopodis pv. manihotis (Xam), is a particularly destructive disease in South America and Africa. The movement of infected asymptomatic stems is a major means of pathogen dispersal as well as infected seeds. The success of a cassava-seed certification program depends on the availability of reliable tests to detect the pathogen in vegetative planting materials and true seeds. We report here the different methods that permitted to detect the pathogen in cassava tissues. A polymerase chain reaction (PCR) test was developed for this pathogen. The PCR assay worked well for pathogen detection in extracts from leaf and stem lesions and the minimum number of cells that could be detected ranged from 3 × 10² to 10⁴ CFU per ml. Nested-PCR worked well for Xam detection from naturally infected seeds. This technique was specific, sensitive, and rapid for detecting Xam in cassava true seeds. The highest detection level found was 1–2 viable cells per reaction. A dot-blot assay was developed by evaluating a 898 bp DNA fragment unique to Xam strains as a diagnostic DNA probe. The probe detected Xam strains in crude extracts of leaf and stem lesions, cassava fruits and sexual seeds that were naturally infected. Overall sensitivity of the dot-blot method was about10³CFU per reaction. The dot-blot hybridization technique can be easily used for culture indexing. A monoclonal antibody (MAb) was also used for an enzyme-linked immunosorbent assay (ELISA) and tested with various infected tissues. Overall sensitivity of the method was about10³CFU per reaction. This revised version was published online in July 2006 with corrections to the Cover Date.     

两轮多重反转录巢式PCR方法扩增全长GalUR基因

研究了两轮多重反转录巢式PCR扩增全长基因的方法.先用多重巢式引物和cDNA进行第1轮PCR扩增;再用纯化的第1轮PCR产物和相同的引物进行第2轮扩增;最后通过改变Mg^2＋浓度、退火时间和退火温度进行PCR条件的优化,以增加PCR扩增的产量,用于克隆.结果显示该方法方便和快速地扩增出了草莓（Fragaria ananassa cv.SweatCharlie）高产量和高质量的全长GalUR基因,也非常快速地扩增出了用于基因构建的带有酶切位点的全长GalUR基因.该方法也可以用于其它全长基因的克隆.     

How representatively can we sample soil mineral nitrogen?

Spatial distribution of soil mineral‐N content (N_min) is a scale‐variant process. Precision farming assumes knowledge about the spatial distribution of N_min. Moreover, sampling in management zones is based on the assumption of spatial dependence between sampling points. In the present study, variability structure of N_min and the sources of variability were investigated. Within an agricultural landscape, N_min was investigated across a field in a nested design over four consecutive years. Temporally unstable structure of individual nests require a sampling with several nests in the field. In the investigated field, 35%–49% of the total variability derived from small‐scale variability observed at spatial distances of <5 m and from sampling and analytical errors. Differences between 10 and 26 kg N ha^–1 for the soil depth increment 0–60 cm can be expected. Uncertainty due to analytical errors were in the order of 5–10 kg N ha^–1 for a 0–60 cm layer.     

三重巢式PCR技术检测抗草甘膦转基因大豆深加工产品

实验以7种具有代表性的含有大豆成分的深加工产品(卵磷脂、大豆蛋白质粉、巧克力饮品、婴儿米粉、大豆粗油、大豆精炼油和大豆色拉油)作为样品进行定性检测，第一轮三重PCR均未能扩增出任何大豆成分，其灵敏度为0.5％。第二轮三重PCR可以扩增出除大豆精炼油和色拉油以外的所有深加工产品的内源基因Lectin、35S-CTP和EPSPS-NOS基因，其检测灵敏度达到0.005％，结果提示三重巢式PCR检测方法适用于大豆深加工产品的定性检测。