Detection of Colletotrichum coccodes from soil and potato tubers by conventional and quantitative real-time PCR

Colletotrichum coccodes is the causal agent of the potato blemish disease black dot. Two PCR primer sets were designed to sequences of the ribosomal internal transcribed spacer (ITS1 and ITS2) regions for use in a nested PCR. The genus-specific outer primers (Cc1F1/Cc2R1) were designed to regions common to Colletotrichum spp., and the species-specific nested primers (Cc1NF1/Cc2NR1) were designed to sequences unique to C . coccodes . The primer sets amplified single products of 447 bp (Cc1F1/Cc2R1) and 349 bp (Cc1NF1/Cc2NR1) with DNA extracted from 33 European and North American isolates of C. coccodes. The specificity of primers Cc1NF1/Cc2NR1 was confirmed by the absence of amplified product with DNA of other species representing the six phylogenetic groups of the genus Colletotrichum and 46 other eukaryotic and prokaryotic plant pathogenic species. A rapid procedure for the direct extraction of DNA from soil and potato tubers was used to verify the PCR assay for detecting C. coccodes in environmental samples. The limit of sensitivity of PCR for the specific detection of C. coccodes when inoculum was added to soils was 3·0 spores per g, or the equivalent of 0·06 microsclerotia per g soil, the lowest level of inoculum tested. Colletotrichum coccodes was also detected by PCR in naturally infested soil and from both potato peel and peel extract from infected and apparently healthy tubers. Specific primers and a TaqMan fluorogenic probe were designed to perform quantitative real-time (TaqMan) PCR to obtain the same levels of sensitivity for detection of C. coccodes in soil and tubers during a first-round PCR as with conventional nested PCR and gel electrophoresis. This rapid and quantitative PCR diagnostic assay allows an accurate estimation of tuber and soil contamination by C. coccodes .     

Detection of Colletotrichum coccodes and Helminthosporium solani in soils by bioassay

The sensitivity of a bioassay in detecting soil inoculum of Colletotrichum coccodes and Helminthosporium solani was examined using potato minitubers and microplants. Tests were conducted on soils which were collected from fields in which the interval after a previous potato crop differed, and which were also artificially infested with conidia or microsclerotia. For C. coccodes , determining plant infection based on the occurrence of infected roots after 9–12 weeks was a sensitive method for detecting and quantifying the amount of inoculum in soil. Infestations of less than 0·4 microsclerotia per g soil were detected in artificially infested soils. A semiselective medium, developed for isolating C. gloeosporioides from pepper, detected soil infestations by C. coccodes as low as nine conidia or one microsclerotium per g soil in artificially infested soil. For H. solani , infection on minitubers was a sensitive measure, with soil inoculum of fewer than 10 conidia per g soil being detected. Soil infestation could be quantified by assessing the percentage surface area of minitubers covered by sporulating lesions, which was strongly related to the amount of soil infestation. The results of these bioassay tests were compared with published results for real-time quantitative PCR assays on the same soils. The two methods were in good agreement in artificially infested soils, but the bioassay appeared to be more sensitive with naturally infested soils.     

行走式自动点灌机的设计分析及应用

从改变点灌机整机的传动原理入手，使整机结构简单、布置合理、造价低廉；将所需要的灌水排量变为可调式，使水能及时、准确地注入穴位；简化了直流永磁电机的制作工艺，从上述诸方面阐述了行走式自动点灌机结构的设计特点，简述了该机的效益及应用前景，并对今后进一步的研究提出了建议。     

地高辛标记cDNA探针检测苹果茎痘病毒

 Partial sequence(314 bp) of ASPV was cloned and used as a probe labelled with digoxigenin-11dUTP. The total RNA extracted from samples with Apple stem pitting virus and a series of dilutions of plasmid with ASPV-cDNA were detected by dot blot hybridization. The results showed that the probe was sensitive and specific. The probe couldn't hybridize with total RNA of Apple stem grooving virus, Apple mosaic virus and Apple chlorotic leaf spot virus samples as well as negative control, only hybridized with that extracted from dormant shoot infected with ASPV. The sensitivity for detection of plasmid contained ASPV-cDNA was 1.64 μg.     

猪蛔虫感染期幼虫抑制消减cDNA文库的构建

为筛选与线虫感染性相关的基因,本研究以猪蛔虫为对象,构建猪蛔虫感染期幼虫差异表达消减cDNA文库,为研究线虫期特异性发育的分子机制奠定基础。分别提取感染期幼虫和其它各期幼虫及成虫的总RNA,纯化mRNA后,采用Clontech公司PCR-selectTM试剂盒进行反转录合成cDNA并进行抑制消减杂交(SSH),构建猪蛔虫感染期幼虫差异表达的消减cDNA文库,并采用Southern斑点杂交进行消减效率的检测。随机从文库中抽取45个克隆进行测序及在线BLAST分析。试验结果表明,感染期幼虫差异表达的消减cDNA文库具有较强的特异性;在得到的41个表达序列标签(ESTs)中,有40个ESTs与已报道的基因有较高的相似性,主要代表猪蛔虫第三期幼虫基因和成虫头部基因,有1个cDNA片段可能代表新基因。猪蛔虫感染期幼虫差异表达消减cDNA文库的成功构建,为进一步研究幼虫发育差异表达基因的功能奠定了基础。     

线粒体nad 6基因在油菜种子发育中的特异性表达

以油菜为材料，研究线粒体ｎａｄ６基因在种子发育中的表达。结果表明，ｎａｄ６基因在叶片中的表达很低，在种子发育过程中各阶段均有较高表达，并随种子发育进程而有加强的趋势。推测线粒体ｎａｄ６基因可能除与呼吸作用有关外还有其他功能     

Production and related variables in bovine leukaemia virus-infected cows

A newly developed milk dot blot test was used to detect anti-bovine leukaemia virus (BLV) antibody in milk samples from 2079 lactating adult cows from among 61 herds. The milk dot blot test was highly repeatable; the concordance rate, compared with the agar gel immunodiffusion test performed on serum, was 83.5%. All herds contained BLV-positive cows; the prevalence rate was 36%. BLV-positive cows tended to come from larger herds and were older and more often later in lactation. Fourteen production and related variables (herd size, age, days open, days in milk, milk somatic cell count, milk, fat, and protein produced in the current lactation, projected production of milk, fat, and protein, and breed class average deviations for milk, fat, and protein) were compared between BLV-positive and BLV-negative cows. Although somatic cell count, milk produced, and projected production of milk and protein were related significantly to BLV status using simple tests of association, once the variables herd size, age and days in milk were controlled, these differences were removed. Further analyses using logistic (outcome: individual cow BLV status) and least-squares regression (outcome:herd proportion of BLV-positive cows) failed to show an association between any of the measured production or related variables and BLV-positivity. We concluded that the effect of BLV on production and related variables in dairy cows was below the sensitivity of our analytical techniques or was non-existent.Abbreviations ABCA herd average breed class average for milk, fat, and protein production - AVGAGE average age of the herd - ADIM herd average for days in milk - AGID agar gel immunodiffusion - AVGSCC herd average milk somatic cell count - BCA breed class average, a milk, fat and protein production index calculated by comparing a cow's actual 305-day lactation production to the corresponding BCA standard for the same breed, age, and month of calving - BLV bovine leukaemia virus - CALVINT calving interval - COWAGE cow age - DBCA breed class average deviation for milk, fat, and protein production, the difference between an individual cow's BCA and the herd average - DIM days in milk - HS herd size corresponding to the number of lactating cows in a herd - LACT actual amount of milk, fat, and protein produced in a cow's lactation - ODHIC Ontario Dairy Herd Improvement Corporation - PCTPOS percentage of herd that is BLV-positive - PROJ projected 305-day production for milk, fat, and protein by fitting to a standard lactation curve adjusted for days in milk and age at calving - RHBCA rolling herd average for breed class average for milk, fat, and protein production, the average for all cows that completed a lactation (cows must have completed a 305-day lactation) during the previous 12 months - SCC milk somatic cell count     

兔防御素cDNA表达质粒的构建及其表达

将兔防御素（MCP－1）cDNA插入真核表达载体pcDNA3的EcorRⅠ和XbaⅠ酶切位点之间，构建了兔MCP－1cDNA的真核表达质粒pcDEF。通过脂质体转染，使兔MCP－1 cDNA在COS－7细胞中表达，在转染60、84、108h后，提取总RNA。采用RT－PCR，在288bp的位置扩增出1条特异性带；RNA斑点印迹杂交表明，兔MCP－1 cDNA在60、84、108h均有表达。     

Monosomic addition lines of Beta corolliflora in sugar beet: plant morphology and leaf spot resistance

Monosomic addition lines in Beta vulgaris from Beta corolliflora were described morphologically and characterized for disease resistance. Monosomic addition plants (2n= 19) were selected among segregating offspring by a squash dot technique in combination with B. corolliflora‐specific probes. Plants carrying an added chromosome were characterized by leaf shape, plant size and plant vigour. In this way, most addition lines could be distinguished from diploid beets, however, to identify those plants unequivocally, molecular marker analysis was also necessary. Transmission frequencies of each addition line were determined to be in the range 13.9% (Cor‐4) to 60% (Cor‐9). High transmission rate of addition line Cor‐9 was assumed to be due to apomictic propagation because transmission rate after selfing cannot exceed 50%. Cercospora leaf spot resistance tests were performed on 167 monosomic plants from seven different addition lines, two fragment addition lines and 89 diploid controls. No line exhibited complete resistance, but the monosomic additions Cor‐3 and Cor‐4 showed significantly lower infection rates than their diploid sibling plants. The identification of monosomic addition lines with apomictic and disease resistance characters offers the possibility of transferring those genes to sugar beet.     

双层量子点阵列浮栅结构纳米存储器

设计了一种新型的存储器结构单元——锗／硅双层量子点阵列浮栅结构纳米存储器．对存储器样品的C—V测量结果显示了这种结构的P沟道器件有着更加优异的存储性能．数值模拟表明了该器件的编程速度在微秒量级，而保留时间长达10年（约10^8S）．这种新型的存储器结构单元有效地解决了目前硅基纳米存储器存在的工作电压和长久存储之间的矛盾，为硅基纳米存储器的实用化拓宽了道路．