用实时荧光定量RT-PCR方法定量绵羊PrP基因的表达

为快速、准确定量绵羊PrP基因的mRNA,建立了绵羊PrP基因实时荧光定量聚合酶链反应检测方法。根据已报道的绵羊PrP基因序列,设计合成引物;采用RT-PCR方法扩增目的片段;构建标准重组质粒制备标准曲线,用于样品检测。结果发现,中枢神经系统组织PrP基因的表达量(copies/ng总RNA,39420)比外周组织(为7845)的高;在中枢神经系统中,脑干的PrP基因的表达量最高(为67020);外周器官中,淋巴结PrP基因的表达量最高(为29086),肾脏的表达量最低(为125)。建立绵羊PrP基因实时荧光定量PCR方法,对PCR扩增反应中每一个循环的产物进行定量分析,为进一步研究绵羊组织器官的PrP表达在传染性海绵状脑病发生中的作用提供基础数据。     

Ion-selective electrodes for quick on-farm determination of ammonium and potassium concentrations in pig slurry

Background

Pig slurries account for a large proportion of the organic manure available worldwide. Because of the large range of slurry compositions, farmers need fast and accurate alternatives to laboratory methods to assess nutrient concentrations in these farm-based fertilizers. In this way, nutrient emissions into the environment can be reduced and targeted nutrition of crops can be implemented, while mineral fertilizers can be saved.

Aim

The objective was to evaluate whether it is possible to rapidly determine ammonium and potassium concentrations in pig slurries on-farm using ion-selective electrodes (ISE) and to compare the measurement with established methods.

Methods

A total of 391 pig slurry samples were collected from farms in north-west Germany, and ammonium and potassium concentrations were analyzed in the laboratory. Then the composition of pig slurries was measured with ISE, the Quantofix-N-Volumeter (QV) and an electrical conductivity (EC) meter and regression models were used to evaluate the on-farm quick tests.

Results

For the 391 pig slurries studied, regressions with an R² of 0.92 for ammonium and 0.91 for potassium were achieved for concentrations measured with the ISE in comparison with laboratory analyzes. The model fits for the electrodes were significantly higher than those based on the EC for ammonium and potassium, and it was equivalent to the QV method for the assessment of the ammonium concentration.

Conclusion

Due to the simple and rapid measurement, ISE offer high potential for on-farm quantification of ammonium and potassium concentrations in pig slurries.     

Real-time PCR for the detection and quantitative analysis of IHNV in salmonids

The rapid identification and quantification of virus in diseased fish is a goal both conservationists and commercial aquaculturists have struggled to attain. Recently a technique for the detection of viral mRNA particles that uses fluorescent tagging and amplification has been developed. Utilizing primers and fluorescent labelled probes generated for the specific identification of the nucleocapsid (N) and glycoprotein (G) genes of infectious haematopoietic necrosis virus (IHNV), and an instrument that measures cyclic emittance of fluorescence, the presence or absence of virus can be easily and rapidly confirmed. This method is not only useful in confirming viral presence but is effective in measuring the relative or absolute quantity of virus present within the sample. This allows for the determination of the health status of a carrier fish by measuring the quantity of viral genomes or transcribed viral genes present. Because this method is based on sequence detection, instead of virus isolation in cell culture, it is also effective in determining the presence of pathogenic organisms from water, fish feeds, or other potential reservoirs of infection.     

高分子量麦谷蛋白亚基表达量与小麦加工品质性状的关系研究

 高分子量麦谷蛋白亚基表达量是影响小麦品质性状的重要原因之一。用251份我国秋播麦区主栽品种和高代品系研究了高分子量麦谷蛋白亚基表达量与全粉蛋白质含量、SDS沉降值、和面时间、耐揉性及弱化值的关系。结果表明,20亚基的表达量最高,而8亚基的表达量最低。高分子量麦谷蛋白亚基表达量与全粉蛋白质含量和SDS沉降值的相关系数较高,与和面时间、耐揉性及弱化值的相关系数较低。就单个亚基的表达量而言,5亚基的表达量与SDS沉降值、和面时间、耐揉性和弱化值的相关性较高,皆达1%的显著水平,相关系数分别为0.38,0.46,0.37和0.37,10亚基的表达量与全粉蛋白质含量、SDS沉降值、和面时间、耐揉性和弱化值的相关系数较小,分别为0.22,0.08,0.03,0.04和-0.03.     

意大利蜜蜂amLDH基因的克隆与表达分析

乳酸脱氢酶(LDH)是糖酵解和葡萄糖异生途径中一种发挥重要作用的蛋白酶。本研究以意大利蜜蜂为研究对象,RT-PCR和生物信息学方法克隆并分析了意大利蜜蜂乳酸脱氢酶基因及其表达产物的分子特征和结构功能,并利用qRT-PCR技术对amLDH基因在意大利蜜蜂不同级型、不同发育阶段的表达模式进行了分析。序列特征结果表明,意大利蜜蜂amLDH基因包含一个1 008 bp的开放阅读框,编码335个氨基酸,该氨基酸序列与中华蜜蜂高度相似(96.4%),并存在13个磷酸化位点、2个乳酸脱氢酶功能域,是一种稳定的疏水性蛋白。qRT-PCR结果表明,amLDH基因在不同级型、不同发育时期的表达差异显著(P<0.05)。工蜂卵期3日龄、雄蜂卵期3日龄、蜂王幼虫期4日龄表达最高,蜂王和雄蜂预蛹期至红眼蛹期表达均高于工蜂。本研究结果推测,amLDH基因在意大利蜜蜂的生长发育以及糖酵解中发挥重要作用,该研究结果能够为进一步探讨amLDH在意大利蜜蜂生殖发育中的功能提供理论依据。     

粮食安全视角下省际耕地生态补偿的标准量化与机制构建

为促进全国耕地利用布局优化,保障粮食安全与生态服务的持续供给,立足粮食安全视角,在厘清省际耕地生态补偿的理论逻辑和补偿标准的测算思路基础上,运用机会成本损失与生态外溢价值法对2019年受偿区粮食安全保障的耕地生态补偿标准进行了量化研究。结果表明:1)研究区有17个省份处于粮食安全保障的耕地生态赤字区(支付区),14个省份为盈余区(受偿区),后者在满足自身粮食需求后,平均约有超过30%的耕地承载的粮食产量和生态服务供给了各生态赤字区;2)受偿区的受偿标准总额约为24 673.4亿元,其中最高的3个省份是河南、山东、安徽,最低的3个省份是宁夏、甘肃、新疆。为此,提出要构建包括补偿主体—目标责任—补偿方式—资金来源构成的补偿机制和配套体系,进一步保障生态补偿的长效实施。     

一种基因编辑位点特异性PCR方法的开发和应用

为了对基因组编辑产品进行精准定性和定量检测，以水稻SP1 基因的编辑植株为材料，在编辑位点上下 游设计通用引物，在编辑位点处设计基因编辑位点特异性TaqMan探针，建立了编辑位点特异性PCR方法。利用该 方法可准确鉴定特异基因组编辑产品，检测灵敏度达到5~10拷贝，可在实时荧光PCR（qPCR）和微滴数字PCR （ddPCR）平台上对基因组编辑产品进行定量检测。由于数字PCR的微反应单元可消除野生型DNA对通用引物的 竞争性消耗，与qPCR的定量结果相比，ddPCR定量结果具有更高的定量准确性。     

Laboratory and clinical findings and their association with viral and proviral loads in cats naturally infected with feline leukemia virus

This study was conducted to correlate clinical, laboratory, and bone marrow (BM) changes in cats naturally infected with feline leukemia virus and their association with viral loads in blood and BM and proviral loads in BM. Cats were classified into five groups based on antigenemia, clinical and/or laboratory findings and viral/proviral loads, according to a prospective study: symptomatic progressive (GI); asymptomatic progressive (GII); regressive (GIII); unclassified (GIV); or healthy (GV). |Correlations between these five groups and viral/proviral loads were evaluated. High viral and proviral loads were detected in GI and GII and viral loads were significantly associated with laboratory signs. Proviral loads detected in BM were significantly lower in GIII and GIV. GI cats were more likely to develop hematopoietic disorders than those from the other groups. Hematological and clinical disorders and disease severity are related to higher viral blood and proviral BM loads.     

转基因玉米NK603转化体/zSSIIb内标基因二重微滴数字PCR方法的建立及应用

【目的】批准进口的转基因玉米NK603是中国转基因生物安全监管的主要对象。转基因安全监管需要标准物质和标准检测方法,建立转基因玉米NK603的数字PCR（dPCR）方法将为转基因玉米NK603的定量检测和标准物质研制提供精准测量技术。【方法】采用人工合成技术构建标准质粒分子pUC57-NK603;将NK603转化体与不同的玉米内标基因引物/探针一一组合,遴选与NK603转化体特异性PCR具有相同扩增能力的玉米内标基因PCR方法;设置二重微滴数字PCR（ddPCR）的退火温度梯度和引物/探针浓度梯度,优化二重ddPCR的反应体系和反应条件;用梯度稀释的标准质粒溶液作模板,考察二重ddPCR的检测极限、定量极限和动力学范围;将转基因玉米NK603种子粉末和非转基因玉米种子粉末混合,配制质量分数分别为100%、10%和6%的盲样,考察二重ddPCR的定量准确性。【结果】用标准质粒分子pUC57-NK603作为二重ddPCR的质控对照,通过考察二重ddPCR反应热图的微滴信号强度、阳性微滴与阴性微滴分辨率、雨滴数量、NK603转化体与内标基因拷贝数比值测量值与预期值的一致性,确定将NK603转化体特异性PCR方法与内标基因zSSIIb PCR方法组合,建立NK603/zSSIIb二重ddPCR方法。二重ddPCR反应体系中NK603转化体和zSSIIb内标基因的引物/探针浓度相同,均为400 nmol·L^-1/200 nmol·L^-1,在60℃退火延伸。NK603/zSSIIb二重ddPCR的检测极限是2 copies DNA模板,定量极限是48 copies DNA模板,动力学范围是10—60 000 copies DNA。应用NK603/zSSIIb二重ddPCR方法可准确定量玉米盲样中的NK603转化体含量,定值结果变异系数小于25%;dPCR定量结果与荧光定量PCR（qPCR）定量结果无显著差异,且具有更高的精确性。【结论】内标基因的选择会影响dPCR定量结果的准确性,在建立dPCR方法的过程中,要用具有准确量值的样品作为质控对照,评估内标准基因的适用性。以人工合成的标准质粒分子pUC57-NK603为质控对照,建立了NK603/zSSIIb二重ddPCR方法。应用建立的二重ddPCR定值方法进行标准物质的研制和定值,已成功研制出转基因玉米NK603有证标准物质。