Molecular investigation on the presence of canine parvovirus in Egypt

Canine parvovirus type 2 (CPV-2) causes a highly contagious gastroenteritis disease of dogs and wild canids. To investigate the CPV-2 prevalence in Dakahlia Governorate, Egypt, a total of 50 fecal swabs were collected from suspected diseased dogs during 2016–2017. Out of 50 collected samples, 35 samples (70 %) presented positive results for CPV-2 using immuno-chromatography (IC) as a rapid test. CPV-2DNA was detected in 42 samples (84 %) by using polymerase chain reaction (PCR). The frequencies of CPV-2 were significantly higher in German shepherd breed (46 %; 23/50) and in age groups less than 6 months (76%; 38/50). We evaluated the breed, age, sex, rapid test results and clinical signs as predictors for classification of animal status into infected and not infected. The best predictors for classification process were rapid test result and clinical signs. Both CPV-2b and CPV-2c subtypes were detected by CPV2-VP2 gene sequences analysis. Deduced amino acid sequences alignment showed substitutions at 3 sites (Arg453Pro, Ala574Glu and Gln457Leu). Further investigations are needed to reveal the genetic and antigenic relation between field and vaccinal strains of CPV-2 in Egypt.     

小麦Glu-3位点基因拷贝数的变异分析

【目的】基因拷贝数变异是一种常见又重要的基因结构变异,往往影响个体表型。低分子量麦谷蛋白（low-molecular-weight glutenin subunit,LMW-GS）是小麦贮藏蛋白的主要组成部分,位于Glu-3位点。小麦作为异源六倍体,其庞大且复杂的基因组结构导致难以利用传统方法检测目的基因的拷贝数,针对小麦基因组,筛选可靠稳定的内参基因和体系,探索适合复杂基因组的拷贝数变异测定技术,测定Glu-3位点LWM-GS基因拷贝数。【方法】以Acc1为内参基因,根据基因序列设计内参引物和探针,通过定性和定量PCR测定内参基因在12个普通小麦品种中的拷贝数,分析该基因拷贝数在不同品种间的稳定性;又以小麦品种篙优2018的5个稀释浓度的基因组DNA为模板,利用qRT-PCR验证Acc1内参系统的重复性和准确性;根据Glu-A3位点LMW-GS基因序列设计特异性引物及探针,利用qRT-PCR和ddPCR 2种方法检测8个小麦品种Glu-A3位点基因拷贝数,比较后选择更优的高通量基因拷贝数检测方法;再根据Glu-B3和Glu-D3位点LMW-GS基因序列设计相应的特异性引物及探针,并利用ddPCR技术检测和分析了231份小麦品种的Glu-A3、Glu-B3和Glu-D3位点上LMW-GS基因拷贝数。【结果】Acc1在12个普通小麦品种间、同一品种5个DNA稀释浓度间的拷贝数测定结果一致,技术重复间的变异系数仅为0.07%—0.77%,所构建的Acc1内参系统稳定;比较qRT-PCR和ddPCR 2种拷贝数检测方法,8个品种所测的Glu-A3位点拷贝数结果一致,分别为3、5、3、4、3、3、3和3;且ddPCR检测重复间的变异系数为0.30%—1.67%,远低于qRT-PCR的3.14%—12.72%,更加可靠;利用ddPCR对231份普通小麦品种的Glu-A3、Glu-B3和Glu-D3位点上LMW-GS基因拷贝检测后分析发现,大多数小麦品种在3个位点上的拷贝数为4,所占频率分别为51.95%、32.03%和28.57%,Glu-3位点总拷贝数变异范围为10—21,变异系数为16.12%。【结论】Acc1内参系统具有良好的稳定性和重复性,可以用作小麦Glu-3位点和其他目的基因拷贝数检测的内参;qRT-PCR和ddPCR均可用于小麦基因拷贝数的检测,但后者更稳定、可靠,且操作简单、检测通量高。     

施氮时期对葡萄叶片光合生理及内源激素水平的影响

以10 a生酿酒葡萄‘蛇龙珠’为试验材料，运用水肥一体化滴灌的方式分别在萌芽期（S1）、新梢旺长期（S2）、开花期（S3）、果实第一次膨大期（S4）和副梢生长旺期（S5）一次性施入300 kg·hm^-2尿素，对照为整个生育期均不施氮肥（CK），分别于花后50 d（DAF50）、花后85 d（DAF85）和花后120 d（DAF120）进行光合特性指标的测定，并采样分析氮肥施用时期对葡萄叶片光合生理及内源激素水平的影响。结果表明：各生育期施氮均能增加叶片净光合速率（Pn）及PSⅡ最大光化学效率（Fv/Fm），在花前施氮肥叶片Pn和Fv/Fm增加最为显著，最高分别达到18.22 μmol·m^-2·s^-1和0.854。S1、S2和S3处理显著增大了不同生育期叶片气孔导度（Gs）和蒸腾速率（Tr），且S2处理在DAF85时最显著，Gs和Tr分别比CK高18.5%和10.8%；S3、S4和S5处理显著降低了叶片的胞间CO₂浓度（Ci）和非光化学淬灭系数（NPQ），S1处理叶片Ci与CK之间无显著差异。果实第一次膨大期之前施入氮肥能够显著增大各生育期叶片的PSⅡ电子传递量子效率（ФPSⅡ）和光化学淬灭系数（qP），最高分别达到0.889和0.959。可见，不同时期施氮肥，通过改变光合荧光相关参数影响叶片光能的吸收、传递、耗散和分配，从而改善叶片光能利用效率。氮肥的施用时期影响各生育期叶片内源激素的含量，施氮肥均显著提高了DAF85后叶片玉米素（ZT）含量，与CK相比至少提高21.9%；花后施入氮肥叶片中IAA含量在果实采收时保持在34.9 μg·g^-1以上，S4和S2处理GA₃含量最高，分别为7.11 μg·g^-1和6.49 μg·g^-1。因此，氮肥施用时期影响葡萄各生育期不同内源激素的积累。     

Assessing the effects of vegetation and precipitation on soil erosion in the Three-River Headwaters Region of the Qinghai-Tibet Plateau,China

Soil erosion in the Three-River Headwaters Region (TRHR) of the Qinghai-Tibet Plateau in China has a significant impact on local economic development and ecological environment. Vegetation and precipitation are considered to be the main factors for the variation in soil erosion. However, it is a big challenge to analyze the impacts of precipitation and vegetation respectively as well as their combined effects on soil erosion from the pixel scale. To assess the influences of vegetation and precipitation on the variation of soil erosion from 2005 to 2015, we employed the Revised Universal Soil Loss Equation (RUSLE) model to evaluate soil erosion in the TRHR, and then developed a method using the Logarithmic Mean Divisia Index model (LMDI) which can exponentially decompose the influencing factors, to calculate the contribution values of the vegetation cover factor (C factor) and the rainfall erosivity factor (R factor) to the variation of soil erosion from the pixel scale. In general, soil erosion in the TRHR was alleviated from 2005 to 2015, of which about 54.95% of the area where soil erosion decreased was caused by the combined effects of the C factor and the R factor, and 41.31% was caused by the change in the R factor. There were relatively few areas with increased soil erosion modulus, of which 64.10% of the area where soil erosion increased was caused by the change in the C factor, and 23.88% was caused by the combined effects of the C factor and the R factor. Therefore, the combined effects of the C factor and the R factor were regarded as the main driving force for the decrease of soil erosion, while the C factor was the dominant factor for the increase of soil erosion. The area with decreased soil erosion caused by the C factor (12.10×10³ km²) was larger than the area with increased soil erosion caused by the C factor (8.30×10³ km²), which indicated that vegetation had a positive effect on soil erosion. This study generally put forward a new method for quantitative assessment of the impacts of the influencing factors on soil erosion, and also provided a scientific basis for the regional control of soil erosion.     

Serologic and molecular survey of horses to Coxiella burnetii in East of Iran a highly endemic area

There are few reports about Q fever in horse populations worldwide. This study aimed to detect the C. burnetii infection by serologic and molecular confirmation using commercial ELISA kit and real-time PCR in the East of Iran a region highly endemic. A total of 177 blood samples and 115 vaginal swabs were randomly collected from horses in East of Iran. The sera samples were analyzed for anti C.burnetii Ig G antibodies by a commercial ELISA kit and nucleic acid extraxted from vaginal samples were used to determine the C. burnetii DNA by real-time PCR assay. Antibodies were detected in 5.64 % (10/177) of sera samples and C. burnetii DNA was detected in 7.82 % (9/115) of horse vaginal samples. There was no significant difference in seroprevalence in different sex, age and breed groups. Our study showed that horses could be considered as a mild potential reservoir of C. burnetii which may be effective on horse health status. However, additional studies are needed to assess whether the horse could be considered as a relevant transmission risk indicator for Q fever.     

牛星状病毒EvaGreen实时荧光定量PCR检测方法的建立及应用

牛星状病毒(BAstV)是我国新发的犊牛腹泻病原,本试验的目的是建立检测BAstV的Real-time PCR方法。根据BAstV流行株的ORF1a基因序列设计引物,通过优化反应条件和体系,成功建立基于EvaGreen检测BAstV的Real-time PCR方法。结果表明,该检测方法的Ct值与标准品模板在1.36×10¹~1.36×10⁸拷贝/μL线性关系良好,相关系数R²=0.999,扩增效率为93.79%;该方法可特异性检出BAstV,对犊牛腹泻其他相关病原呈阴性;最低检测下限为13.6拷贝/μL;批间和批内的变异系数均小于2%,重复性好。对2017年9月至2019年5月采自河南省的221份犊牛腹泻样本进行检测,BAstV的检出率为18.1%(40/221),采样场阳性率为100.0%(14/14)。本试验所建方法灵敏度高、特异性强、稳定性好,为BAstV的检测和流行病学调查提供了有力手段。     

山羊TNNT3基因可变剪切及其对骨骼肌细胞分化的作用

【目的】快速骨骼肌肌钙蛋白T（fast skeletal troponin T3,TNNT3）作为肌钙蛋白(troponin, Tn)家族成员,调节横纹肌收缩、参与骨骼肌的生长发育并影响家畜肉质性状。通过获得山羊TNNT3基因的可变剪切体,分析山羊TNNT3基因可变剪切的表达模式及其在肌细胞分化中的作用,深入解析TNNT3基因在山羊骨骼肌生长发育过程中的作用机制。【方法】基于NCBI已公布山羊TNNT3基因（NM_001314210.1）和牛TNNT3基因（XM_010821200）mRNA序列,使用软件Primer Premier 6.0设计引物,以简州大耳羊胚胎期和出生后7个阶段骨骼肌为试验材料,克隆测序获得山羊TNNT3基因的CDS区可变剪切体,利用软件ORF Finder、EditSeq、DNAMAN、ClustalW和MEGA_X_10.1.8等对序列进行生物信息学分析;进一步设计实时荧光定量（real-time PCR,RT-qPCR）及半定量引物,研究TNNT3基因剪切体在7个不同组织（背最长肌（longissimus dorsi muscle,LD）、半膜肌（semimembranosus muscle,SM）、心、肝、脾、肺、肾）和7个发育阶段（胚胎期E75、E90、E105和出生后B3、B45、B150、B300）肌肉组织（背最长肌和半膜肌）中表达模式;此外,对转录本TNNT3_3进行体外编码能力检测确定其具有编码蛋白的能力,并在山羊骨骼肌卫星细胞（skeletal muscle satellite cells,MuSCs）中过表达,观察细胞形态变化以及检测标志基因的表达变化,研究其对山羊MuSCs分化的作用。【结果】①TNNT3（NM_001314210.1）CDS区全序列主要含有18个外显子,其中外显子16/17相互排斥,转录后单一表达。克隆发现山羊TNNT3基因 5个新转录本（TNNT3_1—5）,其外显子数分别是15、15、20、16、14。②生物信息学分析结果显示山羊TNNT3基因核苷酸序列和氨基酸序列与绵羊、牛、猪等哺乳动物具有很高的一致性,而与鱼类和爬行类动物的一致性较低,说明TNNT3基因序列在哺乳动物高度保守。③TNNT3 mRNA在背最长肌、半膜肌、心、肝、脾、肺、肾7个组织中都有表达,其中在骨骼肌中高度富集(P < 0.01),心脏及肺次之,其余组织中较低;TNNT3 mRNA在背最长肌和半膜肌中的表达始终处于一个动态变化中,胚胎期TNNT3在半膜肌的表达量高于背最长肌（P<0.05）;出生后则背最长肌中高于半膜肌（P<0.05）。④山羊TNNT3基因转录本TNNT3_3重复出现保守的外显子9—11（138bp）,体外翻译实验显示其可编码蛋白且蛋白大小与预期基本相符（37 kD）;相较于对照组,在山羊MuSCs中过表达该转录本使肌分化标志基因Myomaker、MyoG和MyH4 mRNA极显著升高(P < 0.01)。【结论】获得了山羊TNNT3基因具有完整CDS区5个新可变剪切体,TNNT3主要在肌肉组织（背最长肌和半膜肌）中高表达,在哺乳动物中高度保守且促进成肌分化。初步表明TNNT3基因在动物肌肉生长发育中具有重要的生物学功能。     

长期施肥处理对双季晚稻叶绿素荧光特征及籽粒产量的影响

研究施肥模式对水稻叶绿素含量及荧光参数的调节作用,对提高水稻光能利用效率及籽粒产量具有重要意义。本研究以水稻品种赣929为试验材料,比较长期定位施磷钾肥(PK)、施氮磷钾肥(NPK)、等养分条件下70%氮磷钾肥配合施用30%有机肥(70F+30M)、50%氮磷钾肥配合施用50%有机肥(50F+50M)、30%氮磷钾肥配合施用70%有机肥(30F+70M)条件下水稻叶绿素荧光参数和产量变化特征。结果表明,水稻旗叶叶绿素含量从齐穗期到成熟期呈下降趋势,且表现为30F+70M50F+50M70F+30MNPKPK。水稻叶绿素荧光参数Fv/Fm随水稻生育期的推进呈降低趋势,其中PK处理Fv/Fm值最低,而施氮处理比不施氮处理Fv/Fm值增加了2.85%～4.18%。叶绿素荧光参数qL、ΦPSII在NPK处理中表现为齐穗期较高, 20 d之后显著降低;在50F+50M、30F+70M处理中表现为齐穗期较低, 20 d以后显著增加;在PK处理、70F+30M处理中一直处于较高水平。NPQ变化趋势与qL基本相反。ETR-PAR光响应曲线拟合结果表明, 70F+30M处理ETRmax、?和Ek值在齐穗期和20 d后值均最高。综合看来,等养分条件下配施30%有机肥具有最优的叶绿素荧光指标组合及籽粒产量;而配施超过50%有机肥由于前期热耗散增大使得用于光合作用的光能份额减少,而施用氮磷钾肥处理则由于后期的衰老使得光能利用效率下降。     

施氮量对柳枝稷叶片叶绿素荧光特性及干物质积累的影响

研究施氮量对柳枝稷叶绿素荧光特性及干物质积累的影响,对提高盐碱地柳枝稷光能利用效率及生物质产量具有重要意义。在宁夏银北地区采用大田试验,以Cave-in-Rock品种柳枝稷为供试材料,在无氮添加(0 kg·hm^-2,N₀)、施低氮(60 kg·hm^-2,N₆₀)、中氮(120 kg·hm^-2,N₁₂₀)和高氮(240 kg·hm^-2,N₂₄₀)共4个施肥水平下,比较了开花期和成熟期柳枝稷叶片叶绿素荧光参数和干物质积累动态变化,采用灰色关联度分析法对柳枝稷叶片叶绿素荧光参数与干物质积累量进行研究。结果表明:与N₀相比,在N₆₀、N₁₂₀和N₂₄₀处理下柳枝稷开花期和成熟期叶片PSⅡ最大光化学效率(F_v/F_m)、PSⅡ实际光化学量子效率(Φ_PSⅡ)、潜在活性(F_v/F_o)、光化学猝灭系数(qP)、非光化学猝灭系数(NPQ)和干物质积累量均显著性提高,在N₂₄₀处理下达到峰值,而热耗散量子比率(F_o/F_m)显著性降低。在柳枝稷开花期,随着施氮量的增加,柳枝稷叶片PSⅡ潜在活性(F_v/F_o)显现出先上升后逐渐下降的总趋势。在N₁₂₀处理下柳枝稷叶片PSⅡ潜在活性(F_v/F_o)最大,为3.13,较N₀处理显著提高了16.26%。柳枝稷干物质积累量随着生育时期的推进和施氮量的增加,均有不同程度的提高,其中在开花期至成熟期,干物质积累缓慢,在灌浆期达到最大,成熟后期略有降低。柳枝稷成熟期干物质积累量在N₂₄₀处理下最高,每穴干物质积累达378.13 g,较N₀、N₆₀和N₁₂₀处理显著提高了24.33%、20.09%和7.24%。灰色关联度分析结果表明,在本试验条件范围内,N₂₄₀处理下加权关联度与理想施肥水平的关联度最大,有利于促进PSⅡ的光化学活性,从而提高柳枝稷干物质积累量。