凡纳滨对虾感染致急性肝胰腺坏死病副溶血弧菌(VpAHPND)的定量分析

对虾急性肝胰腺坏死病(Acute hepatopancreatic necrosis disease, AHPND)是由致AHPND副溶血弧菌(AHPND-causing Vibrio parahaemolyticus, VpAHPND)携带的pVA1-like质粒所表达的PirAVp和PirBVp毒力蛋白对对虾肝胰腺的急性毒性所致。本研究用2.19×105 CFU/ml VpAHPND分离株20130629002S01对凡纳滨对虾(Litopenaeus vannamei)进行浸泡感染,于感染后2~9 d采集对虾的肝胰腺、鳃、肠道、肌肉组织,采用实时荧光定量PCR方法,检测各组织中的pirAVp拷贝数。结果显示,感染后凡纳滨对虾各组织均能检测到pirAVp,其中,肝胰腺在感染后第4天达到峰值, 为8.71×104 copies/mg,而鳃、肌肉、肠道分别在第3、4、5天达到峰值,分别为9.08×103、2.59×104、5.76×104 copies/mg。早期感染鳃组织中先出现VpAHPND的富集,在高死亡发生期,VpAHPND数量在肝胰腺和肠道出现高峰,在死亡数量逐渐下降的后期,各组织的VpAHPND均快速下降,肠道、肝胰腺和肌肉中的VpAHPND水平趋于接近。对虾肝胰腺组织病理切片显示,同一时间有临床症状的病虾和濒死对虾相比,濒死对虾表现出更严重的AHPND病理特征,且二者的组织病理特征均随着感染时间的延长变得更为严重,但检测到的VpAHPND数量呈下降趋势。研究表明,在VpAHPND感染过程中,组织中的pirAVp基因数量不能代表对虾的发病程度,发病程度及组织病理严重的AHPND样品中VpAHPND的数量不一定处于高水平状态。     

合浦珠母贝实时定量PCR内参基因的稳定性比较

以合浦珠母贝（Pinctada fucata）为研究对象,应用实时荧光定量PCR技术检测了甘油醛-3-磷酸脱氢酶（GAPDH）、β-肌动蛋白（β-actin）和18S核糖体rRNA（18S rRNA）3个管家基因在合浦珠母贝不同组织、性腺发育时期、胚胎发育时期mRNA水平的表达情况。同时比较了BestKeeper、geNorm和NormFinde软件对试验数据处理的差异,从中选出合适的内参基因。结果表明β-actin在不同组织和胚胎发育不同阶段表达最稳定,18S rRNA在性腺发育不同时期表达最稳定。     

马驽巴贝斯虫PCR和qPCR检测方法的建立

马驽巴贝斯虫是一种寄生于马属动物的血液原虫,给马产业造成巨大的经济损失。本研究旨在建立两种高效、可靠的PCR诊断技术,为进一步提高口岸检疫工作效率奠定基础。根据马驽巴贝斯虫Bc48基因序列,设计了一对特异性引物和荧光探针,建立了PCR和qPCR检测方法,测试了两种方法的特异性、灵敏性、稳定性。结果显示：建立的两种方法均具有良好的特异性;PCR方法的最低检出限为4.04×10² copies/μL,qPCR方法的最低检出限为4.04×10¹ copies/μL;对33份马血样品进行检测,结果qPCR检出的阳性样品数为23份(69.7%),而PCR检出的阳性样品数为9份(27.3%)。表明两种方法均可成功用于马驽巴贝斯虫的检验,但是荧光定量PCR方法的灵敏度明显优于PCR方法。     

低温条件下日本医蛭各组织基因表达转录组测序及qPCR验证

为解析日本医蛭(Hirudo nippnica Whitman)繁殖的分子机理,研究不同.温度下饲养的日本医蛭各组织基因表达差异情况.利用转录组学测序检测不同温度饲养条件下日本医蛭肌肉、环带和头部组织的基因表达情况,筛选出11个差异表达基因,并对其进行qPCR验证.qPCR结果显示一种具有去饱和酶功能的膜蛋白编码基因G...     

Effect of nitrogen fertilization on Fusarium head blight in spring barley

Nitrogen fertilization has been shown to influence the occurrence and the impact of Fusarium head blight in wheat. It also plays a key role in adjusting barley quality to the requirements of the malting industry, implying specific contents of protein. The present study investigated the effect of nitrogen input on the incidence of relevant Fusarium species in spring barley under field and greenhouse conditions. Grain material from differently fertilized field plots was analyzed for fungal DNA and mycotoxins by qPCR and LC-MS/MS, respectively. Under natural pathogen pressure no effect of nitrogen on infection was observed. When pathogen pressure (Fusarium culmorum and Fusarium avenaceum) was increased via species-specific soil-surface inoculation, nitrogen application reduced contents of Fusarium DNA and mycotoxins in barley grain. Nitrogen-dependent canopy parameters were recorded over the season and correlated with DNA and mycotoxin data. Apparently, sparser canopy permitted more Fusarium infections in unfertilized plots. In addition, well nitrogen-fertilized plants allowed less fungal development in the barley spike after spray inoculation in the greenhouse. These results suggest that nitrogen fertilization restricts Fusarium grain infection of barley by influencing canopy characteristics and possibly plant physiology.     

山羊卵巢中BMP15、GDF9和BMPR1B基因的表达量研究

山羊产羔数是影响山羊产业的重要性状,因此高产羔数是增加经济效益的有效方式。本文旨在筛选出影响高繁金堂黑山羊多胎主效基因,并探讨影响这两品种山羊多羔性状的分子调控机制。以发情前期的5只高繁金堂黑山羊和5只低繁藏山羊的卵巢组织为材料,提取总RNA,通过RT-PCR技术合成c DNA,采用实时荧光定量PCR技术,检测金堂黑山羊和藏山羊卵巢组织BMP15、GDF9和BMPR1B基因表达量。金堂黑山羊和藏山羊GDF9和BMPR1B基因mRNA在卵巢中的表达量差异不显著,而BMP15基因mRNA在卵巢中的表达量差异显著(P0.05)。说明卵巢中BMP15基因的表达量与金堂黑山羊多羔性状相关。     

胡萝卜SOC1同源基因对光周期的响应及其功能

基于先期抽薹敏感的‘松滋野生’和耐抽薹的‘Amsterdam’胡萝卜转录组测序结果,分析胡萝卜中SOC1同源基因对光周期的响应规律及其功能。胡萝卜中存在2个SOC1同源基因:DcSOC-1和DcSOC-2,具有完整的开放阅读框(ORF),分别编码217和211个氨基酸,均包含MADS-box和K-box结构域,C末端含有保守的SOC1 motif基序。系统进化树分析表明,DcSOC-1和DcSOC-2与猕猴桃Ac SOC1f和葡萄Vv SOC1的亲缘关系较近,属于SOC1/TM3亚家族。在长/短日照处理下,DcSOC-1在‘松滋野生’叶片中的相对表达量均显著高于‘Amsterdam’,且长日照能促进其表达,而DcSOC-2在‘松滋野生’和‘Amsterdam’中的表达水平较低。在春、秋两季生长过程中,DcSOC-1在‘松滋野生’叶片中的最高表达量分别达到522.4和401.7,而在‘Amsterdam’中均呈低水平表达,与先期抽薹率呈极显著正相关,说明DcSOC-1对胡萝卜的先期抽薹起着重要作用。     

Establishment of PMA-qPCR Assay for Detection of Viable E.coli in Milk

In order to detect viable E.coli in milk,a new PMA-qPCR method was established.The influences of different PMA concentration,dark incubation time,exposure time on dead bacteria inhibition effect were determined by detection of the cell numbers of viable and heat-killed E.coli suspensions at concentration of 1×10⁸ CFU/mL through fluorescence quantitative PCR (qPCR) method.The results showed that qPCR assay could specifically detect E.coli,and the viable E.coli must be exposed to 90 ℃ for 30 s in water bath to be lethal.The best treatment was 10 μg/mL PMA with 15 min of dark incubation time and 10 min of exposure time.This treatment could inhibit dead cell signals to a largest extend,while had little impact on aviable cells.The stability of PMA-qPCR assay was kept while the concentration of bacteria was more than 1×10⁸ CFU/mL.The regression equation of standard curve was y=-3.356x+47.413,R²=0.9989,the lowest detection limit was 10³ CFU/mL.The result of adding assay was agreed with the actual situation.This study laid a foundation for using of PMA-qPCR to detect the viable E.coli in food.