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The aim of this study is the development and evaluation of a rapid and accurate quantitative PCR (qPCR)‐based protocol for detection of zoonotic pathogen Streptococcus iniae in bacterial cultures and tissues of diseased fish. For this purpose, the lactate permease‐encoding (lldY) gene was selected as a target for the design of S. iniae‐specific primers based on comparative genomic analysis using 45 sequences retrieved from NCBI genome database. Specificity and applicability of these primers were tested using 115 bacterial strains and fish tissues infected with S. iniae. Sensitivity, reproducibility and efficiency of qPCR assay were also determined. The developed qPCR assay showed 100% specificity with pure bacterial cultures or DNA extracted from S. iniae or tissues of fish infected with the bacterium. The method has high sensitivity with a detection limit of 1.12 × 101 amplicon copies per assay (equivalent to 2 × 10–9 ng/µl) using bacterial DNA and of 1.44 × 101 gene copies in tissues of fish infected with S. iniae. In conclusion, this qPCR protocol provides an accurate and sensitive alternative for the identification of S. iniae and its detection on fish tissues that can be implemented as a routine tool in microbiological laboratories. 相似文献
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Kai Fan Qunfeng Zhang Dandan Tang Yuanzhi Shi Lifeng Ma Meiya Liu Jianyun Ruan 《植物养料与土壤学杂志》2020,183(2):180-191
The contribution of N remobilization is crucial for new shoots growth and quality formation during spring tea shoots development. However, the translocation mechanism of N from source leaves to sink young shoots is not well understood. In the present study, 15N urea was applied to mature tea leaves one week before bud break to track N remobilization in a field experiment. The dynamic changes in plant 15N abundance, contents of amino acids, and the expression levels of genes related to N metabolism and translocation were followed during the 18‐d development of new spring shoots until three expanding young leaves. The results showed that during the growth of new shoots the amount of 15N in the shoots increased, whereas the Ndff (N derived from 15N‐urea) in mature leaves decreased, showing that the foliar‐applied N in mature leaves was readily exported to new shoots. This process was found to be accompanied by decline of chlorophylls. In the mature leaves, expression CsATG18a and CsSAG12 involved in autophagy was dramatically induced (> 4‐fold) at approximately nine days after the bud breaking. The genes involved in the transformation of amino acids, including primarily CsGDH2, CsGDH4, CsGLT3, CsGS1;3, and CsASN2 were upregulated by > 3‐fold after bud breaking. The expression levels of CsATG8A, CsATG9, CsSAG12, CsGS1;1, CsGDH1, and CsAAP6 correlated negatively with the Ndff in mature leaves, but positively with 15N amount and total N amount in new shoots, suggesting these genes played important roles in N export from mature leaves. In the new shoots, the expression of most genes showed two defined peaks, one on six days and one on 12 days after bud breaking. The expression of CsGS2, CsASN3, CsGLT1, and CsAAP4 positively correlated with the 15N amount and total N amount in new shoots. These genes might be involved in the transport and re‐assimilation of N from mature leaves. The overall results demonstrated that the translocation of 15N from mature leaves to new spring shoots was regulated by the genes involved in autophagy, protein degradation, amino acid transformation and transport. 相似文献
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选用分别含有抗番茄黄化曲叶病(TYLCD)基因Ty-1、Ty-2和Ty-3的3种抗病番茄材料(分别简称TY-1、TY-2和TY-3)和感病番茄品种(苏粉9号),利用携带TYLCV的烟粉虱对所选番茄材料幼苗接种病毒,从核酸水平检测抗病品系和感病品种的病毒侵入时间差异,并检测接种病毒前后己糖转运基因(LeHT1)和透性蛋白基因(Permease)在转录水平上的表达量变化。结果显示,感病品种与抗病品系TY-2在接种第2 d便检测到病毒,抗病品系TY-1和TY-3分别在3 d、15 d检测到病毒,说明病毒侵入时间早晚与番茄的抗病性没有直接关系。感病品种和抗病品系TY-1分别在接种后7 d、20 d有明显的发病症状,抗病品系TY-2和TY-3整个生育期均无发病症状。感病品种接种前LeHT1无明表达,接种后随时间增加表达量先上升后下降;抗病品系TY-2、TY-3在接种前有较高的表达量,接种后随时间增加呈下降趋势,抗病品系TY-1在接种后5 d时降至最低,随后稍有增加。接种后,Per-mease基因在感病品种中表达量显著提高,7 d后略有降低;抗病品系TY-1的Permease基因表达量先降低,15 d时表达量回升;抗病品系TY-2的Permease基因在接种后的表达量增加;抗病品系在TY-3中,Permease基因在接种前后表达水平变化不大,直至15 d时略有升高。接种前、后LeHT1和Permease基因在不同抗病材料和感病材料中具有不同的表达特征,表明不同抗源可能含有不同的抗病信号通路和抗病机制。 相似文献
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拟南芥氨基酸透性酶1(AAP1,也叫NAT1)是植物中第一个被分离的氨基酸转运蛋白,在拟南芥主根、侧根、根毛及根表皮细胞中均有增加氨基酸含量的作用。本研究利用RT-PCR的方法,从拟南芥中克隆了该基因,测序结果与GenBank(NM-104616.4)中已发表的拟南芥AAP1(AtAAP1)基因的同源性高达99.93%;并将AtAAP1基因与pCAMBIA3300构建p3300-AAP1植物表达载体,为过表达AtAAP1基因,提高作物氨基酸含量,从而改善作物氮素利用效率提供参考。 相似文献
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转AAP1基因玉米游离氨基酸与可溶性蛋白质含量变化关系的研究 总被引：1，自引：0，他引：1
氨基酸通透酶1(AAP1)是位于细胞膜上与氨基酸转运有关的载体蛋白。以转组成型启动子(UBI)的AAP1玉米植株(N7-30、N7-26)、转胚乳特异性启动子(Glu)的AAP1玉米植株(KF8-33、KF8-39、KF9-22)为材料,探究转AAP1基因玉米中游离氨基酸和可溶性蛋白质含量之间的变化关系。结果表明,转AAP1基因玉米的根部和叶片中游离氨基酸含量在抽丝期最高;根部可溶性蛋白质含量在成熟期最高;叶片中可溶性蛋白质含量在抽丝期最高。叶片中游离氨基酸含量与可溶性蛋白质含量随着生育期的变化均呈现低-高-低的趋势,抽丝期达到最高值;种子中游离氨基酸的含量与可溶性蛋白质含量呈显著正相关性。转胚乳特异性启动子玉米种子中游离氨基酸含量、脯氨酸含量、赖氨酸含量均显著高于转组成型启动子玉米种子。AAP1可以提高植物对游离氨基酸的吸收与转运,进而提高植物对氮素的利用效率。 相似文献
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本研究以大豆Williams 82为实验材料，采用RT-PCR技术克隆到了GmAAP 基因，其开放阅读框长度为1440 bp，编码479个氨基酸。生物信息学分析表明，GmAAP蛋白分子量为53.286 kD，理论等电点为8.93，不稳定指数为34.04，属于稳定的蛋白结构；其二级结构中α-螺旋、β-折叠、β-转角、无规卷曲分别占据47.18%、15.66%、2.71%、34.45%；跨膜区与疏水性分析显示其含有10个跨膜区，且构成跨膜区的氨基酸多为疏水性氨基酸；GmAAP与野生大豆AAP6亲缘关系最为相近，且序列比对的一致性为100%，可能为大豆AAP6 基因。构建融合表达载体，利用PEG-Ca2+介导法转化拟南芥原生质体进行亚细胞定位分析，结果显示GmAAP定位在质膜上。AAP能提高豆科植物对外源氮的吸收与利用率，进而增加种子内贮藏蛋白含量，提升大豆品质。本研究的结果可为GmAAP 功能
的进一步研究奠定基础，同时也为氮素的高效利用提供候选基因。 相似文献
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以Oenococcus oeni苹果酸通透酶基因为目标基因,设计了1对特异性引物PmlepL/PmlepR进行酒酒球菌的快速鉴定研究。结果表明,直接以O.oeni的菌落为模板,通过PmlepL/PmlepR引物的PCR扩增,可得到苹果酸通透酶基因的特异性条带;用此特异性引物进行供试乳酸菌的PCR鉴定,所有O.oeni菌系均得到特异性条带,而供试的其他种类乳酸菌未扩增出目标带。引物PmlepL/PmlepR可用于O.oeni的快速PCR鉴定。 相似文献
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Oenococcus oenj苹果酸-乳酸发酵关键酶基因在酿酒酵母中的转化与表达 总被引：2，自引：0，他引：2
利用西北农林科技大学葡萄酒学院筛选出的酒酒球菌(Oenococcusoeni)优良菌系SD-2a的包含苹果酸-乳酸酶基因(mleA)和苹果酸通透酶基因(mleP)约2.6kb的基因片段,并以PGK1强启动子和ADH1终止子为调控元件,以酵母-大肠杆菌穿梭质粒YEp352为载体,构建了重组表达质粒pYELmleAP,转化酿酒酵母(Saccharomycescerevisiae)YS58,筛选获得酵母转化子YS58/pYELmleAP。斑点杂交检测表明目的基因mleA转化到受体菌中,SDS-PAGE检测表明获得的转化子表达了苹果酸-乳酸酶的目的蛋白。对酵母转化子培养上清进行HPLC检测,采用t检验差异显著性分析,结果表明mleA基因在受体菌中进行了功能性表达。获得的酵母转化子在添加L-苹果酸的SD/-Ura培养基中培养4d,培养液中的L-苹果酸含量降低,培养液上清中L-苹果酸的剩余含量比空载体转化子极显著降低,苹果酸降低19.33%～19.42%。对照未检出乳酸的生成,供试的转化子L-乳酸生成量为1249～1368mg/L,与对照差异显著。 相似文献
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作者通过iCODEHOP在线设计细菌通透酶的简并引物,以发酵黄芪菌FGM基因组DNA为模板进行TouchdownPCR扩增,得到740 bp PCR产物,将产物经pGEM-T Easy载体连接,转化至JM109中,筛选阳性株并测序。序列通过BLAST x检索与GenBank进行同源性比对后,结果表明,此DNA产物序列与其他菌属来源的通透酶蛋白序列具有相似性,所克隆的序列即为FGM通透酶基因片段。用iCODEHOP在线设计的简并引物可信性强,阳性率高。FGM通透酶基因的成功克隆为细菌发酵黄芪机理研究提供了依据。 相似文献
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