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金属硫蛋白是一类富含巯基的低分子量蛋白,在植物的重金属解毒及细胞氧化还原调控等方面起重要的作用。本研究以甘蔗热带种Badila组培苗为材料,分别测定了其在CdCl2、ZnSO4和CuCl2水溶液培养条件下地上部和地下部的重金属含量,结果显示其对上述3种重金属有较强的耐受与富集能力。继而克隆了ScMT1(登录号为KJ504373)、ScMT2-1-5(登录号为MH191346)和ScMT3(登录号为KJ5043704)3个金属硫蛋白家族基因,它们分别属于植物MT亚家族中的MT1、MT2和MT3型基因。ScMT1含有1个内含子和2个外显子,开放阅读框(Open Reading Frame,ORF)长228 bp,编码75个氨基酸;ScMT2-1-5含有2个内含子和3个外显子,ORF长246 bp,编码81个氨基酸;ScMT3含有1个内含子和2个外显子,ORF长198 bp,编码65个氨基酸。RT-qPCR显示,Cd2+胁迫下,在甘蔗地上部和地下部,ScMT2-1-5均连续显著上调表达,而ScMT1的上调应答出现延迟。ScMT3在地上部的上调应答出现延迟,在地下部呈“扬-抑”趋势,提示甘蔗响应Cd^2+胁迫过程中ScMT2-1-5起更积极的作用,ScMT1参与胁迫后期的分子响应,而ScMT3不起主导作用。Cu^2+胁迫下,地上部ScMT1连续显著上调表达,ScMT2-1-5和ScMT3呈总体上调的表达趋势;地下部,ScMT1和ScMT2-1-5的上调表答均出现延迟,仅在胁迫后期显著上调表达,而ScMT3仅在胁迫前期显著上调表达。该结果提示了ScMT1、ScMT2-1-5和ScMT3在Cu2+胁迫响应过程中的协作关系,三者共同参与了地上部的胁迫响应,其中ScMT1起更积极的作用;此外三者还先后参与了地下部对Cu^2+胁迫的分子响应。Zn^2+胁迫下,ScMT1和ScMT3分别仅在地上部和地下部显著上调表达;ScMT2-1-5在地上部和地下部均呈“扬-抑”的应答趋势;提示了在甘蔗响应Cd^2+胁迫应答过程中ScMT1和ScMT3分别在地上部和地下部起主要作用,ScMT2-1-5参与了胁迫前期的分子响应。ScMT1、ScMT2-1-5和ScMT3在甘蔗不同组织中及在重金属(Cd^2+、Zn^2+或Cu^2+)不同累积水平下呈现出相似或互补的应答特性,提示上述甘蔗MT家族不同成员在重金属解毒及细胞氧化还原调控等方面产生了功能分化,且三者在应对过量Cd^2+、Zn^2+或Cu^2+对甘蔗组织造成伤害的过程中存在时空上的协同作用。该研究为深入理解多倍体植物甘蔗中MT家族各成员基因在重金属耐受过程中的协同作用机制奠定了基础。 相似文献
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Identification of internal control genes in milk‐derived mammary epithelial cells during lactation cycle of Indian zebu cow 
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We investigated the abundance and genetic heterogeneity of bacterial nitrite reductase genes (nir) and soil structural properties in created and natural freshwater wetlands in the Virginia piedmont. Soil attributes included soil organic matter (SOM), total organic carbon (TOC), total nitrogen (TN), pH, gravimetric soil moisture (GSM), and bulk density (Db). A subset of soil attributes were analyzed across the sites, using euclidean cluster analysis, resulting in three soil condition (SC) groups of increasing wetland soil development (i.e., SC1 < SC2 < SC3; less to more developed or matured) as measured by accumulation of TOC, TN, the increase of GSM, and the decrease of Db. There were no difference found in the bacterial community diversity between the groups (p = 0.4). NirK gene copies detected ranged between 3.6 × 104 and 3.4 × 107 copies g−1 soil and were significantly higher in the most developed soil group, SC3, than in the least developed soil group, SC1. However, the gene copies were lowest in SC2 that had a significantly higher soil pH (~6.6) than the other two SC groups (~5.3). The same pattern was found in denitrifying enzyme activity (DEA) on a companion study where DEA was found negatively correlated with soil pH. Gene fragments were amplified and products were screened by terminal restriction fragment length polymorphism (T-RFLP) analysis. Among 146 different T-RFs identified, fourteen were dominant and together made up more than 65% of all detected fragments. While SC groups did not relate to whole nirK communities, most soil properties that identified SC groups did significantly correlate to dominant members of the community. 相似文献
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旨在建立快速、高效、精准检测FecB突变的荧光qPCR技术,为滩羊多羔性能研究、群体改良及肉用多羔滩羊新品系培育提供技术支撑。基于荧光qPCR技术基本原理,设计特异性引物对,开发基于荧光qPCR检测绵羊FecB突变的方法;对85只已知FecB基因型的健康、适繁雌性滩羊血液基因组DNA样品分别采用PCR-Sanger测序法、TaqMan探针法和荧光qPCR法进行FecB基因分型,统计3种方法的准确率;采用开发的荧光qPCR方法对939只健康、适繁滩羊进行FecB基因分型,统计不同FecB基因型经产母羊的产羔率。结果表明,使用TaqMan探针法、PCR-Sanger测序法和开发的荧光qPCR法对已知FecB基因型样品检测的比较中,荧光qPCR法对各基因型鉴定的准确度均达100%,高于前两者。939只滩羊FecB基因分型结果显示,6只滩羊为FecB突变纯合型（BB）、57只为杂合型（B+）、876只为野生型（++）,BB型、B+型和++型滩羊的基因型频率分别为0.64%、6.07%和93.29%,其中B等位基因频率为0.04,+等位基因频率为0.96;监测母羊群体的产羔数发现,FecB突变纯合型滩羊产羔率为166.67%,突变杂合型滩羊产羔率为153.12%,野生型滩羊产羔率为105.78%,纯合突变型和杂合突变型母羊群体的产羔率极显著高于野生型群体（P<0.01）。综上所述,与TaqMan探针法及PCR-Sanger测序法相比,本研究建立的绵羊FecB突变荧光qPCR分型方法具有简便易行、廉价高效的优点,在绵羊的分子选育中具有较高应用价值;同时本研究证实了滩羊FecB突变可显著提高母羊产羔率,为多羔滩羊的分子选育提供了坚实的技术支撑。 相似文献
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《Veterinary microbiology》2015,175(2-4):211-217
Squamous cell carcinomas are common feline skin cancers that have been associated with infection with Felis catus papillomavirus type 2 (FcaPV-2). Currently, little is known about the epidemiology of FcaPV-2 infection. The aim of this study was to develop a real-time PCR assay to quantify FcaPV-2 DNA in plucked hairs and skin swabs from 11 healthy breeding queens and their kittens. Samples were taken prior to kittening and then 2, 7 and 28 days after kittening to determine the age at which the kittens were first exposed to the virus. FcaPV-2 DNA was amplified from all of the queens and from 91% of the kittens at 2 days of age. There was a wide range in the quantity of FcaPV-2 DNA detected, from 1 to 92,520 copies per swab, and from 0.01 to 234 copies per copy of reference gene DNA in the hair plucks. The quantity of FcaPV-2 DNA detected in samples collected from the kittens was strongly correlated to that of their respective queens and the mean viral DNA load was similar for cats within a household but varied significantly between households. This is the first time that quantitative PCR has been used to detect FcaPV-2 DNA and the results suggest that the virus is ubiquitous but there is a wide variation of viral DNA loads. Kittens appear to be exposed to FcaPV-2 early in life, presumably from direct contact with their queen. These results are important when determining if FcaPV-2 infection of cats is preventable. 相似文献
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A qPCR assay was developed for rapid and sensitive detection of Flavobacterium psychrophilum, the aetiological agent of bacterial cold-water disease and rainbow trout fry syndrome in salmonid fish worldwide. A set of F. psychrophilum-specific primers based on 16S rRNA gene sequences was designed and validated for specific detection and quantification of DNA isolated from representative strains of F. psychrophilum. The qPCR assay exhibited a high specificity for the 16S rRNA gene of F. psychrophilum (from 4 × 10(8) down to 11 copies per reaction) but not for other Flavobacterium species or other bacteria including fish pathogens. This qPCR-based method proved to be useful in the quantification of the F. psychrophilum titre present within organs dissected out from diseased fish. As the F. psychrophilum genome contains six copies of the 16S rRNA gene, we could infer a limit of detection corresponding to two bacteria per reaction, corresponding to 800 bacteria per fish tissue sample, and therefore 20 F. psychrophilum cells mg(-1) of tissue (for sample weighing 40 mg). The qPCR assay reported here could be a useful tool for veterinary diagnostic laboratories to monitor the F. psychrophilum infection level in fish farms. 相似文献
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WUSCHEL(WUS)基因是植物干细胞的标志基因。采用同源克隆法结合RACE技术从马银花愈伤组织中克隆得到Ro WUS c DNA全长序列,采用染色体步移法克隆得到该基因的启动子序列,登录号分别为KF365488、KF861578。马银花Ro WUS c DNA全长1 123 bp,编码302个氨基酸;DNA序列2 001 bp,包含2个内含子和3个外显子;启动子序列3 122 bp。生物信息学分析表明:Ro WUS亚细胞定位于细胞核,是不稳定的亲水蛋白,无信号肽,具有跨膜结构,包含homeodomain功能位点。系统进化树分析结果表明:Ro WUS单独形成一个分支,与葡萄、大豆和苜蓿的亲缘关系最近。对Ro WUS的启动子进行分析表明,该启动子除了含有丰富的TATA-box和CAAT-box等基本元件以外,还含有多个光响应元件、逆境胁迫响应元件、激素应答元件和其他功能元件。q PCR结果表明:Ro WUS基因在马银花愈伤组织不同生长时期中呈先上升后下降的趋势,在第5个继代周期时表达量最高。外源赤霉素和脱落酸浓度为15 mg/L时表达量最高,说明Ro WUS基因在该浓度时对赤霉素和脱落酸的响应最强。 相似文献
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Strawberry disease (SD) is an inflammatory skin disorder in rainbow trout, Oncorhynchus mykiss (Walbaum). The aetiology of SD is unknown although the 16S rDNA sequence of a Rickettsia-like organism (RLO) has been associated with SD lesions using a nested PCR assay. In this study, we developed a Taqman quantitative PCR assay (qPCR) that targeted the RLO 16S rDNA sequence to examine the distribution of RLO relative to lesion status. We compared 18 lesion samples from 13 fish representing high or low lesion severity as judged by gross examination. QPCR results showed that there was a higher number of RLO sequences in high severity lesions (mean of 12,068 copies) compared with fewer copies of RLO sequence in low severity lesions (mean of 3287 copies, P = 0.012). Grossly normal skin samples (n = 13) from SD-affected fish were all negative by qPCR except two samples (121 and 139 copies). The qPCR assay described herein is a useful tool to investigate the role of RLO in SD in the absence of a culture system for RLO. Our results demonstrate a positive correlation between copy number and lesion severity consistent with the hypothesis that the RLO is the aetiologic agent of SD. 相似文献