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1.
近年来,羊肉及其肉制品的掺假等食品安全事件层出不穷,为了完善市场执法检查法律依据,利用数字PCR定值羊HELZ基因,研制了羊基因组DNA标准物质。由于标准物质可以用于衡量检测方法的准确性,因此可以判定食品及相关制品中羊肉的掺假情况。利用数字PCR对研制的标准物质进行均匀性和稳定性评估,结果表明该批标准物质均匀性良好,在4 ℃、25 ℃可以稳定保存14 d,在-20 ℃可以稳定保存6个月。来自全国9个不同实验室羊源性基因组DNA标准物质(高浓度)和羊源性基因组DNA标准物质(低浓度)的联合定值结果显示,标准值及其扩展不确定度分别为(5.44±0.45)×103 copies·μL-1和(5.68±0.54)×102 copies·μL-1。该羊源性基因组DNA标准物质为动物源性标准物质的市场应用提供了技术基础,完善了羊源性基因组DNA标准物质的制备、定量检测、质量控制和量值溯源技术平台。  相似文献   

2.
体细胞核移植(体细胞克隆)技术在动物生产、医药工业、治疗性克隆以及对珍稀濒危动物的拯救有重要意义,然而克隆效率低下以及克隆动物发育异常,严重制约了克隆技术的发展和应用.在体细胞核克隆中,供体核来自高度分化了的体细胞,发生在核移植后几小时内供体核的重编程,决定了克隆胚胎的发育能力.印记基因是由等位基因表观遗传修饰的不对称导致的基因表达具有亲本选择性,而DNA甲基化是调控印记的一个主要方式.印记基因Mash2在胚胎发育和器官形成过程中起着非常重要的作用.为了探求核移植过程中Mash2基因DNA 甲基化的表观重编程是否充分,利用亚硫酸氢盐测序法对出生48 h内死亡的体细胞核移植牛和正常对照牛肺脏中Mash2基因的DNA甲基化状态进行分析.结果显示,尽管位于Mash2基因启动子和第一个外显子处的CpG岛在正常牛和克隆牛中甲基化水平都不高(20.04%,5.55%),但克隆组的甲基化水平仍显著低于正常对照组 (P < 0.05).甲基化模式正常组中9N3有5种不同的形式,9N4仅1种;而克隆组9C3和9C5也分别是1种.推测Mash2基因的异常DNA甲基化很可能是导致克隆牛肺脏发育异常的一个重要原因.  相似文献   

3.
【目的】新型转基因棉花在进入大规模商业化应用前,需对其生态环境安全性进行评价;同时,经基因改造的新型转基因抗虫棉花可能影响抗虫棉的次生代谢,进而导致一些综合的生态学效应,致使棉花生理上发生改变,这也是转基因植物安全性评价研究的重要内容。【方法】比较了不同关键时期新型转Cry1Ac+Cry2Ab基因棉花与转Cry1Ac基因棉花和非转基因棉花叶片的鲜重、干重和干鲜比、主要酶[超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、过氧化物酶(POD)、抗坏血酸过氧化物酶(APX)和谷胱甘肽还原酶(GR)]活性、营养物质(蛋白质、氨态氮、脯氨酸和可溶性糖)和次生代谢产物(棉酚和单宁)含量的差异及其对棉田不同昆虫营养层昆虫个体总数和物种数的影响。【结果】棉花生长的蕾期、花期和花铃期,转Cry1Ac+Cry2Ab基因棉花、转Cry1Ac基因棉花和非转基因棉花叶片的鲜重、干重和干鲜比呈先升高后降低的趋势;SOD和POD活性在花铃期明显升高,CAT、APX和GR活性无显著变化;蛋白质、氨态氮含量无明显变化,脯氨酸和可溶性糖含量均表现为先升高后下降的趋势;棉酚含量在3个时期无显著变化,而单宁含量呈逐渐升高的趋势。3种棉花叶片中干物质积累、主要酶活性、营养物质和次生代谢产物含量均无显著差异;单株大铃数表现为转Cry1Ac+Cry2Ab基因棉花转Cry1Ac基因棉花非转基因棉花,小铃数则表现为转Cry1Ac基因棉花Cry1Ac+Cry2Ab基因棉花非转基因棉花;昆虫群落和害虫亚群落的昆虫个体总数均表现为转Cry1Ac+Cry2Ab基因棉田转Cry1Ac基因棉田非转基因棉田,天敌亚群落昆虫个体总数无显著变化;3种棉田中昆虫群落、害虫亚群落和天敌亚群落的物种数均未发生显著变化。【结论】转Cry1Ac+Cry2Ab基因棉花叶片干物质积累、产量性状、生化物质含量、酶活性在不同生长期表现不同,但上述参数在3种棉花之间无显著差异;且转Cry1Ac+Cry2Ab基因棉花具有较好的抗虫性,能有效降低棉田害虫数量。  相似文献   

4.
hpc2研究进展   总被引:2,自引:0,他引:2       下载免费PDF全文
生物个体的胚胎发育以及细胞的增殖、分化,都同时受到多种基因的严格调控,PcG基因家族就是一类重要的发育相关基因.而hPc2基因是人PcG基因家族中的一个重要成员,其编码的HPC2蛋白,不仅可以和HPH、BMI-1以及RING1等其他人类PcG蛋白结合形成HPC/HPH PcG复合体,以蛋白复合体的形式参与对homeotic基因的表达抑制,以维持机体的正常发育以及细胞的增殖和定向分化,还发现它能与其他多种蛋白质相结合,提示HPC2可能具有多种功能.因此,对hPc2的深入研究不仅有助于进一步阐明PcG基因家族的作用机理,扩展人们对基因表达调控的认识,还有助于发现PcG基因家族与其他信号转导通路的联系,更好地理解细胞信号网络系统.  相似文献   

5.
为了探究蜡质基因(Wx)与可溶性淀粉合成酶Ⅲ-2基因(SSⅢ-2)等位变异对稻米品质的影响,以籼型高直链淀粉恢复系CG133R和糯性‘爪哇稻22’为亲本杂交,在其F3群体选择仅在Wx和SSⅢ-2基因位点存在多态性的单株为材料,分析各基因型材料的理化指标和粘度速测仪谱特征值。结果表明:(1)SSⅢ-2基因为I型时,Wx基因第一内含子多态性对表观直链淀粉含量有极显著影响。SSⅢ-2基因为Ⅱ型时,Wx基因第一内含子多态性对表观直链淀粉含量和成糊温度有极显著影响。(2)Wxa背景下,SSⅢ-2基因对稻米品质无显著影响;Wxb背景下,SSⅢ-2基因对糊化温度和粘度速测仪谱中最高粘度、崩解值、成糊温度有极显著影响。(3)Wx和SSⅢ-2基因共同作用极显著影响水稻表观直链淀粉含量、糊化温度、最高粘度、崩解值和成糊温度,说明Wx和SSⅢ-2基因之间存在互作,且Wx基因对SSⅢ-2基因具有显性上位性,Wx基因大量表达会遮盖SSⅢ-2基因对稻米品质的效应。  相似文献   

6.
目的 转录因子NFE2的异常表达在许多骨髓增殖性肿瘤患者中被观察到,然而造成这种异常的转录调控机制尚不明确,本研究旨在探究参与NFE2转录调控的元件和分子机制。方法 首先通过公共数据库中ChIP-seq数据和ATAC-seq数据预测NFE2基因的潜在增强子元件,并通过双荧光素酶报告实验进行体外验证。随后,通过PRO-seq和GRO-seq数据结合RACE技术克隆这些增强子RNA转录本,经在线编码潜能预测工具分析认为其为lncRNA,通过RT-qPCR检测该lncRNA在不同白血病细胞系中和这些细胞诱导分化前后的表达变化及其亚细胞定位。最后,通过慢病毒系统在K562细胞中过表达和敲降该lncRNA以探究其功能。结果 鉴定出调控NFE2转录的3个增强子元件,分别位于NFE2转录起始位点-3.6k,-6.2k和+6.3k区域,这些元件插入NFE2启动子上游均能增强下游萤火虫荧光素酶的表达。克隆出-3.6k增强子负链方向的转录本,将其鉴定为-3.6k-lncRNA。本研究发现,该lncRNA在K562、U937和HL-60这3种白血病细胞系中均有一定程度的表达,且均定位于细胞核内。当该lncRNA在K562细胞中过表达,NFE2水平随之提高,细胞增殖和细胞迁移能力受到抑制;当其被敲降时,NFE2水平相应降低而K562细胞增殖能力随之升高。结论 本文鉴定了调控人NFE2基因转录的3个增强子元件和一条增强子lncRNA转录本,并验证了该lncRNA对NFE2转录的正调控作用以及对K562细胞增殖能力具有抑制作用。  相似文献   

7.
Taq DNA聚合酶发现自水生热栖菌(Thermus aquaticus),是一种同时具有逆转录酶活性以及DNA聚合酶活性的工具酶。Colicin E (简称CE)蛋白质是一类以维生素受体BtuB为跨膜受体的大肠杆菌素,其中CE2、CE7、CE8和CE9是非特异性的DNase型大肠杆菌素。Taq DNA聚合酶由5′→3′核酸外切酶结构域、3′→5′核酸外切酶结构域以及聚合酶结构域组成。缺失5′→3′核酸外切酶结构域的Taq DNA聚合酶(ΔTaq)具有更高的产量,但是其进行性很低,无法扩增长片段。为了提高ΔTaq的进行性,本研究融合dCE和ΔTaq,发现dCE-ΔTaq的进行性相比于Taq DNA聚合酶和dCE-Taq显著提升,并且其逆转录酶活性也比ΔTaq更高。dCE8-ΔTaq的提升最明显,不仅能够1 min内扩增8 kb的DNA片段,并且产量高于其他突变体。综上所述,本研究通过将ΔTaq DNA聚合酶和dCE进行融合,提升了Taq DNA聚合酶的PCR效率和逆转录活性,为改造Taq DNA聚合酶提供了新的方法,有望开发出性质更好的Taq DNA聚合酶。  相似文献   

8.
为了探讨金黄色葡萄球菌PVL基因与噬菌体的相关性 ,从 4株含有PVL基因的菌株中分离出DNA ,用HindⅢ或EcoRⅠ酶切后 ,分别与PVLLukM-lukF-PV探针进行Southern印迹杂交 ,以及对含有PVL基因及其下游区域的片段克隆、测序和同源性分析。结果表明3株菌的PVL基因及其下游区域的序列与V8菌株噬菌体∮PVL的PVL基因及其下游噬菌体attsite的序列  相似文献   

9.
尽管研究证明很多克隆动物存在DNA甲基化异常的情况,却很少有研究比较克隆绵羊与自然分娩绵羊之间的甲基化情况,可能是由于克隆绵羊的获得、绵羊基因组、绵羊基因组印记等因素的限制.本研究中,为了证明克隆绵羊重编程的状况,克隆了Peg3基因的差异甲基化区域(differential methylated region,DMR),并且分析了Peg3、Cdkn1c、Gtl2在克隆绵羊和自然分娩绵羊不同组织中的甲基化水平.研究发现,在克隆绵羊和自然分娩绵羊中Peg3呈现为超甲基化水平,在克隆绵羊的肾脏和肺脏中DNA甲基化水平为95.45%、81.18%,相对于正常分娩的绵羊组织中的98.18%、87.27%无显著性差异,而Cdkn1c在两组实验动物中的肾脏和肺脏中表现为非甲基化水平,分别为0%、0.53%、0.53%和0.53%,Gtl2则是低甲基化水平,并且克隆绵羊与正常分娩绵羊之间的DNA甲基化水平无显著性差异(r2 = 0.77).这些结果表明,Peg3、Cdkn1c、Gtl2三个印记基因在克隆绵羊和自然分娩绵羊组织中呈现类似甲基化水平,无显著性差异.  相似文献   

10.
为了探索人工栽培白及的适宜条件,该研究以湖北省十堰市野生白及为对象,采用同源克隆和3'RACE技术,从白及(Bletilla striata)中获得与热激蛋白合成有关的BsHsp17.3基因,并分析BsHsp17.3基因对不同胁迫的响应。结果表明:BsHsp17.3基因开放阅读框长度为453 bp,编码150个氨基酸;蛋白的分子量为17.42 kD,等电点为6.33。进化树分析表明BsHSP17.3蛋白与同为兰科的铁皮石斛进化关系较近,同在一分支上。半定量RT-PCR分析显示BsHsp17.3基因在白及根、叶、鳞茎及花组织中的表达具有特异性,且BsHsp17.3基因在叶中的表达量较高,在鳞茎及花中不表达。实时荧光定量PCR检测显示BsHsp17.3对非生物胁迫高温、低温具有明显应答反应,20%PEG模拟干旱胁迫不诱导该基因表达,推测该基因在白及防止倒苗过程中可能发挥一定作用。  相似文献   

11.
肉和肉制品是人类生活的重要营养来源,但近年来肉制品中发生的掺假使假事件屡见不鲜,使得肉品的质量安全问题已经成为全世界关注的热点话题。以核酸为目标的动物源鉴定是当前普遍使用的方法。在核酸检测中,常用线粒体基因或核基因作为靶标,缺乏统一标准。以绍兴鸭和北京鸭等不同品种及生鲜组织(鸭血、鸭胸肉、鸭肝、鸭皮、鸭心和鸭腿肉)为实验材料,提取DNA后利用微滴式数字PCR开展线粒体和核DNA拷贝数的比较研究,以两者拷贝数及其比值的变异系数为判定依据。结果显示,核DNA的拷贝数在不同品种鸭组织间相对稳定,且变异系数小于线粒体DNA,表明核DNA是开展鸭肉制品掺假定量检测的最适DNA来源。鸭腿肉中线粒体/核DNA拷贝数比值的变异系数最小,表明线粒体DNA作为靶基因的鸭肉掺假比例定量检测时,鸭腿肉来源的肉制品是最佳选择。  相似文献   

12.
Zheng  Si-Jun  Henken  Betty  Sofiari  Eri  Jacobsen  Evert  Krens  Frans A.  Kik  Chris 《Transgenic research》2001,10(3):237-245
Genomic DNA blot hybridization is traditionally used to demonstrate that, via genetic transformation, foreign genes are integrated into host genomes. However, in large genome species, such as Allium cepa L., the use of genomic DNA blot hybridization is pushed towards its limits, because a considerable quantity of DNA is needed to obtain enough genome copies for a clear hybridization pattern. Furthermore, genomic DNA blot hybridization is a time-consuming method. Adaptor ligation PCR (AL-PCR) of genomic DNA flanking T-DNA borders does not have these drawbacks and seems to be an adequate alternative to genomic DNA blot hybridization. Using AL-PCR we proved that T-DNA was integrated into the A. cepa genome of three transgenic lines transformed with Agrobacterium tumefaciens EHA105 (pCAMBIA 1301). The AL-PCR patterns obtained were specific and reproducible for a given transgenic line. The results showed that T-DNA integration took place and gave insight in the number of T-DNA copies present. Comparison of AL-PCR and previously obtained genomic DNA blot hybridization results pointed towards complex T-DNA integration patterns in some of the transgenic plants. After cloning and sequencing the AL-PCR products, the junctions between plant genomic DNA and the T-DNA insert could be analysed in great detail. For example it was shown that upon T-DNA integration a 66bp genomic sequence was deleted, and no filler DNA was inserted. Primers located within the left and right flanking genomic DNA in transgenic shallot plants were used to recover the target site of T-DNA integration.  相似文献   

13.
    
TCR of CD8 T cells recognizes peptides of 8–9 amino acids in length (epitope) complexed with MHC class I. Peptide ligands differing from an epitope by one or two amino acids are thought to modulate the immune response specific to that epitope. H60 is a minor histocompatibility antigen for which the specific CD8 T-cell response dominates during alloresponse after MHC-matched allogeneic transplantation. In the present study, we developed a transgenic mouse (designated H60H Tg) expressing a variant of H60, designated H60H, in which the arginine residue at position 4 of the H60 epitope sequence (LTFNYRNL) is replaced by a histidine residue (LTFHYRNL). Immunization of female C57BL/6 mice with splenocytes from male H60H Tg induced a CD8 T cell primary response and memory response after re-challenge. The response was CD4 help-dependent, demonstrating the potency of H60H as a cellular antigen. The response induced by the H60H cellular antigen was comparable to that induced by H60 in its peak magnitude and overall immune kinetics. H60H challenge recruited broadly diverse TCRs to the specific response, shaping a TCR repertoire different from that of the natural H60 epitope. However, some of the TCRs did overlap between the H60H- and H60-specific CD8 T cells, suggesting that H60H might modulate the H60-specific response. These results may provide a basis for the modulation of the H60-specific CD8 T-cell response.  相似文献   

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Spatial smoothing and hot spot detection for CGH data using the fused lasso   总被引:4,自引:0,他引:4  
We apply the "fused lasso" regression method of (TSRZ2004) to the problem of "hot- spot detection", in particular, detection of regions of gain or loss in comparative genomic hybridization (CGH) data. The fused lasso criterion leads to a convex optimization problem, and we provide a fast algorithm for its solution. Estimates of false-discovery rate are also provided. Our studies show that the new method generally outperforms competing methods for calling gains and losses in CGH data.  相似文献   

17.
人们很早就发现DNA拷贝数变异与特定染色体重组和基因组异常相关这一现象,但最近才知道它与疾病的相关联系。我们对拷贝数变异的原理、最新研究方法,及其与复杂疾病的相关性研究等进展进行了综述;总结了拷贝数变异研究所存在的问题;对拷贝数变异未来的研究重点和需要解决的问题进行了展望。  相似文献   

18.
数字PCR技术及应用研究进展   总被引:1,自引:0,他引:1       下载免费PDF全文
数字PCR是继实时定量PCR之后新兴发展起来的一种绝对定量分析技术。通过将单个DNA分子转移入独立的反应室,PCR扩增反应后,对荧光信号进行检测分析,实现单分子的绝对定量。数字PCR技术摆脱了对标准曲线的依赖,具有更高灵敏度和准确度,在基因突变检测、拷贝数变异检测、微生物检测、转基因食品检测以及下一代测序等方面均得到广泛应用。本文介绍数字PCR技术的定量方法,并评述该技术在主要应用领域的研究进展。  相似文献   

19.
    
Breast cancer is a malignancy harmful to physical and mental health in women, with quite high mortality. Copy number variations (CNVs) are vital factors affecting the progression of breast cancer. Detecting CNVs in breast cancer to predict the prognosis of patients has become a promising approach to accurate treatment in recent years. The differential analysis was performed on CNVs of long noncoding RNAs (lncRNAs) as well as the expression of lncRNAs, microRNAs (miRNAs) and mRNAs in normal tissue and breast tumor tissue based on The Cancer Genome Atlas (TCGA) database. The CNV-driven lncRNAs were identified by the Kruskal–Wallis test. Meanwhile, a competitive endogenous RNA (ceRNA) network regulated by CNV-driven lncRNA was constructed. As the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed, the mRNAs in the dysregulated ceRNA network were mainly enriched in the biological functions and signaling pathways, including the Focal Adhesion-PI3K-Akt–mTOR-signaling pathway, the neuronal system, metapathway biotransformation Phase I and II and blood circulation, etc. The relationship between the CNVs of five lncRNAs and their gene expression in the ceRNA network was analyzed via a chi-square test, which confirmed that except for LINC00243, the expression of four lncRNAs was notably correlated with the CNVs. The survival analysis revealed that only the copy number gain of LINC00536 was evidently related to the poor prognosis of patients. The CIBERSORT algorithm showed that five lncRNAs were correlated with the abundance of immune cell infiltration and immune checkpoints. In a word, by analyzing CNV-driven lncRNAs and the ceRNA network regulated by these lncRNAs, this study explored the mechanism of breast cancer and provided novel insights into new biomarkers.  相似文献   

20.
Female murine mammary tumor virus (MMTV)/neu transgenic mice, expressing a wild-type rat neu oncogene driven by an MMTV promoter, develop focal mammary adenocarcinomas that are pathologically very similar to human breast tumors. Two new cell lines were established from a mammary tumor that arose in a female MMTV/neu transgenic mouse. One of these lines, mammary carcinoma from Neu transgenic mouse A (MCNeuA), has an epithelial morphology, is cytokeratin positive, and expresses high levels of the neu transgene. Karyotyping and comparative genomic hybridization analyses demonstrated genomic alterations in the MCNeuA cell line. The other line, N202Fb3, has a fibroblast morphology, is cytokeratin negative, and expresses the neu transgene at a very low level. This cell line also expresses smooth muscle alpha-actin, suggesting that it is a myofibroblast line. The MCNeuA cell line is tumorigenic when injected into syngeneic MMTV/neu transgenic mice, with an in vivo doubling time of about 14 d. The rationale for establishing this tumor cell line was to provide a tumor transplantation system for rapidly assessing immunotherapeutic interventions before testing in the more cumbersome model of spontaneous tumor development in the MMTV/neu transgenic mice. Mice immunized with a Neu extracellular domain protein vaccine were protected against a subsequent inoculation of MCNeuA cells, indicating that this cell line will be useful for evaluating cancer vaccine strategies. This tumor cell line may also prove useful in studying the biological properties of the neu oncogene and its role in the malignant process. In addition, the tumor-derived fibroblast line may be useful for studying tumor-stromal cell interactions.  相似文献   

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