山东近海习见鱼类DNA条形码及其电子芯片分析

从GenBank下载到13目50科73属77种山东近海鱼类的线粒体细胞色素c氧化酶I(CO I)序列,通过分析其遗传距离及系统进化,并基于CO I序列筛选物种特异性探针来分析DNA芯片技术在进行物种鉴定时的可行性。结果表明,在CO I基因DNA条形码的分析中,77种鱼类的种间遗传距离(平均0.117)明显大于种内遗传距离(平均0.0034),且每个物种的不同个体在进化树上都能聚在一起,提示DNA条形码能全部区分77个物种;根据CO I基因设计的用于芯片的特异性探针中,77个物种最终有64个可以筛选出物种特异性探针,占总物种数的83.1%,本研究旨在为山东近海鱼类物种鉴定提供了技术支持。     

弓形虫感染鼠脾细胞microRNA表达谱的检测分析

MicroRNA（miRNA）是一类约22个核苷酸组成的单链非编码RNA,在分化、发育、肿瘤形成等方面起着重要作用。本研究对弓形虫RH株感染小鼠脾细胞miRNA的表达进行了特异性基因芯片检测分析,并应用荧光定量RT-PCR方法进行验证。结果表明,感染鼠脾细胞中与免疫应答及细胞增殖和肿瘤发生相关的三大类miRNA中,有39种表达下调,同时有36种表达上调。上述结果揭示,弓形虫感染机体后伴随着靶细胞功能性miRNA表达谱的显著改变,这为进一步研究弓形虫感染致病的分子机制开辟了新的方向。     

从干种子中快速获取高质量DNA的高盐提取方法

为寻求一种从植物干种子中简单快速提取高质量基因组DNA的有效方法进行本研究。将种子打磨成粉,取100 mg粉末加入高盐提取液抽提基因组DNA。采用超微量分光光度计检测、PCR及酶切的方法检验DNA的得率和质量。从100 mg 7种常见作物种子粉末中可以得到619.67~1811.21 ng基因组DNA,A260/A280比值在1.87~2.07之间,其纯度和质量适合进行PCR及酶切分析。通过普通PCR方法分别对7种作物的特异性内源基因片段进行扩增,结果均能扩增到明显的目的条带。用EcoRV和HindIII两种核酸内切酶能对所得的DNA充分酶切。结果表明本方法能快速地从干种子中提取到高质量的DNA。     

稻属种间天然异交种的分子细胞学鉴定

利用基因组荧光原位杂交（GISH）技术快速鉴定了栽培稻与野生稻的天然异交种的基因组组成,分析了该杂种在减数分裂中期Ⅰ的染色体配对情况。根据根尖细胞的染色体数目,发现该杂种是具有36条染色体的三倍体;通过减数分裂中期Ⅰ染色体的配对研究,发现该杂种染色体很少发生配对,绝大部分染色体以单价体形式存在;结合GISH技术的分析,证实该杂种是由A、B和C 3个染色体组组成。因此该杂种是栽培稻和小粒野生稻的天然杂交种。     

两株茶尺蠖核型多角体病毒毒株对灰茶尺蠖的毒力及基因组差异

为明确茶尺蠖核型多角体病毒（Ectropis obliqua nucleopolyhedrovirus,EcobNPV）毒株对灰茶尺蠖E.grisescens的毒力差异及其机制,以前期筛选获得的高效毒株EcobNPV-QF4和原始毒株EcobNPV-QV为研究对象,采用PCR扩增技术对其进行分子鉴定,通过叶盘法毒力生测试验测定毒力差异,并用定量PCR技术比较病毒拷贝数及基因组测序方法分析基因组差异。结果表明,EcobNPV-QV经分子鉴定存在3个基因型,而EcobNPV-QF4则存在2个基因型,2株毒株的基因型存在差异;EcobNPV-QV和EcobNPV-QF4对灰茶尺蠖的幼虫毒力分别为4.26×10⁶ PIB/头和9.72×10⁷ PIB/头,EcobNPV-QF4对灰茶尺蠖的毒力是EcobNPV-QV的22.8倍;饲毒0~48 h之间EcobNPV-QF4在灰茶尺蠖体内的增殖拷贝均显著高于EcobNPV-QV,EcobNPV-QF4比EcobNPV-QV具有更快的繁殖能力;比较2株毒株的基因组,EcobNPV-QF4比EcobNPV-QV长315 bp,与EcobNPV-QV相比EcobNPV-QF4基因组的hr1~hr3区域出现倒位,其中核苷酸还原酶小亚基基因在EcobNPV-QF4中重复1次,3个同源重复区的回文序列存在差异但2株毒株仍为α杆状病毒亚家族II亲缘种分离株。表明2株毒株的基因组在同源重复区的序列及回文个数间存在差异,推测这可能与导致其对灰茶尺蠖毒力差异有关。     

水貂犬瘟热CDV_3疫苗株基因组序列测定及H基因遗传稳定性分析

对水貂犬瘟热CDV3疫苗株基因组进行全序列测定与分析,以阐明该疫苗株的来源亲本毒株,基因特征及H基因遗传稳定性。将CDV3疫苗株基因组cDNA分9个重叠片段进行RT-PCR并克隆入pMD 18-T载体中,测定全长cDNA序列。并与CDV野毒参考株和疫苗株N、F和H基因氨基酸序列比对分析其基因变异情况。通过对CDV3疫苗株6个不同生产批次(Vero细胞培养代次)H基因序列测定以分析毒株的分子遗传稳定性。基因序列分析结果表明:CDV3疫苗株基因组全长15 690 bp,与CDV野毒株基因组同源性较低(93.0%~95.3%)。H基因核苷酸和氨基酸序列与Lederle疫苗株(EF418783)同源性最高,分别为99.6%和98.7%。而6个不同培养代次CDV3疫苗株H基因氨基酸序列无任何差异。由此证实CDV3疫苗株的亲本毒株与Lederle疫苗株有着最密切关系,不同培养代次的CDV3疫苗株H基因具有较高的遗传稳定性。     

转小粒野生稻基因组DNA水稻的产量与品质性状研究

野威B是将小粒野生稻(Oryza minuta)的基因组DNA通过穗茎注射法导入杂交水稻亲本V20B中培育出的转基因水稻新种质.与亲本V20B相比,野威B的有效穗数和每穗总粒数小于V20B,但每穗实粒数却高于V20B,平均结实率比V20B增加11.8%,千粒重比V20B的少6 g.野威B倒1叶和倒2叶叶鞘中可溶性糖含量...     

DNA微阵列与基因表达研究概况

DNA微阵列技术是 90年代兴起的一种对成百上千甚至上万个基因同时进行检测的新技术 ,它具有高通量和并行化的特点 ,广泛应用于基因表达、预测基因功能、检测基因突变和多态性分析、发现新药物和药物靶器官以及疫苗设计等方面。文章对 DNA微阵列的基本原理、DNA微阵列制备技术、杂交信号检测以及数据分析。 c DNA微阵列与细胞周期相关基因表达、细菌基因表达、病毒基因表达、肿瘤基因表达进行了概述。     

Conventional and molecular diagnostic testing for the acute neurologic patient

Objective – The aim of this review is to describe and evaluate both conventional and molecular diagnostic testing utilized in dogs and cats with acute neurologic diseases. Various types of polymerase chain reaction (PCR) are explored along with novel molecular diagnostic testing that ultimately may prove useful in the critical care setting. Data Sources – PUBMED was searched to obtain relevant references material using keywords: ‘canine OR feline meningitis AND meningoencephalitis,’‘feline infectious peritonitis,’‘canine distemper,’‘canine OR feline AND toxoplasma,’‘canine neospora,’‘canine OR feline AND rickettsia,’‘granulomatous meningoencephalitis,’‘steroid responsive meningitis arteritis,’‘necrotizing encephalitis,’‘novel neurodiagnostics,’‘canine OR feline AND CNS borrelia,’‘canine OR feline AND CNS bartonella,’‘canine OR feline AND CNS fungal,’‘nested OR multiplex OR degenerate OR consensus OR CODEHOP AND PCR.’ Research findings from the authors' laboratory and current veterinary textbooks also were utilized. Human Data Synthesis – Molecular diagnostic testing including conventional, real‐time, and consensus and degenerate PCR and microarray analysis are utilized routinely for the antemortem diagnosis of infectious meningoencephalitis (ME) in humans. Recently, PCR using consensus degenerate hybrid primers (CODEHOP) has been used to identify and characterize a number of novel human viruses. Veterinary Data Synthesis – Molecular diagnostic testing such as conventional and real‐time PCR aid in the diagnosis of several important central nervous system infectious agents including canine distemper virus, Toxoplasma gondii, Neospora caninum, rickettsial species, and others. Recently, broadly reactive consensus and degenerate PCR reactions have been applied to canine ME including assays for rickettsial organisms, Borrelia spp. and Bartonella spp., and various viral families. Conclusions – In the acute neurologic patient, there are several key infectious diseases that can be pursued by a combination of conventional and molecular diagnostic testing. It is important that the clinician understands the utility, as well as the limitations, of the various neurodiagnostic tests that are available.     

The PFA-AMeX method achieves a good balance between the morphology of tissues and the quality of RNA content in DNA microarray analysis with laser-capture microdissection samples

Recently, large-scale gene expression profiling is often performed using RNA extracted from unfixed frozen or formalin-fixed paraffin embedded (FFPE) samples. However, both types of samples have drawbacks in terms of the morphological preservation and RNA quality. In the present study, we investigated 30 human prostate tissues using the PFA-AMeX method (fixation using paraformaldehyde (PFA) followed by embedding in paraffin by AMeX) with a DNA microarray combined with laser-capture microdissection. Morphologically, in contrast to the case of atypical adenomatous hyperplasia, loss of basal cells in prostate adenocarcinomas was as obvious in PFA-AMeX samples as in FFPE samples. As for quality, the loss of rRNA peaks 18S and 28S on the capillary electropherograms from both FFPE and PFA-AMeX samples showed that the RNA was degraded equally during processing. However, qRT-PCR with 3’ and 5’ primer sets designed against human beta-actin revealed that, although RNA degradation occurred in both methods, it occurred more mildly in the PFA-AMeX samples. In conclusion, the PFA-AMeX method is good with respect to morphology and RNA quality, which makes it a promising tool for DNA microarrays combined with laser-capture microdissection, and if the appropriate RNA quality criteria are used, the capture of credible GeneChip data is well over 80% efficient, at least in human prostate specimens.