水稻OsNHO1基因cDNA序列分离及表达分析

OsNHO1是一种甘油激酶,受多种因素的诱导,在植物先天免疫反应中起着重要的作用。笔者采用同源序列法,根据已报道的拟南芥NHO1基因序列结合水稻基因组测序结果,筛选水稻OsNHO1基因,采用RT-PCR获得水稻OsNHO1基因全长cDNA(1 590 bp,529AA),并通过半定量RT-PCR方法分析OsNHO1基因在SA,PXO99刺激下的表达模式,结果显示OsNHO1基因的表达受SA,PXO99的诱导,为进一步研究OsNHO1的具体功能和作用机理奠定了基础。     

鳜鱼病毒传播途径的初步研究

采用PCR方法,追踪养殖鳜的亲鱼、子代、饵料鱼及与鳜养殖环境相关的水体生物和非生物因子中鳜鱼病毒的存在状况,并探讨鳜鱼病毒的传播途径。本研究中,鳜鱼塘水体中采集的除鳜以外的其他生物、底泥及水样的核酸抽提样本PCR扩增病毒结果呈阴性;感染病毒的亲鱼,其性腺中可检测出病毒,其子代组织病毒检测亦呈阳性。研究获得病毒可以在鳜体内呈潜伏状态存在和病毒可以垂直传播的一些证据。     

Characterization of the DNA nucleotide sequences in the genome of red sea bream iridoviruses isolated in Korea

The nucleotide sequences of DNA fragments amplified by polymerase chain reaction (PCR) from four different genomic regions of nine red sea bream iridoviruses (RSIVs) isolated from different species of fish, different areas and in different years in Korea were compared with the reported reference sequences. One isolate, RSIV Namhae, showed 100% homology to the reference sequences, while the other eight isolates, which appeared to contain identical nucleotide sequences, showed 96.6–98.9% homology with reference sequences depending upon the target regions of PCR gene amplification. However, differences in nucleotide sequences were not apparent between the RSIVs isolated in different locations, in different years or in different host species. We also cloned and sequenced the 3′ end flanking region (K1) of the DNA polymerase (DPOL) gene using the cassette ligation-mediated PCR method. This sequence was 4436-bp long and possessed two open reading frames (ORF-1 and ORF-2) oriented in opposite directions. The putative proteins encoded by these two ORFs could not be characterized by comparison with the proteins of other species in the data banks. The presence of the ribonucleotide reductase small subunit (RNRS) gene at the 3′ end of the K1 region allowed us to determine that these two genes, RNRS and DPOL, are separated 5508 bp and oriented in the same direction in the genome of RSIV. Moreover, it is of interest that a PstI-restriction fragment, of which the sequence but not the location within the RSIV genome had previously been reported, is located at nucleotide positions from 1096 to 2054, extending from within the ORF-1 region, spanning the intervening sequence between ORF-1 and ORF-2, and extending into the ORF-2 region. Various repeating sequences up to 86 bp were present at the 3′ ends of ORFs, especially within the nucleotide sequences at the 3′ terminus of ORF-2. No similarities were detected when the DNA sequences of the K1 region were compared to the DNA sequences of a repetitive element in the genome of other iridoviruses.     

Complete mitochondrial DNA sequence of the Japanese anchovy Engraulis japonicus

ABSTRACT: The complete nucleotide sequence of the mitochondrial genome for the Japanese anchovy Engraulis japonicus (Teleostei: Clupeiformes) was determined. The entire genome was purified by gene amplification using the long polymerase chain reaction (PCR) technique, and products were subsequently used as templates for PCR with 56 fish-versatile primers that amplify contiguous, overlapping segments of the entire genome. Direct sequencing of the PCR products demonstrated that the genome (16 675 base pairs [bp]) contained the same 37 mitochondrial genes (two ribosomal RNA, 22 transfer RNA and 13 protein-coding genes) as those found in other vertebrates, with the gene order being identical to that in typical vertebrates. A major non-coding region between the tRNA^Pro and tRNA^Phe genes (1024 bp) was considered to be the control (D-loop) region, as it has several conservative blocks characteristic to this region.     

定性PCR方法检测转基因玉米MON810转化事件的研究

转基因玉米MON810(Yield Gard R)是孟山都公司通过DNA重组技术和微注射轰击研发的一种具有对欧洲玉米螟(ECB;Ostrinia nublialis)有特殊抗性的转基因玉米品系,目前在世界各地已经得到广泛种植,为加强对该品系玉米的安全管理,本研究旨在建立MON810品系玉米的转化事件特异性定性PCR检测方法。根据MON810的插入序列信息,在3′端的侧翼序列处设计定性PCR检测的引物,检测MON810在其他几种常见转基因作物混合样品的特异性。结果表明,该方法具有很好的特异性。同时检测该引物系统的扩增灵敏度,结果表明,检测引物的灵敏度可达0.1%。建立的MON810特异定性PCR检测方法经全国7家实验室的验证,进一步证实该方法能够特异地检测出样品中的MON810转化事件,检测方法的灵敏度可达0.1%,且检测结果具有良好的可重复性和可重现性。MON810转化事件定性PCR检测方法的建立可满足于抗虫转基因玉米MON810及其衍生品种生物安全管理的需要。     

一种应用LORF11同源子鉴别诊断鸭瘟病毒的PCR方法

为建立一种鉴别诊断鸭瘟病毒的PCR方法,根据已发表的8株鸭瘟病毒株的长区开放阅读框(LORF11)等位基因序列,在其开放阅读框等位基因的上游5 bp和下游101 bp的位置设计1对特异性引物。应用这对引物对鸭瘟病毒LH2011株、v2085株、VAC株、Clone-03株DNA进行PCR扩增,分别获得了4 448 bp、3 277bp、934 bp和2 518 bp的特异性条带。此PCR方法特异性强,敏感性高,可检测到10TCID50或0.1LD50的鸭瘟病毒。在感染鸭瘟病毒的组织(肝脏)中均能检测到鸭瘟病毒DNA。对疑似鸭瘟的临床病料的检测结果也证明,建立的PCR方法不仅能够鉴别样品是否感染了鸭瘟病毒,而且能鉴别感染的鸭瘟病毒的来源。     

Detection of Flexibacter maritimus in fish tissue using nested PCR amplification

A nested polymerase chain reaction (PCR) system was developed for the detection of Flexibacter maritimus from fish tissue. The total procedure for the diagnosis of marine flexibacteriosis, from the point of DNA extraction to the electrophoretic analysis, can be performed in < 4 h. This was achieved by the combination of a short thermal cycling programme with a rapid DNA extraction procedure. The assay was extremely sensitive, capable of detecting as few as 75 cfu mg(-1) fish tissue. The accuracy of the nested PCR was confirmed under field conditions using tissue samples recovered during 1993-2002 from fish suffering marine flexibacteriosis. The nested PCR method proved to be efficient for the rapid and sensitive detection of F. maritimus from fish tissues and can be used for routine diagnosis of the disease caused by this pathogen.