中国对虾杆状病毒PCR扩增产物的DNA序列测定及分析

采用台湾报道的白点综合征相关杆状病毒的一对特异引物，对中国对虾的杆状病毒进行ＰＣＲ扩增，获得了预期的１４４７ｂｐ的扩增产物，将ＰＣＲ产物用ＱＩＡＥＸＩＩ纯化，直接进行序列测定。部分序列测定及分析结果表明，中国对虾的杆状病毒的部分ＤＮＡ序列与台湾ＷＳＢＶ的相关序列的同源性为９８．７％，经计算机分析该段基因序列有６个ＯＲＦ。该ＰＣＲ引物可以用中国对虾杆状病毒及其中相关病毒的检测与比较研究。     

中华绒螯蟹线粒体DNA 12S rRNA基因片段的序列研究

首次进行了中华绒螯蟹（Ｅｒｉｏｃｈｅｉｒ ｓｉｎｅｎｓｉｓ）线粒体ＤＮＡ１２ＳｒＲＮＡ的序列分析。参考果蝇、蚤状序列对中华绒螯蟹线粒体ＤＮＡ１２ＳｒＲＮＡ基因片段做了引物设计、ＰＣＲ扩增及序列测定，结果得到４５７ｂｐ的碱基序列，其Ａ、Ｔ、Ｇ、Ｃ含量分别为１５９ｂｐ（３４．７９％）、１７７ｂｐ（３８．７３％）、５１ｂｐ（１１．１６％）、７０ｂｐ（１５．３２％）、与果蝇、蚤状的相同基因片段的序列含量基     

条纹斑竹鲨基因组的RAPD分析初报

利用１１种随机引物对５要条纹斑竹鲨基因进行了ＲＡＰＤ检测，结果表明，１１种引物在每个个体上扩增的ＤＮＡ片段总数在７７－８４之间，单个随机引物扩增的ＤＮＡ片段数目由至１１条不等，平均为７．５条ＤＮＡ片段，片段的大小在３００－２８００ｂｐ之间，个体之间的相似率在９０％以上。     

中华绒螯蟹线粒体DNA l2S rRNA基因片段的序列研究

首次进行了中华绒螯蟹（ Ｅｒｉｏｃｈｅｉｒ ｓｉｎｅｎｓｉｓ）线粒体 ＤＮＡ １２ＳｒＲＮＡ的序列分析。参考果蝇、蚤状溞序列对中华绒螯蟹线粒体ＤＮＡ １２ＳｒＲＮＡ基因片段做了引物设计、ＰＣＲ扩增及序列测定，结果得到 ４５７ ｂｐ的碱基序列，其Ａ、Ｔ、Ｇ、Ｃ含量分别为 １５９ｂｐ（３４．７９％）、１７７ｂｐ（３８．７３％）、５１ｂｐ（１１．１６％）、７０ｂｐ（ １５． ３２％），与果蝇、蚤状溞的相同基因片段的序列含量基本相似。     

海捕中国对虾杆状病毒的检测

以海捕中国对虾为材料，利用多聚酶链反应（ＰＣＲ）技术和核酸探针技术，对从中国对虾杆状病毒核酸随机文库中筛选的４个片段（大小分别为８００，１１００，１５００，２５００ｂｐ）用地高辛标记作为探针，进行斑点杂交，检测对虾杆状病毒。结果表明海捕中国对虾的鳃、中肠、肝胰腺、卵巢等组织检测为阳性，肌肉组织为阴性。实验表明中国对虾村状病毒存在垂直传播的可能性。     

中华绒螯蟹与日本绒螯蟹线粒体DNA12SrRNA序列的比较

参考果蝇和蚤状Ｚａｏ的线粒体ＤＮＡ １２Ｓ ｒＲＮＡ序列进行了中华绒螯蟹与日本绒螯蟹的线粒体ＤＮＡ１２ＳｒＲＮＡ基因片段的引物设计、ＰＣＲ扩增及序列测定。两种的碱基序列长度相同，为４５７ｂｐ，其Ａ、Ｔ、Ｇ、Ｃ含量相似，分别为１５９ｂｐ（３４．７９％）、１７７ｂｐ（３８．７３％）、５１ｂｐ（１１．１６％）、７０ｂｐ（１５．３２％）和１５９ｂｐ（３４．７９％）、１７８ｂｐ（３８．９５％）５０ｂｐ（１０．９４％）、７０ｂｐ（１５．３２％）。中华绒螯蟹与日本绒螯蟹间有５处碱基序列差异，在９８ｂｐ、１５１ｂｐ、３１７ｂｐ和４１７ｂｐ碱基处为碱基转移，在２９４ｂｐ碱基处为碱基颠换。     

对虾病原菌2—5B菌株16SrRNA基因片段的克隆和序列测定

用引物ＰＬ１－ＰＬ２ＰＣＲ扩增对虾病原菌－坎普氏弧菌２－５Ｂ菌株１６ＳｒＲＮＡ基因－１２２３ｂｐ的片段，采用ｐＵＣ１９质粒构建ｄＴ载体法完成该片段的克隆。部分序列测定及分析结果表明，该菌株与ＧｅｎＢａｎｋ中坎普氏弧菌标准株序列之间同源性为９６．９４％。     

鳜鱼病毒PCR诊断方法的建立

从ＲＡＰＤ扩增的鳜鱼病毒（ＳＣＶ）核酸电泳带中回收了二个片断，克隆子pUC19质粒（称为ＳＣＶＥ３６９和ＳＣＶＥ４５０），序列分析表明插入片段分别为３６９bp和450bp与ＧenBank序列没有显著的同源，根据克隆序列调计两对引物Ｐ１／Ｐ２和Ｐ３／Ｐ４ ，在健康鳜鱼，病鳜以及提纯的ＳＣＶ核酸中进行ＰＣＲ试验，结果表明，Ｐ１／Ｐ２组引物在ＳＣＶ基因组中扩增出特异性核酸片段，可作为鳜鱼病毒ＰＣＲ诊断，检测片段为３６９bp.     

杂交一代（尼罗罗非鱼♀×奥利亚罗非鱼♂）及春亲本基因组ＮＤＡ的比较

采用ＲＡＰＤ技术，用２０个随机引物对尼罗罗非鱼，奥利亚罗非鱼及其杂交一代的精巢ＤＮＡ进行扩增反应，发现引物ＯＰＺ－１４和ＯＰＺ－１８在尼罗罗非鱼和奥利亚鱼之间扩增出有差异的ＤＮＡ片段，作为鉴定两种鱼的分子标记有较高可信度，对这两种鱼的杂交一代基因组ＤＮＡ的ＲＡＰＤ扩增能获得中够的多态性，２０个引物中有８个引物扩增出差异性产物，表明杂交一代基因杂合性增强，这是杂种优势得以形成的重要遗传物质基础之一。     

中国对虾黄渤海沿岸群亲本及子一代RAPD分析

１９９７年３月从山东海阳市近海捕获中国对虾，取两尾单独暂养，产卵后培育出子代，用随机扩增多态性ＤＮＡＡ（Ｒａｎｄｏｍ Ａｍｐｌｉｆｉｅｄ Ｐｏｌｙｍｏｒｐｈｉｃ ＤＮＡ，ＲＡＰＤ）技术对亲本和子代的基因组ＤＮＡ进行多态性研究，目的是在分子水平上解亲本与子一代之间的遗传结构，比较了两个家系的遗传差异。结果表明，使用ＯＰＧ系列２０个引物，有１６个引物获得了谱带清晰且重复性好的产物，扩增的多态性片段分子     

Development of a Penaeus monodon-type baculovirus (MBV) DNA probe by polymerase chain reaction and sequence analysis

Abstract. Penaeus monodon -type baculovirus (MBV) was isolated and purified from the hepatopancreases of MBV-infected Penaeus monodon Fabricius. MBV DNA was extracted and used as a template in a polymerase chain reaction (PCR). The primers were chosen from conserved regions of the polyhedrin gene of Autographa californica nuclear polyhedrosis virus (AcNPV). One DNA fragment (674 base pairs) was amplified after PCR. There was a 65% homology between the predicted amino acid sequence of this PCR product with that of the polyhedrin polypeptide of AcNPV. Nucleotide sequence analysis indicated that the amplified DNA is the open reading frame of the MBV polyhedrin gene. This 674 bp DNA fragment was subsequently used as a probe in a dot blot analysis. The probe was able to hybridize with the DNA extracted from the purified MBV and from the MBV-infected P. monodon , but not from the MBV uninfected P. monodon.     

白斑综合征病毒PCR检测中使用dUTP扩增的PCR产物极限长度

依据白斑综合征病毒的序列设计了7对PCR引物,扩增长度从600bp到1 800bp.结果显示,使用dUTP代替dTTP,用普通Taq酶所能扩增的最大长度约为1 400bp.Mg2+梯度研究发现,扩增片段长度越长,最适Mg2+浓度越低.使用具有3,-5’外切酶活性的Taq酶无法扩增出任何长度的片段.     

湖北地区克氏原螯虾白斑综合征病毒变异区ORF14/15、ORF23/24基因序列比较分析

试验扩增、克隆了在湖北地区采集的19份克氏原螯虾(Procambarus clarkii)白斑综合征病毒(White spot syndrome virus,WSSV)阳性样品的变异区ORF14/15和ORF23/24基因,通过测序比较分析了湖北各WSSV毒株与Gen Bank公布的标准毒株间在变异区ORF14/15及ORF23/24基因的差异性。结果显示,19份WSSV阳性样品中有部分样品在变异区扩增出ORF14/15、ORF23/24基因片段,变异区基因序列分析发现,与Gen Bank已公布的标准毒株相比,存在大片段缺失。在变异区ORF14/15,有3个毒株扩增出1 442 bp的片段,4个毒株扩增出630 bp的片段,基于变异区ORF14/15构建的系统进化树显示,这些毒株归属两个不同的分支。在变异区ORF23/24,有2个毒株扩增出大小为2 096 bp的片段,进化分析发现这2个毒株在变异区ORF23/24的遗传距离较近。     

黄鳝脑芳香化酶基因cDNA的克隆及组织表达特异性分析

根据NCBI数据库报道的黄鳝芳香化酶基因的gDNA序列,设计了一对特异性引物.用Trizol试剂盒提取黄鳝脑总RNA,反转录获得cDNA第一条链,进行PCR扩增.扩增产物经过回收、连接、测序后得到了一条长1514 bp的芳香化酶基因cDNA序列.同源性比较表明该基因属于脑型P450aromB,与其他鱼类脑型P450aromB的同源性较高(>70%),与性腺型P450aromA的同源性较低(<60%),与自身性腺型芳香化酶同源性为59.5%.采用RT-PCR的方法对该基因在雄性黄鳝的各组织表达情况进行了分析,结果表明,该基因的转录本较高的表达于脑和精巢中,较低的在皮肤中表达,而在肝脏、心脏、小肠、肌肉等组织中没有检测到该基因的表达.     

Comparison of dot blot and PCR diagnostic techniques for detection of white spot syndrome virus in different tissues of Penaeus monodon

Comparison of PCR and dot blot diagnostic techniques for detection of white spot syndrome virus (WSSV) was made on different tissues of infected Penaeus monodon including eye stalk, eye stalk with eye, gills, cuticle, pleopod, periopods, uropods and telson. Dot blots of crude DNA extracted from infected tissue samples showed positive reactions with all the samples; however, the sensitivity of the dot blot was reduced with the purification of DNA samples extracted from pleopod, telson and uropod. PCR was found to be more sensitive when compared to dot blot. Both crude DNA and purified DNA samples extracted from all the tissues except for eye stalk with eye showed single step nested PCR positive reaction. The amplification of all or either of the three bands of 941 bp, 525 bp and 204 bp size varied with the tissues analysed. The severity of infection assessed by PCR amplification was found to be maximum in cuticle and telson followed by gill. Other tissues such as eye stalk, pleopod, periopods and uropod were observed to have mild infection. The maximum intensity of the PCR product was for the smallest amplified product of 204 bp followed by 525 bp and the weakest intensity was observed for the 941 bp size. The limitation of PCR due to inhibiting factors present in tissues could be overcome with the use of dot blot which gave positive reaction from the DNA extracted from eye stalk including the eye but yielded no amplification by PCR.     

一个奥利亚罗非鱼雌性特异性SCAR标记的建立

以奥利亚罗非鱼Oreochromis aureus为实验材料,通过对800多条随机引物的筛选,获得了1个奥利亚罗非鱼雌性特异性的、长度为1 488 bp的RAPD标记片段RAPD71699-1488,经琼脂糖凝胶电泳后回收、克隆、测序,并根据测序结果设计PCR特异引物,再经PCR条件优化,成功地将该RAPD标记片段转化为实验结果稳定、操作简便的SCAR标记,即SCAROaF1488。采用双重PCR技术,以mtDNA 16S rRNA基因片段为PCR扩增阳性对照,对该标记的有效性在两个群体共200个个体(雌、雄各100个)中进行验证。结果显示,标记检测结果与表型性别的符合度为100%。SCA-ROaF1 488标记的获得为奥利亚罗非鱼遗传性别鉴定及标记辅助选择提供了有效工具,为深入研究鱼类性别相关基因及性别决定机制提供了重要线索和新的思路。     

Complete mitochondrial DNA sequence of ayu Plecoglossus altivelis

SUMMARY: We determined the complete nucleotide sequence of the mitochondrial genome for ayu, Plecoglossus altivelis . Two large DNA fragments covering the entire genome were amplified using a long polymerase chain reaction (PCR) technique, and the products subsequently used as templates for PCR with 57 fish-versatile and five species-specific primers that amplify contiguous, overlapping segments of the entire genome. Direct sequencing of the PCR products demonstrated that the genome (16 537 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 identical to that in typical vertebrates. A major non-coding region between the tRNA^Pro and tRNA^Phe genes (857 bp) was considered to be the control region (D-loop), as it has several conservative blocks that are characteristic to this region.     

Authentication of flying-fish-meal content of processed food using PCR-RFLP

Nucleotide sequence and polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) analysis of the 3′-portion of the mitochondrial 16S RNA gene (rDNA) coding sequence were used to differentiate between flying fishes and other fishes that are frequently used in ago-noyaki production. In this study, we successfully distinguished between flying fishes and the other fishes by combining amplified DNA fragments with universally designed primers and digesting the PCR products with AfaI and MfeI restriction endonucleases. PCR-RFLP of 15 ago-noyaki, 2 noyaki, and 8 other processed flying-fish products were analyzed to authenticate flying-fish content among processed seafood products. After digestion of the PCR products with both enzymes, we found that all ago-noyaki and processed flying fish products contained either two or three DNA fragments of ~200, 300, and 530 bp, and noyaki samples (which do not contain flying fish) contained only one fragment of ~530 bp. Here, we present a new procedure to confirm the content of flying-fish meal in ago-noyaki.