13种河豚鱼COⅠ基因部分DNA序列分析及在物种鉴定中的应用

应用COⅠ基因聚合酶链式反应(polymerase chain reaction,PCR)扩增通用引物,对福建省和海南省搜集的3属13种河豚鱼样品与9个未知种名的河豚鱼样品的COⅠ基因靶序列片段进行PCR扩增和测序,13个已知物种样品的DNA序列提交基因库(GenBank),取得相应的登录号。各物种COⅠ基因靶序列长度均为681 bp。应用DNAMAN V6软件对样品的DNA序列进行同源性比对分析,建立样品间系统关系同源树。基于COⅠ基因序列,供试13个样品被划分为4个类群组。群间的同源率为84%,群内同源率为90%～100%。根据序列同源性分析结果,9个未知种名的样品被归类到2个类群组中,判定这9个样品为东方鲀属或腹刺鲀属,其中1个样品(HNW2)为月腹刺鲀,4个样品(HNW3、HNW4、FJW2、FJW5)为棕斑腹刺鲀,1个样品(HNW1)为暗鳍腹刺鲀;3个样品(FJW1、FJW3、FJW4)为横纹东方鲀。探讨基于DNA条形码技术的COⅠ基因靶序列片段在河豚鱼种属鉴别中应用的可能性。     

河豚鱼16S rRNA基因部分DNA序列分析及应用

应用16S rRNA基因聚合酶链式反应（polymerase chain reaction,PCR）扩增通用引物,对3 属13 种福建省搜集的河豚鱼样品与9 个未知物种的河豚鱼样品的16S rRNA基因序列中的部分片段进行PCR扩增与脱氧核糖核酸（deoxyribonucleic acid,DNA）碱基序列测定,各物种序列长度在611～614 bp之间。应用DNAMAN V6软件进行样品间DNA序列同源性比对分析,建立样品间系统关系同源树。供试13 个样品被划分为4 个类群组,群间的同源率为87%,群内同源率为94%～100%。根据序列同源性分析结果,9 个未知种名的样品被归类到3 个类群组中,推测9 个未知种名的样品为东方鲀属或腹刺鲀属。探讨了16S rRNA基因部分DNA序列测试及同源性分析技术在河豚鱼种属鉴别中应用的可能性。     

河豚鱼Cyt b基因部分DNA序列分析与应用

建立河豚鱼物种DNA鉴别技术,根据GenBank公布的河豚鱼细胞色素b基因序列,应用软件Primer Premier5.00版设计上游引物HT1-F与下游HT1-R,对3属9种福建省搜集的常见的河豚鱼与2种未知物种名称且外观无法进行形态学判断的河豚鱼样品的细胞色素b基因序列中的部分片段进行PCR扩增,PCR产物通过琼脂糖凝胶电泳确证、纯化后,进行DNA碱基序列测定,序列长度均为423bp。应用DNA MANN软件进行样品间DNA序列同源性比对分析,建立样品间系统关系树状图,供试11个样品被划分为3个类群,第Ⅰ组与第Ⅱ组之间的同源率为89%,这2组与第Ⅲ组之间的同源率为85%。根据序列同源性分析结果,可推测2个未知种名的样品为腹刺鲀属或东方鲀属。     

多基因DNA条形码鉴定6 个鳗鱼物种

建立6?种鳗鱼的物种多基因DNA条形码精准鉴定方法。以鳗鱼DNA为模板,采用3?对通用引物对6?种鳗鱼的3?个基因（16S rRNA、Cyt b、COⅠ）部分DNA片段进行聚合酶链式反应扩增、测序,结果6?种鳗鱼各获得3?条16S rDNA（638～643?bp）、Cyt?b（464～466?bp）、COⅠ（705～707?bp）基因部分DNA序列,从中选取各物种序列同源的片段设计3 对新引物对6 种鳗鱼的16S rRNA、Cyt b、COⅠ基因部分DNA片段进行PCR扩增,其产物大小分别为504～507、400、609?bp,再从各片段中筛选出具有6?种鳗鱼物种特异性强的、碱基数分别为262、280、300?bp的3?条DNA片段序列,作为6?种鳗鱼物种的3?个基因的标准DNA条形码,应用DNAMAN?V6软件进行同源性分析,并通过GenBank数据库的比对验证,制定了供检测实践用的同源率判别指标,建立鳗鱼物种的多基因条形码检测方法。应用该方法对30?个待检鳗鱼样品进行检测,结果表明,各样品基于3?个基因DNA条形码的比对,符合同源率指标,物种判别结果互相吻合,从而精准判别样品的所属物种。该方法稳定、精准、易于操作,可应用于6?种鳗鱼的物种鉴定,值得推广。     

限制性内切酶酶切确证河豚鱼成分PCR检测结果

动植物成分的聚合酶链式反应（polymerase chain reaction,PCR）检测往往出现假阳性结果,为了有效排 除河豚鱼成分检测中出现的假阳性现象,提高检测结果的准确性,应用河豚鱼PCR检测引物进行河豚鱼成分的PCR 检测与限制性内切酶NmeA Ⅲ酶切确证实验。18 个供试样品中3 个样品无PCR扩增产物,判为阴性结果,15 个样品 初步判为疑似阳性。应用限制性内切酶NmeA Ⅲ对疑似阳性样品的PCR产物进行酶切与电泳分析,电泳图谱与河豚 鱼阳性对照不同的2 个样品,判定为假阳性结果,电泳图谱与阳性对照相同的4 个未知学名的河豚鱼加工样品,确 证为阳性结果,即检出河豚鱼成分,PCR产物序列经GenBank同源性序列查询比对（BLAST）予以验证,建立了简 便的河豚鱼成分PCR检测结果确证方法。     

COI及cytb基因对河鲀鱼鱼种鉴定的适用性研究

目的建立河鲀鱼DNA条形码鉴定方法,探讨细胞色素氧化酶亚基I(COI)及细胞色素b(cytb)基因对我国常见东方鲀属、兔头鲀属河鲀鱼鱼种鉴定的适用性。方法野捕河鲀鱼经形态学鉴定后,钓取COI及cytb基因序列并测序。从Gen Bank下载已有河鲀鱼参考序列,分别构建COI及cytb基因分子进化树,确定样品种属并与形态学鉴定比对。应用所建方法对中毒样品进行河鲀成分鉴定。结果 COI和cytb基因分子进化树将57份样品聚类到东方鲀属和兔头鲀属的9个鱼种,除棕斑兔头鲀和暗鳍兔头鲀(COI进化树)、暗纹东方鲀和晕环东方鲀(cytb进化树)外,2种条形码均能对其余鱼种进行有效区分。中毒样品经鉴定均含有月兔头鲀。结论所建立的DNA条形码方法可有效鉴定河鲀鱼鱼种,弥补形态学鉴定的缺陷。     

钝裂银莲花Actin基因片段的克隆与序列分析

提取钝裂银莲花叶片中的基因组DNA,用来克隆其Actin基因序列片段,为研究其生理功能及其他基因在钝裂银莲花中的表达和调控奠定基础。根据GenBan中登录的植物肌动蛋白基因同源核苷酸保守序列设计简并引物。以钝裂银莲花叶片总DNA为模板,利用RT—PCR技术获得了3个Actin基因片段。序列分析表明,3个Actin基因片段长为780bp,含有一段内含子,第一段外显子编码128个氨基酸,具有2个Actin基因的功能位点;与其他植物同源序列进行分析表明,其核苷酸序列同源性在81%-90％之间,氨基酸序列同源性在95％-100％之间。第二段外显子编码84个氨基酸,与其他植物同源性序列进行分析表明其氨基酸序列同源性在94％-99％之间。本研究中得到的3个基因序列是Actin基因的同源片段,分别命名为AOACTI,AOACT2,AOACT3。     

大豆CONSTANS同源基因的克隆及表达

CONSTANS（CO）是调控拟南芥由营养生长向生殖生长转换的关键基因。本研究利用RACE法从晚熟（光周期敏感）大豆品种自贡冬豆中克隆得3个大豆CO同源基因,分别为Glyma04g06240、Glyma13g01290和Gly-ma06g06300。电子克隆法检索发现另外5个同源性较高的基因,这些基因均含一个内含子,其编码氨基酸序列与拟南芥AtCO的B-box和CCT功能结构域高度同源。qRT-PCR分析叶片中Glyma04g06240、Glyma06g06300和Glyma13g01290的表达发现,短日下三者表达均呈现昼夜节律模式,表现为白天降低,夜间积累,凌晨达到表达高峰;长日处理仅影响了这些基因在夜间的积累量。此外,Glyma06g06300和Glyma13g01290在大豆叶片、茎尖、花及嫩荚中有大量表达。短日处理时Glyma13g01290的表达在自贡冬豆由营养生长向生殖生长转化的过程中逐渐增加,而长日处理至13d后其表达逐渐被抑制,表明Glyma13g01290可能参与自贡冬豆开花的调控。     

滁菊类黄酮3′-羟化酶基因的克隆及分子特性

以安徽滁州地区特有的药食两用植物资源——滁菊(Dendranthema morifolium‘chuju’)为试材,采用同源基因克隆方法克隆滁菊类黄酮3′-羟化酶基因(flavonoid 3′-hydroxylase,f3′h),NCBI注册号HQ256697。结果表明:采自不同地点的滁菊样品均能扩增出一条长381bp的f3′h基因片段,该片段与探针序列GU249553第3外显子对应区域的核苷酸序列同源性达98.69%,同源区域内有5个单核苷酸多态性位点(single nucleotide polymorphisms,SNP),其中3个SNPs导致氨基酸编码序列的改变。滁菊与葡萄属(Vitis vinifera)、高粱属(Sorghum bicolor)、芸苔属(Brassica napus)、牵牛属(Ipomoea nil Magenta)、苹果属(Malus×domestica)、番薯属(Ipomoea purpurea)等物种f3′h基因的核苷酸序列和氨基酸序列同源性分别在60%～98%和68%～97%之间。不同植物f3′h基因的分类与物种间的亲缘关系存在明显的相关性,菊属植物f3′h基因在系统进化中处于较高位置。     

烟草CONSTANS同源基因的克隆与分析

日照长度影响植物的生长发育,在拟南芥中CONSTANS是植物光周期开花途径中的关键基因。利用同源序列法结合RACE 技术从短日照烟草品种Kutsaga.Mammoth10 中分离出了开花时间相关的CO(CONSTANS)同源基因,并命名为NtCO1(基因登录号JN022535.1)。经序列分析,NtCO1 全长1493 bp,具有完整的开放阅读框(ORF,81~1292 bp),编码403 个氨基酸;具有CO 蛋白典型的结构域;氨基末端有两个B-box 结构,羧基末端有CCT 保守结构域。氨基酸同源性比对发现,NtCO1 与茄科CO 同源蛋白一致性最高,同马铃薯CO 序列一致性达到86.5%;与拟南芥CO 蛋白和水稻Hd1 氨基酸序列一致性也分别达到50%和43.7%。基因表达分析表明,NtCO1在叶片中优势表达,茎中次之,根中最弱     

Puffer fish-based commercial fraud identification in a segment of cytochrome b region by PCR–RFLP analysis

To identify the mislabeled or fraudulently substituted toxic puffer fish in thermally processed fish products, a polymerase chain reaction (PCR) method using restriction sites and sequence analysis has been developed in this study. A 376-bp fragment of the cytochrome b gene was produced after PCR amplification. Fish tissue samples were prepared under autoclaving conditions at 121 °C for 10–90 min at 10 min intervals. DNA fragments could not be detected after 90 min of autoclaving at 121 °C. For PCR product digestion, BsaJ I, Aci I, Hinf I, Taq I, and Sap I endonucleases were used to yield species-specific profiles for the identification of puffer fish species from 60 commercial market samples. Results from this study showed that the restriction fragment length polymorphism technique can be used to identify 17 puffer fish species from commercial products even after severe thermal processing.     

PCR 方法检测河豚鱼的引物筛选及反应体系优化

根据Genbank 公布的河豚鱼细胞色素b 基因序列,应用软件Primer Premier 5.00 版设计了7 对引物,经过PCR 筛选,确定可以在所有8 个供试河豚鱼样品中检出目的DNA 片段的引物HT-1,用于建立河豚鱼的PCR 检测方法。对该PCR 方法中6 个因素包括退火温度、Mg2+ 终浓度、Taq DNA 聚合酶用量、dNTPs 终浓度、引物终浓度和模板DNA 用量进行优化,确定优化的PCR 扩增体系：10 × PCR 缓冲液2μL,MgCl2 终浓度1.5mmol/L,Taq DNA 聚合酶1.0U,dNTPs 终浓度300μmol/L,引物终浓度0.2μmol/L,DNA 模板400ng,加纯水至总体积20μL。扩增程序定为94℃预变性5min,94℃变性30s,62℃退火30s,72℃延伸30s,40 个循环,72℃延伸5min。据此建立河豚鱼成分PCR 检测方法,并通过河豚鱼与非河豚鱼的PCR 检测结果比较,验证了该方法的河豚鱼特异性。研究结果还表明,该方法的检出限为0.1%,含量为0.1% 的河豚鱼样品PCR 检出率至少在97.5% 以上。     

基于DNA条形码技术对常见石首鱼科鱼胶的物种鉴定

摘　要：目的　运用DNA条形码技术对常见石首鱼鱼胶进行物种鉴定。方法　通过对26份鱼胶样品基因组DNA提取，PCR扩增COI基因、测序，用BOLD物种鉴定系统，与数据库中已有鱼类序列进行比对分析，鉴定出各鱼胶的物种；根据Kimura双参数模型计算样品序列遗传距离，并将所得序列构建NJ和MP系统发育树，进行聚类分析。结果　26份鱼胶样品通过鉴定引物“Fish-F”、“Fish-R”均可实现扩增，条带清晰单一，扩增和测序成功率均为100%；BOLD鉴定结果显示，26份鱼胶样品中23份能够确定物种来源（相似性达98%以上），包括石首鱼科12属15种鱼类，且多数为外来物种，另外3份鱼胶可推测其近缘物种。此外，系统发育树聚类分析结果与物种鉴定结果一致。结论　目前石首鱼类鱼胶来源物种较多，且多为外来基原鱼种。DNA条形码技术与BOLD鉴定系统相结合，可对大部分鱼胶进行准确的物种鉴定。     

鲀毒鱼毒素检测及物种鉴定技术研究进展

鲀毒鱼引起的河鲀毒素(tetrodotoxin,TTX)中毒是中国沿海地区食物中毒致死的主要原因之一.河鲀毒素中毒发病迅速,至今尚无特效药,因此,通过检测TTX含量或鉴定携带TTX的物种可以更好地进行TTX中毒的风险分析、管理和控制.本文综述了鲀毒鱼TTX的检测技术以及鲀毒鱼物种鉴定的方法,以期能预警并减少鲀毒鱼引起的...     

Species Identification and PCR-RFLP Analysis of Cytochrome b Gene in Cod Fish (Order Gadiformes) Products

ABSTRACT: Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) profiles using the PCR product tRNA^Glu-cytochrome b of 9 standard cod fish, all of which are imported into Japan under the name "cod fish," were investigated as a means of rapid species identification of imported cod fish products. Endonuclease digestion using 4 restriction enzymes ( Alu I, Fok I, Mbo I, and Mse I) generated different digestion patterns for the standards, enabling species identification in imported cod fish products by comparison to the standards'RFLP profiles. Additionally, we conducted a more strict check by phylogenetic analysis on imported cod fish products with 13 standards (including 4 additional cod species from GenBank) using the mitochondrial cytochrome b gene (385 nt). Consequently, it was found that the PCR-RFLP method was sufficient for rapidly screening cod products, and in cases that require conclusive identification, phylogenetic analysis was the most suitable.     

Liquid chromatography-tandem mass spectrometry determination of the toxicity and identification of fish species in a suspected tetrodotoxin fish poisoning

Suspected tetrodotoxin (TTX) poisoning was associated with eating unknown fish in April 2009 in Taiwan. After ingestion of the fish, symptoms of the victim included perioral paresthesia, nausea, vomiting, ataxia, weakness of all limbs, respiration failure, and death within several hours. The toxicity in the remaining fish was determined, with the mice exhibiting symptoms of neurotoxin poisoning. The implicated fish and deceased victim tissues were analyzed for TTX by liquid chromatography-tandem mass spectrometry. The urine, bile, cerebrospinal fluid (spinal cord), pleural effusion, and pericardial effusion of the victim contained TTX. In addition, the partial cytochrome b gene of the implicated fish was determined by PCR. The DNA sequence in the partial 465-bp cytochrome b gene identified the implicated fish as Chelonodon patoca (puffer fish). These results indicate that people should avoid eating unknown fish species from fish markets where harvested fish may include toxic species.     

Identification of Toxin and Fish Species in Cooked Fish Liver Implicated in Food Poisoning

ABSTRACT: The cooked fish liver retained by the victims was assayed for toxicity and mitochondrial DNA. Meanwhile, 8 live specimens of puffer Takifugu niphobles were also assayed. The toxicity of cooked fish liver was 280 ± 20 mouse units per gram (MU/g). All specimens of T. niphobles showed high toxicity (more than 850 MU/g) in the liver. The toxin from cooked fish liver and liver of T. niphobles was identified to be tetrodotoxin. The cooked fish liver and fresh liver of T. niphobles showed the same sequence genotype and the same single restriction site for Bsa I. Therefore, the species of cooked fish liver was suggested as T. niphobles.     

Tetrodotoxin poisoning due to smooth-backed blowfish Lagocephalus inermis and toxicity of L. inermis caught off the Kyushu coast, Japan

Food poisoning due to ingestion of a puffer fish occurred in Nagasaki Prefecture, Japan, in October 2008, causing neurotoxic symptoms similar to those of tetrodotoxin (TTX) poisoning. In the present study, we identified the species, toxicity, and toxins using the remaining samples of the causative puffer fish. The puffer fish was identified as smooth-backed blowfish Lagocephalus inermis by nucleotide sequence analysis of the 16S rRNA and cytochrome b gene fragments of muscle mitochondrial DNA. The residual liver sample showed toxicity as high as 1,230 mouse unit (MU)/g by bioassay and TTX was detected by liquid chromatography/mass spectrometry analysis. We therefore concluded that the food poisoning was due to TTX caused by consumption of the toxic liver of L. inermis. This is the first report that the liver of L. inermis caught in Japanese waters is strongly toxic, with levels exceeding 1,000 MU/g. In this context, we re-examined the toxicity of L. inermis collected off the coast of Japan. Of 13 specimens assayed, 12 were toxic, although the toxicity varied markedly among individuals and tissues. Because the intestine and ovary of L. inermis have been considered non-toxic, it is particularly noteworthy that these organs were determined to be toxic, with a maximum toxicity of 43.6 MU/g and 10.0 MU/g, respectively. Furthermore, kidney, gallbladder, and spleen, whose toxicity has been unknown, were frequently found to be weakly toxic with levels ranging from 10 to 99 MU/g. Therefore, further study is needed to re-examine the toxicity of smooth-backed blowfish L. inermis in the coastal waters of Japan.