舟山海域4种鲷科鱼类线粒体Cytb基因全序列克隆分析

摘要：克隆测序了分布于舟山海域的真鲷（Pagrus pagrus）、黑鲷（Sparus macrocephalus）、黄鳍棘鲷（Acanthopagrus latus）和二长棘鲷（Parargyrops edita）4种鲷科鱼类的线粒体Cytb基因1141bp的全序列。通过对4种鲷科鱼类Cytb基因序列的比对分析，发现了292个核苷酸变异位点，其中存在186个信息位点，序列中的转换大于颠换，碱基替换多发生于密码子第3位。遗传距离分析表明，4种鲷科鱼类的遗传差异在0．0967～0．2275之间，其中真鲷与二长棘鲷之间遗传距离最小，为0．0967，黄鳍棘鲷与二长棘鲷的遗传距离最大，为0．2275；序列变异的转换／颠换比值在2．2930～7．3587之间。以25种鲈总科鱼类构建的系统树表明鲷科鱼类与其他科的鱼类存在较远的遗传亲缘关系。     

4种鲷科鱼类的线粒体细胞色素b基因序列及分子系统学分析

从鲷科鱼类中黄猪鲷(Sparus latus)、黑鲷(S.macrocephalusM)、真鲷(Pagrosomus major)和平鲷(Rhabdosargus sarba)的线粒体DNA扩增出约430bp的细胞色素b(Cytb)基因，经Clastal X同源排序后得408bp序列，对该序列进行分析并结合GeneBank中黄鲷(Taius turnifrons)、灰鳍鲷(S.berda)、犁齿鲷(Evynnis japonica)和高背四长棘鲷(Argyrops spinifer)的该区段DNA序列比较，共发现126个核苷酸位点存在变异(30．7％)，序列中的转换大于颠换，碱基替换多发生在密码子的第3位点。黑鲷和灰鳍鲷亲缘关系最近，序列差异为3.8％；而黄鲷和平鲷的亲缘关系最远，序列差异为21．7％。用MEGA2．1软件中的NJ法构建的分子系统树，将上述8种类分为4组，其中黄鲷属、平鲷属和鲷属分类的结果与形态分类学的观点一致；而传统分类上把犁齿鲷、真鲷和高背四长棘鲷归为3个不同属，在本研究分子系统树中，这3个属却聚成一支。本研究目的在于从分子水平上阐明鲷科鱼类的分类与系统进化关系。     

4种笛鲷属鱼类随机扩增多态性DNA及遗传多样性研究

使用RAPD技术分析了红鳍笛鲷、紫红笛鲷、勒氏笛鲷和约氏笛鲷的种内遗传多样性、种间遗传多样性和种间亲缘关系。在120个随机引物中筛选出扩增效果好的引物36个，平均每个引物在每个个体上产生7．7个条带。种内遗传多样性研究结果显示：遗传距离(D)分别为0．2257，0．2228，0．2679和0．2247；遗传多样性指数(H)分别为0．1854，0．1904，0．2015和0．1865。说明目前湛江近海这4种笛鲷的遗传多样性比较丰富，它们的种质资源仍处于一个良好的水平。种间遗传多样性结果显示：勒氏笛鲷与约氏笛鲷遗传距离(Dpq)最小，亲缘关系最密切，勒氏笛鲷与红鳍笛鲷种间Dpq最大，亲缘关系最远。     

鲷科鱼类种质资源与利用

近日，中国水产科学研究院南海水产研究所完成了“鲷科鱼类种质资源与利用”科技项目，并通过了由广东省海洋与渔业局组织的鉴定。该项目采用RAPD，AFLP、线粒体控制区序列分析等技术研究了我国近海黄鳍棘鲷、二长棘犁齿鲷、黑棘鲷、黄牙鲷和真赤鲷的遗传多样性和种群遗传结构；采用RAPD技术、线粒体cyt—b基因序列分析等方法研究了我国近海主要鲷科鱼类的亲缘关系和分子系统；     

鲷科于类属间远缘杂交的发育和生长

鲷科鱼类的肉味鲜美 ,营养价值高 ,生长快 ,其主要养殖品种真鲷 (Ｐａｇｒｏｓｏｍｕｓｍａｊｏｒ)、黑鲷 (Ｓｐａｒｕｓｍａｃｒｏ ｃｅｐｈａｌｕｓ)、黄鳍鲷 (Ｓｐａｒｕｓｌａｔｕｓ)和平鲷 (Ｒｈａｂｄｏｓａｒｇｕｓｓａｒ ｂａ)的人工繁殖及种苗生产已获得成功[1,2 ] ,但未见其杂交育种成功的研究报道。这 4种鲷分属同科不同属。笔者从1 989年开始进行这 4种鲷的杂交育种生产试验 ,均成功孵化出杂种仔鱼 ,并获得全长 6 6～ 86ｍｍ的平鲷与黑鲷杂交后代幼鱼 6 .0万尾。为海水鱼的属间远缘杂交研究及杂交育种生产提供了理论和技术依据1…     

三个不同地理群体真鲷遗传变异的RAPD分析

提要用RAPD技术对取自胶州湾、台湾海峡和北部湾3个野生群体的24尾真鲷进行群体间的遗传变异分析。在事先优化的反应条件下，在使用的60个随机引物中，有31个引物扩增出清晰稳定的片段，共计216条，大小在200—2500bp之间，其中多态性片段176条(多态性片段的比例为81．48％)。群体间的遗传距离分别是0．1645、0．1413、0．1073，台湾海峡与北部湾真鲷的遗传距离最近，胶州湾与台湾海峡其次，胶州湾与北部湾最远。     

人工鱼礁试验总结

一、前言 我区南濒北部湾。北部湾属半封闭海湾，三面大陆环绕，有高山阻挡强风和台风，一般情况下风浪较小。我区沿岸浅海浮游生物丰富，底质平坦，是主要经济鱼类二长棘鲷、红鳍笛鲷、真鲷、平鲷、石斑、石鲈、艮鲈、白姑、蛇鲻、海鲶、沙丁鱼、蓝圆鱼参、青鳞、尤鱼、墨鱼等鱼类和海产经济动物的产卵繁殖场。上述鱼类中的大部份都喜欢栖息在鱼礁内。     

真鲷与黑鲷杂交与多倍体育种系列研究-Ⅰ

真鲷属于鲷科,真鲷属,分布于我国四大海域。黑鲷属于鲷科,鲷属,我国沿海均有分布。真鲷与黑鲷杂交属于属间远缘杂交。本研究利用春季繁殖的真鲷种群与春季繁殖的黑鲷种群进行交配。用HCG与LHRH-A_2催产,网箱内自然受精。正交以真鲷为♀,黑鲷为♂;反交以黑鲷为♀,真鲷为♂。两种杂交组合的雌雄性比均为1:1.5。正交组合的受精率为73％,反交组合为67％。正交组合的孵化率为85％,反交组合为87％。该研究证明海水鱼属间远缘杂交可以成功,并为海水鱼杂交育种推广应用奠定了基础。     

真鲷与黑鲷杂交与多倍体育种系列研究-Ⅰ

真鲷属于鲷科,真鲷属,分布于我国四大海域。黑鲷属于鲷科,鲷属,我国沿海均有分布。真鲷与黑鲷杂交属于属间远缘杂交。本研究利用春季繁殖的真鲷种群与春季繁殖的黑鲷种群进行交配。用HCG与LHRH-A_2催产,网箱内自然受精。正交以真鲷为♀,黑鲷为♂;反交以黑鲷为♀,真鲷为♂。两种杂交组合的雌雄性比均为1:1.5。正交组合的受精率为73％,反交组合为67％。正交组合的孵化率为85％,反交组合为87％。该研究证明海水鱼属间远缘杂交可以成功,并为海水鱼杂交育种推广应用奠定了基础。     

黑鲷与灰鳍鲷杂交育种

黑鲷属于鲷科鲷属，广泛分布于我国沿海，灰鳍鲷同属于鲷科鲷属，在我国仅分布于南海。黑鲷与灰鳍鲷杂交属于种间杂交。本研究利用科季繁殖的黑鲷种群与灰鳍鲷种群进行酱。用HCG与LHRH－A2催产，网相内自然受精。正交以黑鲷为♀，灰鳍鲷为♂；反交以黑鲷为♂，灰鳍鲷为♀。两种杂交组合的雌雄比均为1：1.5。正交组合的受精率为83％，反交组合为86％，正交组合的孵化率为91％，反交为89％，该研究证明海水鲁种间杂交可以成功，并为海水鱼杂交育种推广应用奠定了基础。     

Evaluation of genetic relationship among six Labeo species using randomly amplified polymorphic DNA (RAPD)

Labeo species constitute an important group of fish with intense diversity and potential for commercial aquaculture in many Southeast Asian nations including the Indian subcontinent. The present investigation involves the comparative analysis of randomly amplified polymorphic DNA (RAPD) profiles of six Labeo species viz., L. bata (bata), L. calbasu (calbasu), L. dyocheilus (dyocheilus), L. fimbriatus (fimbriatus), L. gonius (gonius) and L. rohita (rohu) at the nuclear DNA variation level. Fifteen decamer random primers were chosen from 40, which amplified a total of 449 DNA fragments ranging in size from 400 to 3000 bp. Both monomorphic and polymorphic DNA bands were identified based on their presence or absence that could be used for specieswise differentiation. Similarity coefficients were calculated to quantify the genetic variation within and between species. On an average, the highest intra‐species genetic similarity value was found in calbasu (0.93) followed by rohu and fimbriatus (0.91), bata (0.87), gonius (0.86) and dyocheilus (0.77). The interspecies genetic similarity estimates among the species of Labeo were used to deduce their phylogenetic relationships. The cluster analysis showed two main clusters, one with calbasu, rohu, fimbriatus and gonius and another with bata and dyocheilus. The study provides evidence that RAPD could be used for genetic differentiation of closely related species.     

Fresh-Keeping Effects of Melanin-Free Extract From Squid Ink on Yellowfin Sea Bream (Sparus latus) During Cold Storage

Squid ink, a by-product from the squid production process, has been demonstrated to be a multifunctional marine bioactive material that can promote freshness of shiokara and peeled shrimp. However, the preservative effect it has on marine fish as well as its preservative mechanisms are still unknown. In this article, Sparus latus was used as a model to assess the preservative effects and the possible mechanism of the ink through chemical parameters, antibacterial effect, and sensory analysis. Melanin-free extract from squid ink at a concentration of 45 mg/mL and pH 8.5 was revealed to be effective in prolonging cold storage shelf life of Sparus latus, which was confirmed through comprehensive sensory analysis evaluation and measurement of chemical changes including pH, microbial count, total volatile basic nitrogen (TVB-N) value, and nucleotide analysis. It was also discovered that the extract mainly suppressed putrefying bacteria separated from Sparus latus, and the bacteriostatic range was confirmed to be wide. Squid ink significantly prolonged the cold storage period of Sparus latus, indicating that it may be developed to be a natural preservative for marine products.     

Genetic variability of Channa punctatus populations using randomly amplified polymorphic DNA

Genetic variations between Channa punctatus populations collected from three rivers of south India were examined using randomly amplified polymorphic DNA (RAPD). Sixty samples from each population were collected. Genomic DNA was isolated from the blood samples. Out of the 20 primers tested, six primers were used for genetic variation studies. A total of 42 RAPD bands were amplified in C. punctatus by the six primers. The total number of bands observed ranged from 34 in the Quilon population to 37 in the Thirunelveli population. Among the three populations, the highest genetic identity (0.9231) was found between Thirunelveli and Quilon populations. The results of the present study demonstrated that Thirunelveli and Quilon populations are more related to each other than to the Coimbatore population.     

牙鲆和大菱鲆养殖群体的分子标记和遗传变异

用１６个１０碱基随机引物对养殖牙鲆（Ｐａｒａｌｉｃｈｔｈｙｓ ｏｌｉｖａｃｅｕｓ）和大菱鲆（Ａｃｏｐｈｔｈａｌｍｕｓ ｍａｘｉｍｕｓ）进行基因组随机扩增多态性ＤＮＡ（ＲＡＰＤ）遗传分析。发现：（１）多个引物具有种的特异性扩增带。其中引物Ｓ７扩增的６９０和１１６０ｂｐ带,引物Ｓ２扩增的６８０ｂｐ带为牙鲆的种的特异性谱带;而引物Ｓ７扩增的６５３和８８９ｂｐ带,引物Ｓ２扩增的６５０、８７０、９９０及１１     

RAPD fingerprinting of the eel-loaches Pangio filinaris and Pangio piperata: preliminary evaluation

Random amplified polymorphic DNA (RAPD) analysis was applied to two morphologically similar species of the increasingly popular ornamental eel‐loaches, Pangio filinaris and P. piperata, collected from Kemat River, Terengganu and Padang Sanai River, Kedah, West Malaysia. Forty primers were tested in a preliminary screening. Of these, five (OPA‐03, OPA‐09, OPA‐11, OPA‐13 and OPA‐18) were chosen for their ability to provide consistent amplification. RAPD analysis differentiated the two species, with each showing different RAPD patterns for each primer used. The five primers generated a total of 82 scorable loci with up to 83% and 60% polymorphism in P. piperata and P. filinaris respectively. The RAPD banding patterns of these two species ranged from 2 to 14 fragments, with a size range of 250–2000 bp. Genetic similarity within species ranged from 0.610 to 0.985 (mean 0.799±0.14) for P. piperata and from 0.824 to 0.934 (mean 0.885±0.04) for P. filinaris respectively. The interspecies genetic similarity ranged from 0.386 to 0.486 with an average value of 0.434±0.040. Our study revealed RAPDs as useful genetic markers for the taxonomic clarification and assessment of genetic variability of the eel‐loaches.     

Genetic diversity and species identification of Argulus parasites collected from major aquaculture regions of India using RAPD‐PCR

Argulus is one of the most important fish parasites that cause heavy economic loss to aquaculture industry. The present investigation was undertaken to study the genetic diversity of the Argulus sp. collected from 13 locations representing major aquaculture zones in India by RAPD analysis and to develop species‐specific markers. Thirteen random decamer primers were used to amplify DNA fragments from three individual parasites of each location. Of the 172 bands scored by the primers, 168 were polymorphic. The per cent polymorphic loci and gene diversity values varied within a range of 8.14–43.02 and 0.0342–0.1727 respectively. Nei's genetic similarity between populations across all the primers ranged from 0.363 to 0.969. The dendrogram based on Nei's genetic distance showed two clusters; Bangalore and Mandi populations forming one cluster, and the rest in another cluster. The clusters also revealed strong correlation with the species identified as A. japonicus and A. siamensis respectively by morphological method. The study thus indicated A. siamensis as the major prevalent species in carp culture farms in India. Species‐specific primers were designed from unique sequences cloned from RAPD fragments that could able to identify A. siamensis and A. japonicus separately.     

Genetic variation of Rutilus rutilus caspicus (Jakowlew 1870) populations in Iran based on random amplified polymorphic DNA markers: a preliminary study

Rutilus rutilus caspicus is regarded as a valuable fish species both for angling and commercial food in Iran. This fish is also considered as a significant food source for beluga sturgeon. The genetic diversity of this fish species collected from two geographical areas (Gorgan Bay and Anzali Wetland) along the Iranian coastline of the Caspian Sea was examined using the analysis of randomly amplified polymorphic DNA (RAPD). Using 10 decamer primers, the total number of RAPD bands produced in both Gorgan Bay and Anzali Wetland populations were 94 bands. The percentages of polymorphic bands were comparable in Anzali Wetland (41.48%) and Gorgan Bay (43.61%), suggesting similar levels of polymorphism of the two populations to be used for establishing selective breeding programmes. The value of Nei's genetic distance (d=0.04) among populations was small, despite the large geographic separation. The data serve as a baseline analysis of current genetic diversity found among R. rutilus caspicus populations in Iran.     

Induction of apoptosis in sea bream fibroblasts by Vibrio harveyi haemolysin and evidence for an anti‐apoptotic role of heat shock protein 70

In this study, we exposed black sea bream, Mylio macrocephalus (Basilewsky), fibroblast (BSF) and silver sea bream, Sparus sarba Forsskål, fibroblast (SSF) cell lines to a recombinant Vibrio harveyi haemolysin (VHH) and investigated mechanisms involved in apoptosis. A decrease in mitochondrial membrane potential, followed by an increase in caspase 3 activity, occurred within 2–8 h of VHH exposure, in both cell lines; however, VHH did not alter cellular levels of reactive oxygen species. As heat shock protein 70 (HSP70) is known to prevent the onset of apoptosis in certain mammalian cells, we aimed to test whether such a protective effect is operative in VHH‐exposed fibroblasts. The amounts of HSP70 were elevated in SSF and BSF via an acute heat shock or an acute heat shock followed by a 6 h recovery. It was found that the VHH‐mediated reduction in mitochondrial membrane potential was suppressed in cells that had a 6 h post‐heat shock recovery, and the protective effect of heat shock‐induced HSP70 was attenuated following treatment of cells with the HSP70 inhibitor, quercetin. This study demonstrates how haemolysin causes cell death via induction of apoptosis and provides evidence as to the role of HSP70 as an anti‐apoptotic factor.     

Effect of different dietary raw to pre-gelatinized starch ratios on growth performance,feed utilization and body composition of juvenile yellowfin seabream (<Emphasis Type="Italic">Sparus latus</Emphasis>)

The effects of different ratios of dietary raw to pre-gelatinized starch on the growth performance, feed utilization and body composition of juvenile yellowfin seabream (Sparus latus) were evaluated during a 10-week growth trial. Five isonitrogenous, semi-purified diets containing 200 g kg⁻¹ starch comprising different ratios of raw to pre-gelatinized starch [(in g kg⁻¹)100:100 (diet 1), 150:50 (diet 2), 50:150 (diet 3), 200:0 (diet 4), 0:200 (diet 5), raw:pre-gelatinized] were prepared and fed to triplicate groups of juvenile S. latus. Our results showed that fish fed diet 4 (200 g kg⁻¹ raw starch) had the highest weight gain and specific growth rate, followed by those fed diet 2, diet 1, diet 3 and diet 5. Feed efficiency, protein efficiency ratio and protein productive value in the fish on diets 4 and diet 2, respectively, were significantly higher than those on diets 3 and diet 5, respectively. Body and muscle compositions were unaffected by the different dietary raw to pre-gelatinized starch ratios. Values of hepatosomatic index, intraperitoneal fat ratio, viscerosomatic index and condition factor did not vary among experimental treatments. Plasma indices showed variations, but these were not relative to dietary treatments. In conclusion, the partial or total replacement of raw starch by pre-gelatinized starch in diets for yellowfin seabream did not improve its growth performance and feed utilization.     

Genetic diversity of Nile tilapia populations revealed by randomly amplified polymorphic DNA (RAPD)

Genetic diversity of the Nile tilapia Oreochromis niloticus collected from the river Nile (Cairo, Assuit and Qena) and two Delta lakes (Burullus and Manzalla) in Egypt was examined by the analysis of randomly amplified polymorphic DNA (RAPD). Of 25 primers examined,21 primers produced 230 RAPD bands. The percentage of polymorphic bands in Manzalla (29.4%) and Burullus (24%) populations was low compared with Assuit (30.54%), Cairo (33.5%) and Qena (44.84%) populations. The highest percentage of polymorphic bands was observed in the Qena population,suggesting a greater potential for use in breeding programs. The molecular phylogenetic tree constructed by unweighted pair‐group method of analysis shows Manzalla and Burullus populations strongly linked and separate from the Assuit and Cairo populations, with Qena population as outgroup. The data serve as a baseline analysis of the current genetic diversity found among O. niloticus populations in Egypt.