【作者】 吴志刚；

【导师】 戴志远；

【作者基本信息】 浙江工商大学 ， 食品科学， 2010， 博士

【摘要】 虾的种类繁多,仅联合国粮农组织(FAO)列出的具有商业或具有潜在商业价值的虾就有300多种,为重要的经济水产品之一。据FAO统计,全球每年捕捞和养殖虾的产量约为600万吨,其中约60%进行国际贸易。目前虾产品年出口值超过140亿美元,占渔业总出口值的16%,在国际贸易中具有重要地位。当前,中国沿海养殖和捕捞(含海水和淡水)的虾主要有斑节对虾(Penaeus. momodon)、凡纳对虾(Penaeus vannamei)、中国对虾(Penaeus. chinensis)、日本对虾(Penaeus japonica)、罗氏沼虾(Maeobrachium rosenbergii)、墨吉对虾(Penaeus merguiensis)、中华管鞭虾(Solenocera crassicornis)、鹰爪虾(Trachypenaeus curvirostris)克氏原螯虾(Procambarus clarkii)等,年出口量近30万吨,产值近17亿美元。近年来,国际上对贸易品种的要求日益严格,物种鉴定己成为出口农产品、食品的技术性贸易壁垒之一。通常,虾类的品种鉴定主要依据形态学标准(如外形、颜色等)来进行。然而当用于形态辨别的部分被去除、形态非常相近或未形成可辨别形态时,虾类的品种鉴定则显得非常困难,因此采用分子鉴定标记进行品种鉴定的研究日益引起重视,如核糖体转录间隔区、线粒体细胞色素氧化酶C基因、线粒体细胞色素b基因等。本研究首先利用虾类核糖体基因高度保守的18s、28s和中间的5.8s区域,设计出了通用引物,对中国几种主要出口虾类ITS1区域进行PCR扩增并测序。根据测序结果,筛选出了合适的选择性内切酶,针对所研究各虾品种的ITS1序列产生出特异性的酶切图谱,将它们作为分子标签,应用于中国主要出口虾类品种及其产地的快速鉴定,具有特异性强、重复性高、检测时间短、成本低的特点。在该部分研究中,利用所设计的引物,所得到虾类ITS1长度变化在448 bp(戴氏赤虾)到1491 bp(日本沼虾)之间,GC含量为40.95-66.83%,其中戴氏赤虾和日本沼虾的ITS1序列为目前所报道的最短和最长的虾类ITS1序列。各虾品种的ITS1序列比较发现,微卫星序列大多出现在5′端和中间区域。同一种虾品种的微卫星序列差异与群体间ITS1的变异和长度多态性紧密相关,而且对虾科对虾属各虾品种比较发现,亲缘关系越近,其微卫星序列相似度越高。对虾科5个虾品种之间可用ITS1数据进行很好的区分。此外,结合PCR-RFLP技术,不同虾品种的ITS1序列可产生特定的酶切片段,可以此对它们进行有效区分。这些结果表明,ITS1序列作为一个重要的分子标签,在不同分类水平上具有很高的变异性,在虾类品种鉴定和系统发育研究中具有重要的作用。本研究还采用荧光标记引物的AFLP分析方法,对中国主要出口虾类资源进行了遗传变异和品种鉴别研究,依据AFLP标记的数据结果,我们对对虾科下不同属的虾类系统亲缘关系进行了分析。结果显示：在遗传距离上,日本对虾(P.japonicus)和宽沟对虾(P. monodon)之间最小,为0.214；中华管鞭虾(S. crassicornis)和鹰爪虾(T. curvirostris)之间最大,为0.659；遗传一致度上,日本对虾(P.japonicus)和宽沟对虾(P. monodon)最近,为0.787；管鞭虾科的中华管鞭虾(S. crassicornis)和对虾科鹰爪虾属的鹰爪虾(T. curvirostris)之间最远,为0.341。二种分析方法结果基本一致,说明基于AFLP标记揭示的不同虾类之间的遗传距离比较稳定。依据Jaccard遗传相似性系数构建对虾科下所有供试虾样品的UPGMA系统聚类关系图,其中中华管鞭虾(S. crassicornis)作为Outgroup单元对照,独立在外,成一个单独的分支,所有对虾科虾类均聚为一类,在Jaccard遗传相似性系数为0.38处,对虾科内不同属虾类可进一步细分为三大分支。第Ⅰ分支主要由哈氏仿对虾(P.hardwickii)、刀额新对虾(M. ensis)和戴氏赤虾(M.dalei)三类组成；第Ⅱ分支主要由宽沟对虾(P.latisulcatus)、日本对虾(P. japonicus)和凡纳滨对虾(L. vannamei)组成；第Ⅲ分支仅由鹰爪虾(T. curvirostris)组成。这表明利用AFLP标记可以揭示不同虾类之间的遗传差异程度,分析物种之间的系统亲缘关系。本研究中12对引物共扩增出了841条DNA谱带,所有条带的分子量范围在50-550bp之间,其中601条为多态性条带,多态性位点百分率为71.5%,平均每对引物扩增出70.1条总带和50.1条多态性条带。相比于其它研究者采用AFLP标记对对虾科虾类的分析结果来说,多态性位点百分率相对偏高。在引物鉴别效率方面,12对AFLP引物中可将所有供试虾样品100%区分开的引物组合有4对：E-ACG/M-CAA、E-AGG/M-CTA、E-AGG/M-CTC和E-AGG/M-CTT,12对引物的平均鉴别效率达92.8%,可见AFLP标记用于虾种水平上的鉴别效率非常高,这也间接反映出不同虾之间的遗传背景差异较大。本研究采用ITS技术和AFLP技术分析了不同地理区域的凡纳对虾群体的遗传变异和群体遗传分化。研究结果显示,目前我国东南沿海凡纳对虾主要养殖区养殖的凡纳对虾的种质资源与来自美国夏威夷的亲本已有较大的差异,需引起高度重视。建立的技术方法对我国主要养殖虾类的品质退化情况评估、虾苗健康程度的评估,促进虾类养殖业的健康发展,维护中国虾产品出口贸易具有重要的现实和前瞻性研究意义。更多还原

【Abstract】 Shrimp is an important marine animal with various kinds and more than 300 species of shrimp are of economic interest worldwide listed by FAO. According to FAO investigation, world production of shrimp, both captured and farmed, is about 6 million tones, and about 60% enters the international markets. Shrimp is now the most important internationally traded fishery commodity in terms of value. In china, shrimp also has an important postion in export trade, with the export weight nearly 300,000 tones per year and export value nearly $1.7 billion per year (both captured and farmed), including Penaeus momodon, Penaeus vanname, Penaeus chinensis, Penaeus japonica, Maeobrachium rosenbergii, Penaeus merguiensis, Solenocera crassicornis, Trachypenaeus Curvirostris, Procambarus clarkii mainly.With the requirement becoming more strictly, species identification has become one of technical trade barriers for export food and farm commodities. Traditionally, morphological characters are used to identify shrimp of different species. But the gross morphology of some closely related species are so similar that separation of them requires very detailed morphological knowledge known usually only to experts. Moreover, this method is difficult to be implemented for the investigation of processed shrimp products, in which the discriminable morphological characters are unavailable. Therefore, many efforts have been made recently to identify shrimp species using the more accurate molecular markers, such as the internal transcribed spacers (ITS) of nuclear ribosomal DNA (rDNA), mitochondrial cytochrome c oxidase sununit I (COI) gene and the cytochrome b (Cyt b) gene.In this study, the highly conserved 18s,28s and 5.8s ribosomal genes of shrimp were used to design universal primer and amplify the ITS1 region of major exported shrimp species in China. According to the sequencing results, proper restriction enzyme was selected, to produce specific restriction endonuclease map as a molecular label to identify the species and origin of tested shrimp species, with strong specificity, good repeatability and low cost. Using the selected primers, length of the amplified ITS 1 sequences in this study ranged from 448 bp to 1491 bp, with a GC content of 40.95-66.83%. To our knowledge, the ITS1 of M. dalei and M. nipponense were the shortest and the longest sequences obtained in shrimp species up to date. Many microsatellite loci were found at 5’ end and the middle region of ITS 1 sequences. For samples of the same species, these microsatellite loci seemed to be closely associated with intragenomic sequence variation and length polymorphisms, and they showed a higher degree of similarity in composition between species with closer relationship even in the family Penaeidae. This variation might obscure phylogenetic relationship between some shrimp populations, but the separation of five Penaeus species was well supported. In the combination with polymerase chain reaction-restriction fragment length polymerism methods (PCR-RFLP) analysis, ITS1 sequences from shrimp species belonging to different families and genera were also easily discernable. The results suggested that ITS1 was highly variable among different shrimp groups and could be an appropriate marker for species identification and molecular systematic studies.In this study, the fluorescence labeling techniques of AFLP was used to research the species identification and genetic variation level for China’s major export shrimp species and analysis the systemic genetic relationships between different genuses of Penaeidae. The results of AFLP analysis indicated that the minimum genetic distance (0.214) and the maximum genetic identity (0.787) were between P. japonicus and P. monodon, and the maximum genetic distance (0.659) and the minimum genetic identity (0.341) were between S. crassicornis and T. Curvirostris. The results of genetic distance was similar to that by genetic identity, which show that the genetic distance obtained by AFLP labeling is stabilization relatively between different genuses of Penaeidae. Matrix of Jaccard’s coefficients of genetic similarity was used to construct the dendrogram by UPGMA for all sample of Penaeidae. Using S. crassicornis as outgroup, all the tested shrimp species in the family of Penaeidae were clustered together. In the distance of 0.38 Jaccard, the Penaeus species were further subdivision as three obvious clades. M. ensis, M. dalei and P. hardwickii formed the first clade. The P. latisulcatus gathers with P. japonicus first before gathering with L. vannamei, forming the second clade. The group of T. curvirostris clustered to the third clade. These results demonstrate that the AFLP marker could reflect the genetic distances between shrimp species, and analysis their genetic relationships.In this study, a total of 841 DNA bands were detected and 601 bands were the polymorphic bands revealed by the 12 primer pairs, ranging from 50 to 550 base pairs (bp). The polymorphism rate was about 71.5%, the average bands and polymorphic bands amplified by each primer combination were 70.1 and 50.1, respectively. Comparison with the other reports about Penaeus, the polymorphism rate is higher in our AFLP analysis. Among the 12 primer pairs,4 primer combinations (E-ACG/M-CAA, E-AGG/M-CTA, E-AGG/M-CTC and E-AGG/M-CTT) can distinguish all the tested shrimp species. The average identification efficiency rate of 12 primer combinations is 92.8, which indicates that AFLP fingerprinting is effective in species identification, and means the genetic background was obvious different among the studied shrimps species.In this study, ITS sequence and AFLP were used to analysis genetic variation levels and diversity among different geographical populations of P. vannamei and P. japonica. The results indicate an obvious genetic divergence between the farmed P. vannamei populations in southeast China and the broodstock population from Hawaii, USA. Based on.the. obtained results,.we. regarded the established methods.could be used to evaluate the species degeneration and health degree of shrimp larvae, and would have important practical and prospective significance for promoting the development of aquaculture industry in China.更多还原