【作者】 梁君妮；

【导师】 薛长湖； 许加超；

【作者基本信息】 中国海洋大学 ， 水产品加工与贮藏， 2008， 硕士

【摘要】 海参在分类上属于棘皮动物门中现存的六个纲之一的海参纲。海参纲根据海参的形态特征如:触手的形状、管足和呼吸树的有无分为6个目。而在科的水平上,其分类的主要依据是埋没于外皮之下的微小的石灰质骨片,其中海参咽部的钙质骨板(石灰环)的形态在海参分类上很重要。在种的水平上,其形态上的差别较小,对海参进行种的水平上的区分没有非常精确有效的方法。对于已被加工过的海参产品则更缺乏科学的鉴定方法,需要寻找一种简便准确的方法对加工后的海参进行鉴定。采用聚合酶链式反应(PCR)对日本刺参、大连参、日本关西参、北海道刺参、俄罗斯红参、乌枣参、红海参、白肛海地瓜、红腹海参、小梅花参、黑海参的线粒体COⅠ序列进行了扩增,电泳检测在690bp左右得到了清晰的扩增条带,PCR产物纯化后与T载体连接,进行克隆、测序,分别得到了691-692bp之间的碱基序列,这7个种类海参的COI片段的碱基组成差别都不大,GC含量分布在40.0%-44.6%之间。用MEGA软件检测到,以日本刺参为参照数据,白肛海地瓜、黑海参、红腹海参、红海参、乌枣参、小梅花参变异位点比较多,其中小梅花参变异位点最多,有124个变异位点。而日本刺参、北海道刺参、大连参、俄罗斯红参和关西参变异位点很少,在692bp中只有18个变异位点;白肛海地瓜、黑海参、红腹海参、小梅花参与其他海参的遗传距离都较大,分别约为0.196-0.212、0.197-0.225、0.196-0.243、0.179-0.213,红海参约0.095-0.229,其他海参间仅为0.003-0.012之间;通过计算不同个体间的遗传距离构建NJ树和ME树,在NJ树和ME树中,都可以看到两个分支:俄罗斯红参、日本刺参、关西参、北海道刺参、大连参、乌枣参和红海参、白肛海地瓜为一支,其他的海参为一支。采用聚合酶链式反应(PCR)对日本刺参、大连参、日本关西参、菲律宾刺参、俄罗斯红参、乌枣参、红海参、白肛海地瓜、蛇海参、北极参、靴参的线粒体16Sr RNA序列进行了扩增,电泳检测在330bp左右得到了清晰的扩增条带,PCR产物纯化后与T载体连接,进行克隆、测序,7个种类海参的16Sr RNA片段的碱基组成差别不大,长度分布在330-334bp,只有北极参和蛇海参分别是320bp和310bp。GC含量分布在39.4%-48.1%之间,白肛海地瓜、蛇海参、北极参、菲律宾参变异位点比较多,而日本刺参、大连参、俄罗斯红参和关西参、乌枣参和靴参变异位点很少,且变异位点基本一致,关西参的变异位点相对较多,其中俄罗斯红参只有一个变异位点,大连参有四个变异位点;白肛海地瓜、北极参、蛇海参、菲律宾参与其他海参的遗传距离都较大,分别约为0.150-0.291、0.140-0.282、0.140-0.277、0.194-0.291,大连参,日本刺参,关西参和俄罗斯红参遗传距离很小;在NJ树和ME树中,都可以看到两个分支:俄罗斯红参、日本刺参、关西参、大连参、乌枣参、靴参和红海参、白肛海地瓜为一支,其他的海参为一支。通过对COⅠ和16Sr RNA片段的分析,我们发现,COⅠ序列比16Sr RNA具有更好的特异性,因此选择COⅠ序列对海参深加工产品进行鉴定。采用异丙醇与乙酸钠沉淀的改良方法从海参营养液中提取海参的DNA,通过COⅠ引物进行PCR扩增,扩增产物纯化后与T载体进行连接,通过克隆、测序,与已测定的海参序列及Blast进行比对,确认其中所含海参为青岛刺参。干海参及海参产品中COⅠ和16Sr RNA片段的成功扩增,再次表明了两种引物在棘皮类动物中的良好通用性,本实验对海参种类进行准确鉴定,有助于对海参种类鉴定、遗传变异及系统演化的进一步研究,对规范海参市场也有重要意义。更多还原

【Abstract】 The sea cucumbers, class Holothuroidea, constitute one of the six extant classes of the exclusively marine phylum Echinodermata. The approximately 1200 species of sea cucumbers are partitioned into six orders. Sea cucumber orders are distinguished on the basis of gross morphological characters such as the morphology of the tentacles, the presence or absence of tentacular retractor muscles, the presence or absence of an internal respiratory tree, and the presence and distribution of podia on the trunk. At the familial level, taxonomic distinctions are dependent on a finer level of characters such as the morphology of dermal ossicles, the form of the esophageal calcareous ring, the distribution and morphology of the tube feet, etc. At the species level, the differences in morphological can be quite subtle, obscuring taxonomic distinctions. Because of the lack of the scientific, methods for detecting processed dried cucumbers, it’s necessary to find a simple and accurate way to resolve this problem.Amplified the mitochondrial COⅠgenes extracted from Apostichopus japonicus, Actinopyga miliaris, Acaudina leucoprocta, Holothuria atra, Holothuria edulis, Parastichopus californicus, Thelenota ananas by PCR.691-692bp-size products were detected by electrophoresis,then purified for cloning and sequencing.The results of sequencing show that the average GC contain is 40.0% to 44.6% and there are no obvious differences among COⅠfragment of the seven samples.Take the sequencing data of Riben cucumbers as the standard,we find that Actinopyga miliaris, Acaudina leucoprocta, Holothuria atra, Holothuria edulis, Parastichopus californicus, Thelenota ananas have more transition sites in all samples by using MEGA software and Thelenota ananas has 124 transition sites,while all the Apostichopus japonicus only have 18 transition sites. The difference of hereditary distance between 7 sea cucumbers is few, while between Acaudina leucoprocta, Holothuria atra, Holothuria edulis, Thelenota ananas and other samples is obvious(0.196-0.212、0.197-0.225、0.196-0.243、0.179-0.213). Parastichopus californicus is 0.095-0.229,and others is 0.003-0.012.According to the NJ and MP systerm trees based on the calculation of the hereditary distances of samples, we can have a result that Apostichopus japonicus from Russia, Apostichopus japonicus , Apostichopus japonica from GuanXi, Apostichopus japonica from BieHaiDao, Apostichopus japonicus from DaLian formed a close relationship.Amplified the mitochondrial 16Sr RNA genes extracted from Apostichopus japonicus, Actinopyga miliaris, Acaudina leucoprocta, Holothuria coluber semper, Holothuria mexicana, Parastichopus californicus, Actinopyga mauritaana by PCR.330bp-size products were detected by electrophoresis, then purified for cloning and sequencing. The results of sequencing show that the average GC contain is 39.4% to 48.1% and there are no obvious differences among COⅠfragment of the seven samples.Take the sequencing data of Riben cucumbers as the standard, we find that Acaudina leucoprocta, Holothuria coluber semper,Holothuria mexicana have more transition sites in all samples by using MEGA software. The difference of hereditary distance between 7 sea cucumbers is few, while between Acaudina leucoprocta, Holothuria coluber semper,Holothuria mexicana and other samples is obvious(0.150-0.291、0.140-0.282、0.140-0.277). According to the NJ and MP system trees based on the calculation of the hereditary distances of samples, we can have a result that Apostichopus japonicus from Russia, Apostichopus japonicus , Apostichopus japonica from GuanXi, Apostichopus japonicus from DaLian, Actinopyga miliaris, Actinopyga mauritaana, Acaudina leucoprocta, Parastichopus californicus formed a close relationship.We find that the aberrance of COⅠgenes is more obvious than 16Sr RNA genes through the analysis of COⅠgenes and 16Sr RNA genes,so COⅠgenes are used in identifying the products of the sea cucumber. mitochondria of the sea cucumber nutrient solution extracted with optimized method. Agar gelose electrophoresis and PCR were carried out, then purified for cloning and sequencing. The results of sequencing show that the sea cucumber in the sea cucumber nutrient solution is Apostichopus japonicus from Qingdao.The successful amplification of the COⅠand16Sr RNA fragment reconfirms the primers are applicable for Echinoderm. In this paper, we set up a method that is able to identify a number species of sea cucumbers directly, and it is helpful to the study of the identification and molecular evolution on the sea cucumber.更多还原