海参DNA条形码的构建及应用

Construction and application of DNA barcoding in sea cucumber Holothurians

为研究线粒体细胞色素c氧化酶亚基Ⅰ(Mitochondrial cytochrome c oxidase subunitⅠ,COⅠ)基因作为DNA条形码鉴定海参品种的可行性,本实验采集了7种海参(Holothuroidea)43个个体并获得其线粒体COⅠ基因序列,利用DNAstar、DNAMAN和MEGA 4.1软件分析计算了7种海参的碱基组成以及不同海参之间的种间遗传距离和种内遗传距离,利用邻接法和最大简约法分别构建了分子系统树。结果表明,海参的种间遗传距离显著大于种内遗传距离,不同海参在系统树中分别形成各自独立的分支,表明以COⅠ作为海参DNA条形码进行品种鉴定具有一定的可行性。在建立海参DNA条形码的基础上,设计了针对仿刺参(Apostichopus japonicas)的特异性探针。对4种海参(仿刺参Apostichopus japonicas、冰岛参Cucumaria frondosa、加州拟刺参Parastichopus californicus及梅花参Thelenota ananas)进行斑点杂交实验,结果显示,该探针具有较好的特异性和较高的灵敏度,能够实现对仿刺参的鉴定。本研究为后续开展斑点杂交及基因芯片法鉴定海参种类的研究奠定了理论基础。

DNA barcoding is a diagnostic technique in which short DNA sequences can be used for species identi-fication.The mitochondrial cytochrome c oxidase subunit I（CO I） sequence is used in DNA barcoding for species identification in many organisms.In this paper,the validation of the COI gene for DNA barcoding and species identification in sea cucumbers was studied.The CO I genes from seven species of sea cucumbers were amplified and sequenced.Data were analyzed using DNAstar,DNAMAN,and MEGA 4.1.The DNA base composition was analyzed,and the genetic variations among species and within species were calculated.It was found that the ge-netic distance among the seven species was higher than that within a species.Phylogenetic analysis using neighbor-joining and maximum parsimony methods generated tree topologies that indicated that all individuals of each species formed a strong monophyletic group.These results suggested that the mitochondrial CO I gene was a valid DNA barcoding gene for species identification in sea cucumbers.On the basis of the DNA barcoding,spe-cies-specific probes of Apostichopus japonicus were designed.Dot-blot hybridization showed that the probes could identify A.japonicus with high specificity and sensitivity.The design of further probes for different sea cu-cumber species,combined with dot-blot hybridization and microchip assays,will facilitate the identification of sea cucumbers at relatively low costs.