The Construction And Application Of Molecular Methods For Sea Cucumber Species Identification Based On DNA Boarding

Sea cucumbers are regarded as traditional marine food and medicine in Asia.They present a high nutritional value due to their high protein, low fat, amino acidprofile and rich trace elements. It has been reported that sea cucumber was proven toexhibit various biological activities including anti-tumor, immunoregulatory,anti-atherosclerotic and anti-aging properties. Currently, there are a lot ofdeep-processing sea cucumber products, such as dried products, canned products andsea cucumber capsule. However, the rapid expansion and intensification of seacucumber markets has resulted in various problems, such as commercial fraud,adulterating mislabeling, or substituting high-value species with low-value species.According to this, the rapid identification of sea cucumbers species is required forprotecting consumers’ rights urgently.Conventionally, sea cucumber species can be distinguished on the basis ofmorphological and dissection characters, which is difficult when sea cucumbers are inthe state of deep processing. Molecular biology techniques, which permitdiscrimination through the analysis of a small segment of the genome, represent oneextremely promising approach to the diagnosis of biological diversity. Molecularbiology techniques are becoming more and more popular in species differentiation.DNA barcoding is a diagnostic technique in which short DNA sequences can beused for species identification. The mitochondrial cytochrome c oxidase subunit Ⅰ(COⅠ) sequence is used in DNA barcoding for diverse species of organisms. In thisthesis, the validation of COⅠ gene as DNA barcoding for species identification insea cucumbers is discussed. The COⅠ gene in seven species of sea cucumbers isamplified and sequenced. The data are analyzed by using DNAstar6.1, DNAMAN6.0and MEGA4.1. The DNA base composition is analyzed, and the genetic variationamong species and within species are calculated. It was found that genetic distanceamong seven species is higher than the distance within a species. Phylogenetic analysis is carried out with neighbor-joining and maximum parsimony methods. Thetopology of either tree reveals that all individuals of each species form a strongmonophyletic group. Results suggest that mitochondrial COⅠ gene is a valid DNAbarcoding gene for species identification of sea cucumbers.On the basis of DNA barcoding, dot-blot hybridization is developed to identifyfour sea cucumber species (Apostichopus japonicus, Cucumaria frondosa, Thelenotaananas, and Parastichopus californicus). After constructing and sequencing the692bp fragments of COⅠ gene from four sea cucumber species, species-specificprobes are designed using Primer premier5.0. The specificity and sensitivity of thefour probes are tested. The results show that the probes could identify four seacucumbers respectively with high specificity, and the sensitivity achieves100pg.Therefore, dot-blot hybridization provides a convenient, useful, specific and academictechnique to authenticate the species of sea cucumbers. After designing more probesfor different sea cucumbers, dot-blot hybridization and microchip assay will facilitateresearches on identification of sea cucumbers at relatively low costs.Multiplex-PCR is developed to identify the four sea cucumber species. After the692bp fragments of mitochondrial cytochromeⅠ(COⅠ) gene are analyzed andcompared, four sets of species-specific primer are designed with Primer premier5.0software. The amplified lengths of fragments are212bp for Apostichopus japonicas,301bp for Thelenota ananas,358bp for Cucumaria frondosa, and261bp forParastichopus californicus respectively, which can be obviously differentiated onDNA electrophoresis. The four sets of species-specific primer are mixed and appliedto detect sea cucumber species simultaneously. The results show that themultiplex-PCR could identify four sea cucumbers respectively with high specificity,and the sensitivity achieves2ng. Artificially-generated mixtures were analyzed by themultiplex-PCR assay. The results show that two sea cucumber species can besimultaneously detected in the one mixture, even with one species in a highbackground of another one. It also can detect four sea cucumber speciessimultaneously in one vial. The COⅠ gene sequences of Parastichopus californicus, Cucumaria frondosa,Apostichopus japonicas, Thelenota ananas, Actinopyga lecanora are analyzed withDNAMAN6.0software to construct restriction maps of the five sea cucumber species,BamHⅠ, KpnⅠ, PstⅠ, XbaⅠ, Eco31Ⅰ endonucleases are chosen to performPCR-RFLP. The results show that the constructed PCR-RFLP methods candifferentiate the five sea cucumber species in a convenient way and be able to detectand identify the presence of mixed sea cucumber species clearly as well.Different processing samples of the four sea cucumber species are detected withdot blot assay, multiplex-PCR and PCR-RFLP respectively. The result reveals that theprecision rate of multiplex-PCR is100%, the precision rate of PCR-RFLP is87.5%,and the precision rate dot blot assay is75%.Therefore, the study provides three convenient, useful, specific and academictechniques to authenticate the sea cucumber species. These methodologies can be veryuseful for traceability of sea cucumber species.