Identification Of Species Of Pufferfish And Its Product With Molecular Biology Techniques

ObjectTo establish molecular biological methods based on both DNA and protein to identify pufferfish and its product.DNA technique include Cytb sequence analysis with phylogenetic analysis,bootstrap test and RFLP.SDS-PAGE of SDS soluble protein in pufferfish is assessed as a protein technique to identify pufferfish species.MethodMN DNA extract kit(from German)and improved phenol-chloroform extract method are used to extract DNA from pufferfish,ultraviolet spectro photometric analysis is used to evaluate the effect of the methods.Bone is pre-processed first by grinded with liquid nitrogen,then incubation in EDTA to decalcification.Canned fish and roasted fish filet usually contain oil which affect the extraction of DNA and should be removed by incubation in solution (methanol:chloroform:H₂O=2:1:0.8)Estabalish a method to amplification Cytb fragment about 300bp with universal primer.Amplificate the Cytb fragment from pufferfish and its product and send to Invitrogen Co.to sequence.Download Cytb DNA from Genebank.Do cluster alignment to all the sequence,then do phylogenetic analysis and bootstrap test in MEGA sofeware,evaluate the feasibility of this method in identification of pufferfish species.Choose Cytb whole sequence(1200bp) and 300bp fragment as objects,Screen restriction endonuclease which can disgest Cytb of different pufferfish into various part with Primer Primer 5 software.Identify the species of pufferfish by compare the electrophoretic patterns.Extract SDS soluble protein from pufferfish,adjust the concentration to 2mg/mL, and do SDS-PAGE,identify pufferfish by compare the electrophoretic patterns.Result The A260/280 of DNA is near 1.8.Improved phenol-chloroform extract method is prefer with canned fish and roasted fish fillet.DNA from bones can also be extract.The universal primer used in this research can amplify Cytb fragment of pufferfish effectively,the product is only about 300bp,which can be amplified from heated and high pressure processed fish product.Phylogenetic analysis and bootstrap test to all the sequence of Cytb of pufferfish show that pufferfish from the same species cluster into the same branch,and bootstrap＞60.The pufferfish whose species is already known has been clustered into the right branch,which indicate the effectiveness of this method.RFLP of Cytb whole sequence could easily identify all the 7 species pufferfish used in this research,include pufferfish from the same genus.For Cytb fragment about 300bp,Lagocephalus inermis and Lagocephalus gloveri could be easily identified by two endonuclease,but identify species between Takifugu genus is rather difficult.SDS-PAGE of SDS soluble protein show Lagocephalus inermis and Lagocephalus gloveri have special pattern,but electrophoretic patterns between the species of Takifugu genus is almost the same,this method is not suitable for identification species in the same genus.ConclusionBoth MN DNA extract kit(from German)and improved phenol-chloroform extract method can extract DNA effectively from pufferfish.The methods used universal primer to amplify Cytb fragment of pufferfish and phylogenetic analysis and bootstrap test to compare all the sequence of Cytb could distinguish the species of the pufferfish.RFLP of Cytb whole sequence could easily identify pufferfish,include pufferfish from the same genus,while the Cytb fragment about 300bp,could only distinguish the pufferfish of the different species.SDS-PAGE of SDS soluble protein could distinguish Lagocephalus inermis and Lagocephalus gloveri,but could not distinguish the pufferfish in the species of Takifugu genus.