Molecular Authentication of Endangered Reptiles for Chinese Medicinal Materials Wong Ka Lok A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Philosophy in Biochemistry ©The Chinese University of Hong Kong July 2001 The Chinese University of Hong Kong holds the copyright of this thesis. Any person(s) intending to use a part or whole of the materials in the thesis in a proposed publication must seek copyright release from the Dean of the Graduate School. f/統系It書因 I \ UPR 12 V •F.，"- —-™ 一 � .\ �vii^W—...�/ Acknowledgements I would like to express my pleasure to my supervisor Dr RC. Shaw whc offered guidance and advice on my project. His thoughtful comments, questions and suggestions were invaluable. I also acknowledge to my other supervisor, Dr J. Wang. This work could not have been completed without the support provided by him. Special thanks goes to Mr. F.C.F. Yau for the general technical advice and support. I would like to thank the Agriculture, Fisheries and Conservation Department of HKSAR and the Kadoorie Farm & Botanic Gardens for supplying some of the samples and Environment and Conservation Fund for providing partial financial support for my work. t - i Abstract Selected DNA sequences of cytochrome b and 16S rRNA genes were amplified and sequenced from eight snake and four crocodile species. Sequence homology among these species and individuals of the same species was compared. It was found that interspecific variation was much higher than intraspecific variation and the identity of the concerned reptiles could be revealed by their DNA sequences. Cytochrome b and 16S rRNA gene sequences of snake species recorded in GenBank were retrieved. Using the available sequences, cytochrome b and 16S rRNA sequence databases of snake were constructed and this provides a convenient reference for other researchers to retrieve and analyze DNA sequences of snake species. PCR-RFLP DNA fingerprints were generated from eight snake and four crocodile species. Each of these species has a characteristic pattern that distinguishes from each other. DNA fingerprints for three medicinal important snake species, Agkistrodon acutus, Bungarus multicinctus and Zaocys dhumnades, were generated by RAPD. The polymorphic RAPD marker generated was then sequenced and converted into SCAR. A pair of SCAR primers specific to four crocodile species was designed based upon the 16S rRNA sequences and used to discriminate the four crocodiles. ii Ten unknown fresh and dried meat samples were obtained from the Agriculture, Fisheries and Conservation Department of HKSAR to test the applicability of the protocols developed. DNA was extracted from these samples and the concerned cytochrome b gene fragments were sequenced. The identity of the nine samples was revealed to high reliability, and the remaining one sample failed to do so owing to the lack of available cytochrome b sequences in the database for comparison. This confirms that the DNA sequencing is a reliable tool for authentication of endangered and medicinal important materials. •；t ‘ iii 摘要 利用PCR技術擴增八種蛇和四種鱺魚線粒體細胞色素b和16S核糖體基因 片段，然後進行DNA序列分折並計算種內及種外的序列差異0結果表明種間序 列差異遠較種內差異高。因此，可利用這些序列鑒別蛇和鱷魚樣本°又從 GenBank基因庫中匯集其他品種蛇的細胞色素b和16S核糖體DNA序列並構建 一公眾數據庫，方便其他硏究人員取得並分析有關蛇類的基因資料° 由硏究所得的DNA序列，應用PCR-RFLP檢測了八種蛇及四種鱷魚基因 的多態性0結果顯示相關的蛇及鱷魚可由其獨特指紋圖譜分辨出來。 利用RAPD技術對三種中藥常用蛇一五步蛇，銀腳帶和烏梢蛇進行分析， 首先得出隨機擴增多態DNA指紋圖譜，並將所得之多態性DNA片段轉化成 SCAR�又比較四種常見動物、人類及此項硏究中八種蛇、四種鱷魚樣本的16S 核糖體基因序列，成功找出一段鱷魚獨有之保守序列，然後由該段序列設計出 可成功鑒別該四種鱷魚的一對SCAR弓丨物。 爲了確定此項硏究之實用性，從香港特區政府漁農及自然護理署取得十個 新鮮及乾肉樣本。利用細胞色素b弓丨物擴增基因及進行序列分析，成功地辨別 其中九個樣本的來源，其中一個樣本因缺乏參考序列而無法辨認。結果表明我 所建立的方法有助於執法機關_別瀕危動物° iv Table A. Tables in this thesis Table ‘ Description Page no. Table 1.1 Snake species in the world. ^ Table 1.2 Snake species listed in appendices of CITES. 4 Table 1.3 Twenty-three species of crocodile in the world. 6 Table 3.1 Eight snake species used in this study 32 Table 3.2 Sequence homology of cytochrome b among six snake species. 37 Table 3.3 16S rRNA gene sequence homology among eight snake species. 46 Table 3.4 A partial cytochrome b sequence database of 90 species of snakes. 51 Table 3.5 Cytochrome b sequence homology of 90 species of snake. 52 Table 3.6 A partial 16S rRNA gene sequence database of 63 species of snakes. 53 Table 3.7 16S rRNA gene sequence homology of 63 species of snakes. 54 Table 4.1 Restriction fragments of partial cytochrome b gene of six snakes. 61 Table 4.2 Restriction fragments of 16S rRNA gene of eight snakes species. 66 Table 5.1 The four crocodile species examined in this study. 75 Table 5.2 Cytochrome b sequence homology among six crocodile species. 82 Table 5.3 16S rRNA gene sequence homology among five crocodile species. 87 Table 5.4 Size of fragments generated after digestion of partial cytochrome b of 89 four crocodile species with restriction enzymes. Table 5.5. Size of fragments generated after digestion of partial 16S rRNA gene 91 fragments of four crocodile species with restriction enzymes. Table 6.1 Ten unknown samples received from AFCD. 101 Table 6.2 Revelation of identity of the ten unknown samples. 110 Table 6.3 Cytochrome b sequence homology among sample F2 and three animal 110 species. Table 6.4. Cytochrome b sequence homology among dried unknown samples and 111 common animals and human. Table 6.5. Cytochrome b sequence homology of dried unknown samples and 112 crocodiles. Table 6.6 Cytochrome b sequence homology among crocodiles. 112 T able 6.7. Cytochrome b sequence homology among dried unknown samples and 113 Python. Table 6.8 Cytochrome b sequence homology among seven species in 114 Osteoglossiformes. V Table B. Figures in this thesis. Figure Page number � Figure Page number Fig. 1.1 7 Fig. 5.2 78 Fig. 1.2 8 Fig. 5.3 79 Fig. 1.3 9 Fig. 5.4 80 Fig. 1.4 10 Fig. 5.5 81 Fig. 3.1 34 Fig. 5.6 83 Fig. 3.2 35 Fig. 5.7 84 Fig. 3.3 36 Fig. 5.8 85 Fig. 3.4 38 Fig. 5.9 86 Fig. 3.5 39 Fig. 5.10 88 Fig. 3.6 40 Fig. 5.11 90 Fig. 3.7 41 Fig. 5.12 90 Fig. 3.8 42 Fig. 5.13 92 Fig. 3.9 43 Fig. 5.14 92 Fig. 3.10 44 Fig. 5.15 93 Fig. 3.11 47 Fig. 5.16 94 Fig. 3.12 48 Fig. 5.17 95 Fig. 3.13 49 Fig. 5.18 96 Fig. 4.1 59 Fig. 5.19 96 Fig. 4.2 60 Fig. 6.1 100 Fig. 4.3 60 Fig. 6.2 102 Fig. 4.4 62 Fig. 6.3 103 Fig. 4.5 62 Fig. 6.4 103 Fig. 4.6 63 Fig. 6.5 103 Fig. 4.7 63 Fig. 6.6 104 Fig. 4.8 64 Fig. 6.7 104 Fig. 4.9 64l Fig. 6.8 105 Fig. 4.10 65 Fig. 6.9 106 Fig. 4.11 67 Fig. 6.10 107 Fig. 4.12 68 Fig. 6.11 108 Fig. 4.13 69 Fig. 6.12 109 Fig. 4.14 70 Fig. 6.13 113 Fig. 4.15 71 Fig.Al 130 Fig. 4.16 72 Fig. A2 131 Fig. 5.1 I Ti\ J vi Table of Contents Acknowledgments i Abstract u Table A v Table B vi Table of Contents vii 參 Abbreviations Chapter 1 Molecular authentication of endangered crocodiles and snakes 1.1 Introduction 1 1.2 Traditional method of snake and crocodile identification 1.2.1 Morphology 7 1.2.2 Chemical Analysis 9 1.3 Molecular Technology in Authentication 1.3.1 Polymerase Chain Reactions (PCRs) 11 1.3.2 Random-primed amplification reaction 12 1.3.3 Sequence Characterized Amplified Region (SCAR) 13 1.3.4 PCR-RFLP 13 1.3.5 DNA sequencing 1.4 Objectives and strategies of the study 15 Chapter 2 Materials and General Methods 2.1 Reagents and Buffers 2.1.1 Buffers for Total DNA Extraction 17 2.1.2 Reagents for Agarose Gel Electrophoresis 17 2.1.3 Reagents for Plasmid DNA Preparation 18 2.1.4 Medium for Bacterial Culture 18 2.1.5 Reagents for Preparation of Competent Cells 19 2.2 DNA Isolation 2.2.1 Extraction of DNA from meats 20 2.2.2 Extraction of DNA from blood 20 2.3 Phenol/Chloroform Extraction 21 2.4 Ethanol Precipitation H vii 2.5 DNA Concentration/Purity Estimation 22 2.6 Mitochondrial DNA amplification 23 2.7 Random-Primed Polymerase Chain Reactions 24 2.8 SCAR for Snake samples 24 2.9 SCAR for Crocodile samples 25 2.10 Restriction fragment length polymorphism analysis 25 2.11 Agarose Gel Electrophoresis of DNA 26 2.12 Purification of PGR product 26 2.13 Preparation of Escherichia coli Competent Cells 27 2.14 Ligation and transformation ofE. coli 27 2.15 Plasmid preparation 28 2.16 Screening of Plasmid DNA by Restriction Digestion 29 Chapter 3 DNA sequencing of snakes & construction of snake database 3.1 Introduction 30 3.2 Materials and methods 3.2.1 Snake samples 32 3.2.2 DNA Extraction, mitochondrial gene amplification and DNA sequencing 33 3.2.3 Construction of database 33 3.3 Results 3.3.1 Cytochrome b gene amplification and sequencing 34 3.3.2 Gene amplification and sequencing of 16S rRNA 42 3.3.3 Cytochrome b sequence database 50 3.3.4 16S rRNA sequence database 53 3.4 Discussion 3.4.1 Cytochrome b and 16S rRNA genes of snake species 55 3.4.2 Cytochrome b and 16S rRNA databases 55 Chapter 4 Application of PCR-RFLP and SCAR in snake species identification 4.1 Introduction 57 4.2 Material and Methods 4.2.1 DNA extraction and PCR-RFLP 58 4.2.2 RAPD and SCAR 58 viii 4.3 Results 4.3.1 PCR-RFLP of cytochrome b genes of snakes 59 4.3.2 PCR-RFLP of 16S rDNA 61 4.3.3 RAPD & SCAR analysis 67 4.4 Discussion Chapter 5 Application of DNA sequencing, PCR-RFLP and SCAR to identify crocodile species 5.1 Introduction 74 5.2 Materials and methods 5.2.1 Crocodile, human and four animal samples 75 5.2.2 DNA Extraction, mitochondrial gene amplification and DNA sequencing 75 5.2.3 PCR-RFLP and SCAR 76 5.3 Results 5.3.1 Isolation of crocodiles DNA 77 5.3.2 Isolation of DNA from Human and four animal species 78 5.3.3 Cytochrome b gene amplification and sequencing 78 5.3.4 16S rRNA gene amplification and sequencing 84 5.3.5 PCR-RFLP of cytochrome b 89 5.3.6 PCR-RFLP of 16S rRNA 91 5.3.7 SCAR primers for four crocodile