- 學位論文
利用DNA編碼技術以psbKI及psbA-trnH基因標誌進行木鱉果品種鑑定
Identification of Momordica cochinchinensis (Gac Fruit) Species by DNA Barcoding Technique Using psbKI and psbA-trnH Gene Markers
摘要
Momordica cochinchinensis Spreng (木鱉果) 是葫蘆科屬之一。他生長於熱帶氣候國家，尤其是東南亞。木鱉果有高含量胡蘿蔔素，有助於人體健康，如抗癌、抗氧化、抗發炎、抗微生物活性是眾所皆知的。但其功能會因來源及種類而有異。因此在鑑定木鱉果品種的方法上是必要的。在2003年時，Paul Hebert開發出的分子生物學技術幫助解決短鏈DNA序列鑑定生物體的相關問題，也稱為DNA編碼技術。在這想研究中，5個DNA片段matK, ITS, rbcL, psbA-trnH, 及psbKI被用來做DNA編碼技術之DNA鑑定，鑑定越南及臺灣的木鱉果種類。從越南及台灣不同的地點採集了25個樣本，並對5個DNA片段進行DNA分離、擴增及定序。親緣關係樹利用MEGA X軟體中的Neighbor-Joining (JN) 方法建造的。利用MEGA X軟體中的p-distance計算p距離百分比。結果顯示，在比對25個樣本時，相似序列百分比最高的為rbcL (99.59%) ，其次是matK (98.84%)、psbA-trnH (95.88%), psbKI (92.88%)，最低的為ITS (79.86%)，來自越南及臺灣，只有參考序列。親緣關係樹結果顯示，本研究中的25個樣本中有10個不同序列的百分比高於2%。其中有4個樣品(VN7, HL1, VN3, 及 VN8)與木鱉果種有密切關係；2個樣品 (HL4, 及 T1) 與M. friesiorum voucher密切相關；3個樣本 (CY2, CY3,及 CY6) 與M. anigosantha voucher密切相關，最後一個與M. suringrii voucher密切相關的標本為VN6。重組基因psbA-trnH與psbKI在親緣關係樹分析中表現優異於單基因分析。重組基因可以更清楚被區別來自臺灣及越南的25個樣品，特別是在Cluster V.1中psbA-trnH的基因親緣關係樹分析。
並列摘要
Momordica cochinchinensis Spreng (gac fruit) is one of the members of the Cucurbitaceae family. It is found in tropical climate countries special in South East Asia. The gac fruit was known with high carotenoid content and benefited human health, such as anti-cancer, anti-oxidant, anti-inflammatory, anti-microbial activities. However, the functionality varies due to their sources and varieties. Therefore, finding a method to identify the species of gac is essential. In 2003 a molecular biology technique developed by Paul Hebert helped solve problems related to identifying an organism based on short DNA sequences, also called the DNA barcoding technique. In this study, 5 DNA fragments matK, ITS, rbcL, psbA-trnH, and psbKI were used as a DNA marker for the DNA barcoding technique to identify gac fruits species in Vietnam and Taiwan. Twenty-five samples were obtained from different places in Vietnam and Taiwan, and then DNA was isolated, amplify and sequencing 5 DNA fragments. The phylogenetic tree built using the Neighbor Joining (JN) method in MAGA X software. The p-distance percent calculated using the p-distance method in MEGA X software. The results showed the highest percent of the similar sequence is rbcL (99.59%) following by matK (98.84%), psbA-trnH (95.88%), psbKI (92.88%), and lowest by ITS (79.86%) when alignment 25 samples from Vietnam and Taiwan with reference sequences only. The phylogenetic tree results showed, 10 in all 25 samples in this study has a percent of the different sequences is higher than 2%. In which four samples (VN7, HL1, VN3, and VN8) have closely related to M. cochinchinensis species; 2 samples (HL4, and T1) closely associated with M. friesiorum voucher; 3 samples (CY2, CY3, and CY6) closely related to M. anigosantha voucher and finally 1 sample closely related to M. suringrii voucher is VN6. The recombined two genes psbA-trnH and psbKI in phylogenetic tree analysis showed bester than single gene analysis. The recombination genes support to discrimination more clearly 25 samples from Vietnam and Taiwan especially 15 samples in Cluster V.1 of psbA-trnH gene phylogenetic tree analysis.