番木瓜種子不同部位所萃取核酸之PCR擴增效果

DNA Extraction from Different Parts of Papaya (Carica papaya L.) Seeds for PCR Analysis

陳哲仁(Jen-Ren Chen) ； 陳淑惠(Shu-Hui Chen) ； 周明燕(Ming-Yenn Chou) ； 周佳霖(Jia-Lin Chou) ； 張惠如(Hui-Ju Chang) ； 孫永偉(Young-Wei Sun) ； 鍾文全(Wen-Chuan Chung)

《植物種苗》 17卷1期 (2015/03) Pp. 47-58

摘要

番木瓜是全球性重要水果，由於現有基因改造番木瓜存在，為了正確檢測番木瓜種子是否為基因改造產物，良好的核酸樣品製備方法及PCR反應條件是檢測的重要基礎。本試驗以市售番木瓜’台農2號’之成熟種子整粒、種皮以及胚(含胚乳)作為試驗材料，經過核酸萃取後分析波長260、280及230 nm吸光值，以評估核酸品質，其中所抽取DNA之A260/A230比值均介於0.57-0.87之間，明顯低於標準值(2.0-2.4之間)，顯示由各種子部位所萃取核酸中存有豐富的干擾物質，可能會影響PCR擴增效果。進一步以相同PCR條件、在20 ul反應體積以100ng DNA作為模板，進行三者核酸品質的測試，結果顯示自胚(含胚乳)萃取核酸具有最佳之PCR擴增效果，且產量達3.45-13.31 ng/μl，然而完整種子及種皮所萃取核酸則多無擴增任何PCR產物。為降低胚(含胚乳)萃取核酸的雜質濃度以獲得最佳之PCR擴增，透過DNA模板用量系列稀釋方式，得知以15-25 ng DNA模板可得到最佳之PCR擴增效果。此外，在未成熟種子萃取核酸中PCR干擾物含量較低。本研究所建立之番木瓜胚(含胚乳)核酸萃取標準程序應可有效用於基改番木瓜種子之檢測。

關鍵字

番木瓜 ； 基因改造 ； 種子 ； 核酸品質

並列摘要

Papaya is a major fruit crop in tropical and subtropical regions worldwide. Genetically modified papayas with resistance to ringspot virus have been commercialized in the world. To effectively detect GM papaya, DNA quality and polymerase chain reaction conditions are critical influences. The quality of DNA extracted from intact seed, seed coat and embryo (+endosperm) of papaya cv.‘Tainung No. 2 was evaluated by absorbance values of 260, 280 and 230 nm. Results showed that the ratios of A260/230 ranged from 0.57-0.87 were lower than the expected values (2.0-2.4), indicating that the genomic DNA of papaya seed is rich in organic compounds and that may affect PCR amplification. Evaluations of PCR efficacies for DNA samples extracted from different parts of the seed were carried out with the same amount of DNA template and PCR conditions. PCR product was able to amplify DNA templates extracted from embryo (+endosperm), but not from intact seed and seed coat in most cases. To further reduce impurities in the PCR mixtures, a serial dilution of DNA samples extracted from embryo (+endosperm) was carried out. The optimal quantity of DNA template was 15-25 ng in 20 ul reaction volume. Furthermore, there are relative less PCR inhibitors accumulated in immature papaya seeds. The findings in this study would be helpful to establish standard detection method for genetically modified papaya seeds.

並列關鍵字

Carica papaya ； seeds ； genetic modified ； DNA quality ； polymerase chain reaction