基因改造蘋果Real-Time PCR檢驗方法之建立   全文下載

Development of Real-Time PCR Detection Methods on Genetically Modified Apple

Okanagan Specialty Fruits （OSF）公司所研發的基因改造蘋果，產品名為北極（Arctic）蘋果，其基改特性為抗褐變，利用RNA干擾（RNA interference）的方式抑制蘋果多酚氧化酶（polyphenol oxidase, PPO）的表現，降低PPO催化蘋果氧化形成褐色產物。目前北極金冠蘋果GD743和北極翠玉蘋果GS784已獲得美國及加拿大核准上市，主要以新鮮袋裝切片和蘋果乾的商品形式在美國販售，由於OSF公司的北極蘋果尚未向我國申請基因改造食品查驗登記，北極蘋果在台灣仍屬於未核准的基因改造食品，為了避免國際非同步許可所造成的食安議題，本研究開發基因改造蘋果PCR及real-time PCR檢驗方法以因應檢驗所需。針對pSR7基因與NOS終結子區域設計的構造特異性（construct-specific） PCR及real-time PCR檢驗方法可檢測北極蘋果，TFDA現有CaMV 35S與NOS定性方法也適用於北極蘋果的篩檢。進一步分析金冠北極蘋果GD743的轉殖基因及其跨接區域，除了確認北極金冠蘋果GD743的轉殖基因序列全長，亦找到2個不同的5'端轉殖基因片段跨接蘋果基因體區域源自GD743中2個不同的殖入片段拷貝，本研究已確認2個不同片段中的1個拷貝轉殖基因片段位於蘋果chromosome 14具特異性可做為基改蘋果檢測，並進一步確認3'端跨接蘋果chromosome 14的基因序列，運用解析出5'端及3'端跨接蘋果chromosome 14的核酸序列，建立北極金冠蘋果GD743轉殖品項特異性（event-specific） real-time PCR檢驗方法。

The genetically modified （GM） apple, which developed by Okanagan Specialty Fruits Inc. （OSF） and called Arctic apple, has a modified characteristic of browning resistance. Arctic apple's browning resistance is achieved by suppressing the expression of polyphenol oxidase （PPO） through the method of RNA interference. This methodology can avoid the oxidation of apples by PPO catalyzed and enzymatic browning reduced. Nowadays, Arctic Golden Delicious apple event GD743 and Arctic Granny Smith apple event GS784 have been approved and legally marketed in the United States and Canada. They are mainly sold in the form of fresh bagged slices and dried apples in the United States. As OSF's Arctic apples have not yet applied for GM food registration in Taiwan, Arctic apples are still unauthorized GM foods in Taiwan. In order to avoid food safety issues caused by international non-synchronized GM food approval, this study developed GM apple PCR and real-time PCR methods to meet the needs for testing. The constructive-specific PCR and real-time PCR methods that focused on the designated region between pSR7 gene and the NOS terminator can be used to detect Arctic apples. The Taiwan Food and Drug Administration （TFDA）'s existing CaMV 35S and NOS qualitative methods are also suitable for GM apple screening as well. By further analyzing the Artic apple's transgenic genes and its flanking regions, we have confirmed the GD743's full transgenic gene sequences. In addition, we have also found two different 5' flanking junction region sequences that come from 2 different copies of the insertion fragments in GD743. One of these two fragments is located on chromosome 14 of the apple and its uniqueness can be used for GM apple testing. The transgenic sequences of 3' junction on apple chromosome 14 were further confirmed. Finally, we have developed and established the methods for detecting GD743 apple based on the event-specific levels of 5' and 3' junction.