基因轉殖木瓜種苗檢測技術之研發
Other Title
The Development of the Detection Method for Genetically Modified Seedling of Papaya
Journal
中華農學會報
Journal Volume
6
Journal Issue
2
Pages
194-205
Date Issued
2005-02
Date
2005-02
Abstract
本研究旨在硏發基因轉殖木瓜與傳統木瓜(非基因轉殖木瓜)於基因層次之鑑別方法。傳統木瓜使用了台農一、二、五、六號、日陞、紅妃、穗中紅、馬大瓜、泰國種等品種系，基因轉殖木瓜分別編號16-0-1、17-0-5、18-2-4三個基因轉殖木瓜株系。本研究採PCR方法進行鑑定檢測，第一層次為初步篩檢是否為基因轉殖木瓜，其係針對轉殖構築中之35S啟動子基因、nos終結子基因，及kanamycin抗藥篩選基因npt II序列設計引子，基因轉殖木瓜可分別於870bp、126bp及810bp處得到專一性條帶，而未經轉殖的品種系全無此條帶。第二層次為基因特異性之檢測，係針對所轉入之PRSV鞘蛋白結構基因序列設計引子，PCR反應結果顯示三個基因轉殖木瓜株系均可於820bp處產生專一性的產物，而未經轉殖的品系全無此條。第三層次為結構特異性之檢測，引子設計所擴增的區域係跨於啟動子與外來基因兩區之間，亦可於基因轉殖木瓜得到專一性之條帶。因此本究結果可以使用第一次之基因轉殖樣品篩檢，第二層次之基因特異性檢測及第三層次的結構特異性檢測等三道程序，逐步漸進的鑑定基因轉木瓜並予以確認。同時為加速檢測之效率，以9 (傳統木瓜)：1 (基因轉殖木瓜)的混合樣品中，亦可檢測出基因轉殖木瓜所具有的專一性條帶。
The purpose of this study was to establish a standard protocol to discriminate the transgenic papaya from the traditional papaya (non-transgenic papaya). The traditional papaya used in this research includes Tamung No. l, Tainung No.2, Tainung No.5, Tainung No.6, Sunrise, Red Lady, Red in Ear, Mai Tai Kua and Tailand varieties, while the transgemc papaya are 16-0-1, 17-0-5 and 18-2-4 transgenic lines. The PCR (Polymerase Chin Reaction) analysis has been applied within this research.
For category I we looked at identification, where the primer sets were designed to detect the 35S promoter gene, nos terminator gene and the kanamycin selection marker gene npt ll. The results showed the transgenic papaya to have specific bands of 870, 126 and 810 bp, respectively, whilst all the traditional varieties did not have any of those bands. This protocol can be used for the identification of transgenic papaya. Category 2 looked at gene specific detection, we used a primer set based on
PRSV coat protein gene sequence to detect the transgenic A specific PCR product of 820 bp can be found in the transgenic papaya. Furthermore, a primer set named category 3, was designed for the detection of construction specificity, to amplify the region Crossing 35S promoter and PRSV coat protein gene. Those specific fragments described above can be used, step by step, to identify transgenic papaya from traditional papaya. In order to enhance the efficiency of detection, we poled the DNA of traditional and transgenic papaya in different ratios. The results showed that the specific fragment for transgenic lines could be detected even if the ratio was from nine (traditional papaya) to one (transgenic papaya).
Subjects
基因轉殖
木瓜
木瓜雗點病毒
檢測
gene transfer
papaya
PRSV (papaya ringspot virus)
detection
Publisher
中華農學會
Type
journal article