- 學位論文
基因轉殖木瓜之分子檢測及花粉流佈之研究
The research of molecular identification and pollen flow of transgenic carica papaya L.
摘要
本研究為測試抗木瓜輪點病毒(Papaya ringspot virus, PRSV)的轉殖木瓜花粉散佈程度，評估花粉對生態環境的影響範圍，並開發分子鑑別基因轉殖株系之方法。針對PRSV鞘蛋白結構基因、nptΠ抗性基因分別以PCR方法鑑別基因轉殖木瓜。評估花粉流佈試驗則是使用16-0-1兩性株作為授粉源，18-2-4則作為母本，其種苗皆由國立中興大學植物病理學系葉錫東教授實驗室所提供，並種植於農業試驗所之隔離田中。依據不同方位及距離採集雌株之成熟果實，並紀錄果實重量以及種子數目。於每顆果實中取30顆種子播種發芽，以PCR方法檢測每個果實內後代之基因型及其分離率。利用種子數及親本分析之方法來推測基因轉殖花粉傳播。結果顯示，果實授粉率會依距離的增加而有遞減情形，但卻無達顯著性差異，然而雖然於方位有顯著性不同，但卻與風向無關，另在隔離試驗田中捕捉之蜜蜂帶有花粉，並設置網罩試驗其結果發現果實內均無種子。由此可知，木瓜受粉主要應為蟲媒。利用親本分析來計算花粉流佈之頻率，發現花粉流佈會隨著距花粉源處的距離增加而頻率遞減，於兩公尺測得最高頻率為38.5 %，五公尺及十公尺分別為15.3%及14.8%。而我們也由後代分離率發現，攜有16-0-1 RB flanking標幟之後代會隨著距離的增加其比率的改變情形，另外16-0-1 RB flanking出現的比率偏低，可能因16-0-1 RB flanking之花粉傳播率較低。由以上結果得知，單抗基因轉殖木瓜花粉流佈的主要媒介應該主要為昆蟲，且基因流佈距離至少可達10公尺。
並列摘要
• This research is to evaluate the pollen flow of transgenic papaya in the open field and to develop a transgenic papaya detection and identification method based on molecular markers. The molecular identification method was carried out by designing a set of primers in the coding area of Papaya ringspot virus (PRSV) coat protein gene and npt II antibiotic gene and then uses polymerase chain reaction (PCR) method to amplify a specific product for transgenic papaya detection and identification. The transgenic papayas used in this trail are hermaphroditic plants (16-0-1) as the pollen donor source while female plants (18-2-4) as a receptor, those seedlings were provided by Dr. Yeh, Shyi-Dong and planted in the isolation field of Taiwan Agricultural Research Institute. We sampled the fruits of female plants located in different directions and distances, and recorded their fruit weights and numbers of seed in each fruit. Thirty seeds in each one fruit were sampled for determine their genotype and segregation rate by PCR method. The spreading range and direction of pollen were estimated by analyzing seeds number and their genotypes. The result shows the spreading range of pollen from hermaphroditic plants (16-0-1) declined with the increase the distance of the female plants (18-2-4) from hermaphroditic, but there was no significant relationship by the statistic analysis. Furthermore, according to our field observation, the insect like honeybee, were considered an important pollinator instead of wind. There was a high frequency of gene flow (38.5%) at 2 m from central transgenic papaya as pollen donor, 5 m and 10 m are 15.3% and 14.8% respectively, the gene flow frequency is steep decline as the distance increased. We discovered that the segregation rate of 16-0-1 RB flanking sequence was low when the distance increased, probably due to the reason that the spread range was low with the pollen contained 16-0-1 RB flanking sequence. According to our results, it was concluded that insect affects the pollen flow of transgenic papaya, and the gene flow of distance can be up to 10 m.