利用T-DNA插入分子標記監測基因轉植木瓜之花粉流佈

Development of T-DNA Insertion Molecular Markers to Assess Pollen Flow of Transgenic Papaya

Marita Anggarani(Marita Anggarani)

指導教授 ： 范宗宸

亞洲大學/健康學院/生物科技學系碩士班/碩士(2009年)

摘要

基因轉殖番木瓜攜帶著PLDMV /PRSV之抗性（木瓜畸葉輪點病毒/番木瓜輪點病毒）已研究成功。本研究旨在開發T - DNA插入兩翼序列之分子標記，來測量基因轉殖番木瓜之花粉流佈。試驗的田地面積為27 .5公尺× 27.5公尺，其中包括 了代號12-4計9株, 為雌雄株的基因轉殖番木瓜， 種於田間的中心點作為花粉源者； 而四周圍繞的是代號10-4的112株， 為雌株基因轉殖番木瓜，作為花粉的接收者。本研究第一部分，根據雌株果實中所收穫之種子數量來監測授粉率，然後再利用PCR分析其後代基因型，來監測花粉流佈。授粉率意味著外來花粉使雌株受孕，但往往隨著距離的增加而減少授粉率，在距離5公尺和10公尺的結果已證實。授粉媒介不只是風，本研究結果也證實昆蟲，亦為授粉媒介，如蜜蜂和蛾一樣。經由Adaptor ligation PCR所獲得的T – DNA兩翼序列，可辨別出12-4和10-4這兩種不同的轉基因品系。特定位點設計的引子提供了PCR分析，其設計之位點被發展作為分子標記，來檢測基因轉殖的雌株後代。在後代植株的基因型中，T-DNA的插入來源，它可能是從12-4，為1個T-DNA插入的2個拷貝數或是10-4的兩個T-DNA插入所貢獻的。藉由檢測位於T-DNA兩邊的側翼序列來檢測後代的花粉流佈。每三個不同的距離，有28個雌株合計有816個後代，透過PCR分析他們的T-DNA分離率。基因流佈次數的測量，由於12-4 RB flanking是為提供花粉捐贈者，所以，以檢測其後代的flanking數量做為次數計算。結果顯示，基因流佈次數在2,5和10公尺，分別為45.24,51.06和46.74。在本篇研究的缺點為試驗田地的有限大小，10公尺是不足夠精確測量基因流佈的距離。因此，建議較大的試驗田地以了解實際的分佈距離的。然而，這項研究 提供了寶貴的工具，分子標記來自T - DNA插入側翼序列，可用以評估花粉流布之距離。

關鍵字

HASH(0xa9f6c80) ； HASH(0xa9f6cbc) ； HASH(0xa9f6cf8)

並列摘要

Transgenic papayas conferring the resistance to PLDMV/PRSV (Papaya Leaf-Distortion Mosaic Virus/Papaya Ringspot Virus) had been developed. This study aims to develop molecular markers T-DNA insertion flanking sequences which are used to measure the pollen flow of transgenic papaya. The experimental plot of 27.5 m x 27.5 m consisted of 9 plants of 12-4 which are hermaphrodite transgenic papaya as the pollen donor in the center and surrounded by 112 plants of 10-4 which are female transgenic papaya as the pollen recipient. The first part of this study is to monitor the pollination rate based on the seed number collected from the female fruits, and the latter is to monitor the pollen flow by PCR analysis of the progeny genotypes. The pollination rate implies the pollen that fertilized the female plants, which tends to decrease as the distance increase except the similar result in the 5 and 10 m. Instead of wind which was known as pollinator, this result also supports the presence of insect as another pollinator such as the honeybee and moth. Flanking sequences of T-DNA insertion have been generated by adaptor ligation PCR which can distinguish these two different transgenic lines. Specific sets of primer for PCR analysis have been developed as molecular markers to detect the presence of transgene in the progenies of those female plants which might be contributed from the 12-4 which has 1 insert of two copies of T-DNA and 10-4 which has 2 inserts of T-DNA in the progenies. The pollen flow on the progenies was measured by detecting the flanking sequences that located in the border of T-DNA insertion. A total of 816 progenies of 28 female plants in each three different distances have been assayed by PCR for their T-DNA segregation ratio. The gene flow frequency was measured as the number of detected flanking from pollen donor of 12-4 RB flanking to the number of tested progenies. The result showed that the gene flow frequency for 2, 5 and 10 m were 48.33, 54.55 and 49.17, respectively. The shortcoming of this research is the limited size of experimental field, where 10 m is not sufficient to precisely measure the gene flow distance. Therefore, a larger experimental field to know the actual distribution distance is recommended. Nevertheless, this study has offered valuable tools, the molecular markers derived from T-DNA insertion flanking sequence, to assess the pollen flow in the opened area.

並列關鍵字

Pollen flow ； PRSV/PLDMV ； Flanking sequence

參考文獻

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