文心蘭乙烯訊息傳導雙重基因默化轉殖延長花期之研究

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题名:	文心蘭乙烯訊息傳導雙重基因默化轉殖延長花期之研究 Studies on Dual Silencing of Genes Involved in Ethylene Signal Transduction to Prolong Vase Life of Oncidesa Flowers
作者:	黃鵬林杜宜殷
贡献者:	生物科技研究所
关键词:	核醣核酸干擾文心蘭農桿菌 RNA interference Oncidesa Agrobacterium tumefaciens
日期:	2022
上传时间:	2023-03-16 11:43:35 (UTC+8)
摘要:	＜P＞文心蘭 (＜em＞Oncidesa＜/em＞＜em＞) ＜/em＞是目前臺灣外銷第一大切花作物，約占總產量 80%，其切花經過繁複的採後處理及分級篩選，花藥蓋容易脫落，導致乙烯大量產生，使花朵壽命減短，造成嚴重的經濟損失。本計畫利用 RNA 干擾 (RNA interference, RNAi) 策略，同時默化＜em＞EIN2＜/em＞及其下游蛋白質＜em＞EIN3＜/em＞兩個基因，期望阻斷文心蘭花朵中的乙烯訊息傳導，育成延長花期的文心蘭品種。先前計畫已完成＜em＞EIN2＜/em＞及＜em＞EIN3＜/em＞雙重基因默化農桿菌轉殖用質體之構築，所得到的最終質體經轉型至農桿菌並確認後，利用此農桿菌，進行文心蘭＜em＞EIN2＜/em＞及＜em＞EIN3＜/em＞雙重基因默化質體之基因轉殖，接著持續以含 G418 的培養基進行文心蘭＜em＞EIN2＜/em＞及＜em＞EIN3＜/em＞雙重基因默化質體轉殖細胞之篩選。篩選存活的癒傷組織，經再生得到的文心蘭轉殖植株，本年度將持續溫室栽培，使文心蘭轉殖株幼苗長成成株且開花，進行園藝性狀調查及外源基因的邊界序列分析。預期轉殖株體內可形成髮夾結構之雙股 RNA，達到默化文心蘭內生＜em＞EIN2＜/em＞及＜em＞EIN3＜/em＞基因的效果，以延長文心蘭切花壽命。＜/P＞＜P＞＜em＞Oncidesa＜/em＞ orchids are the first export cut flowers in Taiwan, amounting 80% of its total production. The operation of harvest and classification of cut flowers after harvest result in ethylene production induced by pollinia cap dislodgment and petal senescence. Therefore, it is important to delay ＜em＞Oncidesa＜/em＞ flower senescence. To prolong vase life of cut ＜em＞Oncidesa＜/em＞ flowers, two different specific 21 bp from ＜em＞EIN2＜/em＞ and ＜em＞EIN3＜/em＞ genes were constructed together in antisense-loop-sense orientation to obtain hairpin-type siRNA-expression RNAi plasmid. ＜em＞EIN2＜/em＞ and ＜em＞EIN3＜/em＞ RNAi-mediated dual silencing construct was transformed into＜em＞ Oncidesa ＜/em＞calli by ＜em＞Agrobacterium tumefaciens ＜/em＞previously. Transformed calli were screened on selection medium containing antibiotics. The transformed calli have been regenerated and the transgenic ＜em＞Oncidesa＜/em＞ seedlings have been developed. These transgenic ＜em＞Oncidesa＜/em＞ plants were further proved by GUS histochemical staining and polymerase chain reaction. In this year, these transgenic ＜em＞Oncidesa＜/em＞ plantlets will be continuously cultured into mature plants in the isolated greenhouse. During cultivation the horticultural characteristics will be investigated. Furthermore, the inverse polymerase chain reaction (IPCR) technique will be employed to determine the flanking sequences harboring the transgene in transgenic ＜em＞Oncidesa＜/em＞ plants. We expect that expression of ＜em＞EIN2＜/em＞/＜em＞EIN3 ＜/em＞dual gene RNA-mediated silencing construct would block the ethylene signal transduction in ＜em＞Oncidesa＜/em＞ and thereby prolong vase life of cut ＜em＞Oncidesa＜/em＞ flowers.＜/P＞
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