文心蘭EIN3基因默化轉殖與功能互補試驗

Abstract

為了確定文心蘭EIN3 (ETHYLENE-INSENSITIVE 3) 之功能，本研究構築 2XCaMV35S::OgEIN3質體，以農桿菌花序浸染法轉入阿拉伯芥突變株ein3 中，進行功能性互補分析。經分子檢測確認轉殖株後，觀察其外表型、株高、鮮乾重及葉綠素含量，均恢復與野生型之相同型態，顯示文心蘭OgEIN3具有和阿拉伯芥EIN3相同之功能。 在文心蘭癒傷組織再生方面，以不同醣類組合測試對文心蘭癒傷組織再生的影響，結果以10 g/L Sucrose和20 g/L Trehalose組合的培養基，對文心蘭癒傷組織增長量、擬原球體增殖和抽芽較好，於光照環境下兩個月，即可再生成植株，而Maltose和Trehalose的組合對生成植株有較好的影響，醣類濃度較高的培養基，對於植株增生、再生成擬原球體和抽芽方面都有較佳的影響。分析不同醣類組合培養基對文心蘭植株葉片總葉綠素、類胡蘿蔔素及花青素之影響，培養在10 g/L Sucrose和5 g/L Maltose組合培養基，總葉綠素含量最多，類胡蘿蔔素最高為培養在5 g/L Maltose和10 g/L Trehalose組合培養基的植株，培養在10 g/L Sucrose和20 g/L Maltose組合培養基六個月的葉片花青素含量最高。 將功能確認後之文心蘭OgEIN3，構築為基因默化質體，以農桿菌媒介法轉入文心蘭癒傷組織或葉片中，希望能阻斷乙烯訊息傳導路徑，延長切花壽命，以提高其經濟價值。轉殖後之擬轉殖細胞經長時間篩選後，以GUS活性染色確認擬轉殖細胞，具GUS反應。但文心蘭癒傷組織經轉殖後死亡率高且轉殖效率不佳，抽取經轉殖之癒傷組織基因組DNA，顯示有小片段DNA (DNA laddering) 的發生，但如進行農桿菌轉殖前，先對癒傷組織Heat shock預處理，可有效減緩農桿菌對癒傷組織造成之PCD反應。

The cut flower of Oncidium Gower Ramsey is one of the most important export flowers in Taiwan. After cut flowers are harvested, the operation of classification and selection of flowers results in the dislodgment of pollinia cap and petal senescence induced by ethylene production. Therefore, it is important to delay Oncidium senescence and decrease flower wilt efficiency. To confirm EIN3 of Oncidium function, the 2XCaMV35S::OgEIN3 construct was transformed into Arabidopsis ein3 mutant for complementation test. The phenotype, length, weight and chlorophyll content of complementation line were the same as wild-type. These results indicate that function of OgEIN3 is the same as Arabidopsis EIN3. MS medium in half strength supplemented with either 10 g/L sucrose and 20 g/L trehalose or 10 g/L sucrose and 20 g/L maltose was more effective for the formation of PLB, development and growth of plantlets than supplementation with other carbohydrate sources. Effect of carbohydrate source and concentration on chlorophyll, carotenoid and anthocyanin content in 6 month old Oncidium Gower Ramsey calli was carried out. Plantlet cultured on 10 g/L sucrose and 5 g/L maltose had highest chlorophyll content. The highest carotenoid content was cultured on 5 g/L maltose and 10 g/L trehalose, and plantlet cultured on 10 g/L sucrose and 20 g/L maltose had highest anthocyann content. To prolong vase life of Oncidium, OgEIN3 RNA-mediated silencing construct was transformed into Oncidium calli or leaves by Agrobacterium tumefaciens. Oncidium transformation efficiency was low. Agrobacterium-induced nuclear DNA fragmentation was found in Oncidium calli, and this phenomenon could be partially inhibited by heat shock.