香蕉果實後熟相關基因MhDnaJ及苦瓜鈣離子依賴型核酸分解酶基因McCAN1之研究

Abstract

第一部分 香蕉果實後熟相關基因MhDnaJ 香蕉 (Musa spp.) 為世界重要經濟果樹之一，了解其果實後熟生理有助改善櫥架壽命。MhDnaJ基因自香蕉第三級後熟果實之cDNA庫篩選而得，其胺基酸序列屬type III之J-protein，利用即時定量反轉錄聚合酶連鎖反應 (real-time reverse transcription polymerase chain reaction) 檢測北蕉 (Musa `Pei Chiao') 不同組織內屬type III J-protein之MhDnaJ基因表現，結果顯示後熟果實基因表現量最高，推測此基因與香蕉果實後熟相關。基因過量表現及默化MhDnaJ基因之轉殖阿拉伯芥及菸草經聚合酶連鎖反應分析及南方氏雜交分析，證明目標序列已插入植物之基因組中。分析過量表現MhDnaJ之轉殖菸草對於鹽類逆境之抗性，結果顯示CaMV 35Spro:MhDnaJ-1轉殖系於高鹽環境下具有較佳的耐受性。藉分析前人研究有關香蕉後熟相關轉錄體資料，選取10個後熟相關之J-protein基因，進行未轉殖北蕉、及默化ACC oxidase (MhACO1和MhACO2) 之轉殖株果實基因表現量分析，以進一步探討J-protein與香蕉果實後熟之相關性。 第二部分、苦瓜鈣離子依賴型核酸分解酶基因McCAN1 McCAN1基因選殖自苦瓜開花後16天果實所製備之cDNA庫，其胺基酸序列與鈣離子依賴型核酸分解酶 (Ca2+-dependent nuclease) 具同源性，本研究利用大腸桿菌表達McCAN1重組蛋白，並利用His•Bind Resin (Novagen Co.)進行純化，經檢測酵素活性結果顯示，McCAN1於37℃分解基因組DNA之活性最佳，而70℃高溫下仍有活性，且在pH5~7環境下具有最佳活性。為了分析McCAN1之基因功能，將過量表現及基因默化質體轉殖至阿拉伯芥 (Arabidopsis thaliana ecotype Columbia) 及菸草 (Nicotiana tacacum L. cv. W38)，且已獲得轉殖植物。同時本研究亦利用花藥特異性之文心蘭金魚草素合成酶基因 (Oncidium ‘Gower Ramsey’ aureusidin synthase, OgAS1) 啟動子於菸草表達McCAN1 之cDNA，以期獲得雄不稔 (male-sterile) 性狀，經聚合酶連鎖反應 (polymerase chain reaction) 分析及南方氏雜交分析 (Southern hybridization analysis)，證明目標片段已整合入植物基因組中，且完成拷貝數檢測。

PartI Banana (Musa spp.) is one of the most important economic fruits, and the elucidation of fruit ripening has been an important issue for improving the shelf life. The MhDnaJ gene was isolated from a cDNA library constructed from the ripening fruit at stage 3. Gene expression of MhDnaJ, classified to type III J-protein, in different tissues of banana was analyzed by real-time reverse transcription PCR. MhDnaJ is proposed to be involved in the regulation of fruit ripening because its mRNA was most abundant in ripening fruit. Both overexpressing- and silencing- MhDnaJ transgenic Arabidopsis and tobacco plants have been identified. To analyze the resistant of salt tolerance for overexpression MhDnaJ transgenic tobacco, the results show CaMV 35Spro: MhDnaJ-1 transgenic line at high salt environment with better tolerability. To explore the relationship of J-protein with banana fruit ripening, MhDnaJ and ten more J-protein genes were chosen for gene expression analysis in wild type and ACC oxidase gene silenced transgenic fruits based on previous transcriptome analysis. PartII McCAN1 gene was cloned from the cDNA libray of the bitter gourd fruit of 16 days after flowering and genomic library. In our study, McCAN1 recombinant protein was expressed in E. coli to examine the enzyme acitivity. The results showed that McCAN1 best activity at 37 ℃, still active at 70 ℃ high temperature, and in the pH 5 ~ 7 environment with optimal activity. Both overexpression and silencing plasmids of McCAN1 were transformed separately to Arabidopsis thaliana ecotype Columbia and Nicotiana tacacum L. cv. W38 for analysis of gene function, and have obtained transgenic plants. Furthermore, McCAN1 cDNA driven by the anther-specific promoter of Oncidium `Gower Ramsey' aureusidin synthase gene (OgAS1) was transformed to generate the posible male-sterile tobacco plants. Polymerase chain reaction and Southern analysis have been demonstrated the target fragments were integrated into the plant genome and copy numbers of transformants have been identified.