熱休克處理對番木瓜種子激勃素生合成之影響及GA-20、GA-2 氧化酶 cDNA 選殖與表現分析

Abstract

種子為番木瓜（Carica papaya L.）最主要的繁殖體，其種子具生理休眠性，熱休克處理具有提高其發芽率、縮短發芽時間之效果，然而其生理機制仍待釐清。採自‘台農2號’番木瓜完熟果實乾燥2週種子，於26℃水中浸潤至16天之發芽率僅0~3%；溫水34℃熱休克處理3小時及36℃熱休克處理5小時皆能顯著促進種子發芽，浸潤16天發芽率分別為44±4%與67±5%。36℃熱休克處理第5小時生物活性GA4含量顯著累積至103.8 ng•g-1 DW，高於水浸潤對照組則為45.0 ng•g-1 DW。熱休克處理前24小時至熱處理期間種子浸潤於10-2~10-3 M GA生合成抑制劑調環酸並無法顯著抵銷熱處理促進發芽效果。以RT-PCR策略自番木瓜種子組織分離GA代謝路徑關鍵酵素GA20 oxidase（GA20ox）、GA2 oxidase（GA2ox）cDNA選殖系各一，GA20ox全長為1705 bp，衍繹385個胺基酸，具GA基質、Fe2+結合位及4個保守胺基酸，命名為CpGA20ox。GA2ox全長1362 bp，衍繹335個胺基酸，具三個保守區域 5 個保守胺基酸，包含Fe2+結合位，屬第Ⅰ類GA 2-oxidase，命名為CpGA2ox。番木瓜種子於36℃熱休克處理1~5小時期間並未顯著促進CpGA20ox mRNA累積，而熱休克處理後培養於26℃第1與第4天CpGA20ox表現量分別增高307與2.4倍。熱休克處理後第4天CpGA2ox表現量降低至0.38倍。由於GA代謝路徑關鍵酵素皆為多基因家族，本試驗數據仍無法明確解釋熱休克促進番木瓜種子發芽機制。

Seeds are the main propagules for papaya (Carica papaya L.), which exhibit physiological dormancy. Although the germination percentage was enhanced and the germination time was reduced via heat shock treatment, the underlying mechanism is still unrevealed. The germination ratio of 2 week-dry papaya seeds extraced from fully ripe ‘Tainung no. 2’ papaya was only 0-3% after imbibition in water for 16 days in dark at 26℃; however, the ratio increased to 44±4%% and 67±5%, respectively, after immersion in water with 34℃ for 3 h and 36℃ for 5 h. The content of GA4, a bioactive GA, in the seeds treated with 36℃ water immersion for 5 h was 103.8 ng•g-1 DW, which was higher when compared with 45.0 ng•g-1 DW of control. However, the stimulating effect of germination was not offset in papaya seeds transferred to 10-2 ~ 10-3 M prohexadione, a GA biosynthetic inhibitor, between 24 h before and during the heat shock procedure. By ultilizing RT-PCR strategy, two cDNA clones of GA 20-oxidase (GA20ox) and GA 2-oxidase (GA2ox), two key enzymes of GA metabolic pathway in planta, were obtained from the tissures of papaya seeds. The full-length GA20ox cDNA was 1705 bp, deriving 385 amino acids, with binding sites for gibberellin substrate and Fe2+ as well as four conserved amino acids and was named CpGA20ox. The GA2ox cDNA clone had 1362 bp, deriving 335 amino acids, with three conservative domains and five conserved amino acids, including a gibberellin substrate, and was named CpGA2ox, a Classes I GA 2-oxi gene,. CpGA20ox was not up-regulated during the 5 h of 36℃ heat shock treatment, but the gene expression was induced to 307 and 2.4 times at the first and the fourth day at 26℃, respectively, after the heat treatment. On the other hand, CpGA2ox expression of the heat-shocked papaya seeds was suppressed to 0.38 fold on the fourth day after heat shock. Since the key enzymes in GA biosynthetic pathway were multi-gene family, the data obtained in this research were unable to clearly depicted the mechanism of the germination enhancement in papaya seeds via heat shock treatment.