由於植物於冬季及早春時期容易受到大陸冷氣團和寒流的侵害,為了更加瞭解甘藷在低溫逆境時的調節機制,本研究利用抑制性扣減雜交法,篩選甘藷葉片在低溫處理30 min (早期)及6 hr (中期)具差異性表現的基因,以了解甘藷在低溫逆境下基因的調控。
使用甘藷TN71為試驗材料,25℃常溫處理作為控制組,低溫7℃處理0 min、30 min、60 min、6 hr、24 hr作為實驗組,並以葉綠素營光儀測量各參數,之後抽取各低溫處理時間之葉部RNA,以subtractive hybridization之分析技術將低溫7℃處理30 min、6 hr與常溫處理30 min、6 hr之cDNA分別進行forward 與 reverse subtractive hybridization,然後以DNA dot blot確認所選之colony為putative positive,再將其送定序分析,與資料庫比對,而找出其具差異性表現的功能性基因,並以Real-time PCR技術來分析這些功能性基因mRNA之相對表現量。
結果發現Fv/Fm在15℃及7℃處理6小時後有顯著差異,而可做為甘藷耐寒之生理指標。將低溫7℃處理30 min及6hr與常溫處理做正、反扣減後之DNA序列,經資料庫比對分析過後,篩選到2個具差異性表現的基因: metallothionein-like type 1 protein以及NADP-dependent malic protein-like mRNA。而NADP-dependent malic protein-like mRNA在15℃及7℃處理6小時及30分鐘有最大差異表現量。
The objective of this work was to study the function of expressed genes induced by chilling stress. Sweet potato cultivar “TN 71” was used to measure chlorophyll fluorescence under 25、15℃ and 7 ℃ for 0 min、30 min、1 hr、6 hr and 24 hr. RNA was isolated from the leaf of sweet potato for both 7 and 25 ℃ at 30 min and 6 hr followed by RT-PCR. The cDNA clones of forward and reverse and suppression subtractive hybridizations (SSH) were employed, and the positive colonies were then confirmed by the dot blotting technique. After DNA sequencing of these differential expression genes, these cDNA clones were compared with NCBI-blast for homology and known genetic functions. After that, the relative levels of identified genes were expressed against actin house-keeping gene using real-time PCR.
The results show that compared to Fm’、Fo、qP and qN, Fv/Fm may be used as useful parameter due to significantly different at 25 and 7 ℃ under various stressed times. The PCR products of forward and reverse SH at 30 min (as early stage) and 6 hr (as late stage) were present in 100~800bp and 100~500bp, respectively. Two possible functions of SSH genes under chilling stress were identified: metallothionein-like type 1 protein and NADP -dependent malic protein-like mRNA. The expression of NADP -dependent malic gene at 6h and 30m under 15℃and 7℃ showed significantly higher amounts than other treatments using actin gene as internal control.