水稻抗病能力異常 T-DNA 突變株 M0023454及高分蘗 M0111350 突變株之基因功能探討

Abstract

本研究自中研院團隊建立的臺灣水稻T-DNA插入性突變體資料庫中篩選出兩種具外表特異性之突變株M0023454和M0111350，希望藉由其特異之外表性狀與基因型，探討水稻於調控抗病以及分蘗之基因表現及功能。 M0023454於前人研究中發現其於營養生長期與生殖生長期轉換期間，出現極似病原菌攻擊產生之嚴重病斑，並於黃熟期逐漸枯萎。T-DNA插入位附近僅發現454-10基因 (LOC_Os04g32620) 有被活化之情形，然而將454-10以ubiquitin大量表現於水稻中，並無發現具有與原突變株相同之外表性狀，是以本研究重新檢視其外表性狀以及基因表現分析。首先擴大分析T-DNA插入位完整BAC clone之基因表現，發現454-10為唯一活化之基因。進一步觀察田間突變株病斑分布情形，發現病斑數量以及分佈與T-DNA插入事件呈正比，而於溫室之突變株卻沒有病斑的形成。另外，由於發現突變株幼苗具葉緣白化之情形，及部分突變株於營養生長期即出現枯萎現象，是以設計暗誘導試驗誘發提早老化，而進一步比較突變株與野生種之差異。發現除外表性狀並無顯著差異外，基因層次上，分別偵測早、中和晚期之枯萎指示基因，結果亦顯示突變株與野生種並無明顯差別，是以排除此現象為老化相關分子調控所造成。綜合以上推斷M0023454確為易感病之突變株，且須暴露於外在生物環境壓力下才會導致病斑形成。將Ubi::454-10轉殖株進行序列比對，以及經由不同資料庫比對及基因表現確認，雖454-10上游492 bp處仍具轉錄活性，但轉殖株之構築並無錯誤。欲更進一步瞭解調控此外表性狀之基因究竟為何，便進行miRNA及RNA micro array的分析。其中一可能與抗病相關之miR1858b，其目標基因RST1之表現與野生種並無明顯區別。而RNA micro array的結果顯示，部分可能具抗病功能之基因於幼苗時期即具顯著之活化，結果亦以RT-PCR做進一步確認，是以推測M0023454之病斑形成可能為病原菌攻擊後產生之過敏反應。另以Tryban blue，NBT以及DAB分別進行死細胞及過氧化物之染色，均發現顯著呈色於病斑形成處，證實該病斑形成處確有死細胞與過氧化物堆積。 M0111350為一矮化及高分蘗之突變株，且具有葉長較短及葉寬較窄之現象。農藝性狀調查結果顯示，進入生殖生長期後，該突變株仍有新分蘗產生；然而其穗長、單株稔實種子數、稔實率以及單株穀粒重均顯著低於野生種。分析其T-DNA插入位附近基因表現，發現350-3具活化之情形，然而建立構築定序後發現該基因306 bp處產生突變並造成提早終止。將插入位序列送至miRNA資料庫 (miRBase) 發現插入位上游583及207 bp處各具有osa-miR156，進行stem-loop RT-PCR分析表現量發現有活化之情形，且其主要目標基因Squamosa promoter binding like protein (SPL) 14及16均受到抑制，與前人研究成果相符合。

M0023454 and M0111350 are T-DNA mutants constructed by TRIM database. Genomic functionalizations of these phenotypically specific mutants were expected to shed lights on disease resistance and tillering regulation in rice. M0023454 was previously demonstrated pathogen susceptible and lesion formation prone in the transition from vegetative growth stage to reproductive stage. The flanking gene 454-10 was activated in mutant, however, the Ubi::454-10 transgenic line did not phenocopy that of M0023454. To reveal its exact mechanism, all predicted genes in T-DNA inserted BAC clone were analyzed, 454-10 was the only activated still. The further observation on phenotype assured the positive relation of lesion formation and T-DNA insertion. The potential of early-senescence was also excluded based on its phenotypic reflection and molecular level comparing to TNG67. Sequencing of Ubi:454-10 proved transgene identical to database prediction. Genetically characterization showed 454-10 is transcriptionally active from at least 492 bp upper-stream. The miRNA and total RNA micro array analysis revealed several potential defensive related genes regulated early in its seedlings. Histochemical staining with trypan blue for dead cells and NBT and DAB for ROS, indicated the defense system regulated lesion formation in M0023454. M0111350, the dwarf and unstoppable tillering T-DNA mutants was characterized as shorter and narrower leaves, with extremely lower yield production. Molecular evidence suggested a pre-mature termination of the activated 350-3 gene at 306 bp. On the other hand, the flanking non-coding osa-miR156 were activated and led to both SPL14 and SPL16 knockdown in M0111350.