利用轉殖菸草分析甘藷sporamin?動子之受傷誘導調控機制

Abstract

Sporamin為甘藷塊根中含量豐富的儲存性蛋白質及具抗蟲活性的胰蛋白?抑制因數，平時在非塊根組織如葉部及莖部的表現極少。本實驗針對sporamin基因，在植物防禦系統上扮演的角色與植物受傷後其被誘導的機制進行探討，藉由轉殖菸草中報告基因β-glucuronidase的表達及甘藷葉部sporamin基因的表現，來探討植株受傷或相關訊息傳導試劑處理時sporamin啟動子被活化的情形，並分析其在不同植物組織中之調控能力。 將含有sporamin啟動子（簡稱PROSPOA）及GUS基因的雙向載體(binary vector)pBI101，轉型入農桿菌(Agrobacterium tumefaciens) LBA4404菌株中，再以轉型菌株進行葉圓片轉殖法(leaf disc transformation)感染栽培菸草，誘導植物再生，藉由抗生素kanamycin篩選及聚合?連鎖反應的確認後得到五十一株轉殖植物(TSPOA1?51)，各轉殖株在一般時期與受傷時期其GUS的表現量皆有很大的差異，其中以TSPOA11、TSPOA25與TSPOA37轉殖株在受傷後GUS活性有較強且顯著的表現。在位於植株中間高度的葉片上用刀片劃出傷口，並測量受傷前後植物各組織GUS活性的差異，得知植物受傷後各組織的GUS活性強度較受傷前高出許多且有差異性的表現，葉脈和莖部的反應尤甚，而葉齡越大的葉片GUS之表現亦越明顯，結果顯示sporamin基因在植株受傷後有全株性的表現，而此現象或許是藉由微管束組織來傳遞訊息，使植物體各部位組織皆有反應；另外，以葉部及種子小苗為材料，進行受傷誘導後之GUS活性染色的分析，可見到植株葉片於受傷後表現出很強的GUS活性；而於機械受傷後不同時間取樣進行GUS的螢光活性分析，發現GUS的活性在四十五分鐘即有顯著的上昇情況，在八?十二小時達到最高點，往後四十八小時內即維持穩定狀態，顯示sporamin基因在非塊根部位的表現確實與植物之受傷反應息息相關，且在約一小時即有明顯的表現以應付外來傷害。 以各種訊息傳導相關試劑對sporamin基因的活化效應進行試驗，利用北方雜合分析法測試甘藷葉片中sporamin的表現，GUS螢光活性分析和GUS活性染色分析法研究轉殖菸草中PROSPOA-GUS的活化情形，發現茉莉酸、離層素對PROSPOA啟動子有誘導作用，且不同濃度活化的效果不同，以T1種苗進行誘導劑處理後之GUS活性染色分析，結果發現，植株各部位亦如受傷誘導之反應，具有差異性的表現情形（葉脈與莖部的GUS活性較明顯，葉齡越大的葉片反應越強），此現象說明PROSPOA啟動子的受傷調控情形似乎會因生長時期不同而表現有所差異，或具有組織差異性；水楊酸對此啟動子則不具誘導能力，結果證實sporamin基因的防禦調控機轉為受茉莉酸調控(JA-dependent)的誘導途徑。另外，分析葉片的失水效應對PROSPOA的影響，則發現失水逆境對此啟動子亦具有誘導能力，反應較受傷誘導者微弱而緩慢。結果顯示sporamin與甘藷植株之耐旱、抗蟲特性密切相關，為甘藷植株中十分重要的儲存性及防禦性蛋白質。

Sporamin is an abundant storage protein in tuberous roots of sweet potato. Its gene is not expressed in leaves and stems at the normal stage. This protein exhibits the trypsin inhibitor activity and was proved to have pest-resistant ability in transgenic tobacco plants. Because of these characteristics, it is suggested to be a protein playing an important role in plant defense response. In this report, the role of sporamin on the plant defense system and the signal pathway to induce its promoter were studied. To initiate this study, the PROSPOA::pBI101 construct which contain the sporamin promoter sequence (1.3 kb) was transformed to tobacco plants (Nicotiana tabacum cv. Wisconsin 38) via Agrobacterium-mediated method. Then the transformants were selected by kanamycin (200 μg/ml) containing medium and detected by PCR analysis. After selection, fifty-one transgenic plants were obtained. Most of these plants express strong GUS activities after wounding, especially the TSPOA11, TSPOA25 and TSPOA37 transgenic lines. Time course analysis of the wound response of the promoter showed that it will be induced in forty-five minutes and highlighted at eight to twelve hours after wounding. GUS activities in different tissues of wounded transgenic tobacco plants express differentially. The maturer leaves show stronger activities than the younger ones. When treated with various elicitors, like jasmonic acid (JA), abscisic acid (ABA) and salicylic acid (SA), sporamin gene have different responses. It's prominently induced by 50 μM JA solution compared to 100 μM JA, 100 μM ABA and 200 μM ABA. It seems that SA doesn't activate this gene. Therefore, the signal pathway which regulate the activation of sporamin gene was thought to be JA-dependent. Additionally, the expression of sporamin gene is also found to be activated by water-deficit stress. Conclusionally, sweet potato is pest- and dry- resistant plant, sporamin may be an important protein associated to these characteristics.