水稻PDR類ABC轉運蛋白分子特性分析及其對重金屬鎘、銅及鋅之反應

Abstract

金屬離子是植物體內生化反應進行時重要的因子之一，植物藉由許多金屬蛋白進行許多重要的生理活動，但是金屬被吸收後，要通過膜系必須藉由許多轉運蛋白的輔助，其中ABC(ATP-Binding Cassette Transporters)轉運蛋白也參與其中。ABC轉運蛋白中，已知有些PDR(pleiotropic drug resistance)類的ABC轉運蛋白和金屬進出膜系相關，但水稻的資訊相當少，本研究透過研究水稻PDR類ABC轉運蛋白之分子特性及對於鎘、銅及鋅的反應，對水稻PDR 基因家族基因表現與鎘、銅及鋅和金屬吸收的關係進行初步探討。試驗中以耐重金屬的台農六十七號水稻(TNG67)及不耐重金屬的台中在來一號(TN1)為材料，先測定在鎘、銅及鋅處理下，水稻的重金屬吸收量，並利用即時定量聚合酶連鎖反應分析水稻PDR基因家族二十個基因的組織專一性，及對鎘、銅及鋅這三種重金屬的反應。鎘、銅及鋅處理四十八小時後，TN1的第二葉較TNG67有明顯萎凋現象，TN1地上部中鎘、銅及鋅累積量較TNG67高，TNG67根部鎘、鋅含量則高於TN1，TN1根部銅含量則 高於TNG67。以TNG67為材料的組織專一性分析結果顯示，幼苗時期OsABCG35、OsABCG41及OsABCG48在根部的表現量較高，OsABCG40、OsABCG44及OsABCG50則在地上部有較高的表現；在生殖生長期中，OsABCG36、OsABCG43、OsABCG48在葉鞘的表現量很高，OsABCG31、OsABCG33、OsABCG34、OsABCG38、OsABCG46、OsABCG47、OsABCG49、OsABCG50則在幼花序中有較高的表現；在乳熟期或糊熟期種子中，則有OsABCG31、OsABCG33、OsABCG45、OsABCG46及OsABCG47等五個基因表現量較高。在重金屬誘導性方面，OsABCG34、OsABCG35、OsABCG36、OsABCG37、OsABCG39、OsABCG43、OsABCG44及OsABCG48等八個基因會同時在TNG67及TN1根部受到鎘、銅及鋅的誘導，其中OsABCG36的誘導性高達500倍；OsABCG33、OsABCG40、OsABCG46及OsABCG47等四個基因在TNG67的根部只受銅誘導，OsABCG49只在TN1根部受鎘誘導；另外，OsABCG42在TNG67根部的基因表現會受到銅的抑制。在地上部方面，OsABCG35、OsABCG36、OsABCG41、OsABCG43、OsABCG47及OsABCG48等六個基因在TNG67同時受到鎘、銅及鋅的誘導，其中OsABCG36受到銅誘導6倍表現量，TN1的OsABCG48會受到高濃度銅及鋅的誘導。OsABCG33、OsABCG34、OsABCG42、OsABCG49及OsABCG50則是在兩個品種的地上部皆表現較微弱或受抑制。將受銅誘導表現的水稻PDR基因啟動子序列進行序列相似性比對，結果發現一個可能是與銅反應相關的新型保守性區域GACAAC。為了釐清重金屬對PDR基因的調控，我們選擇受重金屬高度誘導的OsABCG36，進行進一步的功能分析，半定量反轉錄聚合酶連鎖反應結果顯示，除 了重金屬外，OsABCG36在根部亦受鹽分及PEG誘導，但不受缺水誘導。將轉殖水稻OsABCG36轉譯起始點上游2Kb的啟動子片段，接上GUS報導基因，GUS染色結果顯示此基因會在葉身(Blade)、幼花序的花藥及維管束組織中表現，葉鞘則是在切口處出現GUS訊號；在幼苗時期，OsABCG36在不完全葉及側根中表現，但銅處理後，GUS則累積在在主根。生物資訊分析結果顯示OsABCG36屬膜蛋白，GFP-OsABCG36融合蛋白的次細胞定位分析，結果發現於細胞膜及細胞核皆有此 蛋白的存在。為了解OsABCG36之功能，試驗中將OsABCG36基因大量表現在水稻中，並利用RNA干擾技術產生OsABCG36基因靜默的轉殖水稻；目前共得到28個不同的大量表現OsABCG36轉殖癒傷組織，並已獲得15個不同轉殖系的幼苗，利用RNA 干擾技術獲得的基因靜默轉殖株，目前已有種植七株於溫室，待獲得種子後，將進行功能分析。總的來說，此金屬吸收及基因表現差異的相關先導性研究，為探討PDR 類ABC 轉運蛋白的功能提供相當豐富的資訊，有助於未來的功能探討。

Metal ion is one of the important factors that involved in many biochemical processes in plants. A lot of plant physiological responses are modulated by metalloproteins. In plants, metals are uptaken by many kinds of transporters. The PDR-type ABC transporters (ATP-binding Cassette transporter) have been known to be involved in metal transport in Arabidopsis. However, the function of the PDR-type ABC transporters of rice is largely unknown. In this pilot study, we use a heavy metal tolerant rice cultivar TNG67 and a heavy metal sensitive rice cultivar TN1 as plant materials. After two days treatment with Cd, Cu, and Zn, the second leaf of TN1 showed severe yellowing phenotype. In contrast, no obvious changes could be observed in TNG67. Metal uptake analysis shown that TN1 accumulates higher Cd, Cu, and Zn than TNG67 in shoot tissues. However, except Cu, TNG67 accumulates higher Cd and Zn than TN1 root tissues. The tissue specific gene expression analysis in two-week-old seedlings indicated that OsABCG35, OsABCG41, and OsABCG48 are highly expressed in root tissue, while OsABCG40, OsABCG44 and OsABCG50 are highly expressed in shoot tissue. In the reproductive stage, OsABCG36, OsABCG43, and OsABCG48 are highly expressed in the sheath tissue, while OsABCG31, OsABCG33, OsABCG34, OsABCG38, OsABCG46, OsABCG47, OsABCG49 and OsABCG50 are expressed in the young spikes. Furthermore, OsABCG31, OsABCG33, OsABCG45, OsABCG46 and OsABCG47 are highly expressed in the immature seeds. Analysis of the transcriptional profiles of twenty rice PDR-type ABC transporter gene family and their response to Cd, Cu, and Zn by quantitative real-time PCR showed that eight genes including OsABCG34, OsABCG35, OsABCG36, OsABCG37, OsABCG39, OsABCG43, OsABCG44, and OsABCG48, could be induced by Cd, Cu, and Zn either in TNG67 or in TN1. Among these genes, The OsABCG36 gene was dramatically induced five hundred folds in root tissues upon Cd treatment. The OsABCG33, OsABCG40,OsABCG46 and OsABCG47 are solely induced by copper in roots of TNG67, while the OsABCG49 is solely induced by cadmium in roots of TN1. In addition, the OsABCG42 is repressed by copper in roots of TNG67, and the OsABCG45 is repressed by cadmium or zinc in both cultivars. In shoot tissues of TNG67, six genes were induced by Cd, Cu, and Zn including the OsABCG35, OsABCG36, OsABCG41, OsABCG43, OsABCG47, and OsABCG48. But it seems that few genes could be induced by metals in TN1, except the OsABCG48. To dissect the molecular mechanisms underlying the heavy metal induced PDR-type ABC transporter gene expression , the promoter sequence of Cd, Cu, and Zn induced genes were analyzed. We found a novel putative copper response element ”GACAAC” presented at the promoter region of these genes. Since the OsABCG36 is highly induced by heavy metals, we chose this gene for functional study related to heavy metals. The results shown that the OsABCG36 is not only induced by heavy metals, but is also induced by salt and PEG. To study the promoter activity, 2Kb promoter of OsABCG36 was isolated and fused with GUS reporter gene. The GUS protein of POsABCG36/GUS transgenic rice was mainly accumulated in blade and anthers of immature flower. The GUS signal could also be detected in coleoptile, incomplete leaves, and lateral roots of rice seedlings. Upon copper treatment, GUS signals moved from lateral roots to main roots and showed more strong signal in root tissues. Subcellular localization assay demonstrated that GFP-OsABCG36 fusion protein not only located at the plasma membrane, but also located at nucleus. In order to explore the function of the OsABCG36, OsABCG36-OE and OsABCG36-RNAi transgenic rice plants were generated. Totally, 28 putative OsABCG36-OE transformed calli were obtained, and 15 putative transgenic rice were regenerated so far. For the OsABCG36-RNAi plants, 7 transgenic rice plants were obtained. Taken together, our pilot study on the differential metal uptake ability and the gene expression patterns to heavy metal in two rice cultivars offer a lot of information for functional studies on PDR-type ABC transporters in the future.