Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/30961
標題: 番茄過敏性反應中硝酸還原酵素在一氧化氮自由基生合成之參與作用
The involvement of nitrate reductase in production of nitric oxide radical from tomato undergoing hypersensitive response
作者: 顧芳如
Ku, Fang-Ju
關鍵字: tomato
番茄
nitrate reductase
nitric oxide
hypersensitive response
enzyme purification
硝酸還原酵素
一氧化氮
過敏性反應
酵素純化
出版社: 植物病理學系所
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摘要: 本研究旨在探討番茄過敏性反應中硝酸還原酵素(nitrate reductase, NR)於NO生合成所扮演之角色,以農友301品種番茄(Lycopersicon esculentum L. cv. Known-You 301)懸浮細胞與植株分別接種青枯病無毒力菌株Ralstonia solanacearum(Ps61-)作為模式系統,並以NO分析儀(NOA280)檢測反應系統氣相空間中氣體NO之產生。本研究發現於接種Ps61-之番茄懸浮細胞中添加不同濃度之NaNO2,可導致其快速大量NO之產生,以添加20 mM NaNO2之處理組為例,其於接種8小時後NO之累積產量達1,900 nM以上,約為未添加NaNO2對照處理組NO生成量的40倍以上;同一反應系統中,NR抑制劑K2WO4之添加,則可抑制NO之產生。外加NaNO2與K2WO4下分別對NO生合成之促進或抑制作用顯然與添加濃度有關,在同時添加時兩者之作用會有相互抵銷的現象;光照對此一NO生合成作用明顯有促進效果,光照處理下,其NO生成量約為黑暗下培養之處理組高出2-5倍。NaNO2添加之促進性及K2WO4添加之抑制作用明白顯示此一NO生合成作用應與NR之活性有關,光照之立即促進作用則進而顯示其為細胞中constitutive form NR參與之作用。另於番茄植株則發現,接種Ps61-時NaNO2之添加可加速注射接種部位細胞膜之滲漏作用、細胞死亡以及過敏性反應典型壞疽斑病徵之顯現,相反的K2WO4之添加則可延緩此些病徵顯現並降低NaNO2添加之促進效果。此些生體內所見之試驗結果再度顯示NR於NO生合成過程之參與作用及其對過敏性反應病徵發展之關鍵性。本研究另外探討鈣離子之存在對上述NR酵素所參與NO生合成作用之影響,於番茄細胞懸浮培養系統,利用上述NO氣體檢測方式,本研究發現,鈣離子或calcium ionophore的添加對於NO氣體之產生只有些微之促進作用,而無如陳(2002)所見明顯的促進效果,隨Ca2+添加濃度的提高,對番茄懸浮細胞培養NO之產生以及植株過敏性反應病徵之發生亦未見有明顯之促進效應;然當以NOA檢測反應系統液相中亞硝酸含量以反應NO之生成量時,則外加Ca2+或calcium ionophore處理均有明顯促進NO生合成之效應,與陳(2001)之研究結果相符,其與上述氣相中NO檢測結果差異明顯原因顯為檢測方法所致。由於液相與氣相中NO之存在其作用部位與造成之生理衝擊亦可能不同,其於反應系統中之分佈情形很值得進一步探討釐清。 另一方面本研究亦嘗試由正在進行過敏性反應之番茄細胞中純化與一氧化氮自由基產生有關之硝酸還原酵素(NR),以經接種Ps61-之農友301品種番茄細胞懸浮培養作為模式系統,酵素純化過程各步驟所收取蛋白樣品,經添加NR專一性的受質亞硝酸與輔助因子(cofactors)後,先後以NOA及於NO專一性DAF-2螢光色基添加反應下,以螢光光電比色儀監測其NO產生活性;在酵素萃取上,經由比較蛋白質收量及NO產生活性檢測檢測,證實以Rockel氏有關報告中所用之萃取緩衝液應用效果最好;所萃取獲得酵素蛋白粗萃取液(crude extract)旋經於ÄKTA之FPLC系統以Superdex 75 HR 26/60 膠體層析管柱及Hitrap blue親和層析管柱進一步層析純化所含之NR,經多次重複嘗試卻發現無法如預期般由反應系統中純化出足夠純度與收量之標的蛋白;另外嘗試利用Amicon Ultra-15離心管濃縮裝置,在系列不同分子量過濾膜的配合處理下,則證實確可於分子量大於100 kDa部分檢測到標的蛋白之活性,此些蛋白質樣品經以Native PAGE電泳純化配合活性染色確定具標的NR活性之蛋白帶繼而以SDS-PAGE分析顯示含100與120 kDa之兩條蛋白。其中100 kDa蛋白樣品經濃縮後,以Hitrap Blue管柱進行層析分離,回收樣品中具明顯NR活性之兩fraction經進一步SDS-PAGE分析證實分別含55/37與40/30 kDa之蛋白分子,此些經電泳分離並確定具NR活性之條帶蛋白(包括100、120 kDa部分)最後送請蛋白質體中心利用Maldi-TOF質譜分析進行氨基酸解序結果,經與既有蛋白氨基酸序列資料比對尚未見有與既有NR序列具同源性部分。本研究中雖者所獲結果尚未能成功獲得與NO生合成有關NR蛋白之純化製備,唯由既有結果已初步建立可供自番茄細胞萃取濃縮此酵素蛋白之方法,所獲粗萃取蛋白樣品分子量以及所需cofactors均與已知NR相似,進一步的酵素純化工作有待未來繼續努力。
In order to explore the role of nitrate reductase (NR) in the production of nitric oxide (NO) in tomato undergoing hypersensitive response, the suspension cell culture or pot grown plants of tomato (Lycopersicon esculentum L.cv. Known-You 301) were challenge inoculated with an avirulent Ps61- strain of Ralstonia solanacearum and used as a model system. The NO produced by the Ps61- inoculated tomato suspension cell was collected by a syringe from the gaseous phase of the reaction system and the accumulated yield was determined by a Nitric oxide analyzer (NOA280, Sievers). Upon Ps61- inoculation, the supplementation of NaNO2 as substrate greatly increased immediately the yield of NO from the suspension cells. The accumulated yield of NO with 20 mM NaNO2 supplementation 8 hours after inoculation reached 1,900 nM that was more than 40 folds comparing to that of non-treated control (CK). In contrast to this, the addition of NR inhibitor K2WO4 was greatly inhibitory to the NO production. The promotive effect of NO2- and the inhibitory effect of K2WO4 both appeared to be dose dependent. When both were added simultaneously, their effect appeared to be counteractive. The production of NO was greatly stimulated by light illumination (7.5 w/m2). The accumulated yield of NO was appoxinately 2-5 folds compared to that incubated in the dark. The stimulative effect of NO2- and the inhibitory effect of K2WO4 indicated clearly the functioning of NR in the NO production. Whereas the light stimulation reflect clearly the activation of NR and its constitutive states. As for the whole plant system, the addition of NO2- upon Ps61- inoculation accelerated the membrane leakage and cell death of the infiltrated tissue and thus the progress of the necrotic hypersensitive response (HR) symptom development. The addition of K2WO4, on the contrary, inhibited all these observed damaging effects and counteracted the stimulative effect of NO2- supplementation. The stimulative effect of NO2- supplementation and the counteractive effect of K2WO4 on HR symptom development in the in vivo system reflected again the involvement of NR in NO production and indicated further its critical importance in HR symptom development. As Ca2+ has been implicated to stimulate NO production in the studied system in our previous work, the role of Ca2+ in the NR involved NO production was also explored. The adapted methodology which detected primarily NO production from the gaseous head space of suspension culture, however, failed to show a significantly stimulated NO production from the test tomato cells as that demonstrated by Chen (2001). The addition of Ca2+ provided only a small scale promotive effect on the NO production of the tomato cells and the necrotic symptom development on the tomato plants; the effectiveness was best shown at 0.1 mM supplementation. The accumulation of nitrite in the liquid phase of the reaction system was later examined by NOA as a reflection of NO production; the great stimulation of Ca2+ on NO production shown by Chen (2001) was demonstrated. The observed discrepancy of the results obtained was due clearly the change of applied methodology. As NO in gaseous phase may exert discrete physiological effectiveness comparing to that in liquid phase, its partitioning in the reaction system worth further attention. Otherwise, purification of nitrate reductase (NR) relating to nitric oxide (NO) production in tomato undergoing hypersensitive response (HR) was attempted. The enzyme proteins were extracted from tomato (Lycopersicon esculentum L. cv. Known-You 301) suspension cell culture challenge-inoculated with an avirulent strain of Ralstonia solanacearum (Ps61-). The activity of NO production of the purified enzyme preparation at each purification step was monitored by spectrofluorometry detection with the use of DAF-2 as a fluorochrome. The detection was processed with the provision of NO2- substrate and the known NR cofactors. The extraction buffer used by Rockel, P. (2002) was chosen from a screening trial for the attempted work as its yielded better protein with the activity of NO production. The purification of the crude extract by Superdex 75 HR 26/60 gel filtration column chromatography and followed Hitrap Blue affinity column chromatography with the use of an ÄKTA FPLC system (Amersham) turned out not successful in regard to the resolution and the yield of the target enzyme. The fractionation of the crude extract by using of Amicon Ultra-15 centrifugal device with the application of membrane at serial molecular weight cut-offs, however, was successful for the concentration of the target enzyme protein with molecular weight greater than 100 kDa. A protein fraction with NR activity was obtained from the continued native gel electrophoresis, and a followed SDS-PAGE revealed that it contained 2 major protein bands with molecular weight at approximately 100 and 120 kDa, respectively. The protein concentrates obtained from 100 kDa molecular cut-off centrifugation was then resolved by Hitrap Blue affinity column chromatography. The 2 sample fractions with conspicuous NR activity appeared to contain proteins with molecular weight at 55/37 and 40/30 kDa respectively. The amino acid sequence analysis by Maldi-TOF mass spectrometry, however, revealed that none of the resolved protein bands (including 100, 120 kDa's bands) shown homology with known NR sequences. The results obtained provide some useful messages for extraction and concentration of the NR-like enzyme relating to NO production during HR of tomato. The molecular weight and required cofactors are similar to that of known NR. The purification of the enzyme requires further efforts.
URI: http://hdl.handle.net/11455/30961
其他識別: U0005-2808200601503000
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-2808200601503000
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