光強度對水稻榖粒蛋白質表現及品質形成之影響

Effects of light intensity on the expression of grain proteins and quality formation in rice (Oryza sativa L.)

王湘儀(Hsiang-Yi Wang)

指導教授 ： 盧虎生

國立臺灣大學/生物資源暨農學院/農藝學研究所/碩士(2005年)

https://doi.org/10.6342/NTU.2005.01315

摘要

光是光合作用的能量來源，也是調控作物從種子萌發到開花結實的生長發育過程。在實際栽培上作物品種的株型及田間的種植密度皆可影響植冠（canopy）的光照環境。良質米的栽培需要適宜的環境條件，台灣一、二期稻作栽培環境的光強度變化很大，被認為是影響一、二期稻米品質的主因之一。蛋白質對米粒的理化性質、營養價值及外觀性狀有決定性的影響，因此研究蛋白質生合成的過程有助於稻米品質的改進。
本試驗主要的目的為探討光強度對水稻榖粒蛋白質表現與貯藏性蛋白質生合成與累積的影響及其與品質形成的相關性。以蛋白質分析技術上具有高解析度的蛋白質雙向電泳分析，配合proteomics之觀念，將在遮蔭處理下具有差異的蛋白質分子進行胺基酸序列鑑定。本試驗使用三個良質米品種(台中秈10號號、台稉9號、越光)，日夜溫25/20 ℃，對照組與處理組（遮蔭50％），三個生育時期，共18個處理組合。試驗分析包括光強度紀錄、穗重、葉綠素含量、光合作用速率、米粉性質（RVA）、蛋白質含量、積溫、各貯藏性蛋白質的變化及稻米理化性質。再以蛋白質雙向電泳圖譜在各處理條件下的變化，藉以建立生理與蛋白質雙向電泳圖譜間的關聯。
試驗結果顯示，在生理分析上，越光、台中秈10號、台稉9號在遮蔭處理下，穗重、粒重的累積速率減緩；葉綠素含量（SPAD值）則降低2~3、光合作用速率下降約50％；積溫需求以越光增加最明顯，其次為台中秈10號，最低為台稉9號，分別增加213、202、157 degree-day；抽穗到成熟累積天數延長約7~8天，反應低光照會延遲水稻穀粒的充實生理過程。米粉性質之Peak Viscosity、Final Viscosity、Setback、Breakdown值因遮蔭處理而下降；直鏈性澱粉含量在遮蔭處理下含量皆下降；而鹼性擴散程度在遮蔭處理下，越光屬於中間糊化溫度，台稉9號屬於中高糊化溫度，台中秈10號則仍和在對照組時相同，為中高糊化溫度；膠體展延性方面，沒有明顯的差異；成熟種子總蛋白質含量則因遮蔭增加約50％。
電泳圖譜分析顯示，單向 SDS-PAGE分析方面，越光、台中秈10號、台稉9號在遮蔭處理下，其穀粒發育期之prolamins、glutelins之表現皆增加；而雙向 SDS-PAGE方面，越光、台中秈10號、台稉9號其穀粒發育期間大約可以觀察到400~500個蛋白質點，台中秈10號其蛋白質點在遮蔭處理下受到較大的影響，而且蛋白質總量上較越光、台稉9號大，可能是低光照影響了貯藏性蛋白質累積的種類及生合成途徑；此外，三品種經遮蔭處理，各prolamins、glutelins之各單元分子（subunit）表現也增加，但增加不同步。台中秈10號之prolamins、glutelins累積量最明顯，台稉9號之glutelins累積量增加，但prolamins則無明顯變化，此結果與總蛋白質含量測定的結果符合。進一步以RT-PCR方法分析在遮蔭處理下相關基因之RNA表現情形，Prolamin、Glutelin、SSII均有明顯量上的差異，GBSS、NDK則在對照組與處理組之間沒有量上的差異；此結果與蛋白質電泳膠片所顯示的結果相符。將差異表現之30個蛋白質點收集後，以MALDI-TOF MS/MS或ESI-Q-TOF進行蛋白質身份鑑定，再將其分析後所得之資料以Mascot Search進行MS/MS Ions Search比對，得到定序並確定的蛋白點包括：5個prolamins、4個glutelins、3個glutelin precursor、2個glyceraldehyde 3-phosphate dehydrogenase（G3PDH）、fructose-bisphosphate aldolase isoenzyme C-1（FBPA）、glyoxalase I、cytoplasmic malate dehydrogenase、glycogen starch synthase、peroxiredoxin、putative stress-related protein、alanine aminotransferase、bifunctional subtilisin/alpha-amylase inhibitor（BSAI）、group 3 LEA （type I） protein、allergen RA5B precursor、triose-phosphate isomerase（TPI）、major allergenic protein （MAP）、UDP-glucose pyrophosphorylase，這些蛋白質大多因遮蔭而表現量增加，少數隨遮蔭而表現量減少或消失，這可能跟該蛋白質在穀粒發育期間所扮演的角色相關，本研究將各蛋白質的表現與穀粒發育期的過程進行分析歸納，推論這些蛋白質與光照及稻米品質形成的關係。

關鍵字

水稻 ； 光強度 ； 蛋白質 ； 米質

並列摘要

Light is one of crucial environmental factors affecting rice yield and quality. The purpose of the present approach was to study the effects of light on the quality formation of rice grains, utilizing proteomic related techniques.
Three quality rice cultivars were grown in a phytotron and shading（50％）treatment were performed at flowering stage. Rice grains were sampled at 6 days, 15 days and mature after flowering（DAF）for analysis. Physiological characteristics, yield traits, and physical-chemical properties of rice grains were determined. For molecular analysis, changes of proteome expression pattern were monitored by one or two dimension electrophoresis（SDS-PAGE or 2-DE）. Differential expressed proteins were selected for putative function identification using MALDI-TOF and ESI-Q-TOF. The relationships between functions of proteins and the effects of light intensity on rice grain quality were analyzed and discussed.
The results showed that, plant growth and all physiological processes were slowed down under shade. Cultivar TCS10 was more sensitive to the light intensity reduction than Koshihikari and TK9. Under shading treatment, three cultivars panicle and grain weight accumulation rate was slow down. Chlorophyll content (SPAD value) was decreased, and photosynthesis activity was dropped by about 50%. Under shading the time to mature was prolonged for about one week, suggesting more heat units is necessary to complement the low light effect. Low light intensity increased grain protein content and decreased amylose content. In physical-chemical related properties revealed by RVA, peak viscosity, breakdown, and final viscosity were decreased by low light treatment.
In proteomic analysis a total of 400 to 500 protein spots could be resolved by 2-DE gels. Expression patterns of protein spots for three cultivars were significantly affected by shading. More than 30 differential expressed proteins were selected, identified with their pI and mass, and quantified. During rice grain development the amount of 7 proteins in Koshihikari, 18 proteins in TCS10 and 10 proteins in TK9 were increased. And the levels of 20 proteins in Koshihikari, 5 proteins in TCS10 and 14 proteins in TK9 were decreased during rice grain development. Shading increased the expression of prolamins, glutelins, G3PDH, FBPA, and alanine aminotransferase, but decreased the expression of glyoxalase I, cytoplasmic malate dehydrogenase, glycogen starch synthase, and several allergen proteins. The decrease of glycogen starch synthase might be associated with the decrease of amylose content of rice grains grown under low light. And the increase of the amount of glutelins and prolamins may influence the physical-chemical characteristics of rice flour.
In conclusion, the present study provided a proteomic and functional basis that can be used as a tool to monitor and rationalize the effect of light on the rice grain quality formation.

並列關鍵字

rice ； light intensity ； proteins ； quality

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