類別:

麩醯胺酸對阿拉伯芥生長與發育的影響

2021-01-01T00:00:00Z

標題: 麩醯胺酸對阿拉伯芥生長與發育的影響; Effects of glutamine on the growth and development of Arabidopsis seedlings 作者: Hong-Sheng Liao; 廖紘陞摘要: "麩醯胺酸 (Gln) 是植物中第一個同化生成的有機氮化合物，相對於其在代謝上的功能，我們對Gln在訊息傳導所扮演的角色所知甚少。相較於硝酸銨，當阿拉伯芥幼苗生長在以Gln為氮源的培養基時，會有更多的側根及更長的根毛，根毛發育的關鍵基因也會被Gln所誘導。茉莉酸合成抑制劑或乙烯作用抑制劑的處理可以阻斷Gln促進根毛延長的效果。因此，Gln可能作用於茉莉酸以及乙烯的上游來調控根毛的生長。為了找出阿拉伯芥中與Gln代謝、運送或訊息傳導相關的基因，我們以Gln作為唯一氮源，篩選具有生長缺陷的突變株。其中一個分離自阿拉伯芥種源中心編號CS19945種子庫中的突變株，生長在Gln的培養基時會大量累積花青素。遺傳分析與基因圖譜定址選殖 (map-based cloning) 的結果顯示，該突變株花青素的性狀可能是由兩個不同的基因突變所造成。其中一個隱性的突變是因為T-DNA插入At3g18520基因 (組蛋白去乙醯酶15, HISTONE DEACETYLASE15, HDA15)，使其功能喪失所致。另外一個則是顯性的突變，由未知功能基因At3g21480發生T-to-C點突變造成F332L胺基酸的改變，此突變是否會導致Gln誘發花青素的累積，尚待進一步研究。透過分析獨立的hda15-1突變株及其互補株，確認了hda15-1生長在Gln的培養基時，花青素的含量比野生株高。在其他已知會誘導花青素合成的生長條件下，例如高糖、低氮或甲基茉莉酸處理，hda15-1花青素的含量也都比野生株高，一些花青素生合成基因以及調控花青素合成的轉錄因子基因在hda15-1中的表現也比較高。組蛋白免疫沉澱分析的結果發現這些在hda15-1表現較高的基因，有較高的組蛋白乙醯化程度。總結來說，阿拉伯芥幼苗生長在高糖、低氮或甲基茉莉酸處理時所誘發的花青素合成，有一部分是透過組蛋白乙醯化修飾，來促進花青素生合成基因及相關調節基因的表現。另外，硝酸銨比Gln更能有效地抑制阿拉伯芥幼苗中花青素的合成，部分與HDA15所調控的染色體修飾有關。"

鹽碘與尿碘檢驗方法之調查與應用

2013-01-01T00:00:00Z

標題: 鹽碘與尿碘檢驗方法之調查與應用; Application of Methods to Survey Iodine Content in Salt and Urine 作者: Pin - Chu Chen; 陳品竹摘要: 甲狀腺腫曾列臺灣十大最常見疾病，食鹽加碘後成功降低罹病率。然臺灣於 2002 年加入 WTO，於 2004 年廢止鹽政條例，許多廠商由國外進口天然鹽品，民國 93 - 97 年國人尿碘中位數降至臨界值。缺碘導致甲狀腺腫，更造成胎兒智能低下，此時評估碘營養狀況指標更顯重要，因此本研究建立鹽碘與尿碘檢驗法，應用於調查鹽品供碘狀況與癌存者碘營養狀況。市場調查發現進口鹽傾向天然無添加，國產鹽強調有複合礦物質。83 項鹽品中僅 7 項加碘，平均碘含量約為 13 ppm，檢測值與標示相符。檢測方法為 iodometric titration，回收率標準為 100 ± 15%、精密度為 < 5%，結果碘酸鉀強化鹽之 CV 與回收率皆符合；未加碘鹽回收率範圍為 -11–120%；CV 範圍由 0–69%，是因為濃度過低導致；碘化鉀強化鹽 CV 為 4%，回收率 < 79%，表示此部分檢驗方法建立不佳。結果岩鹽之碘酸鉀皆低於偵測極限；海鹽含約 0.5 ppm 碘。尿碘檢驗方法是Ammonium persulfate digestion on microplate method (APDM) 法，選用 Quality control (QC)、精密度與回收率做為判斷標準，QC 進行 12 天的檢驗後，訂定後續檢驗 QC 落於 mean ± 1 SD 為佳，回收率與精密度依照濃度區分，< 100 ug / L 者 CV 需 < 10%，回收率為 100 ± 15%，>100 ug / L 者則各標準皆減少 5%。三項標準皆達到，才可作為該受試者尿碘檢驗結果。收尿採用86 位癌存者尿碘之隨機尿液，部分受試者捐尿時不斷喝水幫助排尿，因此範圍為 7.5 ~ > 143384 ug / L，中位數為 156.5 ug / L，屬於正常範圍。

鹽方扁平古菌中兩種被預測為細菌視紫紅質之特性研究

2012-01-01T00:00:00Z

標題: 鹽方扁平古菌中兩種被預測為細菌視紫紅質之特性研究; Characterization of two predicted bacteriorhodopsins in Haloquadratum walsbyi 作者: Hung-Yi Liu; 劉鴻毅摘要: 微生物視紫紅質自 1973 年開始，在模式生物嗜鹽古細菌Halobacterium salinarum 發現四種利用光源為能量行使生理功能的視紫紅質，其中分為調節離子濃度的離子幫浦型與感應光趨性的感光受器型，細菌視紫紅質即為前者，其為最早且研究最為透徹的古生菌視紫紅質，利用光源為能量將氫離子送出胞外，造成氫離子濃度梯度，進而帶動三磷酸腺苷生成系統產生能量。古生菌 Haloquadratum walsbyi 在 2006 年基因體解碼資料中，有三種預測會表達視紫紅質的基因，包括兩種類細菌視紫紅質及一種類感光型視紫紅質。其中視紫紅質基因bop1 和 bop2 皆被預測為類細菌視紫紅質，其轉譯後蛋白被分別命名為HwBR 和 MR( middle rhodopsin )。本研究旨在研究上述兩類細菌視紫紅質間之特性，並探討為何 H. walsbyi 內擁有兩細菌視紫紅質系統。經序列比對與演化樹比對，HwBR 和 MR有很高的相似度，且基因經大腸桿菌表達系統表達純化後，兩者之特徵吸收峰分別為552 奈米和 488奈米。HwBR 經氫離子幫浦測試後，擁有如細菌視紫紅質般的能力，惟 MR 即使經其特徵吸收峰波長 470奈米雷射光源刺激亦無明顯反應。在光週期測試方面，HwBR 光週期如細菌視紫紅質般，約為200毫秒，然而MR光週期達2秒，類似感光型視紫紅質。進一步利用點突變研究，分別對兩基因突變兩處細菌視紫紅質保守且重要之序列，其影響結果HwBR 與細菌視紫紅質相同，MR則非。爰此，整體研究可以清楚推論H. walsbyi與H. marismortui之兩細菌視紫紅質系統相異，前者只包含一種細菌視紫紅質和一種稱為MR之獨特視紫紅質，其被獨立分類於離子型與感光型間，其生理功能尚需研究與探討，而Asp96之保守性也不再是判斷為細菌或感光型視紫紅質之依據。; Studies in microbial rhodopsins have been focused on those found in Halobacterium salinarum since 1973. In the past, the studies in halophilic archaea identified four kinds of retinal-binding proteins and they use light as energy source to mediate different physiological functions. Among them, two main functions are identified: light-driven ion transporters and sensory receptor for phototaxis responses. Bacteriorhodopsin belong to ion-transporter type and is the first and best-understood archaeal rhodopsin which can pump protons out of cell upon light illumination to induce a proton gradient, which further lead to ATP synthesis via F1Fo ATP synthase system. Haloquadratum walsbyi genome was completed in 2006 and a total of three retinal-binding proteins were predicted, including two bacteriorhodopsins-like and one halorhodopsin-like chloride pumping photoreceptor. The two opsin genes in H. walsbyi, bop1 and bop2, were those that identified and assumed to encode two bacteriorhodopsin-like proteins, and their protein products are named HwBR and MR, respectively. The goal of this study is to compare the features between HwBR and MR to further understand whether H. walsbyi indeed posses a two-bacretiorhodopsin system. The protein sequence alignment and phylogenetic tree analysis showed the high identity of HwBR and MR. The genes HwbopI and HwbopII were cloned and expressed in E. Coli C43(DE3) for biochemical property studies, and the maximum absorbance were 552 nm and 488 nm, respectively. The light-driven proton pumping activity showed that HwBR to have light-driven proton transportation function as that found in bacteriorhodopsin, while MR showed no such activity at all, even when being activated with the 470nm laser beam. The photocycle kinetic measurements showed HwBR to have a 200 msec photocycle time just like bacteriorhodopsin, while it was a 2-second for MR, a time course similar to those found in sensory rhodopsins. Further different mutagenesis studies at Asp85 and Asp96, two conserved and functionally critical residues found in bacteriorhodopsin, found only HwBR showed¬- the same impact in photocycle kinetics as those reported in HsBR but not in MR. The overall results in this study generated three conclusions: i) H. walsbyi does not contain a two-bacteriorhodopsin system as that identified in Haloarcula marismortui ; it has one bacteriorhodopsin-like protein and one unique rhodopsin, MR, ii) MR possesses biochemical properties that can only be classified as one that between ion-type and sensory-type. Further experiments are needed to perform to determine the function of MR. iii) The existence of a corresponding resiude Asp96 as in HsBR no longer clearly separates a bacteriorhodopsin-type retinal-binding protein from a sensory rhodopsin-type.

鹽方扁平古菌上氯視紫蛋白質光驅動離子傳遞能力之探討

2015-01-01T00:00:00Z

標題: 鹽方扁平古菌上氯視紫蛋白質光驅動離子傳遞能力之探討; Investigation on light-driven ion translocation capability of halorhodopsin from Haloquadratum walsbyi 作者: Xiao-Ru Chen; 陳筱儒摘要: 嗜鹽古細菌 (haloarchaea) 生存在高鹽的嚴苛環境中，菌體中存在許多蛋白質協助抵抗極端環境，其中一類為微生物視紫蛋白質(Microbial rhodopsin)，當受到光刺激後會有不同的功能，主要可分為兩大類，光驅動離子幫浦及光感受器。在光驅動離子幫浦中，其中一種為氯視紫蛋白質 (halorhodopsin, HR)，目前已知是一種光驅動氯離子幫浦，可以維持古生菌的細胞滲透壓。當受光刺激後，氯離子會由胞外送至胞內，氫離子被動運輸至細胞內。在本研究中，透過對於氫離子敏感的光電流測試，進一步測量光驅動氫離子釋出的活性。結果發現當 Halobacterium salinarum (Hs) 及 Haloarcula marismortui (Hm) 上的氯視紫蛋白質 (HR) 以 E.coli 表現時，質子的確被動進入膜內。但是若將蛋白質純化後，以蛋白質溶液進行光電流測試，照光前後並不會產生質子梯度。然而，由結果發現在 Haloquadratum walsbyi 中的氯視紫蛋白質 (HwHR) 有別於 HsHR 及 HmHR，無論是以表現於 E.coli 的菌液或是純化後的蛋白質溶液，進行光電流測試，皆有質子梯度的產生。為了找出在 HwHR 中，與細菌視紫蛋白質 (bacteriorhodopsin) 相似的質子幫浦活性所涉及的胺基酸，找出帶負電胺基酸進行點突變後，發現 D254N 質子釋出的活性消失。D254N-HwHR在特徵吸收波長上往短波長移動 15 nm，光週期則比 wild type 慢 6 倍。綜合上述結果， Asp254 對於光驅動質子釋出及視黃醛重新異構化的效率上扮演重要角色。; Microbial rhodopsins response to light and function as light-driven ion transportation or light sensor. Halorhodopsin (HR), one of these microbial rhodopsins, is known to be a light-driven inward chloride pump for osmolarity maintenance at least in haloarchaea. During inward transportation, chloride ions are proposed to passively carry protons across the membrane. In this study, we used a proton sensitive assay, photocurrent measurement, to measure light-driven proton releasing activity. Here we found halorhodopsin in Halobacterium salinarum (HsHR) and Haloarcula marismortui (HmHR), the protons indeed passively transported across the membrane when they were expressed in E. coli cells, but in the purified protein level, no light-driven photocurrent was recorded, indicating no proton gradients were formed inside and outside protein upon illumination. However, halorhodopsin in Haloquadratum walsbyi (HwHR) generated positive in both cell-based and protein photocurrent measurement. In order to find the residue(s) in HwHR mediating such bacteriorhodopsin-like proton activity, we mutated several negative residues and found D254N eliminate such activity in both cell-based and protein experiments. D254N-HwHR underwent a 15-nm blue-shifted in maximum absorbance and the recovery time of photocycle was ~6 time slower when compared to wild type. According to these results, we conclude Asp254 as the critical residue for light-driven proton releasing and the efficiency of retinal reisomerlization.