文心蘭類胡蘿蔔色素生合成相關基因選殖與功能性分析

Cloning and Functional Characterization of Carotenogenic Genes of Oncidium Gower Ramsey

李文立(Wen-Li Lee)

指導教授 ： 陳福旗

國立屏東科技大學/農學院/熱帶農業暨國際合作系所/博士(2012年)

https://doi.org/10.6346/NPUST.2012.00288

摘要

「南西」品種文心蘭(Oncidium Gower Ramsey)由於其花朵主要累積氧化態的胡蘿蔔素-9’順式堇黃質素(9’ cis-vaolaxanthin)而具有明亮的黃顏色，是一種具有高經濟價值的全球性切花。我們選殖了八氫番茄紅素聚合酶 (phytoene synthase, PSY)、八氫番茄紅素去氫酶 (phytoene desaturase, PDS)及ζ 胡蘿蔔素去氫酶 (ζ -cartoene desaturase, ZDS)等3個胡蘿蔔素生合成路徑中重要的關鍵酵素。這3個酵素分別具有412、531及563個氨基酸。
此外，當轉殖35S::OncPSY於煙草進行異體表現（ectopic expression）時，造成包括矮化、葉片畸形及花朵顏色變淡等數種嚴重的外表型改變；當異體表現35S::OncZDS時，轉基因煙草植株也會造成葉片畸形及增加總胡蘿蔔素含量；而異體表現35S::OncPDS的煙草植株卻無明顯的外表型改變。
我們也研究了11個涉及胡蘿蔔素生合成的主要基因對光的反應。利用即時定量PCR技術 (real-time PCR)進行不同光質條件下，不同基因的表現情形。研究資料顯示，白光處理下OncPSY、OncLCYb及OncNCED受到正調節 (up-regulated)但是對多數基因，包括OncPDS, OncZDS, OncCrtISO, OncLCYe, OncCHYB, OncZEP, OncCCD1及OncSDR受到負調節 (down-regulated)。紅光可以增強OncPSY, OncLCYb, OncLCYe 及OncCCD1的表現量，紫外光可以增進OncPSY, OncPDS, OncLCYb, OncZEP及OncNCED等基因的表現量；而OncCHYB和OncSDR在所有組織中，以及OncLCYe在花朵中的表現量較低；OncPSY在花朵及葉片中具有高表現量，在而根部較少。另外，我們也選殖分析了OncPSY的啟動子(promoter)，此區域除了具有許多光反應因子 (light-responsive elements)，像是G盒 (G-box)、I盒 (I-box)、GT-1基序 (GT-1 motif)、GAG基序 (GAG-motif)及H盒 (H-box)外，也具有2個順式調節因子（cis-regulator elements），TATA盒 (TATA box)及CAAT盒 (CAAT box)。
我們對於光線調節胡蘿蔔素生合成及啟動子的研究結果或許可以對如何操控胡蘿蔔素含量及生產高品質「南西」文心蘭切花有所助益。

關鍵字

文心蘭 ； 胡蘿蔔素 ； 色素定量分析 ； 功能性分析 ； 異體表達 ； 光質

並列摘要

Oncidium Grower Ramsey is a valuable cut flower plant around the world because of its bright yellow color due to the accumulation of 9’ cis-violaxanthin, an oxidated carotenoid. Three key enzymes, phytoene synthase (PSY), phytoene desaturase (PDS), and ζ-carotene desaturase (ZDS) which are responsible to carotenoid biothesis were isolated with polypeptides of 412, 531 and 563 amino acid residues, respectively.
In the transgene experiments with tobacco plant, ectopic expression of 35S::OncPSY resulted in dwarfing, altered leaf morphology and color fading; ectopic expression of 35S::OncZDS resulted in altered leaf morphology and carotenoid increase; and ectopic expression of 35S::OncPDS resulted in minimal effects in the plant morphology.
In addition, eleven genes which may involve in the regulations of carotenoid biosynthesis were studied in response to the light. These experiments were performed with different light sources and expression profiles of different genes were determined using real time PCR. It showed that white light increased the expression of OncPSY, OncLCYb, OncNCED but decreased the expression of OncPDS, OncZDS, OncCrtISO, OncLCye, OncCHYB, OncZEP, OncCCD1 and OncSDR; red light increased the expression of OncPSY, OncLCYb, OncLCye and OncCCD1; UV light increased the expression of OncPSY, OncPDS, OncLCYb, OncZEP, and OncNCED while OncCHYB, OncSDR expressed in low level in all tissues and OncLCYe expressed in low level in the flower in all treatments. OncPSY was highly expressed in flowers and leaves but less in roots. Furthermore, the promoter region of PSY were cloned and sequenced. Several light responsive elements such as G-box, I-box, GT-1 motif, GAG-motif and H-box were identified besides two cis-regulatory elements, TATA box and CAAT box.
The data presented here may help to find ways to manipulate carotenoid content and to produce high quality cut-flower of Oncidium Grower Ramsey orchid.

並列關鍵字

Oncidium Gower Ramsey ； carotenoids ； pigment quantification ； functional analysis ； ectopic expression ； light quality

參考文獻

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