受光調控因子TDR4影響番茄果實中茄紅素含量之功能性研究

Functional study of a light-induced factor TDR4 affecting lycopene levels in tomato fruit

曾鈺媛(Yu-Yuan Tseng)

指導教授 ： 謝旭亮

國立臺灣大學/生命科學院/植物科學研究所/碩士(2010年)

https://doi.org/10.6342/NTU.2010.10490

摘要

番茄中豐富的類胡蘿蔔素在植物體中扮演重要角色，能提供植株呈現不同顏色形態，作為天然抗氧化劑，以抵抗光氧化所產生的自由基傷害。先前文獻研究指出，不同光質及光照強度會改變番茄果實質地、風味、外觀及抗氧化物質含量。而參與光調控的類胡蘿蔔素路徑之因子，以及其作用的分子機制目前仍尚未明朗。
前人以不同耐熱及非耐熱品系番茄果實(CL5915、CL5675、L18 及CHT1200)進行試驗，發現與黑暗相比較藍光可增加耐熱番茄品系(CL5915、L18)果實中茄紅素的含量。選擇CL5915 超耐熱蕃茄品系，利用Suppression Subtractive Hybridization (SSH)；得到4 個在藍光下表現量較高的基因: (1) Solanum lycopersicum vacuolar proton ATPase proteolipid subunit (SlV-ATPase)；(2)
Solanum lycopersicum TDR4 transcription factor (SlTDR4)；(3) Solanum lycopersicum phytoene synthase (SlPSY)；(4) Solanum lycopersicum S-adenosyl - l-homocysteine hydrolase (SlSAHH)，針對這些基因進一步利用Virus-induced gene silencing (VIGS)降低果實中的基因表現，發現能改變其外觀性狀及減少果實中茄紅素含量。
由於VIGS造成之性狀無法遺傳保留至後代，本研究利用農桿菌感染方式建立穩定的轉殖系統，以確立先前的研究結果。在四個候選基因中，先前文獻報導指出Solanum lycopersicum TDR4 transcription factor (SlTDR4)，會表現在子房、心皮外膜及果實外表皮部分，後者與番茄果實中茄紅素生合成位置相符。本研究進一步選定SlTDR4進行功能性分析，以cauliflower mosaic virus (CaMV) constitutive 35S promoter 將CL5915耐熱番茄品系中SlTDR4大量表現在番茄(Solanum lycopersicum cv Micro-Tom)中，卻發現35S: SlTDR4果實顏色普遍變淺，檢測果實中LeTDR4表現量確實下降。在野生型Micro-Tom不同組織部位中，利用RT-PCR檢測基因的表現，結果顯示SlTDR4與類胡蘿蔔素生合成相關基因，如： SlPSY1、SlPDS、ZDS表現量呈現正相關。另外，35S: SlTDR4果實顏色淺者其茄紅素含量減少，果實中SlTDR4及SlPSY1表現量相對降低。預測SlPSY1啟動子區域，發現具有MADS-box 轉錄因子辨認的順式DNA序列(CArGCW8)，可能因某種機制造成其基因的表現量顯著下降，因此影響SlPSY1的表現。而SlTDR4大量表現的阿拉伯芥分別在藍光、紅光與遠紅光下，呈現長下胚軸長度的外表型。綜合這些結果顯示，SlTDR4調控番茄果實類胡蘿蔔素生合成途徑，進而影響果實外觀色澤及茄紅素含量，並且也參與在光訊息傳遞過程。

關鍵字

番茄果實 ； 茄紅素 ； 光調控

並列摘要

Carotenoids rich in tomato are important and key components in plants. Different light qualities and intensities can change the quality of tomato fruit, the favor, the visible phenotype, and the levels of antioxidants. But, how light affects the biosynthesis of carotenoids remains to be elucidated. Previous studies using heat-tolerant and heat-intolerant tomato lines (CL5915, CL5678, L18 and CHT1200) revealed that blue light can increase lycopene levels in the fruit of heat-tolerant tomato lines (CL5915 and L18). Thus, the CL5915 was used for the isolation by Suppression Subtractive Hybridization (SSH) of light-induced genes that regulate the levels of lycopenes, which leads to the isolation of 4 blue light-induced genes: (1) Solanum lycopersicum vacuolar proton ATPase proteolipid subunit (SlV-ATPase), (2) Solanum lycopersicum TDR4 transcription factor (SlTDR4), (3) Solanum lycopersicum phytoene synthase (SlPSY), (4) Solanum lycopersicum S-adenosyl-l-homocysteine hydrolase (SlSAHH). These genes have been demonstrated to be responsible for the reduction of lycopenes in tomato fruit by virus-induced gene silencing (VIGS). This thesis is using the SlTDR4 for further functional studies. Transgenic tomato lines harboring a TDR4-overexpressed construct were generated by agrobacterium-mediated transformation method and exhibited a reduced color of fruit, which was further shown to be due to a reduction of the transgene TDR4. In addition, the results from RT-PCR analysis indicated that TDR4 levels in Micro-Tom were positively correlated with the levels of carotenoid biosynthetic genes such as SlPSY, SlPDS and ZDS. Moreover, the light color of fruit in transgenic tomato lines 35S::SlTDR4 was resulted from a reduction of lycopenes as well as the levels of SlTDR4 and SlPSY1. Further examination of the SlPSY1’s promoter revealed that it contained a conserved cis-element CArGCW8 recognized by so-called MADS-box transcription factor. It is very likely that the reduced level of SlTDR4 encoding a MADS-box protein in transgenic tomato lines resulted in decreased levels of SlPSY1 transcripts by direct targeting. Besides, Arabidopsis transgenic lines containing the SlTDR4-overexpression construct exhibited a long-hypocotyl phenotype under blue, red and far-red light conditions. Taken together, these data indicate that SlTDR4 indeed regulates carotenoid biosynthesis and affects the levels of lycopene in tomato fruit.

並列關鍵字

TDR4 ； carotenoids ； light

參考文獻

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