選殖番茄果實中受藍光誘導而影響茄紅素累積之相關基因

Abstract

在成熟的番茄果實中含有豐富的類胡蘿蔔素，包含：茄紅素(~90%)、β胡蘿蔔素(~5-10%)及葉黃素(~1-5%)，可作為其他維生素之前驅物或抗氧化劑。這些類胡蘿蔔素生合成相關的酵素目前已有相當詳盡之報導，不過在調控方面目前所知不多。另外，番茄屬溫帶作物，對溫度很敏感，溫度太高則類胡蘿蔔素無法正常累積。是否耐熱與非耐熱番茄品系在果實的色素累積上有差異，目前亦無相關報導。在本研究中，我們想要了解耐熱性與非耐熱性的番茄品系在不同光照處理下，茄紅素的累積是否會有差異。並進一步探討，那些受光調控的因子影響茄紅素的累積。 我們以不同耐熱及非耐熱品系綠熟期的番茄果實(CL5915、CL5675、L18及CHT1200)進行試驗，發現與黑暗相比較除了已被證實的紅光之外，藍光亦可增加耐熱番茄品系(CL5915、L18)果實中茄紅素的含量。選擇CL5915超耐熱番茄品系，利用Suppression Subtractive Hybridization (SSH)將藍光處理的番茄果實中表現之基因扣減掉黑暗處理者，確定找到16個基因在藍光及黑暗中具有差異性的表現，其中4個在藍光下的表現量較高，其餘12個在黑暗中具有較高表現量。藉由文獻之搜尋，選擇下列4個可能的候選基因並進行進一步的功能性研究：(1) Lycopersicon esculentum vacuolar proton ATPase proteolipid subunit (LeV-ATPase)；(2) Lycopersicon esculentum TDR4 transcription factor (LeTDR4)；(3)Lycopersicon esculentum phytoene synthase (LePSY)；(4) Lycopersicon esculentum S-adenosyl-l-homocysteine hydrolase (LeSAHH)。利用RT-PCR檢測候選基因的組織專一性表現，發現四個候選基因在紅熟期之果實中表現量皆有增加。另外，LeTDR4、LePSY及LeSAHH可以受到光誘導表現，其中LeTDR4及LePSY在果實中的基因表現量與茄紅素含量成正相關。利用VIGS降低果實中的基因表現，證實LeTDR4、LePSY及LeSAHH確實能夠影響番茄果實中茄紅素的累積。

Carotenoids are rich in ripening tomato fruit and made up of lycopenes (~90%), β-carotenes (~5-10%) and luteins (~1-5%), providing nutritional benefits as precursors to essential vitamins and as antioxidants. Although genes involved in carotenoid biosynthesis have been extensively studied, regulatory mechanisms responsible for the process remain largely unknown. Lycopenes are not accumulated under high temperature. Whether lycopene accumulations are different between heat-tolerant and nonheat-tolerant tomatoes remain to be elucidated. Thus, the thesis is intented to understand the differences in lycopene accumulations between heat-tolerant tomato and nonheat-tolerant tomato under various light treatments and also to identify light-regulated factors involved in the control of lycopene levels of tomato fruit. Tomato fruits of different cultivars (CL5915, CL5675, L18 and CHT1200) at mature green stage were treated with different light sources and found that blue light, in addition to red light, can increase lycopene levels, especially in heat-tolerant tomato lines (CL5915、L18). So we chose the super heat tolerant CL5915 line for further studies. Use blue light treated tomato fruit cDNA as tester, dark treated tomato fruit cDNA as driver to perform suppression subtractive hybridization (SSH). Sixteen genes were shown to have differential expression patterns between dark and blue light treated tomato fruits. Four of these 16 genes were expressed in higher levels under blue light and other 12 genes were expressed more in the dark than in the blue light. We chose 4 candidate genes for further functional studies based on literature search : (1) Lycopersicon esculentum vacuolar proton ATPase proteolipid subunit (LeV-ATPase), (2) Lycopersicon esculentum TDR4 transcription factor (LeTDR4), (3) Lycopersicon esculentum phytoene synthase (LePSY), (4) Lycopersicon esculentum S-adenosyl-l-homocysteine hydrolase (LeSAHH). Tissue specific expression studies indicated that all of them were expressed at the red ripe stage of tomato fruit. Besides, the expression of LeTDR4, LePSY and LeSAHH can be induced by light and the expression levels of LeTDR4 and LePSY are positively correlated with lycopene accumulations. Further, using virus-induced gene silencing (VIGS) to knock down the expression of LeTDR4, LePSY and LeSAHH genes in tomato fruits indeed reduced lycopene levels. Taken together, LeTDR4, LePSY and LeSAHH genes isolated by SSH are involved in the regulation of lycopene levels of tomato fruit.