在番茄與阿拉伯芥中參與耐熱之基因的分析

Analyses of genes involved in thermo-tolerance of tomato and Arabidopsis

沈毓星(Yu-Hsing Shen)

指導教授 ： 鄭石通

國立臺灣大學/生命科學院/植物科學研究所/博士(2013年)

https://doi.org/10.6342/NTU.2013.10479

摘要

全球暖化造成的溫度升高所影響之植物生長與發育，尤以生殖組織為嚴重，並降低了作物的產量。藉由對不同溫度下耐熱蕃茄品系(CL5915)之雄蕊筒RNA所進行的suppression subtractive hybridization，兩個分別可轉譯轉錄出β-ureidopropionase (SlUPB1, EC 3.5.1.6) 及 sucrose synthase (SlSUS3, EC 2.4.1.13)的基因被篩出並進行進一步的分析。SlUPB1可催化產生自然界中唯一的β型胺基酸β-alanine。在開花時期，SlUPB1於生長在35/30 oC (日/夜溫)之CL5915雄蕊筒中的表現量，與生長在30/25 oC或25/20 oC之不耐熱番茄品系(L4783)的雄蕊筒相比，分別是他們的2.16及2.93倍。在以L4783為背景的上升表現，及以CL5915為背景的下降表現SlUPB1番茄轉殖株中，以LC-MS-MS測量其β-alanine的含量。發現其β-alanine的含量與SlUPB1的表現呈正相關。在不同的花粉發育溫度中，轉殖株與其對應的背景品系的花粉萌發率在不同的萌發溫度下被檢測。過量表現SlUPB1轉殖株之花粉萌發率高於L4783，而下降表現SlUPB1轉殖株之花粉萌發率則低於CL5915。若在花粉萌發液中加入了β-alanine，則可在下降表現SlUPB1之轉殖株中部份回復其花粉萌發率。這些結果顯示了SlUPB1的表現對於花粉萌發是很重要的，且在適溫與高溫的的花粉萌發中，β-alanine扮演了一個重要的角色。而SlSUS3可催化可逆的蔗糖分解反應。在30/25 oC下生長的CL5915雄蕊筒中，其SlSUS3的表現量高於生長於同溫度下的L4783之雄蕊筒。經過轉殖後，得到三個在CL5915中上升表現SlSUS3的轉殖株(35S:SlSUS3-1，-2，和-5)。在35S: SlSUS3-1葉子中，其澱粉含量較CL5915為高。在不同的花粉發育溫度中，轉殖株與CL5915的花粉萌發率在不同的萌發溫度下被檢測。結果顯示過量表現的轉殖株其花粉萌發率高於CL5915之花粉萌發率。在35/30 oC的生長溫度下，35S:SlSUS3-2的著果率則與CL5915的著果率有著顯著差異。在未開花之花苞的雄蕊筒中，35S:SlSUS3-2的蔗糖含量並未被HPLC偵測到。然而其果糖及葡萄糖的含量則較CL5915為高。這些結果顯示SlSUS3的表現可能影響葉澱粉及雄蕊筒之可溶性醣類的含量，這些改變對於在35/30 oC下的花粉萌發及著果率有重要的影響。
熱對阿拉伯芥的影響有使葉柄延長、加速花軸抽高、降低著果率及影響種子發育等。為了調查在35/30 oC高溫下花軸中基因表現為何，本研究以22/20 oC及35/30 oC的花軸RNA為材料進行了微矩陣分析。數個假定的基因被選出並進行進一步的分析。myo-Inositol oxygenase (MIOX, EC 1.13.99.1)可促進抗壞血酸的合成，但其在小苗及生殖組織的角色則鮮為人知。在藉由轉殖所得到的上升表現AtMIOX4之阿拉伯芥植株(35S:AtMIOX4-1、-2)中，其生長在22/20 oC的小苗中抗壞血酸的含量與野生型阿拉伯芥(WT)相比有顯著上升，且其過氧化氫的含量有顯著下降。但在向種子中心所購買的下降表現植株(AtMIOX4-KD)中，則無差異。當轉殖株及WT小苗生長在35/30 oC時，其小苗中抗壞血酸的含量則皆無差異。35S:AtMIOX4-2的種子萌發較WT早，而TD約有10%的種子不會萌發。在生殖時期，上升表現AtMIOX4之植株其花的大小、花粉萌發率及果莢數均較WT及AtMIOX4-KD大、低及多，但其花藥的開裂則較WT及AtMIOX4-KD小，這代表了其所釋出的花粉數目較少。這些結果顯示過量表現AtMIOX4可促進種子萌發、提高抗壞血酸含量及減少過氧化氫的含量，且其花的大小、花粉萌發率及果莢數會有顯著提升。
SUCROSE SYNTHASE 1 (AtSUS1, EC 2.4.1.13)參與在澱粉及可溶性醣類的代謝中，但其對於在高溫下發育的生殖組織的影響則少為人知。而在轉殖後得到了三株上升表現AtSUS1之阿拉伯芥植株(35S:AtSUS1-3、-5及-7)，但其澱粉含量與WT在各種生長溫度下均無差異。在花藥開裂的部份，35S:AtSUS1-7生長在35/30 oC的植株則較WT開裂的較完全。當以30/25 oC下所發育的種子進行種子萌發的測試時，35S:AtSUS1-3及35S:AtSUS1-7的種子萌發率在各種萌發溫度下均較WT來的高。在鹽及滲透壓逆境下，35S:AtSUS1-3與WT的根長則無差異。因此，AtSUS1的表現可能影響了在高溫下的種子發育及花藥開裂。其他如AtYUC6、AtYUC11、AtWRKY54及AtWRKY70等基因，皆為可能的耐熱基因。他們的表現在30/25 oC下均較22/20 oC為低，這可能代表他們參與在熱逆境下所造成的一些缺陷。

關鍵字

熱逆境 ； 番茄 ； 阿拉伯芥 ； 花粉萌發率 ； 醣類 ； β-alanine ； MIOX ； sucrose synthase ； 花藥開裂 ； 種子萌發率 ； 澱粉

並列摘要

Global warming has seriously decreased world crop yield. High temperatures affect development, growth, and particularly, reproductive tissues in plants. Two genes encoding β-ureidopropionase (SlUPB1, EC 3.5.1.6) and sucrose synthase (SlSUS3, EC 2.4.1.13) were isolated from the stamens of a thermo-tolerant tomato (CL5915) using suppression subtractive hybridization. SlUPB1 catalyzes the production of β-alanine, the only beta-form amino acid in nature. In the anthesis stage, SlUPB1 expression in CL5915 stamens, growing in 35/30 oC (day/night), was 2.16 and 2.93 times greater than that in a thermo-intolerant tomato (L4783) cultivated in 30/25 oC or 25/20 oC, respectively. Transgenic tomatoes, up-regulating SlUPB1 in L4783 and down-regulating SlUPB1 in CL5915, were constructed. The amounts of β-alanine were measured by LC-ESI-MS in these plants, indicating that the β-alanine in transgenic overexpression of SlUBP1 was higher than that of L4783 and also that the β-alanine in the transgenics down-regulating SlUBP1 was significantly lower than the β-alanine of CL5915. Pollen germination rates of these transgenics were analyzed under different developmental and germinating temperatures. The results indicated that germination rates of transgenics overexpressing SlUBP1 were higher than germination rates of the background tomato L4783. Germination rates of transgenics down-regulating SlUPB1 were significantly lower than germination rates of background tomato CL5915, indicating the necessity of functional SlUPB1 for pollen germination. Pollen germinating in the buffer with the addition of β-alanine further indicated that β-alanine effectively enhanced pollen germination in tomatoes with low SlUPB1 expression. Together these results showed that the expression of SlUPB1 is important for pollen germination, and β-alanine may play a role in pollen germination under both optimal and high temperatures. Furthermore, SlSUS3 catalyzed a reversible cleavage reaction of sucrose. In the anthesis stage, SlSUS3 expression in CL5915 stamens, growing in 30/25 oC (day/night), was greater than that in L4783. Three lines of transgenic tomatoes up-regulating SlSUS3 in CL5915 (35S:SlSUS3-1, -2, and -5) were obtained, and the amount of starch in the transgenic overexpression of SlSUS3 35S:SlSUS3-1 was higher than that of CL5915. Pollen germination rates of these transgenics were analyzed under different developmental and germinating temperatures. The results indicated that germination rates of transgenics overexpressing SlSUS3 were higher than those of CL5915. The fruit setting rate of 35S:SlSUS3-2 developed under 35/30 oC was higher than that of CL5915. In the stamen of closed floral buds, the sucrose content could not be detected by HPLC in 35S:SlSUS3-2. However, the content of fructose and glucose of 35S:SlSUS3-2 was higher than that of CL5915. Together these results showed that the expression of SlSUS3 might alter the starch content in leaves and the soluble sugar content in flowers. These changes might be important for pollen germination and fruit set under high temperatures.
Heat severely affects Arabidopsis growth and development including elongation of petioles, acceleration of inflorescence bolting, fruit setting rate, and seed development. To investigate the gene expression pattern of Arabidopsis inflorescence under 35/30 oC, microarray analyses were proceeded using the RNAs of inflorescence cultivated under 22/20 and 30/35 oC. Several putative thermo-tolerant genes were selected for further analysis. The myo-inositol oxygenase (MIOX, EC 1.13.99.1) is involved in the biosynthesis of ascorbate (AsA), and its role in seedling and reproductive stages is scantly known. Therefore, transgenic Arabidopsis 35S:AtMIOX4 and AtMIOX4-knockdown (KD) were constructed and obtained from seed stock center, respectively. The amounts of AsA increased in 35S:AtMIOX4-1 and 35S:AtMIOX4-2 than those in WT under 22/20 oC, and the amounts of H2O2 reduced in 35S:AtMIOX4 plants under 22/20 oC. The seeds of 35S:AtMIOX4 plants germinated earlier than those of wild-type (WT) and AtMIOX4-KD plants, and approximately 10% seeds from AtMIOX4-KD plants did not germinate. In the reproductive stages, the flower sizes, pollen germination rates, and silique numbers of 35S:AtMIOX4 plants were larger, lower, and more than those of WT and AtMIOX4-KD plants, respectively. Nevertheless, the dehiscences of 35S:AtMIOX4 anthers were less wide than those of WT plants, indicating that pollens released from 35S:AtMIOX4 plants decreased. These results suggested that overexpressing AtMIOX4 could promote seed germination, increase AsA levels, and reduce H2O2 content in seedlings. The pollen germination rates, flower sizes, and silique numbers of the 35S:AtMIOX4 plants might rise during the reproductive stages. Therefore, the expression of AtMIOX4 may affect Arabidopsis in both seedling and reproductive stages.
SUCROSE SYNTHASE 1 (AtSUS1, EC 2.4.1.13) particepates in the starch and soluble sugar metaboliam, and its role in reproductive stages under high temperature is scantly known. Hence, three transgenic Arabidopsis, 35S:SUS1-3, -5, and -7, overexpressing AtSUS1 were constructed. The starch contents in plants decreased under high temperature. However, there were no difference among WT and transgenics. The dehiscence of 35S:SUS1-7 was better than that of WT under 35/30 oC. Seed germination rates of 35S:SUS1-3 and -7 which developed under 30/25 oC were higher than those of WT. However, the root lengths of WT and 35S:SUS1-3 showed no difference under salt and osmotic stresses. Hence, the expression of AtSUS1 may affect anther dehiscence and seed development under high temperature. There were several putative thermo-tolerant genes including AtYUC6, AtYUC11, AtWRKY54, and AtWRKY70. Their gene expression decreased under 35/30 oC, implying that they might participate in the heat-inducible defects.

並列關鍵字

heat stress ； tomato ； Arabidopsis ； pollen germination rate ； sugar ； β-alanine ； MIOX ； sucrose synthase ； anther dehiscence ； seed germination rate ； starch

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