具不同蔗糖合成酶 RSuS3 表現量之轉殖水稻性狀分析

Characterization of Transgenic Rice Plants Expressing Various Abundance of Rice Sucrose Synthase RSuS3

李秉衡(Bing-Heng Lee)

指導教授 ： 王愛玉

共同指導教授 ： 宋賢一

國立臺灣大學/生命科學院/生化科技學系/博士(2013年)

https://doi.org/10.6342/NTU.2013.10014

摘要

在植物中，蔗糖合成酶可催化蔗糖與 UDP 形成果糖與 UDP-glucose 的可逆反應。在水稻中，有六種不同的蔗糖合成酶基因 (RSus1-RSus6)，而其中 RSus3 主要表現於水稻種子內。為了瞭解 RSus3 之生理功能，本實驗室先前已建構在水稻中恆常表現 RSus3 及在發育種子中過量表現 RSus3 之水稻轉殖株，然而在轉殖株中，RSus3 轉基因具有一個胺基酸突變 (F680S)，會導致轉基因表現之 RSuS3 活性降低。本論文藉由 RSuS3(F680S) 轉殖水稻研究異位表現 (ectopic expression) 失活之RSuS3(F680S) 突變株對水稻生長的影響。另外也建構 RSus3 表現受抑制的 RNAi 轉殖株來探討抑制種子內 RSus3表現對水稻的影響。
針對不同轉殖水稻，其子代已篩選出具單拷貝轉基因之同型合子，並找出其轉基因插入染色體位置。由野生型水稻 (台農 67 號，TNG67) 種子發育時期的蔗糖合成酶異構酶表現分析，RSus3 主要在種子發育初期表現。以 real-time reverse transcription-polymerase chain reaction (qPCR) 與免疫呈色分析 RNAi 轉殖水稻中 RSus3 表現量的差異，TNG67 水稻植株之內生性 RSus3 主要表現在授粉後第 7 天的種子中，而在 RNAi 轉殖株中其內生性 RSus3表現則是受到抑制。在授粉後第 7 與 14 天的種子中，RNAi 轉殖株之蔗糖合成酶活性分別比野生型下降 53-63% 和 43-52%。RNAi 轉殖株之授粉後 7 天種子內果糖含量比 TNG67增加，但蔗糖含量與成熟種子內澱粉含量則較野生型下降。RNAi 轉殖水稻的生長外觀與 TNG67 相比並無差異，只有充實率比野生型低 5-11% 以及種子發芽率出現 24 小時之延遲。以 actin 啟動子控制RSus3(F680S) 轉基因表現之轉殖水稻，其葉片分別以qPCR 與免疫呈色分析顯示具有轉基因表現之 mRNA 與蛋白質。轉殖株之葉片內蔗糖合成酶活性與植株高度均比野生型下降。除此之外，轉殖株葉片外觀也出現改變。轉殖水稻之農藝性狀也與 TNG67 不同。由這些結果推測 RSuS3 對於種子發育初期內蔗糖/六碳糖濃度的動態平衡與澱粉合成具有重要的影響，而且 RSuS 活性對於水稻正常生長也扮演重要的角色。

關鍵詞：蔗糖合成酶、RSus3、轉殖株、基因表現、酵素活性、糖含量、農藝性狀

關鍵字

蔗糖合成酶RSus3 ； 轉殖株 ； 基因表現 ； 酵素活性 ； 糖含量 ； 農藝性狀

並列摘要

In plants, sucrose synthase (SuS) catalyzes the reversible conversion of sucrose and UDP into UDP-glucose and fructose. In rice, six genes (RSus1-6) encoding SuS have been identified and RSus3 is predominantly expressed in the developing seeds. To elucidate the physiological roles of RSus3, our laboratory had previously constructed different rice transgenic plants, which constitutively expressed RSuS3 in various tissues and overexpressed RSuS3 in developing seeds, respectively. However, in these transgenic plants, the RSus3 transgene had an amino acid mutation (F680S), which resulted in a decrease in enzymatic activity of SuS. This study is aimed at investigating the effect of ectopic expression of an inactive RSuS3(F680S) mutant on the growth of rice using RSus3(F680S) transgenic plants. In addition, the effect of suppression of the RSus3 expression in rice seeds was also investigated by using the RSus3 RNAi transgenic plants.
For different transgenic rice plants, the homozygous progeny lines with single-copy transgene inserted were selected, and the location of the transgene insertion in chromosome was identified. According to the expression analysis of different RSus genes in the wild-type rice (TNG67), RSus3 is mainly expressed at the early stage of seed development. Real-time reverse transcription-polymerase chain reaction (qPCR) and western analysis were conducted to determine the expression levels of RSus3 in TNG67and RSus3-RNAi transgenic plants, respectively. In TNG67 rice plants, the transcripts of RSus3 were mainly expressed in the developing seeds at 7 day after pollination (DAP), while they were reduced in the RNAi transgenic lines. SuS activities were reduced by 53-63% and 43-52% in the 7-DAP and 14-DAP seeds of RNAi plants as compared to the wild-type plant. The level of fructose in seeds at 7- DAP of RNAi mutants was higher than that of TNG67, but the levels of sucrose in 7-DAP seeds and starch in mature seeds were less than wild type. The phenotype of RNAi transgenic plants showed no difference from that of the wild type, except that the seed fertility was 5-11% lower and the germination had a 24-hour delay. In the leaves of transgenic rice plants carrying the RSus3(F680S) transgene under the control of actin promoter, the mRNA and proteins expressed from the transegnes was detected by qPCR and western analysis, respectively. The SuS activity in the leaves and the plant height in the mutant plants were lower than those of the wild type. Besides, the leaf morphology of the transgenic plants was altered. The agricultural properties of transgenic rice plant also differenent from those of TNG67 plants. These results suggest that RSuS3 is important for homeostasis of sucrose/hexose ratio and starch synthesis in seed development, and SuS activity is important for the normal development of rice plants.

Keywords: sucrose synthase, RSus3, transgenic plants, gene expression, enzyme activity, sugar content, agricultural properties

並列關鍵字

sucrose synthase ； RSus3 ； transgenic plants ； gene expression ； enzyme activity ； sugar content ； agricultural properties

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