探討SlSWEET5b於番茄雄不稔品系建立的應用與熱逆境下之表現

Abstract

番茄產業面臨兩大瓶頸，F1種子生產的高勞力成本與高溫導致果實減產。第一瓶頸以應用雄不稔親本為重要策略，然而鮮少應用於番茄。SlSWEET5b (Sugar Will Eventually Exported Transporter 5b) 為番茄花粉糖供給的重要糖轉運蛋白，其靜默會導致花粉不稔。因此，本論文第一目的為利用CRISPR-Cas9基因編輯系統，突變番茄SlSWEET5b功能，建立可應用的番茄雄不稔親本。在番茄品系TM01~06，不管環控或田間種植，TM01及TM03中SlSWEET5b皆高表現於花苞發育後期，特別是雄蕊，與先前研究一致。因此針對TM01及TM03進行基因編輯。建構GUS標誌基因質體進行農桿菌轉殖條件測試，發現TM01及TM03之子葉與下胚軸培殖體皆可轉入GUS，在轉殖後6周表現。TM01子葉培殖體的再生率可達7成，然而TM03相較TM01培殖體皆褐化。後續建構CRISPR-Cas9質體以進行TM01轉殖，至目前已成功建立46株T0轉殖株，其中36.4%有Cas9基因轉入表現，並得到2株具片段缺失的slsweet5b突變株。後續將篩選無外源基因之T1¬子代，並分析性狀。而轉殖株獲得率偏低，未來需改善突變效率。 在第二瓶頸方面，則以建立耐高溫品種為主要策略。若能找到花粉中抗高溫的機制，將能利用分子育種加快品種培育。研究顯示花粉耐熱性與糖含量呈正相關，推測花粉專一SlSWEET5b糖轉運蛋白可能參與熱逆境下的糖累積。因此本論文第二目的為，探究SlSWEET5b在花粉耐熱性的角色。當番茄模式品種Micro-tom歷經2天35℃熱逆境後，花粉活力與萌發率顯著降低50及75%，証實番茄花粉對熱逆境的高敏感。熱逆境下，相較其它Clade II SlSWEET基因，SlSWEET5b仍高度表達於後期花苞的雄蕊中，而熱逆境顯著抑制其在花粉的表現。此與全花中糖含量，尤其是單糖，熱逆境下降低相呼應。此外，主要糖代謝基因中，Susy2在雄蕊中表現也顯著下降。綜合以上結果可推論，熱逆境會抑制花粉中SlSWEET5b轉運活性，進而降低花中的糖運移及儲存能力，造成花粉有碳缺乏的現象，而抑制其成熟。這些成果也顯示提高熱逆境下SlSWEET5b的表達可能協助花粉的耐熱性。

The tomato industry is facing two obstcales- high labour cost in F1 seed production and low yield due to high temperature. To overcome the first problem, ulitlization of male-sterile parents is the main strategy. However, the application in tomato is still limited. SlSWEET5b (Sugar Will Eventually Exported Transporter 5b) is an important sugar transporter to provide sugars for tomato pollen. Silencing of SLSWEET5b would lead to pollen sterility. So, the first purpose of this study is to establish applicable tomato male sterile parent via mutation of SlSWEET5b function through CRISPR-Cas9 gene editing system. Among TM01~TM06 6 inbred lines, under both environmental control or field grown, SlSWEET5b in TM01 and TM03 lines was highly expressed in late stage of flower bud, especially in stamen. The trend is consistent with previous study. Therefore, TM01 and TM03 were choosen for gene editing. Constructing GUS reporter gene plasmid was proceeded to examine conditions for Agrobacterium transformation. Results demonstrated that the GUS gene could be successfully transfered into cotelydon and hypocotyl explants from two lines and expressed after 6 weeks of transformation. The TM01 cotelydon explants exhibited 70% of regeneration rate, while all TM03 explants became browning. Then, the CRISPR-Cas9 plasmid was constructed and transferred into TM01. Till today, 46 T0 transgenic plants were generated and 36.4% of them contained Cas9 gene expression. Two slsweet5b deletion mutant lines were obtained. Subsequently, the selection of transgene free plants will be conducted and analyze the traits from T1 progeny. The mutant line generation rate is low, however; the mutant efficiency would be improved in the future. Regarding the second obstacle, the main strategy is to establish heat tolerance cultivar. Discovering the mechanism for of high temperature resistance in pollen can be found, molecular breeding can speed up the breeding process. Previous study shows that pollen heat tolerance is positively correlated with sugar contents, suggesting that the pollen-specific SlSWEET5b sugar transporter may be involved in sugar accumulation under heat stress. Therefore, the second purpose of this study is to explore the role of SlSWEET5b in pollen heat tolerance. When tomato modle Micro-tom was treated with 35℃ for 2 days, the pollen viability and germination rate were significantly reduced by 50 and 75%, indicating that high sensitivity of pollens to heat stress. Compared with other CladeII SlSWEET genes, SlSWEET5b was still highly expressed in the stamen of late flower bud under heat stress, which significantly inhibited SlSWEET5b expression in pollen grains. The trend was nicely corresponding to reduced sugar contents, especially monosaccharides, in whole flowers under heat stress. Moreover, among the major sugar metabolism genes, expression of Susy2 was significantly reduced in stamens as well. Combining all these results, we propose that heat stress would inhibit the transport activity of SlSWEET5b in pollen, thereby reduce the sugar transport and storage capacity in flowers. That would result in carbon deficiency and retarded maturation in pollen. These results also reveal the possibility that increasing SlSWEET5b expression under heat stress may contribute the heat tolerance of pollen.