全球暖化帶來的地表層溫度上升在二十二世紀前預計達到攝氏1.8度。而番茄作為世界第二高產蔬菜作物其大多數產量卻僅源自北半球與溫帶地區。反映出嚴峻的氣溫對這項廣泛食用作物所帶來的巨大限制。我們藉由比對熱耐受性栽培種 (CLN1621L) 與熱敏感性栽培種 (CA4) 可以觀察到減數分裂和四倍體期的絨氈層(花葯內部營養層)與花粉細胞在CA4內提早死亡。這也造成其花粉在熱逆性下活性與發芽率降低。相對結果而言，CLN1621L保有更多具活性與發芽能力的花粉進入授粉階段。轉錄體分析也顯示花發育早期階段CLN1621L較為集中表現與耐熱性相關的基因富集而CA4傾向廣泛性表現基因。因此我們透過篩選CLN1621L早期顯著性熱誘導的基因和RNA-seq結果來建構共表現網路。在此關係網中，揭示了在熱逆境下，對絨氈層功能和花粉發育相關的 MYBs (例如AtTDF1-like, AtTDF1-like2, AtTDF1-like) 和 bHLH (例如 ms32, ms10, AtAMS-like) 轉錄因子於CLN1621L花發育早期能保有較高的表現量。qRT-PCR結果證實這些必要基因的穩定表現僅在CLN1621L被觀察到。根據共表現網路和RNA-seq的疊加分析，我們認為苯丙烷和黃酮素生合成路徑在絨氈層功能和花粉發育扮演著重要角色。我們也透過親緣性分析和qRT-PCR發現一新穎R2R3MYB轉錄因子，R2R3MYBS10-X (Solyc12g049300)，具有調控黃酮素生合成基因如FLS的潛能。目前對R2R3MYB轉錄因子的認知不僅是在調控初級及次級代謝也包括植物生物性及非生物性逆境的抗性。未來研究將著重在探討此轉錄因子在花發育早期階段調控苯丙烷和黃酮素生合成對番茄耐熱性的影響。此研究也架構此基因過表現與CRISPR/Cas9 基因剔除載體和轉殖方法用於驗證R2R3MYBS10-X對植物熱耐受性的幫助。

The effect of Global warming keeps contributing the increasing surface temperature which is expected to over 1.8 ℃ by twenty-second century. Even tomato is the second most important vegetable crop worldwide, most of the production come from northern hemisphere and temperature zone. It implies one of the huge limits for this popular crop is its sensitiveness to the adverse heat. By comparing a heat tolerant cultivar (CLN1621L) and a heat sensitive cultivar (CA4), we observed pollen cell death and pre-mature death of tapetal cells at meiosis and tetrad stages in CA4. This contributes to low pollen viability and germination rate under heat stress. In contrast, CLN1621L held higher portion in available pollens to fertilization. Transcriptomic analysis showed GO terms related to thermotolerance in CLN1621L during early stage but CA4 tended to extensively expression pattern. Significantly heat-induced genes during entire early events were extracted and constructed co-expression network overlapping with RNA-seq. We revealed some MYBs (e.g. AtTDF1-like, AtTDF1-like2, AtTDF1-like) and bHLH (e.g. ms32 and ms1035, AtAMS-like) transcription factors important for tapetum function and pollen development were able to maintain high levels of expression in CLN1621L during SI-SIII stages of flower development. qRT-PCR results validated stable expression of necessary genes only in CLN1621L. Based on results of overlapping analysis, we hypothesize synthesis of phenylpropanoid and flavonoid might play important role in tapetum function and pollen development under heat stress. We have also identified a novel R2R3MYB transcription factor, R2R3MYBS10-X (solyc12g049300) with potential regulation on late biosynthesis genes such as FLS by phylogenetic and qPCR analysis. R2R3MYB transcription factors are known not only involving in primary and secondary metabolism but also in abiotic and biotic stress tolerance. Future study will explore its function in thermotolerance by regulating phenylalanine and flavonoids biosynthesis during early stages of flower development. Over-expression and CRISPR/Cas9 gene knockout transgenic construct will be used to validate the function of this R2R3MYB transcription factor in thermotolerance.

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