"在傷害誘導的JA訊息傳遞過程中，WRKY26, WRKY38 和 MYC2是調控ORA47基因表現的上游轉錄因子"

Abstract

目前為止的研究報導已經知道有很多轉錄因子參與茉莉酸(JA)訊息傳遞的調控，像是MYC2和一些ERFs。ORA47是一個ERF轉錄因子，並且在wounding和JA誘導的情況下會大量表現，我們研究室的研究也已經發現ORA47是很多JA生合成基因的上游調控者。我的研究主題是找到可能的ORA47上游調控者。在核甘酸序列分析發現ORA47 promoter序列上含有一些保守性序列，像是MYC2結合的保守性序列(CACGTG, or G-box)和推測的WRKY26和WRKY38結合序列(TTGATC, or W-like box)，因此我們想知道它們在wounding訊息傳遞中是否會直接調控ORA47表現。ORA47 pro:GUS 感染(inoculated) 實驗觀察到myc2突變株的GUS活性比野生型植物來的低，代表MYC2對wounding誘導的訊息傳遞是必要的。ORA47和一些JA生合成基因在35S::MYC2-GFP轉植株中的表現量都被誘導表現，代表MYC2是ORA47的上游調控者。染色體免疫共沉澱(ChIP)分析實驗增加MYC2可以和ORA47 promoter上兩個位置相近的G-box結合，接著電泳遷移改變分析法(EMSA)也看到MYC2只能結合到G-box1，並且酵母單雜交分析(yeast one-hybrid)結果也看到MYC2會和G-box1結合而不是G-box2；關於WRKY轉錄因子的部份，WRKY26和WRKY38在電泳遷移改變分析法實驗中都可以結合到W-like-box2 (WL-box2)、W-like-box3、W-like-box4位置上，並且也有微弱的結合到W-like-box1的能力。在酵母單雜交分析分析則看到WRKY26和WRKY38可以和WL-box1還有WL-box4結合。染色體免疫共沉澱分析則看到WRKY26和WRKY38只能和ORA47 promoter的WL-box1結合。本篇研究的結論是：MYC2，WRKY26和WRKY38在JA訊息傳遞路徑上是ORA47的直接上游調控者。

Jasmonates (JAs) are plant signaling molecules that play important roles in defense of biotic stress. Many transcription factors, such as MYC2 and some ERFs are reported to be involved in the regulation of the signaling pathways mediated by JA. ORA47 is an ERF transcription factor and its expression is highly induced by wounding and JA. ORA47 is also known from our lab to upregulate many JA biosynthetic genes. My research aim is to study the potential upstream components of ORA47. Nucleotide sequence analysis revealed that the promoter of the ORA47 gene contained conserved sequence motifs, which are analogous to the MYC2 binding consensus sequences (CACGTG, or G-box) and putative WRKY26 and WRKY38 binding sequences (TTGATC, or W-like box). We therefore asked whether they could directly regulate ORA47 expression in wounding signaling. The GUS activity of ORA47 pro:GUS inoculated plants was lower in myc2 mutant line than in WT indicating wounding induced ORA47 expression is MYC2-dependent. ORA47 and several genes involved in JA biosynthesis were induced in the 35S::MYC2-GFP mutants compared with WT plant indicating MYC2 is an upstream regulator of JA biosynthesis. Chromatin immunoprecipitation (ChIP) assay showed MYC2 could interact with two closely linked G-box elements in the ORA47 promoter. Furthermore, electrophoretic mobility shift assay (EMSA) analysis showed MYC2 could only bind to G-box1 and yeast one-hybrid assay also exihibted MYC2 could interact with the G-BOX1 fragment but not G-BOX2. With respect to WRKY transcription factors, both WRKY26 and WRKY38 could bind to W-like-box2 (WL-box2), W-like-box3, W-like-box4 and much less the W-like- box1 cis-elements in the EMSA experiment. Moreover, yeast one hybrid assay showed WRKY26 and WRKY38 could interact with the WL-box1 and WL-box4 DNA fragments. ChIP assay revealed WRKY26 and WRKY38 could interact only with the WL-box1 element in the ORA47 promoter. In conclusion, MYC2, WRKY26, and WRKY38 directly regulate ORA47 in the JA signaling pathway.