藉由IbWRKY與Calmodulin的蛋白質交互作用探討甘藷sporamin受傷誘導路徑

Study of sporamin wounding-induced pathway by protein-protein interaction of IbWRKY and Calmodulin in sweet potato ( Ipomoea batatas cv. Tainong 57 )

何牧庭(Mu-Ting Ho)

指導教授 ： 葉開溫

國立臺灣大學/生命科學院/植物科學研究所/碩士(2010年)

https://doi.org/10.6342/NTU.2010.01755

摘要

Sporamin是甘藷塊根中含量豐富的蛋白質，已經被證實它具有胰蛋白酶抑制因子的活性，這類的蛋白質被認為在植物具有抗蟲及抗病的防禦功能。而經由我們實驗室對sporamin的啟動子區域的研究，發現其中的一個片段SWRE( Sporamin wounding response element )具有被受傷誘導的能力，並且經由yeast one-hybrid篩選法找到了會與此片段結合的兩個轉錄因子：IbNAC與IbWRKY。有被發現在其他植物中，攜鈣素( calmodulin )與IbWRKY同源的WRKY Ⅱd subgroup的蛋白質具有結合能力。
本論文要探討攜鈣素與IbWRKY兩者在甘藷中是否也具結合能力，以及觀察sporamin、 IbNAC、 IbWRKY和IbCaM 這些基因在受傷逆境及相關處理之下的表現情形，以進一步瞭解sporamin被受傷誘導的訊息傳遞機制。結果如同實驗室前人的研究，sporamin的表達會受到受傷與植物荷爾蒙SA、 MeJA的誘導，並且轉錄因子在受傷、SA和MeJA的處理下也都有表達量上升的現象，顯示這兩個轉錄因子可與sporamin的表達有相關或直接參與調控sporamin的表現。負責與IbWRKY結合的IbCaM則在受傷時其RNA有明顯表達的增加。表示第二信使Ca2+藉由Camodulin與IbWRKY結合來調控下游基因的路徑可能在受傷時最為顯著，因此Ca2+也可能參與了受傷後的訊息反應。另外藉由Gel Shift Assay和CaM Overlay Assay的方式，顯示在甘藷中IbWRKY與IbCAM會在有鈣離子訊號的情形下結合，鈣離子可能間接參與了sporamin的調控機制。另外雖然有報導NAC基因家族有部分成員會與攜鈣素結合，但是IbNAC本身並不會與攜鈣素結合，因此它的轉錄能力並不受Ca2+訊息路徑的調控。

關鍵字

甘藷、受傷、sporamin、NAC、WRKY、攜鈣素

並列摘要

Sporamin is a storage protein which is abundant in the tuberous root of sweet potato, Which has been identified with trypsin inhibitor activity, and considered as a defender against predators and pathogens. According to the research of the promoter region of sporamin on which our lab focused, we identified a sequence element, SWRE ( sporamin wounding response element ) ,which was wounding inducible. Through yeast one-hybrid assay, we found two transcriptional factors: IbNAC and IbWRKY , which could bind to SWRE. It was reported that in other plant species, Calmodulin can bind with WRKY Ⅱd subgroup proteins to which IbWRKY is attributed.
We verified whether the binding of IbWRKY and Calmodulin also works in sweet potato, and for the signaling pathway of induction of sporamin through the expression pattern of sporamin、 IbNAC、 IbWRKY and IbCaM under treatment of wounding and several defense relevant hormones. It revealed that sporamin would be induced by wounding, SA and JA, as shown by our lab. And additionally the two transcription factors were induced by these treatments. It suggests that these two transcription factors are relevant to the regulation of sporamin expression.
IbCaM is induced in wounding , and functions through its binding to IbWRKY. It suggests that the pathway of second messenger Ca2+ could be involved in wounding response.
In Gel Shift Assay and CaM Overlay Assay, IbCaM can bind to IbWRKY in the existence of Ca2+, suggesting Ca2+could be involved in regulation of expression of sporamin. Although some NAC family members have the domain for binding Calmodulin, IbNAC lacks the domain and its transcription ability seems not regulated by Ca2+.
Key words: sweet potato, wounding, sporamin, NAC, WRKY, Calmodulin.

並列關鍵字

sweet potato、wounding、sporamin、NAC、WRKY、Calmodulin

參考文獻

林沂(2009)甘藷sporamin基因受傷調控轉錄因子之選殖及功能分析。台灣大學

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