阿拉伯芥組蛋白去乙醯化酶HDA6和HDA19對於離層酸與逆境反應之功能研究

Role of Arabidopsis histone deacetylases HDA6 and HDA19 in ABA and abiotic stress responses

陳俐婷(Li-Ting Chen)

指導教授 ： 吳克強

國立臺灣大學/生命科學院/植物科學研究所/碩士(2009年)

https://doi.org/10.6342/NTU.2009.01534

摘要

組蛋白乙醯化(Histone acetylation)與去乙醯化(Histone deacetylation)對於真核生物的轉錄活性非常重要，而組蛋白乙醯化程度是由組蛋白乙醯化酶(Histone acetyltransferase)與去乙醯化酶(Histone deacetylase)所調控。組蛋白乙醯化通常會造成基因表現的提高，而組蛋白去乙醯化則會降低基因表現。近幾年的研究報告指出，組蛋白乙醯化參與於植物對於離層酸(ABA)與高鹽、乾旱、低溫等非生物逆境之反應與抗性當中。然而到目前為止，植物中組蛋白乙醯化與去乙醯化與逆境反應之間的分子機制尚未明朗化。因此在本研究中，我將針對離層酸與高鹽、乾旱、低溫等非生物逆境，來探討組蛋白乙醯化酶HDA6與HDA19在阿拉伯芥中的功能。 HDA6與HDA19屬於RPD3/HDA1家族中的組蛋白去乙醯化酶，兩者具有類似的氨基酸序列，而且兩者在阿拉伯芥不同的生長階段具有相似的表現。在本次研究裡，我使用了hda6的突變株axe1-5和RNA干擾株 (RNA interfering line) CS24039以及 hda19的T-DNA插入株athd1-t1來研究HDA6和HDA19在阿拉伯芥中對於離層酸與非生物逆境反應的訊號傳遞扮演著何種角色。axe1-5是一種splicing mutant，此突變株在HDA6 剪切位(splicing site)上發生點突變。athd1-t1是HDA19的T-DNA插入株，有一T-DNA插入在HDA19的第二個外顯子(exon)中。
axe1-5，CS24039和athd1-t1在種子萌發階段對於離層酸皆表現出高度敏感性，而且與離層酸反應相關的基因都呈現較低的表現量(ABI1, ABI2, KAT1, KAT2 和RD29B)。在高鹽逆境下，axe1-5和CS24039呈現較低的種子萌發率與存活率，但是athd1-t1與野生型植物並無明顯差異。根據RT-PCR的結果，我發現axe1-5和CS24039中與高鹽有所反應的相關基因(MYB2, RD29B, DREB2A和RD29A)的表現量較低，但是在athd1-t1中只有RD29B和DREB2A呈現較低的表現量。從植物對於高鹽逆境的外表現型與相關基因表現量的結果發現，HDA6相較於HDA19在高鹽逆境的機制中扮演著比較重要的功能。同時，我也觀察了植物在低溫逆境中的外表形與相關基因表現的差異，發現HDA6和HDA19的突變株與野生型植物並無明顯差異。根據這個結果可推論出，HDA6與HDA19並無參與於低溫逆境的機制中。
我利用組蛋白免疫沈澱法(Chromatin immunoprecipitation assay)瞭解了與離層酸和高鹽逆境相關基因的組蛋白3(H3)乙醯化與甲基化程度。透過這種實驗方法得知離層酸與高鹽可以提高其相關基因 (ABI1, ABI2, KAT1, KAT2,DREB2A, RD29A和RD29B)的H3乙醯化與甲基化程度，而Col野生型與axe1-5 之間的差異亦可證明HDA6參與其中。由這個結果可推論出離層酸與高鹽環境透過改變H3乙醯化與甲基化程度的方式來增加其離層酸與高鹽逆經相關基因的表現量，而HDA6扮演著重要的角色。

關鍵字

阿拉伯芥 ； 組蛋白 ； 去乙醯化酶離層酸 ； 非生物逆境

並列摘要

Acetylation and deacetylation of nucleosomal core histones play important roles in regulation of eukaryotic transcriptional activity. Histone acetylation levels are determined by the action of histone acetyltransferases and histone deacetylases. Acetylation of the histone is often associated with increased gene activity, whereas deacetylation of histones is correlated with transcriptional repression. Recent studies indicated that histone acetylation is involved in plant response to ABA and abiotic stresses including salt, drought and cold stresses. However, little is known about the molecular mechanisms of how histone acetylation and deacetylation are involved in stress response in plants. In this study, I focus on ABA, salt, drought and cold stresses to study the role of Arabidopsis histone deacetylases HDA6 and HDA19 in ABA response and abiotic stress signaling. HDA6 and HDA19 are members of RPD3/HDA1 histone deacetylases family, and they have similar amino acid sequence and gene expression patterns in different development stages. A hda6 mutant line, axe1-5, and a HDA6 RNA interfering line, CS24039, as well as a HDA19 T-DNA insertion line, athd1-t1, were used to study the role of HDA6 and HDA19 in ABA and abiotic stress signaling. axe1-5 is a splicing mutant which carries a point mutation in the HDA6 splicing site, athd1-t1 has a T-DNA inserted in second exon of HDA19.
Compared with wild-type, axe1-5, CS24039 and athd1-t1 displayed higher sensitivity to ABA during the seed germination stage, and lower expression level of ABA-responsive genes (ABI1, ABI2, KAT1, KAT2 and RD29B). In high salinity stress, axe1-5 and CS24039 displayed lower germination rates and survival rates compared with wild-type. However, athd1-t1 has no significant difference with wild-type in high salinity stress. Compared with wild-type, axe1-5 and CS24039 displayed lower expression of salt-responsive genes (MYB2, RD29B, DREB2A and RD29A) under salt stress, but athd1-t1 displayed lower expression of RD29B and DREB2A only under salt stress. These results suggested that HDA6 might plat an important role in salt stress signaling pathway. Phenotypic comparison and RT-PCR analysis of cold stress responsive genes indicated that there was no significant difference between wild types, hda6 and hda19 mutants, suggesting that HDA6 and HDA19 are not involved in cold stress signaling pathways.
I further analyzed the H3 acetylation and methylation levels of the salt and ABA response genes by chromatin immunoprecipitation assay. It was found that ABA and salt can increase the H3 acetylation and methylation level of ABA and salt response genes (ABI1, ABI2, KAT1, KAT2, DREB2A, RD29A and RD29B), and wild-type and axe1-5 displayed different level of H3 acetylation and methylation. These results suggested that ABA and salt can induces the response gene expression by altering the H3 acetylation and methylaiton level, and HDA6 is required for this process.

並列關鍵字

Arabidopsis thaliana ； histone ； histone deacetylase ； ABA and abiotic stress

參考文獻

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