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標題: | AtWRKY54 和 AtWRKY70 基因表現與調控的研究 Expression and regulation AtWRKY54 and AtWRKY70 |
作者: | Chun-Chung Wang 王群中 |
指導教授: | 鄭石通(Shih-Tong Jeng) |
關鍵字: | AtWRKY54,AtWRKY70,包涵體,染色質免疫沉澱法, AtWRKY54,AtWRKY70,Chromatin immunoprecipitation,inclusion body, |
出版年 : | 2015 |
學位: | 碩士 |
摘要: | WRKY蛋白質為植物所具有的一個轉錄因子家族,其對於生物性逆境的功能廣為人知。而近期的研究發現,WRKY家族也在植物面對非生物逆境的基因調控上扮演者重要的角色。阿拉伯芥中WRKY家族的兩名成員:AtWRKY54和AtWRKY70已經被證實參與了許多植物的抗菌以及植物葉片老化的相關基因調控,並可能參與植物耐熱反應。在本次的研究中,嘗試利用protein-DNA pull down assay找出AtWRKY54和AtWRKY70所辨認的基因上游啟動子序列,以推測其所調控的基因。並希望藉由染色質免疫沉澱法(Chromatin immunoprecipitation)確認AtWRKY54和AtWRKY70與此基因的關係。在利用大腸桿菌大量表現AtWRKY54或AtWRKY70時,蛋白質表現會出現在包涵體(inclusion body),並在經過不同溫度、不同誘導時間、以及降低誘導物的濃度以表現AtWRKY54和AtWRKY70時,包涵體的情況並沒有因此而降低,以至於無法使用protein-DNA pull down assay尋找AtWRKY54和AtWRKY70所調控的基因。而在利用染色質免疫沉澱法進行釣取,並測試多樣實驗的步驟,也無法獲得下游所調控的基因。在前人研究上,已發現多個基因會受到AtWRKY54和AtWRKY70所以影響,並利用PLACE網站分析這些基因是否含有類似W-box的序列,其中有21個基因具有W-box, 並利用即時定量聚合酶連鎖反應,調查植物AtWRKY54大量表現株、AtWRKY70大量表現株、wrky54突變株和wrky70突變株中的這些基因表現,結果發現,wrky54突變株和wrky70突變株裡面的五個基因COR47(Cold regulated gene 47)、ERD7(Early-response to dehydration 7)、LEA14(late-embryogenesis abundant protein)、LTI78(Low-temperature-induce 78)和RD2( dessication responsive protein 2)的RNA表現量明顯低於野生型植物珠(Wild Type)。這些基因都不會被熱所誘導,但是已被報導會被諸如光逆境、鹽逆境、滲透壓逆境、乾旱逆境和冷逆境等各種不同的非生物性逆境所誘導,其中的COR47(Cold regulated gene 47)或和RAB18(Ras-related protein Rab-18)共同大量表現的話會使植物有抗寒的作用。這可能意味著AtWRKY54和AtWRKY70雖然在熱的情況會被抑制,但在一些非熱的非生物逆境上扮演者一定程度的角色。 WRKY family, a transcription factor family in plant, is well-known for its regulation in biotic stresses. In recent studies, genes in WRKY family also play roles in abiotic stresses. WRKY54 and WRKY70, two members of WRKY family, are involved in the regulation of various processes including plant defense and senescence, and may also participate in plant thermotolerance. In this study, protein-DNA pull down assays were used to find the promoter sequences that are recognized by WRKY54 or WRKY70 protein and to speculate what genes would directly be regulated by WRKY54 or WRKY70. Chromatin immunoprecipitation was designed to confirm this speculation. Expression of AtWRKY54 or AtWRKY70 by transforming E.coli produces inclusion body, and no detectable AtWRKY54 and AtWRKY70 was found in the soluable phase for further study. This problem obstructs the using of protein-DNA pull down assay. In addition, chromatin immunoprecipitation in several ways was performed to find genes regulated by WRKY54 or WRKY70 without predicted primers, but it fails. In recent study, wrky54wrky70 double mutant would change several gene expression under osmotic treatment. Analysis promoters of these genes by PLACE to find if promoters of these genes contain W-Box-liked sequence. Six genes with W-Box were analyzed by RT-qPCR in WRKY54 overexpression line, WRKY70 overexpression line, wrky54 T-DNA insertion line and wrky70 T-DNA insertion line. As the result, the gene expression of these five genes, COR47(cold regulated gene 47)、ERD7(early-response to dehydration 7)、LEA14(late-embryogenesis abundant protein)、LTI78(low-temperature-induce 78) and RD2( dessication responsive protein 2), are much lower in wrky54 T-DNA insertion line and wrky70 T-DNA insertion line than wild type. These five genes are not induced by heat but induced by other abiotic stresses including light stress, salt stress, osmotic stress, drought stress and cold stress. It means that WRKY54 and WRKY70 may play roles in several abiotic stresses. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/54716 |
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顯示於系所單位: | 植物科學研究所 |
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