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標題: 阿拉伯芥WD40蛋白質之特性和功能分析
Characterization and Functional Analysis of a WD40 Containing Protein in Arabidopsis thaliana
作者: 張溢麟
Chang, Yi-Lin
Contributors: 王國祥
生物科技學研究所
關鍵字: WD40蛋白
WD40 protein
日期: 2013
Issue Date: 2013-11-18 10:44:03 (UTC+8)
Publisher: 生物科技學研究所
摘要: 外在環境的變化對植物生長與發育會造成深遠的影響,在高等植物中醣類不僅提供能量外,也是細胞內重要的訊號分子。利用3%葡萄糖生長培養基篩選到數個具葡萄糖高敏感 (hypersensitivity)性狀的阿拉伯芥T-DNA插入突變株。本研究以其中一個突變株ghs40-1進行進一步的研究。在3%與4.5%葡萄糖下, ghs40-1的子葉綠化和擴張率較野生株低。AtGHS40基因可編譯出615個胺基酸,分子量為66.8 kDa。蛋白質序列比對結果得知AtGHS40 含有四段WD40 repeats 序列。透過與阿拉伯芥中其他含有WD40 domain 蛋白質序列比對發現AtGHS40為一獨特的WD40蛋白質。利用大腸桿菌表現AtGHS40,純化並當作抗原對兔體免疫產生抗體。免疫墨點法(immunoblot)分析阿拉伯芥總蛋白顯現出一個60 kDa與一個大於100 kDa的蛋白質可被偵測到。利用親和純化(affinity purification)之純化抗體明顯偵測到大於100 kDa的蛋白質,可能是AtGHS40的二聚體。藉由基因槍將AtRH57-EGFP和AtGHS40-ERFP表現載體打入洋蔥表皮細胞,AtGHS40融合蛋白被定位在細胞核與核仁,然而在細胞質中仍發現到些微螢光訊號。由於AtGHS40蛋白主要位在細胞核與核仁上,故推測AtGHS40的功能可能影響pre-rRNA之成熟過程。利用即時定量聚合酶鏈鎖反應(quantitative real-time polymerase chain reaction)分析,在4.5%葡萄糖長及短時間處理下,醣類調節基因CHS和APL3表現於短時間內未改變,但於長時間處理下,ghs40-1中CHS和APL3 的mRNAs表現量明顯高於野生株,而ghs40-1中ASN1、bZIP63、CAB1與PC2基因不論長短時間的處理,其表現皆明顯受到抑制。在短時間4.5%葡萄糖處理下,ghs40-1中離層酸生合成基因ABA2和ABA3表現較野生株低,長時間下僅有ABA3表現較野生株低。而離層酸訊息傳遞基因隨時間的延長,其ABI3、ABI4與ABI5 mRNAs的表現量均高於野生株。此外,AtGHS40基因除了受到4.5%葡萄糖的誘導之外,也受不同逆境所誘導。利用農桿菌轉殖法篩選到四株35S::AtGHS40轉基因植物,其中一株能夠抵抗4.5%以上葡萄糖之逆境,轉植株的種子萌發率及子葉綠化率都較野生株高。
The change in external environment may cause profound effects on plant growth and development. In higher plants sugars not only provide energy but also act as important signaling molecules. A number of Arabidopsis T-DNA insertion mutants that exhibit glucose hypersensitivity in the presence of 3% Glc (glucose) were identified, one of which, ghs40-1, was selected for further study. ghs40-1 exhibited impaired cotyledon greening and expansion compare with WT (wild-type) either in 3% or 4.5% Glc. AtGHS40 encodes a polypeptide of 615 amino acids with a calculated molecular mass of 66.8 kDa. Sequence analysis indicated that it contains four WD40-repeat motifs. Alignment with other WD40 domain containing proteins in Arabidopsis revealed that AtGHS40 is a protein distinctive from the other WD40 proteins. The protein was over-expressed in E. coli, purified, and used to immunize a rabbit for antibody production. Immunoblot analysis of total Arabidopsis protein indicated that a 60 kDa protein and a protein with a molecular mass larger than 100 kDa were detected. Affinity-purified antibody distinctly recognized the protein larger than 100 kDa, possibly a dimer of AtGHS40. The use of particle bombardment of AtRH57-EGFP and AtGHS40-ERFP into onion epidermal cells showed that the fusion proteins predominantly were localized in nucleus and nucleolus. However, less fluorescent signal in the cytoplasm was also detected. It suggests that AtGHS40 may be involved in the process of pre-rRNA maturation. Quantitative real-time PCR analysis revealed that the expression levels of glucose responsive genes, CHS and APL3 in ghs40-1 did not alter for a short time of Glc stimulation, but increased for 9-d treatment; however, the expression levels of ASN1, bZIP63, CAB1 and PC2 genes decreased in ghs40-1 in 4.5% Glc compared to WT. As to ABA biosynthetic genes, ABA2 and ABA3 decreased their levels of expression in ghs40-1 for a short time of Glc treatment; the expression level of ABA3 was further reduced for 9-d treatment. The ABA signaling genes, ABI3, ABI4 and ABI5 increased their levels of expression in ghs40-1 no matter which in a short time or 9-d Glc treatment. The AtGHS40 is not only induced by 4.5% Glc but also by different kinds of stress. Moreover, we have identified four 35S::AtGHS40 transgenic plants using Agrobacterium-mediated transformation. Of those, only one transgenic plant showed significant resistance to Glc higher than 4.5%. The transgenic plant exhibited better germination and cotyledon greening than WT.
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