阿拉伯芥GLR3.7突變株中根毛發育之研究

Study of root hair development in Arabidopsis GLR3.7 mutant

林怡妡(Yi-Sin Lin)

指導教授 ： 張英峯

國立臺灣大學/生命科學院/植物科學研究所/碩士(2017年)

https://doi.org/10.6342/NTU201701162

摘要

植物體內具有一類蛋白質與在哺乳類神經訊息傳導方面扮演重要角色之離子型麩胺酸受體 (ionotropic glutamate receptors，iGluRs) 相似，被命名為glutamate receptor-like proteins (GLRs)。在阿拉伯芥中具有20個GLRs，目前研究普遍認為GLRs為植物細胞膜上的鈣離子通道，調控鈣離子動態並參與許多生長發育過程。另外，14-3-3s為廣泛存在於真核生物中的鷹架蛋白，結合磷酸化的受質蛋白後調控其功能，進而參與許多細胞內反應與生理作用。本實驗室先前於in vitro的研究證實AtGLR3.7可被鈣離子依存性蛋白激酶 (CDPK) 磷酸化並與At14-3-3ω結合，且發現atglr3.7突變株的根毛生長受到嚴重抑制。因此本研究欲探討AtGLR3.7是否參與調控阿拉伯芥根毛發育，以及At14-3-3ω是否在植物體內調控AtGLR3.7功能並影響根毛生長。實驗結果顯示AtGLR3.7啟動子的GUS表現在幼苗許多組織，包括根毛，暗示AtGLR3.7對於植物生長發育可能相當重要。atglr3.7突變株於根毛生長受到嚴重抑制，生物晶片與gene ontology (GO) 分析顯示其在根毛細胞分化與延長兩階段中受到干擾，並挑選出下游可能影響此根毛發育缺陷之候選基因，而SHAVEN3 (SHV3) 可能是其中最關鍵的基因。然而AtGLR3.7互補轉殖株根毛缺陷的表型並未回復，且以Tail PCR找出另一個T-DNA插入位點。此外，At14-3-3ω過量表現轉植株之根毛生長亦被抑制，顯示At14-3-3ω可能於根毛發育中扮演負調控者的角色，而atglr3.7突變株與At14-3-3ω過量表現轉植株之雜交後代卻部份回復根毛生長，然詳細機制未明。綜合以上實驗結果，本研究發現一根毛發育嚴重缺陷之阿拉伯芥突變株，但具有兩個突變基因，並以生物晶片分析可能影響此根毛發育缺陷之下游基因，然而本研究尚無法證實AtGLR3.7是否參與根毛發育，且仍未釐清At14-3-3ω如何影響根毛發育。未來須進行單一基因突變株的根毛表型檢測以釐清其中造成根毛發育缺陷的機制。

關鍵字

GLR3.7 ； 14-3-3ω ； 根毛 ； 生物晶片 ； 交錯式熱不對稱PCR

並列摘要

In Arabidopsis, there are 20 glutamate receptor-like proteins (GLRs), which are homologous to ionotropic glutamate receptors (iGluRs) as important neurotransmission component in animals. To date, it is noted that GLRs are calcium channels on the cell membrane, which mediate calcium dynamics and regulate many plant developmental processes. On the other hand, 14-3-3s are scaffold proteins which can bind to phosphorylated client proteins and participate in many intracellular responses and physiological processes in eukaryotes. Our previous studies indicate that AtGLR3.7 can be phosphorylated by calcium ion-dependent protein kinase (CDPK) and bind to At14-3-3ω in vitro. In addition, the root hair growth of atglr3.7 mutant was greatly inhibited, suggesting that AtGLR3.7 may be involved in root hair development. Therefore, this study aimed to investigate whether AtGLR3.7 is involved in root hair development in Arabidopsis, and whether At14-3-3ω regulates the function of AtGLR3.7 in root hair development. The results show that the AtGR3.7 promoter-GUS expression was distributed in many tissues including root hair, suggesting that AtGLR3.7 may be important in plant development. Microarray and gene ontology (GO) analysis reveal that the atglr3.7 mutant was arrested in root hair cell fate decisions and root hair elongation. Candidate genes related to root hair defective phenotype were selected. Among them, SHAVEN3 (SHV3) appears to be the most critical genes. However, the root hair defective phenotype of AtGLR3.7 complementation lines was not recovered. And additional T-DNA insertion site was identified by Tail PCR. In addition, the root hair growth of At14-3-3ω overexpression lines was also inhibited, indicating that At14-3-3ω may be a negative regulator in root hair development. However, the offsprings of atglr3.7 mutant crossed to At14-3-3ω overexpression lines exhibited partially recover of root hair growth. Taken together, these results reveal the identification of an Arabidopsis mutant which has two T-DNA insertion sites in two genes, exhibited a seriously root hair defective phenotype. The downstream candidate genes related to the root hair defective phenotype were also be selected through microarray. However, it’s still cannot be confirmed whether AtGLR3.7 is involved in root hair development, and how At14-3-3ω affects root hair development. In the future, root hair phenotype of single gene mutant lines should be investigated to clarify the mechanism regulating root hair development.

並列關鍵字

GLR3.7 ； 14-3-3ω ； root hair ； microarray ； tail PCR

參考文獻

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