- 學位論文
麩醯胺酸對阿拉伯芥生長與發育的影響
Effects of glutamine on the growth and development of Arabidopsis seedlings
摘要
麩醯胺酸 (Gln) 是植物中第一個同化生成的有機氮化合物，相對於其在代謝上的功能，我們對Gln在訊息傳導所扮演的角色所知甚少。相較於硝酸銨，當阿拉伯芥幼苗生長在以Gln為氮源的培養基時，會有更多的側根及更長的根毛，根毛發育的關鍵基因也會被Gln所誘導。茉莉酸合成抑制劑或乙烯作用抑制劑的處理可以阻斷Gln促進根毛延長的效果。因此，Gln可能作用於茉莉酸以及乙烯的上游來調控根毛的生長。為了找出阿拉伯芥中與Gln代謝、運送或訊息傳導相關的基因，我們以Gln作為唯一氮源，篩選具有生長缺陷的突變株。其中一個分離自阿拉伯芥種源中心編號CS19945種子庫中的突變株，生長在Gln的培養基時會大量累積花青素。遺傳分析與基因圖譜定址選殖 (map-based cloning) 的結果顯示，該突變株花青素的性狀可能是由兩個不同的基因突變所造成。其中一個隱性的突變是因為T-DNA插入At3g18520基因 (組蛋白去乙醯酶15, HISTONE DEACETYLASE15, HDA15)，使其功能喪失所致。另外一個則是顯性的突變，由未知功能基因At3g21480發生T-to-C點突變造成F332L胺基酸的改變，此突變是否會導致Gln誘發花青素的累積，尚待進一步研究。透過分析獨立的hda15-1突變株及其互補株，確認了hda15-1生長在Gln的培養基時，花青素的含量比野生株高。在其他已知會誘導花青素合成的生長條件下，例如高糖、低氮或甲基茉莉酸處理，hda15-1花青素的含量也都比野生株高，一些花青素生合成基因以及調控花青素合成的轉錄因子基因在hda15-1中的表現也比較高。組蛋白免疫沉澱分析的結果發現這些在hda15-1表現較高的基因，有較高的組蛋白乙醯化程度。總結來說，阿拉伯芥幼苗生長在高糖、低氮或甲基茉莉酸處理時所誘發的花青素合成，有一部分是透過組蛋白乙醯化修飾，來促進花青素生合成基因及相關調節基因的表現。另外，硝酸銨比Gln更能有效地抑制阿拉伯芥幼苗中花青素的合成，部分與HDA15所調控的染色體修飾有關。
並列摘要
Glutamine (Gln) is the first organic nitrogen (N) synthesized in the primary N assimilation pathway in plants. In addition to its metabolic functions, Gln can affect the growth and development of Arabidopsis seedlings. When grown on medium containing Gln as the sole N source, Arabidopsis seedlings have more lateral roots and longer root hairs compared with those grown on NH4NO3. The expression of key genes involved in root hair development was up-regulated by Gln. Treatment of jasmonic acid (JA) biosynthesis inhibitor or ethylene action inhibitor was able to block the effect of Gln on root hair elongation, indicating that Gln may act in the upstream of JA and ethylene to regulate root hair development. To identify genes involved in Gln metabolism, transport or signaling, we screened for Arabidopsis mutants that grew poorly on medium containing Gln as the sole N source. One of the isolated mutants, line 19945, has high levels of anthocyanins when grown on medium containing Gln. Genetic analyses and map-based cloning revealed that two different loci are linked to the anthocyanin-related phenotype: A recessive mutation caused by a T-DNA insertion in the At3g18520 gene encoding histone deacetylase 15 (HDA15), and a dominant mutation caused by a T-to-C point mutation resulting in an F332L amino acid change in the unknown function gene At3g21480. We are in the process of verifying if the mutation in At3g21480 is responsible for the anthocyanin phenotype of the 19945 mutant. An independent hda15-1 T-DNA insertion mutant and its complementation line were used to confirm that anthocyanins accumulated in the mutant when grown on medium containing Gln as the sole N source. Furthermore, treatments of high sucrose, low N, and methyl JA also enhanced anthocyanin accumulation in the hda15-1 mutant. The expression of some anthocyanin biosynthetic and regulatory genes was up-regulated in the mutant. Chromatin immunoprecipitation assays indicated that the up-regulated genes also have increased levels of histone acetylation in hda15-1. These results suggest that histone acetylation plays a role in the activation of anthocyanin biosynthetic and regulatory genes. The phenomenon that ammonium nitrate is more effective than Gln in reducing anthocyanin accumulation could be partly explained by the repression of anthocyanin biosynthetic and regulatory genes caused by histone deacetylation mediated by HDA15 in Arabidopsis seedlings.
參考文獻
延伸閱讀
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