阿拉伯芥缺氧反應突變株之遺傳分析

淹水為常見的環境逆境，當植物遭逢淹水逆境時，增加不定根、通氣組織以及改變代謝機制都可增加植物對淹水逆境的適應性。在淹水情況下，植物根部附近的土壤空隙充滿水分，造成土壤通透性不佳，形成缺氧的環境。當缺氧逆境發生時，植物體內的代謝途徑將從有氧呼吸轉換為無氧發酵。前人的研究已證實在缺氧時，糖類的代謝、糖解作用以及無氧發酵中的主要基因都會被誘導表現，像是SUCROSE SYNTHASE (SUS)、PYRUVATE DECARBOXYLASE (PDC) 和ALCOHOL DEHYDROGENASE (ADH)，其中，ADH表現量的上升有助於增加植物對缺氧的耐受性。本實驗室在先前研究中，將ADH啟動子(promoter)驅動GUS 報導基因(reporter gene) 以轉基因方式轉入阿拉伯芥，並以ethyl methanesulfonate (EMS)處理產生突變株，再使用丙烯醇(allyl alcohol)篩選出對丙烯醇具有抗性且GUS基因表現量下降的植株，命名為allyl alcohol resistance1 (aar1)突變株。以期利用此突變株了解在淹水逆境下訊息傳導路徑。本研究的主要目的為利用遺傳圖譜(genetic mapping) 找出aar1突變株所突變的位置以及分析其於分子層次之特性。利用aar1 (C24 background)與另一種生態型(Lansberg)進行雜交後，收集其F2植株進行遺傳圖譜分析，接著使用分別於不同染色體上的DNA分子標記將可能的突變區域縮小，根據目前的結果我們推測aar1突變株可能存在兩個突變位置，且這兩個位置都位於第三條染色體上，而其中一個位置為一段NON-SPECIFIC PHOSPHOLIPASE C- 6 (NPC6) 基因的缺失，而另一個位置位於NPC6基因之前。在分子特性方面，我們檢測了一些缺氧逆境時主要會被誘導表現的標誌基因，結果發現ALLYL ALCOHOL RESISTANCE 1(AAR1) 基因只影響了ADH表現，表示AAR1可能參與在其他的代謝途徑之中。因此，我們進一步也利用了微矩陣基因表現技術探討於缺氧逆境下，尋找可能受AAR1 調控的基因群。結果顯示表現於缺氧情況、水分傳導的基因在aar1突變株中有較高程度的表現；然而過氧化酶的活性在aar1突變中表現量較低。因此，我們推論AAR1基因扮演負面調控的角色，抑制過氧化酶等基因表現，另外AAR1可能藉由一個抑制者來調控缺氧時的水分傳導與基因表現。雖然本篇研究尚未知道aar1突變株的確切突變位置與在訊息傳導中所扮演的確切角色，但藉由遺傳圖譜已經知道位於第3條染色體上，而經由微矩陣基因表現技術得到一些線索關於AAR1的位置及其可能參與的訊息傳導路徑。

關鍵字

缺氧逆境；酒精去氫酶遺傳圖譜

並列摘要

Flooding is a common environmental stress. When it happened, the catabolism in plants switched from aerobic respiration to anaerobic fermentation. Previous studies showed that oxygen deficiency induced several genes in sugar metabolism, glycolytic and fermentative pathways, including ALCOHOL DEHYDROGENASE (ADH). Our lab has used a negative-selection approach to isolate mutants that were defective in regulating the expression of ADH gene during seed germination and in seedlings under hypoxia stress. This approach used a transgenic line, AG2, in which expression pattern of ADH::GUS gene reflecting the endogenous ADH gene, for ethyl methanesulfonate (EMS) mutagenesis. In order to screen mutants that affect ADH gene expression, seeds were treated with allyl alcohol and selected for survivors. The surviving plants, designed allyl alcohol resistance (aar) mutants, were candidates for hypoxic regulatory mutants. To map and characterize one of these aar mutants, aar1, which is a recessive mutant, we first checked the sequence of the endogenous ADH gene to confirm that the mutation is not located at the ADH locus. Then, we crossed another ecotype Lansberg and aar mutant (C24 background), and collect the F2 plants for genetic mapping. We used markers on different chromosomes, and already narrowed down to two possible regions both at chromosome 3. Through its segregation rate, we hypothesize that there are two mutation sites in the aar1 mutant. One is a deletion in NPC6 locus, and the other one locates at a locus that is proximal to NPC6. To characterize aar1 mutant, we examined the expression level of several key glycolytic and fermentative genes. The results showed that AAR1 only affects ADH gene, indicating that AAR1 may be involved in other signaling pathway. We also used microarrays to identify the differential gene expressions in aar1 mutant under hypoxic condition. We found groups of genes, including core hypoxia responsive genes and water transport genes, are down regulated in aar1 mutant, while genes that incode peroxidase are up regulated in aar1 mutant. According to this result, we hypothesize that AAR1 plays a negative role, and represses activity of peroxidases under hypoxia. On the other hand, AAR1 regulates ADH and other genes that encode water transporters may be mediate via repressors. Although the identity of the aar1 mutation is still not determined in this study, the genetic mapping and microarray results gave us some clues about the location of AAR1 on chromosome III, and the role that AAR1 plays under hypoxia stress.

並列關鍵字

Hypoxia ； aar1 ； ADH ； mapping

參考文獻

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國際替代計量

阿拉伯芥缺氧反應突變株之遺傳分析

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