丙胺酸消旋酵素轉基因阿拉伯芥之特性分析及釐清D-amino acid對植物具有毒性之機制

Abstract

植物基因轉殖需要適當的篩選標記，目前大多利用抗藥性與抗殺草劑基因作為基因重組的篩選標記，將這些標記基因使用於植物基因轉殖，可能會對人類或環境有不利的影響。最近，我們從土壤多元體基因庫中選殖出L-lysine racemase基因(Appl. Environ. Microbiol., 2009)，並發現此基因可以取代抗藥性基因，以L-lysine為篩選劑，作為植物基因轉殖時之篩選標記，可應用於菸草及阿拉伯芥這兩種模式植物的基因轉殖(Plant Mol. Biol., 2010)。然而，此種篩選系統僅適用於對L-lysine敏感的植物種類，因此必需再建立其他非抗藥性的篩選系統。上半年度的國科會計畫中，我們發現Corynebacteriumglutamicum NCHU 87078的alanine racemase基因(alr)可作為篩選標記，搭配D-alanine作為篩選劑，利用模式植物Arabidopsis為材料，已成功的發展出Alr篩選系統，或可作為其他植物基因轉殖之用。惟alr轉殖之阿拉伯芥的T1植株莖較為纖細，株高較低，花期早且花苞及莢果數目較少，種子產量較低，且具有早熟現象。由於D-amino acid造成植物生長抑制及其他生理上的失調之原因，尚未釐清。本研究的主要目的在於利用alr轉基因阿拉伯芥為材料，觀察野生型及alr轉基因阿拉伯芥經D-alanine處理後之農藝性狀變化，來釐清D-amino acid對植物生長具有毒性之可能機制。在上半年度的國科會計畫中，我們已由核酸微矩陣分析經D-alanine處理或未處理的模式植物-Arabidopsis幼苗，以了解其整體基因表現之差異。未來將以胺基酸轉運蛋白、胺基酸生合成蛋白、轉錄因子...等為標的，進行real-time RT-PCR及基因產物生化特性分析，來協助解析D-amino acid對植物的毒性機制。研發工作將在三年內完成，具體工作如下：第一年一、 阿拉伯芥同質alr 轉基因品系之遺傳、外表型、生化及分子特性的分析二、 轉基因阿拉伯芥alr 基因表現後的胺基酸組成份分析三、 利用具有毒性的D-alanine 來篩選gek1 突變株及gek1-overexpression 轉殖株，並觀察他們的胺基酸組成變化四、 根據核酸微矩陣分析結果及real-time RT-PCR，推測deacylase 酵素，胺基酸轉運蛋白，胺基酸的生合成基因及轉錄因子...等與D-amino acid 毒性之間的關係第二年一、 與D-alanine 毒性有關聯性的基因產物之生化特性分析二、 利用CaMV 35S 啟動子，在Arabidopsis 植物中大量表現與D-alanine 毒性有關的基因三、 觀察gene overexpression 之Arabidopsis T1 轉殖株在含有D-alanine 的培養基之生理反應與農藝性狀，並解析目標基因在D-amino acid 毒性上所扮演的角色四、 利用透射電子顯微鏡，觀察D-alanine 處理或未處理的alanine-racemase-transgenic 和野生型Arabidopsis 植株之根部細胞結構之差異第三年一、利用RNA interference 技術，降低與D-alanine 毒性有關的基因在Arabidopsis 植物之表現二、觀察gene knockdown 之 Arabidopsis T1 轉殖株在含有D-alanine 的培養基之生理反應與農藝性狀，並解析目標基因在D-amino acid 毒性上所扮演的角色

Public concerns on the biosafety and regulatory issues over the use of antibiotic and herbicide-basedselectable markers have necessitated the scientists to develop alternative approaches for transgenic plantselection. Very recently, we have successfully isolated a lysine racemase gene from soil metagenomic library(Appl. Environ. Microbiol., 2009) and demonstrated the utilization of lysine racemase as a non-antibioticmarker using L-lysine as selection agent for the transformation of Arabidopsis and tobacco (Plant Mol. Biol.,2010). However, the use of our system is limited to lysine-sensitive plants. Therefore, it is imperative toestablish other non-antibiotic marker system for wider use in the development of transgenic plants. We havethen successfully used of alanine racemase (alr) gene from Corynebacterium glutamicum NCHU 87078 as aselectable marker and D-alanine as the selection agent for the plant transformation. However, the T1 plantsexhibit phenotypic variations including reduced number and small size of rosette leaves, decrease in seedyield, and early maturity. It has been known that plants are highly sensitive to exogenously applied D-alanineand D-serine, resulting in the growth inhibition and other physiological disorders, but the toxicity of D-aminoacids to plants remains to be elucidated. This proposal is primarily aimed at the characterization of wild-typeand alanine-racemase-transgenic Arabidopsis plants after treating D-alanine to study the underlyingmechanisms of D-amino acid toxicity in plants. The main thrust will be to gain new insights into themechanisms of toxicity based on the studies of amino acid transporters, amino acid biosynthetic genes, andtranscription factors using the methods of real-time RT-PCR and biochemical characterization. The researchwork in the coming three years is as follows:First grant year1. Genetic, phenotypic, biochemical and molecular characterization of selected homozygous alanine-racemase-transgenic lines.2. Analysis of endogenous amino acid composition in the alr-expression transgenic plants.3. Screening of gek1 mutants and gek1-overexpressing transgenic plants on toxic D-alanine and observationof differences in their amino acid profile.4. To correlate deacylase, amino acid transporters, amino acid biosynthetic genes and transcription factorsetc. to D-amino acid toxicity based on microarray data and real-time RT-PCR.Second grant year1. Biochemical characterization of the gene products involved in D-amino acid toxicity.2. Overexpression of the target genes related to D-amino acid toxicity in Arabidopsis plants using CaMV35S promoter.3. Determination of the phenotypic and agronomic traits of the Arabidopsis T1 transgenic plantsoverexpressing target genes in the medium containing D-alanine to elucidate the roles of target genes inD-amino acid toxicity.4. Examination of ultrastructural changes/differences in the root cells of control and D-alanine treatedalanine-racemase-transgenic and wild-type Arabidopsis plants using transmission electron microscopy.Third grant year1. Knockdown of the target genes related to D-amino acid toxicity in Arabidopsis plants using RNAinterference technique.2. Determination of the phenotypic and agronomic traits of the gene knockdown Arabidopsis T1 transgenicplants in the medium containing D-alanine to elucidate the roles of target genes in D-amino acid toxicity.