綠竹異戊烯基轉移酶之分子生物學與生化學研究

Abstract

細胞分裂素 Cytokinins (CKs) 為植物賀爾蒙，在植物生理上具有調控植物發育的重要功能，與生長素 (auxin) 協同作用促進細胞分裂、植株發芽，並影響癒瘡組織的生長型態。CKs亦可促進植株側芽生長與延遲老化。DMAPP: ATP路徑被認為是植物中 CKs 主要合成的路徑，該合成路徑是將 DMAPP 中的異戊烯側鏈 (isopentenyl side chain) 轉移至 AMP 中的 N6 位置而形成 isopentenyladenosine-5’-monophosphate (iPTP)。此反應是由 Adenylate isopentenyltransferases (AIPT) 所催化的。AIPT 所催化反應是 CKs 生合成速率決定步驟，因此 IPT 被認為是 CKs 生合成路徑之關鍵酵素。 以阿拉伯芥與水稻的 AIPT 序列為基準，設計出 degenerate primer，利用 degenerate PCR 方式得到核酸探針，以此探針進行綠竹 (Bambusa oldhamii) 基因組庫的篩選，得到具有 AIPT 基因的 DNA片段，該片段已知序列為 4.7 kb，內包含一個全長 1,035 bp 之 open reading frame (ORF)，此 ORF 不具有 intron，轉譯成蛋白質分子量預估為 37,599 dalton，具有 344 的胺酸組成，命名為 BoAIPT1。BoAIPT1 與其他物種 AIPT 在親源相似程度具有47-71% identities。利用網路 ExPAsy 蛋白質體分析工具進行 BoAIPT1 生化特性的預測，發現 BoAIPT1 中的 Ser、Thr 與 Tyr 可能具有磷酸化與醣基化之轉譯後修飾作用。 BoAIPT1 等電點為 8.47，胞器定位預測結果顯示 BoAIPT1 應存在細胞質中。以 PLACE 啟動子分析工具進行 BoAIPT1 啟動子序列分析，發現 BoAIPT1 啟動子中可能具有生長素 (auxin)、吉貝素 (gibberellin) 與光感應相關的調控區。 利用北方雜合法分析 BoAIPT1在綠竹不同生長時期表現情況，發現出土後的綠竹筍 BoAIPT1表現量比未出土時高，經由Real-Time RT-PCR比較出土與未出土綠竹中，BoIPT1在不同部位的表現情況，發現綠竹出土後莖頂之BoAIPT1表現量最高。BoAIPT1與GFP融合進行洋蔥表皮細胞定位實驗，顯示BoAIPT1主要表現位置可能存在質體中。以E.coli 表現系統表現BoAIPT1並分析BoAIPT1酵素活性，由酵素動力學研究推測綠竹BoAIPT1以ATP與DMAPP為受質，啟動iP合成途徑。

Cytokinins (CKs) are a class of plant hormones that play a pivotal role in plant development. They induce cell division in the presence of auxins, and induce shoot formation on calli. They also release axillary buds from apical dominance, increase sink strength, and delay senescence. In the CKs synthesis pathway, the isopentenyl group is transferred from DMAPP to the N6of AMP, resulting in the production of isopentenyladenosine-5’-monophosphate (iPMP). This reaction is thought to be catalyzed by isopentenyltransferases (IPT). IPT is a key enzyme in CKs biosynthesis pathway. A DNA fragment encoding isopentenyltransferase (IPT) was cloned and sequenced from genomic library of Bamboo (Bambusa oldhamii). Library screening by IPT specific probe from degenerate PCR using degenerate oligonucleotide primers based on the conserved sequences of Arabidopsis thaliana AtIPT and Oryza sativa OsIPT isozymes. The 4.7 kb genomic DNA fragment contains a 1,035 bp open reading frame encoding a molecular mass of 37,599 dalton protein with 344 amino acid named BoIPT1 revealed absence of intron in the frame. BoAIPT1 deduced amino acid sequence shares 47-71% identity to OsIPTs. Prediction of biochemical properties of BoAIPT1 amino acid sequence using ExPAsy (Expert Protein Analysis System) proteomics server revealed phosphorylation and glycosylation sites in Ser、Thr and Tyr residues. Isoelectric point of BoIPT1 is 8.47. Subcellular localization prediction of BoIPT1 is a cytosolic protein. Analysis BoAIPT1 promoter by PLACE promoter scanning tool revealed cis-acting regulatory DNA elements involved auxin、gibberellin and light response. Using northern blot analysis on BoAIPT1 at various growth stages, demonstrated the expression level of BoAIPT1 is higher in bamboo shoot as compared to the etiolated shoot. Using Real-Time RT-PCR to compare the BoAIPT1expresion level in various parts of bamboo shoot and etiolated shoot, results revealed that the expression level was highest in the shoot. Results from GFP revealed that BoAIPT1 was located in the plastid of onion epidermal cell. Expression of BoAIPT1 in E.coli was used to perform enzyme assay, this revealed to ve similar with other reports regarding IPTs of different plants. This suggests that BoAIPT1 uses ATP and DMAPP as substarte to initiate the iP sysnthesis pathway.