Molecular Characterization of Isopentenyltransferase (BoAIPT1) from Bambusa oldhamii Expressed in Escherichia coli

以大腸桿菌系統分子鑑定綠竹異戊烯基轉移酶

葉傳山(Chuan-Shan Yeh) ； 謝陸盛(Lu-Sheng Hsieh) ； 楊健志(Chien-Chi Yang) ； 李平篤(Ping-Du Lee)

《Botanical Studies》 52卷3期 (2011/07) Pp. 249-256

摘要

細胞分裂素Cytokinins(CKs)爲植物賀爾蒙，具有調控植物發育的功能，例如細胞分裂與植物生長過程的調節，CKs生合成路徑關鍵酵素爲腺嘌呤核苷酸異戊烯基轉移酶Adenylate isopentenyltransferase(AIPT)。本研究中，根據蛋白質保守性序列設計出DNA引子，進行PCR得到部分綠竹AIPT DNA序列。由染色體基因庫篩選出基因全長核酸序列。利用專一性引子進行5'-RACE與3'-RACE得到全長cDNA序列名爲BoAIPT1，BoAIPT1讀碼框內序列含1,035bp，可表現出具有344個胺基酸，分子量37.7kDa的蛋白質。BoAIPT1基因不含插入序列(intron)。分析BoAIPT1與其他物種AIPT相似在N端具有ATP/GTP結合區。大腸桿菌表現BoAIPT1重組蛋白具有活性可將DMAPP中異戊烯側鏈(isopentenyl side chain)轉移至腺嘌呤核苷酸中N^6位置。Real-time PCR分析結果顯示BoAIPT1表現情況因綠竹生長而增加，且莖頂具有最高表現量。

關鍵字

綠竹 ； 腺嘌呤核苷酸異戊烯基轉移酶 ； 分子選殖 ； 大腸桿菌表現系統

並列摘要

Cytokinins (CKs) are a group of phytohormones involved in stages of plant development, such as cell division and various other growth processes. The key enzyme in de novo biosynthesis of naturally occurring cytokinins is adenylate isopentenyltransferase (AIPT). This study amplified a PCR product, containing partial sequence encoding putative AIPT, using primers based on conserved protein sequences. Genomic DNA containing a full-length of AIPT was obtained by screening a genomic DNA library. This study used 5'-RACE and 3'-RACE, using gene specific primers, to clone and sequence cDNA encoding BoAIPT1. The open reading frame of BoAIPT1 was 1035 bp in size, encoding 344-amino acids, with a molecular mass of 37.7 kDa. No introns were found by comparing AIPT from bamboo with AIPT sequences from other species. BoAIPT1 contains a putative ATP/GTP binding motif at the N-terminal as observed in other known AIPT. Recombinant BoAIPT1 expressed in Escherichia coli catalyzed the isopentenyl transfer reaction from dimethylallyldiphosphate (DMAPP) to the N^6 adenylate amino group. Real-time PCR analyses revealed BoAIPT1 expression increased simultaneously with green bamboo shoot growth, with the top green shoot section having the highest expression level.

並列關鍵字

Adenylate isopentenyltransferase AIPT ； Bambusa oldhamii ； Molecular cloning ； Gene expression ； Escherichia coli