綠竹中兩種推定為細胞分裂素受體BoCRE1及BoCRE2p之選殖及分子生物學研究

Abstract

綠竹具生長快速的特性，此現象可能與細胞分裂素的生理特性有關。前人經由綠竹 cDNA 庫篩選到兩個推定為細胞分裂素受體基因 (CRE, cytokinin response element)，分別為 BoCRE1 和 BoCRE2p，然而所得到的 BoCRE1 和 BoCRE2p 與阿拉伯芥或水稻之 CRE 相比，少了 N 端的部分序列，因此為了獲得 BoCRE1 和 BoCRE2p 之完整序列，故以 TAIL-PCR、5’-RACE、及 RT-PCR 進行 5’端序列之延長。序列比對結果顯示，BoCRE1 的初級序列與水稻的 OsCRE1 相似，而 BoCRE2p 的初級序列與 ZmHK 相似，但 BoCRE2p 缺少 histidine kinase (HK) 和 receiver domain (RD)。為了後續可以進行 BoCRE1 之生化研究，利用大腸桿菌 BL21(DE3) 表現不同長度的 BoCRE1，分別為 HK、 RD，和 HKRD 進行重組蛋白質表現。目前發現 HK 及 HKRD 的重組蛋白質會分布在不溶體。而 RD 則分布在可溶部份，並將其進行蛋白質純化，作為抗原免疫小鼠生產多株抗體。另一方面，為驗證 BoCRE2p 在綠竹中的身分，以 3’-RACE 確認其轉譯終止碼，並以 BoCRE1 和 BoCRE2p 保守性序列設計探針進行北方墨點法，觀察這兩個基因在 RNA 層次的表現情形，並以 real-time PCR 偵測綠竹筍出土前後 BoCRE1 和 BCRE2p 的表現差異，發現 BoCRE1 在出土後基部和頂部表現量會有明顯差異，而 BoCRE2p 在出土後中部表現量也會有明顯差異。

Bambusa oldhamii is characterized by its fast growth. This feature might relate to physiological role of cytokinin. Two putative cytokinin receptors genes (CRE, cytokinin response element) are cloned from bamboo cDNA library, designated as BoCRE1 and BoCRE2p. However, both genes lack the DNA fragment encoding the N-terminal sequences by comparing with CRE1 from other species, such as rice and Arabidopsis. To obtain the full-length cDNA encoding BoCRE1 and BoCRE2p, TAIL-PCR, 5’-RACE and RT-PCR were used to amplify the fragment of the 5’-end. The deduced protein sequences of BoCRE1 is similar to the OsCRE1. The deduced protein sequences of BoCRE2p is more similar to the ZmHK, but lacks of histidine kinase (HK) and receiver domain (RD). In an attempt to analyze BoCRE1 biochemical properties, recombanant protein of HK, RD and the HKRD domains were heterologously expressed in BL21(DE3). Recombinant HK and HKRD were expressed in inclusion bodies, whereas recombinant RD was expressed in soluble fraction. Polyclonal antibody against purified recombinant RD was prepared. To confirm the identity of BoCRE2p in Bambusa oldhamii, 3’-RACE were employed to identify the stop codon. Probes designed based on the conserved sequences of BoCRE1 and BoCRE2p were prepared for Northern blot analysis to determine RNA expression level of these genes. The expression profiles of these two genes in the different bamboo tissues and the different growth stages were analyzed by quantitative real-time PCR. The levels of BoCRE1 mRNA showed significant changes in the base and the top regions of bamboo at different growth stages, whereas the levels of BoCRE2p mRNA showed significant changes in the middle regions at different growth stages.