綠竹BoMSP41轉殖水稻之建立

Abstract

BoMSP41 (Monocot-specific protein-41 in Bambusa oldhamii) 為綠竹之未知功能基因，其基因表現於綠竹快速生長時期之節間會大幅提升，有可能在綠竹快速生長時具生理意義。此基因所轉譯出的蛋白質被預測為一個 intrinsic disorder protein (IDP)。為了建立可用於探討 BoMSP41 之生理功能與調控機制的平台，本研究以 genome walking 法選殖 BoMSP41 轉譯起始點上游的序列，並建構了以玉米 Ubiquitin 1 啟動子驅動恆常性表現的 BoMSP41-2-Flag 與 Flag-BoMSP41-2 水稻轉殖株。分析上游序列發現，BoMSP41 上游基因所轉譯的蛋白質為 COP10-like ubiquitin-conjugating enzyme，而 BoMSP41 的啟動子僅有約 600 bp。以細胞免疫化學定位分析轉殖株原生質體，並以共軛焦顯微鏡觀察的結果顯示，BoMSP41-2 可能被運送至成熟的葉綠體中，且推測水稻中MSP41 homolog (簡稱為 MSP41H) 也會被運送至成熟葉綠體中。免疫呈色分析綠竹出土之幼竹、無菌培養苗與水稻野生型及轉殖株的細胞質、葉綠體、細胞核蛋白質，結果顯示在綠竹無菌培養苗、水稻轉殖株中的 BoMSP41 和水稻野生型中的 MSP41H 都會位於葉綠體中。然而，在綠竹出土之幼竹中，BoMSP41 在細胞質、葉綠體、細胞核中有許多不同的分子量大小。因此推測，在不同生長時期、不同組織中可能會有不同的 BoMSP41 異構型被表現出，也有可能與不同的蛋白質有交互作用後，將 BoMSP41 帶到不同的胞器中執行不同的功能。

BoMSP41 (Monocot-specific protein-41 in Bambusa oldhamii), an unknown function gene in bamboo, is highly up-regulated in the internode region of rapidly elongating bamboo shoots and might participate in bamboo growth. The protein encoded by this gene was suggested to be an intrinsic disorder protein. To establish the platforms to investigate the possible regulatory mechanism and the physiological function of BoMSP41, the sequence upstream of the translation initiation site of BoMSP41 was cloned using genome walking method and transgenic rice constitutively expressing Flag-tagged BoMSP41 proteins under the control of maize ubiquitin 1 promoter were constructed. Sequence analysis revealed that the gene upstream of BoMSP41 encoded a COP10-like ubiquitin-conjugating enzyme; the promoter region of BoMSP41 was about 600 bp. Immunocytochemical analyses of protoplasts isolated from seedlings of transgenic rice plants indicated that BoMSP41 was transported into mature chloroplasts, and so was its homolog in rice (designated MSP41H). Western analyses of cytoplasmic, chloroplastic and nuclear fractions of developing bamboo shoots, in vitro-cultured multiple shoots of bamboo, wild-type and transgenic rice plants showed that BoMSP41 in in vitro-cultured multiple shoots of bamboo, Flag-tagged BoMSP41 in transgenic rice seedlings and MSP41H in wild-type rice seedlings were all localized in chloroplasts. However, in developing bamboo shoots, BoMSP41 with different apparent molecular sizes could be detected in cytoplasm, chloroplast, and nuclei. It was proposed that different isoforms of BoMSP41 were expressed in different growth stage and various tissues of bamboo and/or interactions of BoMSP41 with different binding partners might results in different functions of BoMSP41.