與綠竹 BohLOL1 具交互作用之蛋白質探討

Abstract

BohLOL1 為綠竹中與阿拉伯芥 LSD1 (Lesions Simulating Disease resistance 1) 及 LOL1 (LSD-One-Like 1) 同源的基因，參與在竹的快速生長與抵禦生物逆境。本研究的目的，在生長時期的綠竹筍中找出與 BohLOL1 有交互作用的蛋白質。藉由酵母菌雙雜交系統 (yeast two-hybrid, Y2H) 於綠竹筍之 cDNA library 中，篩選得到 25 株與 BohLOL1 具交互作用的候選蛋白質。依據其於其它物種中同源基因的功能與細胞內定位資訊，挑選其中 7 株以進行進一步的確認。以綠竹筍 total RNA 為模版進行反轉錄聚合酶連鎖反應 (reverse transcription-polymerase chain reaction, RT-PCR)，以選殖其全長編碼序列 (coding sequence, CDS)，並以 BohLOL1 為釣餌進行 Y2H 分析。結果顯示僅有 2 株與 BohLOL1 有交互作用。此 2 株於阿拉伯芥與水稻中的同源蛋白質分別為 12-oxophytodienoate reductase (OPR) 與 cysteine protease precursor (CYSEP)；因此，將其分別命名為 BoOPR 與 BoCYSEP。於大腸桿菌中表現重組BohLOL1 與 BoOPR 蛋白質並進行共免疫沉澱分析 (co-immunoprecipitation, Co-IP)，結果再次證實兩種蛋白質具交互作用。此外，細胞內定位分析顯示 BohLOL1 位於細胞核與細胞質、BoOPR 位於細胞核與過氧化體、BoCYSEP 位於細胞核與細胞質中未明的聚集。進一步於雙分子螢光互補分析 (bimolecular fluorescence complementation, BiFC) 中，驗證了 BohLOL1 與 2 株候選蛋白質的交互作用，並觀察到 BohLOL1 與 BoOPR 之交互作用位於過氧化體及其它未明胞器；BohLOL1 與 BoCYSEP 之交互作用位於細胞質中未明的聚集。依據上述之結果以及 BoOPR 與 BoCYSEP 於阿拉伯芥及水稻中同源蛋白質的功能，推測 BohLOL1 參與在多種細胞路徑中：藉由與 BoOPR 產生交互作用，BohLOL1 可能與抵禦病源菌時訊息傳遞分子的合成有關；藉由與 BoCYSEP 產生交互作用，BohLOL1 可能與植物生長時蛋白質的降解有關。

BohLOL1, a homolog of Arabidopsis LSD1 (Lesions Simulating Disease resistance 1) and LOL1 (LSD-One-Like 1) in Bambusa oldhamii, participates in bamboo growth and in the response to biotic stress. The objective of this study is to identify proteins that interact with BohLOL1 in growing bamboo shoots. By using yeast two-hybrid (Y2H) screening, 25 candidates that putatively interacted with BohLOL1 were obtained from a bamboo shoot cDNA library. According to the functions and subcellular localization informations of their homologous genes in other plant species, 7 candidates were selected for further confirmation. The full-length coding sequence (CDS) were cloned by reverse transcription-polymerase chain reaction with total RNA from bamboo shoots as a template, and then subjected to Y2H analysis with BohLOL1 as the bait. The results showed that only two candidates did interact with BohLOL1. Their homologous proteins in Arabidopsis thaliana and Oryza sativa were 12-oxophytodienoate reductase (OPR) and cysteine protease precursor (CYSEP), respectively; therefore, these two candidates were named as BoOPR and BoCYSEP, respectively. The BohLOL1 and BoOPR recombinant proteins were expressed in E. coli and subjected to co-immunoprecipitation (Co-IP) analysis. The result again confirmed the interaction between each other. Besides, subcellular localization analysis showed that BohLOL1 was localized in the nucleus and cytoplasm, BoOPR was localized in the nucleus and peroxisomes, and BoCYSEP was localized in the nucleus and forming unknown aggregates. Furthermore, the interactions between BohLOL1 and BoOPR or BoCYSEP were confirmed by bimolecular fluorescence complementation (BiFC) analysis and the results showed that the interaction between BohLOL1 and BoOPR occurred in peroxisomes and in other unknown organelle and the interaction between BohLOL1 and BoCYSEP occurred in unknown spots present in the cytoplasm. According to the above results and the finctions of the homologs of BoOPR and BoCYSEP in Arabidopsis thaliana and Oryza sativa, BohLOL1 was proposed to participate in multiple cellular pathways: by interacting with BoOPR, BohLOL1 may be involved in synthesis of signaling molecules for pathogen defense; by interacting with BoCYSEP, BohLOL1 may be involved in protein degradation for supporting plant growth.