探討大豆幾丁質酶於植物促生根際細菌預啟防禦反應之基因表現與功能

Abstract

植物可辨識植物促生根際細菌 (plant growth-promoting rhizobacteria, PGPR) 釋出的激發子 (elicitor) 以預啟 (prime) 植物防禦反應相關基因，進而提升植物的抗病能力。在預啟反應所表現的蛋白中，病程相關蛋白 (pathogenesis-related protein, PR-protein) 之表現扮演了重要的角色。其中隸屬病原性相關蛋白PR-3、PR-4、PR-8及PR-11的幾丁質酶 (chitinase) 為一種廣泛存在於多種生物的糖苷水解酶 (glycoside hydrolase)，可透過水解幾丁質之糖苷鍵，進而影響真菌細胞壁等由幾丁質所構成的組織。由於幾丁質酶提供良好抗病潛力，加上近年高通量定序技術的發展，目前已有數種植物以全基因體角度切入探討幾丁質酶相關基因。此外，結合植株受病原菌感染之轉錄體分析，亦可進一步研究受到土壤微生物預啟的幾丁質酶基因是否受到病原菌調控，並以基因轉殖技術驗證其功能性。大豆 (Glycine max) 作為重要經濟作物之一，目前鮮有研究對其基因體中的幾丁質酶基因數量進行預測，故本研究透過大豆全基因體分析找到37個幾丁質酶基因，並經由大豆接種Burkholderia. ambifaria及Fusarium oxysporum之轉錄體分析，發現了5個幾丁質酶基因會受二者影響而上調。結合來自22種植物中已受功能性驗證之幾丁質酶序列進行親緣樹分析後，本研究挑選3個大豆幾丁質酶基因 (GmChi01、GmChi02及GmChi16) 透過農桿菌介導的阿拉伯芥轉殖技術，獲得了三個大豆基因的過表達子代。相較於阿拉伯芥Col-0，過表達大豆幾丁質酶基因之阿拉伯芥在整體生長勢上並無顯著差異，然而以離葉接種及土壤接種F. oxysporum f.sp. rapae後，計算葉片黃化程度換算病害發生進程下之面積 (AUDPC) 及幼苗存活率，發現過表達GmChi02及GmChi16之阿拉伯芥葉片可展現較強之的抗病能力。雖然GmChi02與GmChi16皆具提升抗病能力，但僅有GmChi02受到B. ambifaria預啟，說明幾丁質酶基因可能受到不同PGPR影響。透過接種6種不同PGPR之轉錄體分析，發現9個幾丁質酶基因不受到所測試的6種PGPR影響，另有10個幾丁質酶基因受到至少一種PGPR影響而顯著上調。綜合上述，本研究以全基因體分析大豆幾丁質酶基因，並搭配轉錄體學及阿拉伯芥轉殖技術驗證大豆幾丁質酶之抗真菌能力，同時紀錄6種PGPR預啟大豆幾丁質酶基因表現能力具有差異性。

Plants recognize elicitors released by plant growth-promoting rhizobacteria (PGPR) to initiate pathogenic-related proteins (PR-proteins) responses in disease priming. PR-proteins such as PR-3, PR-4, PR8, and PR-11 are chitinases that hydrolyze the glycosidic bond of chitin, which is the main component of fungal cell wall. In advantage of high-throughput sequencing technologies, it becomes powerful to perform genome-wide characterization of chitinases in plant genomes. Together with transcriptomic analyses to find PR-protein genes under biotic stresses such as fungal infection, validating gene functions in resistance through heterogeneous transformation has been robust and approachable. Soybean (Glycine max) is one of the most important economic crops, but limited researches have focused on soybean chitinase genes in the genome or validated their functions in defense responses. In this study, we identified 37 chitinase genes in the soybean genome, and we integrated the transcriptomic data in response to Burkholderia ambifaria and Fusarium oxysporum infection to prioritize 5 candidate chitinase genes. According to the phylogenetic analysis with previously functionally characterized chitinase genes from 22 plant species, we chose 3 genes (GmChi01, GmChi02, and GmChi16) to obtain the over-expression Arabidopsis lines by Agrobacterium‐mediated transformation. Compare to the Col-0, transgenic Arabidopsis lines did not show significant differences in phenotypes of agronomic traits. Furthermore, the results of detached leaf assay and soil inoculation showed that over-expression of GmChi02 and GmChi16 in Arabidopsis reduced area under disease progress curve (AUDPC) and enhanced the survival rate of seedlings upon F. oxsporum f.sp. rapae infection. Although GmChi02 and GmChi16 significantly enhanced defense to F. oxsporum f.sp. rapae, only GmChi02 was significantly primed by B. ambifaria. This observation indicated plant chitinases may be induced by different PGPR in defense responses. According to the transcriptomic analysis of soybean in response to 6 PGPR, 9 chitinases were not induced by any of PGPR and 10 chitinases were significantly primed by at least one of PGPR. In conclusion, this study verified the functions of GmChi01, GmChi02 or GmChi16 in disease responses and provided a comprehensive analysis of 37 soybean chitinase genes in PGPR-induced defense priming.