水稻抗白葉枯病突變體SA0423抗性表現數量性狀基因座(eQTL)之研究

Study on the Resistance eQTLs in the Rice Bacterial Blight Resistant Mutant, SA0423

林大鈞(Da-Gin Lin) ； 林玉鈴(Yu-Ling Lin) ； 黃巧宜(Chiao-Yi Huang) ； 曾馨儀(Hsin-Yi Tseng) ； 曾文彬(Wen-Bin Tseng) ； 陳純葳(Chun-Wei Chen) ； 王強生(Chang-Sheng Wang)

《台灣農業研究》 65卷1期 (2016/03) Pp. 54-69

https://doi.org/10.6156/JTAR/2016.06501.06

摘要

林大鈞、林玉鈴、黃巧宜、曾馨儀、曾文彬、陳純葳、王強生。2016。水稻抗白葉枯病突變體SA0423 抗性表現數量性狀基因座 (eQTL) 之研究。台灣農業研究65(1):54–69。為開發新的白葉枯病抗性基因，本研究針對轉錄體分析所得17 個抗性候選基因，利用GRAMENE 網站，檢索基因兩側5 cM 內的SSR 標誌，篩選於2 親本 ‘TN1’ (感性) 及SA0423 突變體 (抗性) 間具多型性的標誌，針對177 個‘TN1’ × SA0423 F2 個體進行基因型分析，並配合白葉枯病菌XF89b 接種後病斑之外表型調查。所得資料利用R/qtl 以單一標誌回歸分析法，分析基因型與抗性外表型的連鎖關係。結果發現，僅有鄰近Ankyrin 基因之RM6838 的LOD 值 (6.86) 超過閥值 (LOD = 3.0)，顯示Ankyrin 基因極有可能參與SA0423 的抗性表現。生物資訊學分析發現，Ankyrin之蛋白質序列與阿拉伯芥RING型E3接合酶(XBAT32)具有76%的相似性，顯示其可能透過調控1-aminocyclopropane-1-carboxylate synthase (ACS)之生合成，改變植物體內的乙烯量，而延緩植物的免疫系統。即時定量逆轉錄聚合酶鏈鎖反應(real time RT-PCR)分析發現，接種XooXF89b後，Ankyrin基因在SA0423突變體中的表現低於‘TNG67’。同時，OsACS1及OsACS3在SA0423 突變體的表現量顯著高於‘TNG67’。這些結果顯示，SA0423突變體在接種後可能改變乙烯生合成路徑，因而具有較強的抗病性。經選殖Ankyrin基因上下游共11.8 kb的DNA片段進行定序結果顯示，在編碼區有15個突變位點，導致胺基酸置換 (Ser280Pro與Thr381Ala)。綜合試驗結果說明，Ankyrin不僅在表現量與SA0423白葉枯病抗性間具有高度相關，其蛋白質特性亦與‘TNG67顯著不同。因此，Ankyrin基因應是參與SA0423突變體白葉枯病抗性機制的表現數量基因座(eQTL)之一。

關鍵字

水稻 ； 葉枯病抗性 ； 變體 ； 錄體 ； 現數量性狀基因座

並列摘要

Lin, D. G., Y. L. Lin, C. Y. Huang, H. Y. Tseng, W. B. Tseng, C. W. Chen, and C. S. Wang. 2016. Study on the resistance eQTLs in the rice bacterial blight resistant mutant, SA0423. J. Taiwan Agric. Res. 65(1):54-69. To explore the novel bacterial blight resistance gene for rice disease resistance breeding, 17 candidate resistance genes found from previous transcriptomic analysis were characterized in this study. The SSR markers flanking 5 cM region of them were retrieved from GRAME web site, and analyzed for the polymorphic markers between ‘TN1’ (susceptible parent) and SA0423 (resistant parent). Genotyping analysis of 177 ‘TN1’ × SA0423 F2 individuals was performed using polymorphic SSR markers. The lesion of these F2 individuals were also investigated following the inoculation of Xanthomonas oryzae pv. oryzae XF89b (Xoo XF89b) as the resistance phenotype. The linkage between genotyping and resistance was analyzed by R/qtl using the single marker regression model. The result displayed that only RM6838 adjacent to Ankyrin showed a significantly high LOD (6.86) comparing with the threshold LOD (3.0). It is suggested that Ankyrin have high potential to be involved in providing the resistance of SA0423. The bioinformatic analysis demonstrates that the Ankyrin protein shares a similarity of 76% with the Arabidopsis RING type ligase, XBAT32, an Arabidopsis XB family. Results suggested that Ankyrin may change the ethylene biosynthesis pathway by regulation of 1-aminocyclopropane-1-carboxylate synthase (ACS), and then compromised the immune system. The real-time RT-PCR displayed that the expression profile of Ankyrin in SA0423 was less than that of ‘TNG67’; however, higher expressions of OsACS1 and OsACS3 were found in SA0423. These findings indicated that the biosynthesis of ethylene might change in the SA0423 mutant leading to resistance against pathogens. Cloning and sequence comparison on Ankyrin gene found that 15 mutations were located in the coding region and resulted two amino acid mutations, Ser280Pro and Thr381Ala. Therefore, Ankyrin is considered to be one of the expression quantitative trait loci (eQTLs) involved in the bacterial blight resistance of SA0423.

並列關鍵字

Rice ； Resistance of bacterial blight ； utant ； ranscriptome ； xpression quantitative