β-氨基丁酸诱导马铃薯对晚疫病的抗性组织化学及信号传导途径分析

doi:10.3864/j.issn.0578-1752.2014.13.009

摘要/Abstract

摘要： 【目的】β-氨基丁酸（BABA）田间或离体叶片喷洒能够有效诱导马铃薯防卫反应，增强对晚疫病的抗性。研究从组织化学层面进一步探究BABA诱导马铃薯晚疫病抗性过程中诱发的防卫反应及相关信号途径，为深入研究BABA诱导马铃薯产生晚疫病抗性的可能机制奠定基础。【方法】用4 mmol?L-1 BABA或蒸馏水（对照，Mock）喷洒马铃薯植株叶片，3 d后取离体叶片接种晚疫病菌（Phytophthora infestans），接种后不同时间用打孔器取接种点叶盘用于织化学染色和过敏反应（HR）观察。采用二氨基联苯胺（DAB）组织化学染色方法检测接种点周围活性氧（H2O2）累积情况；叶盘用苯胺蓝（aniline blue）染色，然后在荧光显微镜下观察接种点周围胼胝质积累和表皮细胞HR发生情况；利用干涉了StCOI1（阻断了茉莉酸JA信号传导途径）和超量表达细菌水杨酸羟化酶基因NahG（阻断了水杨酸SA信号传导途径）的转基因马铃薯株系为材料，通过研究BABA能否在这些特殊马铃薯材料上有效诱导晚疫病抗性，进而推断BABA诱导马铃薯晚疫病抗性可能参与的信号途径。【结果】用清水作为空白接种时，Mock和BABA预处理叶片各时间点均未观察到H2O2积累，而病原菌接种处理24 h后，Mock和BABA预处理叶片接种点周围都出现H2O2积累，随着病原诱导时间的延长，H2O2积累增强。但是，BABA预处理叶盘中接种点周围H2O2累积比对照提前12 h，且累积量显著强于对照。苯胺蓝染显示Mock和BABA预处理叶片在接种清水时检测不到胼胝质的沉积，而接种晚疫病菌24 h后，Mock和BABA预处理叶片上均观察到胼胝质的沉积，BABA预处理叶片在侵染点及其附近胼胝质积累量要显著高于对照。荧光显微观察结果显示，BABA预处理和Mock叶片晚疫病接种点周围表皮细胞均有HR发生，BABA预处理叶片早在接种24 h后，接种点周围就已出现HR反应；接种48 h时，BABA预处理85%叶盘的接种点周围表皮细胞出现HR，而Mock叶片只有30%叶盘上观察到HR，BABA预处理叶片HR发生频率比Mock叶片高出55%。BABA不能在超量表达NahG的马铃薯叶片上有效诱发抗性，但能够在干涉了StCOI1的马铃薯叶片上正常诱导产生抗性。【结论】BABA能够从H2O2和胼胝质累积以及HR发生多个层面有效诱发马铃薯基础防御反应，BABA预处理能够在病原菌侵染点周围显著提高胼胝质和H2O2积累水平，诱发接种点发生较高频率的HR，从而提高马铃薯对晚疫病的抗性；BABA诱导马铃薯对晚疫病的抗性依赖水杨酸信号传导途径，但不依赖茉莉酸信号传导途径。

关键词: 马铃薯 , β-氨基丁酸诱导抗性 , 晚疫病 , 组织化学 , SA信号途径

Abstract: 【Objective】 β-aminobutyric acid (BABA) can effectively induce defense responses in potato against late blight disease by means of spray in field or on detached leaves. The objective of this study is to investigate the histochemical aspects of BABA induced resistance (BABA-IR) and signaling pathway involved in BABA-IR in potato. The present study will provide knowledge for further elucidating the mechanisms of BABA-IR.【Method】Potato plants were sprayed with 4 mmol?L-1 BABA or distilled water (Mock, as control). Three days after treatment, the sprayed leaves were detached and inoculated with Phytophthora infestans. Then leaf discs were punched out from attached leaves at different time points after inoculation and used for histochemical staining and hypersensitive response (HR) observation. Diaminobenzidine (DAB) staining method was used to detect hydrogen peroxide (H2O2) generation around inoculated site. Callose deposition and HR were detected by aniline blue staining combined with fluorescence microscopic observation. For investigating the signaling pathway in which BABA induced late blight resistance involved, transgenic potato plants which disturbed the jasmonic acid (JA) or salicylic acid (SA) signaling pathway by interferencing StCOI1 or overexpressing NahG were used. 【Result】 No deposition of H2O2 was observed when Mock and BABA pretreated leaves were inoculated with water. While H2O2 deposition appeared around inoculation sites on both Mock and BABA pretreated leaves 24 h after P. infestans inoculation. It was obvious that BABA pretreated potato leaves accumulate H2O2 strongly and early ahead of Mock (control) 12 h after P. infestans inoculation. No obvious callose deposition was observed when Mock and BABA pretreated leaves were inoculated with water. But strong callose deposition was observed around the P. infestans inoculation sites in BABA pretreated potato leaves compared to that of Mock leaves. Fluorescence microscopic observation showed that HR occurred around the P. infestans inoculation sites on BABA pretreated leaves as early as 24 h. Although HR appeared on both Mock and BABA pretreated leaves, high frequency (85%) of HR occurred around the inoculation sites on BABA pretreated leaves, which was about 55% higher than it occurred on the Mock leaves. Treatment with BABA could not effectively induce late blight resistance in the potato leaves overexpressing NahG, while it was capable of inducing resistance in the StCOI1 silenced potato leaves, which meant that BABA-IR needs SA signaling pathway. 【Conclusion】 Various of defense responses, including H2O2 accumulation, callose deposition and HR, were rapidly and strongly activated in BABA pretreatment potato leaves after P. infestans inoculation. SA signaling pathway involved in BABA-IR in potato against P. infestans.

Key words: Solanum tuberosum , β-aminobutyric acid induced resistance , late blight , histochemical assays , SA signaling pathway

王静, 王海霞, 田振东. β-氨基丁酸诱导马铃薯对晚疫病的抗性组织化学及信号传导途径分析[J]. 中国农业科学, 2014, 47(13): 2571-2579.

WANG Jing, WANG Hai-Xia, TIAN Zhen-Dong. Histochemical Assays and Signaling Pathway Analysis of β-Aminobutyric Acid Induced Resistance in Potato Against Phytophthora infestans[J]. Scientia Agricultura Sinica, 2014, 47(13): 2571-2579.

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