etr1-1在矮牵牛中诱导表达可提高叶片对灰霉病的抗性

Induced etr1-1 Expression in Petunias is Responsible for Its Tolerance to Botrytis cinerea

【目的】研究etr1-1诱导表达对矮牵牛抗灰霉病的作用。【方法】以转GVG:etr1-1矮牵牛为材料,地塞米松(DEX)处理,接种灰霉病菌,通过调查发病程度和定量半定量基因表达分析揭示etr1-1在矮牵牛抗灰霉病中的作用。【结果】与对照叶片相比,DEX处理的矮牵牛叶片表现出延缓衰老和减轻病害的症状。接种病原菌后,DEX处理的叶片发病率增加比例为0,对照叶片为66.77%;DEX处理的叶片平均病斑扩展速率为4.69 mm·d-1,对照叶片为6.29 mm·d-1。qRT-PCR和semi-qRT-PCR基因表达分析表明,DEX处理的叶片etr1-1与Bcact表达呈相反趋势。在DEX处理叶片中,DEX诱导了叶片etr1-1表达,随着诱导时间延长,基因相对表达量上升,诱导后第3天达到最大,无菌水接种叶片的表达量是病原菌接种叶片的10.71倍。DEX处理的叶片,Bcact在接种后第1天表达量最大;对照叶片,Bcact在接种后第2天表达量最大;其中处理叶片Bcact的最大表达量比对照叶片降低了20倍。在DEX处理的叶片上随着etr1-1表达增强,Bcact表达量下降。DEX处理的叶片,CP10仅在接种后第3天轻微表达;对照叶片上,随接种时间延长,CP10表达量上升,接种后第3天达到最大,处理最大表达量比对照降低了23.6倍。随接种时间延长,DEX处理的叶片和对照叶片,ACO表达均先升高后降低,在第2天达到最大,处理叶片上最大表达量仅为6.75,比对照低7.33倍。DEX处理的叶片,乙烯途径基因ETR2、ERS1、EIL1和EIN2表达受到抑制。ERS1和ETR2在DEX处理和对照叶片上均有表达,并在接种后第3天达到最大,但处理的最大表达量比对照低51倍。DEX处理的叶片,接种后第1—2天EIN2表达被抑制,第3天表达上升,在对照叶片上,EIN2表达量随接种时间而增加,第3天达到最大,DEX处理的叶片EIN2最大表达量比对照低31.58倍;DEX处理的叶片和对照叶片,EIL1与EIN2有相似的表达趋势,且DEX处理的叶片EIL1最大表达量低于对照两倍。AOC和COI1在JA生物合成和信号转导途径中起重要作用。DEX处理的叶片,AOC和COI1表达均被减弱,AOC在接种后第1—2天被抑制,第3天表达,对照叶片上,随接种时间延长AOC表达量增加,在第3天达到最大,处理叶片AOC最大表达量比对照低12.96倍;COI1在DEX处理和对照叶片上均有表达,且在第3天表达量最大,DEX处理叶片COI1最大表达量比对照低6.14倍。病原菌侵染植物时,ERFs会被乙烯或JA调控以激活病程相关基因表达。经DEX处理的叶片ERF4表达先升高后降低,第2天表达量最大;对照叶片随接种时间延长ERF4表达量上升,在第3天达到最大;处理最大表达量比对照低2.96倍。无论处理还是对照,ERF8表达量都随接种时间延长而上升,在第3天达到最大,但处理的最大表达量比对照低3.52倍。接种病原菌后,DEX处理的叶片上,所有病程相关基因表达微弱,最大表达量均低于10;而对照叶片上,所有病程相关基因均有表达,并在接种后第3天表达量最大。其中以PR1表达量最大为10 521.11,其次是EX-CHI和OSM,分别为184.95和184.96,Defense1和AC-CHI表达量最小为23.39和14.58。【结论】etr1-1诱导表达通过延缓灰霉病菌引起的叶片衰老,从而提高转基因矮牵牛对灰霉病的抗性。

[Objective]The objective of this study is to clarify the effect of induced etr1-1 expression on petunia response to infection by Botrytis cinerea, the causal agent of gray mold disease. [Method]Detached leaves of GVG：etr1-1 transgenic petunias treated by DEX were inoculated with B. cinerea, then the regulation mechanism of etr1-1 was investigated by observing disease symptoms, measuring disease severity and analyzing gene expression by quantitative RT-PCR and semi-quantitative RT-PCR. [Result]Induced etr1-1 expression by dexamethasone resulted in the retarded senescence and reduced disease symptoms on detached leaves. During the period of inoculation, the percentage of increased disease incidence was 0 on DEX-treated leaves, whereas 66.77%on control leaves. The growth rate of the leaf lesions on DEX-treated leaves was 4.69 mm＆#183;d-1, whereas 6.29 mm＆#183;d-1 on control leaves. qRT-PCR and semi-qRT-PCR provided the following results. The extent of decreased disease incidence was negatively correlated with the etr1-1 expression. Following prolonged induction time, the level of etr1-1 expression was enhanced on DEX-treated leaves and obtained the peak at 3rd day after DEX treatment. 10.71-fold changes were increased on mock-inoculating leaves than that on B. cinerea-inoculating leaves. The maximum expression of Bcact was obtained at 1 dpi on DEX-treated leaves, whereas, at 2 dpi on control leaves. The maximum level of Bcact expression was 20-fold lower on DEX-treated leaves than that on control leaves. Infection of B. cinerea activated CP10 expression and caused the senescence symptom. In comparison, 23.6-fold changes were decreased on DEX-treated leaves than on control leaves. The expression of ACO exhibited a decline after an up on both DEX-treated and control leaves. The peak of expression level was observed at 2 dpi. The peak of expression of ACO was only 6.75 on DEX-treated leaves, which suggested 7.33-fold lower than that on control leaves. On DEX-treated leaves with induced etr1-1 expression, repressed genes expressions in ethylene/JA pathway decreased ERF4 and ERF8 expression and consequently reduced pathogenesis-related gene expression. The expressions of ERS1 and ETR2 were observed and the peak of expression came out at 3 dpi on both DEX-treated and control leaves, however, 51-fold lower expression was observed on DEX-treated leaves than that on control leaves. The expression was suppressed at 1 and 2 dpi, and activated at 3 dpi on DEX-treated leaves. Following the prolonged inoculation time, the level of EIN2 expression was enhanced on control leaves. It was found that the maximum expression on DEX-treated leaves was 31.58-fold lower than that on control leaves. The expression of EIL1 showed the similar patterns as that of EIN2. It was observed that 2-fold lower expression of EIN2 on DEX-treated leaves than that on control leaves. AOC and COI1 played important roles in the biosynthesis and signal pathway of jasmonic acid. The expression of AOC and COI1 was repressed at 1-2 dpi and activated at 3 dpi on DEX-treated leaves, however, the expression level was increased following the prolonged inoculation time on control leaves. It was found that 12.96-fold lower expression on DEX-treated leaves than on control leaves. The expression of COI1 was discovered and obtained the peak at 3 dpi, regardless of DEX-treated leaves and control leaves. It was observed that 6.14-fold lower expression of COI1 on DEX-treated leaves than on control leaves. The expression of ethylene response factors （ERFs）, as downstream components, integrated the ethylene and JA signaling pathway. The expression of ERF4 showed up before a decrease pattern on DEX-treated leaves. The expression level of ERF4 showed rising patterns following inoculation. The expressions of ERF8 displayed ascend patterns following the prolonged inoculation time, regardless of DEX-treated and control leaves. It was found that 2.96-and 3.52-fold lower expression of ERF4 and ERF8 on DEX-treated leaves than on control leaves. Little expression of pathogenesis-related genes was monitored on DEX-treated leaves and the peak of all genes was lower than 10, whereas, the expression was enhanced on control leaves, such as, the maximum level of expression of PR1, EX-CHI, OSM, Defense1 and AC-CHI was 10 521.11, 184.95, 184.96, 23.39 and 14.58, respectively.[Conclusion]Induced etr1-1 expression delayed the senescence of leaves of petunia caused by B. cinerea and was consequently responsible for tolerance of GVG：etr1-1 transgenic petunias to B. cinerea.