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白菜型冬油菜抗寒生理响应与抗氧化酶基因表达分析
Anti-cold Physiology Response And Antioxidant Enzyme Genes Expression on Winter Turnip Rape (Brassica Campestris L.)
【作者】 曾秀存；
【作者基本信息】 甘肃农业大学 ， 作物遗传育种， 2015， 博士
【摘要】 白菜型冬油菜是我国西北寒旱区主要的油料生态作物,但该地区冬季酷寒干燥,极端低温天气频发,使其不能安全越冬。本研究以超强抗寒冬油菜(陇油6号、陇油7号)、强抗寒品种(陇油8号、陇油9号)和耐寒品种(天油4号、天油7号)为试材,利用植物生理生化手段和分子生物学技术(基因克隆、实时荧光定量PCR)分析冬油菜冬前光合特性和越冬期根部生理生化对低温的响应;并克隆和实时荧光定量PCR检测抗氧化酶基因,分析其在低温胁迫下的表达变化。将生理生化与分子生物学研究结果相结合,揭示白菜型冬油菜抗寒机制,为冬油菜抗寒品种选育提供理论依据。主要结果如下:1.冬前低温胁迫后,气孔导度(Gs)、光合速率(Pn)、气孔限制值(Ls)、蒸腾速率(Tr)、羧化效率(CE)、光能利用效率(LUE)和水分利用效率(WUE)均下降,而胞间二氧化碳浓度(Ci)和胞间二氧化碳浓度/大气中二氧化碳浓度(Ci/Ca)上升,冬油菜Pn下降主要由非气孔限制所致。Pn、Gs、LUE和CE在品种间均表现为:耐寒品种(天油4号、天油7号)>抗寒品种(陇油9号)>强抗寒品种(陇油8号)>超强抗寒品种(陇油6号、陇油7号);Ci和Ci/Ca的升幅为:超强抗寒品种>强抗寒品种>抗寒品种>耐寒品种;WUE在品种间存在较大差异。2.冬油菜Pn日变化呈不规则的“单峰”曲线,光合进程平稳,Pn在12:00-14:00时达到峰值;Tr、CE和Ls日变化呈“单峰”曲线;LUE日变化呈“V”型曲线;Ci和Gs在品种间存在较大差异。3.低温胁迫后,冬油菜PSⅡ最大光化学效率(Fv/Fm)、PSⅡ潜在光化学活性(Fv/Fo)和PSⅡ的电子传递(Fm/Fo)明显下降,超强抗寒品种降幅最大、光抑制最强;冬油菜初始荧光产量(Fo)和最大荧光产量(Fm)呈“下降-上升-下降”的变化;稳态荧光产量(Fs)、光适应下最大荧光(Fm’)、PSⅡ实际光化学效率(φPSⅡ)和PSII电子传递速率(ETR)均下降,超强抗寒品种降幅最大;非光化学淬灭(NPQ)升高,而耐寒品种(天油4号、天油7号)的升幅最小。4.低温使冬油菜叶绿素荧光启动过程中各参数发生明显变化,超强抗寒品种启动最快,PSⅡ电子传递活性最强,光抑制程度最小,而耐寒品种热耗散最大。5.低温胁迫后,超强抗寒和强抗寒品种根部MDA含量呈“上升-下降”的趋势,SS含量为“上升-下降-上升”趋势,SP含量下降,超强抗寒品种根部MDA含量最低,SS和SP含量均为超强抗寒品种(陇油6号、陇油7号)>抗寒品种(陇油8号、陇油9号)>耐寒品种(天油4号、天油7号)。6.低温胁迫后,冬油菜根部T-SOD、Cu/Zn-SOD、CAT、APX和GR活性增强,而POD活性减弱,超强抗寒品种中的各种酶活性最强,T-AOC能力为超强抗寒品种(陇油6号、陇油7号)>抗寒品种(陇油8号、陇油9号)>耐寒品种(天油4号、天油7号)。7.主成分分析表明Pn和CE是反映白菜型冬油菜低温响应的主要因素,CE和WUE是反映冬油菜光合日变化的主要指标;暗适应下的Fv/Fm、Fv/Fo和Fm/Fo与光适应下的φPSⅡ和Fs是冬油菜叶绿素荧光响应低温的主要指标;Fs和φPSII是影响冬油菜叶绿素荧光启动的主要指标;APX、Cu/Zn-SOD和GR活性是影响冬油菜根部抗寒能力的主要因素;超强抗寒品种光合能力和叶绿素荧光参数低温响应最弱,但叶绿素荧光启动和根部抗寒能力综合评价为:超强抗寒品种(陇油6号、陇油7号)>抗寒品种(陇油8号、陇油9号)>耐寒品种(天油4号、天油7号)。8.从陇油7号中克隆到APX、Cu/Zn-SOD和Fe-SOD,实时荧光定量PCR分析为,低温下APX和Cu/Zn-SOD的表达量和酶活性均提高,且超强抗寒品种高于耐寒品种,APX和Cu/Zn-SOD是低温诱导表达基因;Fe-SOD半定量和相对定量分析,在4℃时,Fe-SOD上调表达,在-4℃和-8℃时,其表达量急剧下降,且耐寒品种的表达量高于超强抗寒性品种;根部总SOD酶活性高于叶片,超强抗寒品种总SOD高于耐寒品种。
【Abstract】 Winter turnip rape(Brassica campestris L.) is a valuable ecologically beneficial oil crop in cold and arid region of northwestern China. However, coldness and extremely low temperature can make winter turnip rape fail to overwinter and as a result limit its production.In this study, by applying technology of plant physiology and biochemistry,combined with gene cloning and Real-time quantitative PCR, we analyzed effect of the low temperature stress on leaf photosynthetic characteristics, root physiological and biochemical responses on winter turnip rape, and carried out the expression analysis of genes related to cold-resistance in response to coldness stress. The aim of this study is to explore the mechanism of cold resistance and lay a foundation for resistance breeding of winter turnip rape. The specific findings are as follows:1. Diurnal variation of Pn, Tr, CE and Ls were one-peak curve, which reached peak between 12:00 and 14:00, but Ci, Gs and LUE showed “V” curve,2. With decreases of temperature, Fv/Fm, Fv/Fo and Fm/Fo of winter rape obviously decreased, but NPQ rised. The super cold-tolerance winter rape(Longyou 7and Longyou 6) showed the largest decline and the strongest photoinhibition. The Fo and Fm showed the tendency of “drop-rise-drop”. Fs, Fm’, φPSⅡand ETR showed the tendency of drop and The super cold-tolerance winter rape(Longyou 7 and Longyou 6)appeared to decline most steeply.3. During the chlorophyll fluorescence startup, chlorophyll fluorescence parameters had seen significant change under the lower temperature. The fastest startup of chlorophyll fluorescence was super cold-tolerance winter rape, which had the minimum photoinhibition, but poor cold-tolerance winter rape showed the maximum heat dissipation.4. With decreases of temperature, MDA content of super cold-tolerance and cold-tolerance winter rape showed the tendency of “rise-drop”, SS content showed the tendency of “rise-drop-rise”, SP content showed the tendency of drop. The root MDA content of super cold-tolerance winter rape was the lowest. SP and SS content were super cold-tolerance rape>cold-tolerance rape>poor cold-tolerance rape.5. With decreases of temperature, root activity of T-SOD、Cu/Zn-SOD、CAT、APX and GR increased, but POD activity decreased. The enzymatic activity was the strongest.T-AOC showed super cold-tolerance winter rape > cold-tolerance winter rape>poor cold-tolerance winter rape.6. Principal component analysis showed WUE, CE and Pn were the key factors which had an effect on photosynthetic capacity of winter rape. Fv/Fm, Fv/Fo, Fm/Fo,φPSⅡ and Fs were the key factors which had an effect on chlorophyll fluorescence of winter rape. Fs andφPSIIR were the key factors which had an effect on the chlorophyll fluorescence startup. APX, Cu/Zn-SOD and GR activity were the key factors which had an effect on root cold-resistant capability of winter rape. Composite scores for photosynthetic capacity and chlorophyll fluorescence parameters was super cold-tolerance winter rape<cold-tolerance winter rape<poor cold-tolerance winter rape. Composite scores for chlorophyll fluorescence startup and root cold-resistant capability of winter rape was super cold-tolerance winter rape>cold-tolerance winter rape>poor cold-tolerance winter rape.7. The cDNA sequence of APX, Cu/Zn-SOD and Fe-SOD were isolated from Longyou7 by RT-PCR. Semi-quantitative and real time RT-PCR were used to assess the genes expression in response to lower temperature stress. The APX and Cu/Zn-SOD could be induced and enzymatic activity of could be changed by lower temperature and the two genes was a differential expressed gene induced by lower temperature. Fe-SOD was expressed upregulatedly in response to early low temperature stress(4℃). However, the expression of Fe-SOD was inhibited at-4℃and-8℃. SOD activity in roots was higher than that of leaves and super cold-tolerance winter rape was higher than that of poor cold-tolerance winter rape.