油菜BN5和BN10基因的诱导表达和病毒抗性鉴定及拟南芥遗传转化
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摘要
油菜是我国重要的油料作物。病毒病是危害油菜的主要病害,芜菁花叶病毒(TuMV)、黄瓜花叶病毒(CMV)、油菜花叶病毒(YOMV)和烟草花叶病毒(TMV)是油菜病毒病的主要毒源。在油菜生产上,种植抗病品种是防治病毒病的有效措施,但目前尚缺乏对病毒病高抗或免疫的油菜品种,并且寄主对病毒病抗性的分子机制研究相对滞后。
本研究利用基于病害防御反应信号分子诱导处理的油菜叶片制备的UniGene库和对应的油菜cDNA芯片,通过基因表达谱分析和序列分析,结合参阅相关文献,挑选出与抗病抗逆相关的油菜BN5基因和BN1O基因；利用农杆菌介导的泡花法(Floral-dip)将油菜BN5和BN10基因转化到Columbia野生型拟南芥；利用荧光定量PCR检测BN5和BN10基因在油菜不同组织中的表达差异,以及病毒和化学处理对该基因的诱导表达。取得的主要研究结果如下：
1.YOMV、TuMV和CMV三种病毒在油菜感病品种华杂6号上活化后的病状表现基本一致,表现为花叶、斑驳、疱斑、明脉和皱缩等症状。病毒接种鉴定结果显示,转BN5基因RNAi载体的转基因油菜(pRNAi-BN5)比非转基因对照(苏油1号)对病毒更敏感。在三种油菜病毒中,转基因油菜对CMV最为敏感,接种CMV后发病最重,其次是YOMV和T'uMV。
2.pRNAi-BN5转基因油菜中BN5基因荧光定量PCR分析表明：BN5基因的表达受到明显抑制,表达量仅为非转基因对照的22%-45%。BN5基因在转基因和非转基因油菜根、茎、叶中均有表达,其中在茎的表达量最高,叶居中,根的表达量最低。油菜接种YoMV、CMV和TuMV,不管是转基因油菜还是非转基因油菜,三种病毒均能诱导BN5基因的上调表达,但不同时间点的表达量和上调表达幅度有所不同。三种病毒中,接种CMV时BN5的诱导表达量最高,且最先达到峰值,但之后迅速下降,降幅大于接种YOMV和TuMV时BN5的表达量。说明BN5基因对CMV的刺激最敏感,可能在油菜抗病毒过程中发挥了重要作用。
3.BN10基因在油菜根、茎、叶中均有表达,其中在根的表达量最高,茎居中,在叶中的表达量最低。用甲基茉莉酸(MeJA)和草酸(OA)处理油菜叶片,荧光定量PCR分析表明：MeJA诱导处理后BN10基因表达先升后降,在24h表达量最高(为诱导前的2.58倍),之后有所下降,但诱导后的表达量始终高于诱导前,72h时的表达量仍为诱导前的1.13倍。OA诱导处理后,BN10表达量也是先增加后降低,在12h达到最大值(为诱导前的2.5倍),72h下降至最低(仅为诱导前的25%)。研究结果显示,甲基茉莉酸和草酸均能诱导BN10的表达,推断BN10可能在油菜抗逆过程中具有一定的作用。
4.通过化学法(CaCl2法)将油菜BN5和BN10基因的RNAi载体和过表达载体,即pRNAi-BN5、pG4A-BN5、pRNAi-BN10和pG4A-BN10 4种质粒DNA成功转化农杆菌EHA105。通过除草剂筛选及PCR检测,分别获得含pRNAi-BN5、pG4A-BN5、pRNAi-BN10和pG4A-BN10质粒的T0代转基因拟南芥20株、10株、7株和10株。
Rapeseed (Brassica napus) is one of the most important oilseed crops in China. Virus disease is the main disease in oilseed rape. Turnip mosaic virus(TuMV), Cucumber mosaic virus(CMV), Youcai mosaic virus(YoMV) and Tobacco mosaic virus(TMV) are major virus that infecte oilseed rape. At present, the cultivation of resistant varieties is the best way to resist virus diseases. But we are lacking in highly resistant or immune varieties of rape and the molecular mechanism of resistance to virus is also lagging behind.
In this research, we selected two genes (named BN5 and BN10) form oilseed rape genome based on UniGene libraries and cDNA microarray induced by disease defense response signaling molecules on leaves. BN5 and BN10 genes were transformed into Arabidopsis thaliana by way of floral-dip mediated by Agrobacterium tumefaciens; The difference in expression of BN5 and BN10 genes in different tissues were detected by fluorescence quantitative PCR(Q-PCR). Viruses and chemical treatment were also analysed. The main results are as following:
1. The plants of Huaza No.6 which is a hypersensitive variety showd mosaic, mottle, blister spot, clear vein and shrinking symptoms infected by TuMV, CMV and YoMV. Majority of positive transgenic plants of pRNAi-BN5 were more susceptible to the viruses than the non-transgenic ones after inoculated with Cucumber mosaic virus (CMV), TuMV and YoMV viruses. Furthermore, transgenic plants were more susceptible to CMV than to YoMV and TuMV.
2. Gene expression profiles of transgenic plants transformed with BN5 RNA interference vector were detected by real-time quantitative PCR. The results showed that the expression level of BN5 gene was inhibited. The relative expression of BN5 gene in transgenic plants was only 22%-45% relative to the non-transgenic control. BN5 can be detected in roots, stems and leaves of transgenic plants and non-transgenic ones with the highest expression level in stems and the lowest in roots. TuMV, CMV and YoMV can induce up-regulated expression of BN5 gene in transgenic and non-transgenic plants although their degree is various. The expression of BN5 gene was highest and first reached the peak and then declined rapidly induced by CMV infection than by YoMV and TuMV. This result showed that BN5 gene was hypersensitive to CMV infection. BN5 gene may have important function in resistant to viruses.
3. BN10 can be detected in roots, stems and leaves in non-transgenic oilseed rape plants with the highest expression level in roots and the lowest in leaves. Methyl jasmonate (MeJA) and oxalic acid (OA) can induce up-regulated expression of BN10 gene in leaves analyzed by quantitative fluorescent PCR. The results suggested that BN10 gene expression up-regulated firstly and then decline induced by mathyl-jasmonate treated. BN10 had the highest expression level at 24h (2.58 times before the induction), downregulated to 72h (1.13 times before the induction). After oxalic acid induced, BN10 gene expression reached the maximum at 12h (2.5 times before the induction), dropped to the lowest at 72h (only 25% before the induction). These data suggested MeJA and OA could induce BN10 gene expression. It can be inferred tha BN10 may play a role in stress resistance pathway.
4. RNA interference vectors and plant over-expression vectors of BN5 and BN10 genes (namely pRNAi-BN5, pG4A-BN5, pRNAi-BN10 and pG4A-BN10) were transformed into Agrobacterium tumefaciens EHA105 by using CaCl2 treated method.20, 10,7 and 10 of TO generation of transgenic Arabidopsis thaliana plants of four vectors were obtained tested by herbicide screen and PCR test.
本研究利用基于病害防御反应信号分子诱导处理的油菜叶片制备的UniGene库和对应的油菜cDNA芯片,通过基因表达谱分析和序列分析,结合参阅相关文献,挑选出与抗病抗逆相关的油菜BN5基因和BN1O基因；利用农杆菌介导的泡花法(Floral-dip)将油菜BN5和BN10基因转化到Columbia野生型拟南芥；利用荧光定量PCR检测BN5和BN10基因在油菜不同组织中的表达差异,以及病毒和化学处理对该基因的诱导表达。取得的主要研究结果如下：
1.YOMV、TuMV和CMV三种病毒在油菜感病品种华杂6号上活化后的病状表现基本一致,表现为花叶、斑驳、疱斑、明脉和皱缩等症状。病毒接种鉴定结果显示,转BN5基因RNAi载体的转基因油菜(pRNAi-BN5)比非转基因对照(苏油1号)对病毒更敏感。在三种油菜病毒中,转基因油菜对CMV最为敏感,接种CMV后发病最重,其次是YOMV和T'uMV。
2.pRNAi-BN5转基因油菜中BN5基因荧光定量PCR分析表明：BN5基因的表达受到明显抑制,表达量仅为非转基因对照的22%-45%。BN5基因在转基因和非转基因油菜根、茎、叶中均有表达,其中在茎的表达量最高,叶居中,根的表达量最低。油菜接种YoMV、CMV和TuMV,不管是转基因油菜还是非转基因油菜,三种病毒均能诱导BN5基因的上调表达,但不同时间点的表达量和上调表达幅度有所不同。三种病毒中,接种CMV时BN5的诱导表达量最高,且最先达到峰值,但之后迅速下降,降幅大于接种YOMV和TuMV时BN5的表达量。说明BN5基因对CMV的刺激最敏感,可能在油菜抗病毒过程中发挥了重要作用。
3.BN10基因在油菜根、茎、叶中均有表达,其中在根的表达量最高,茎居中,在叶中的表达量最低。用甲基茉莉酸(MeJA)和草酸(OA)处理油菜叶片,荧光定量PCR分析表明：MeJA诱导处理后BN10基因表达先升后降,在24h表达量最高(为诱导前的2.58倍),之后有所下降,但诱导后的表达量始终高于诱导前,72h时的表达量仍为诱导前的1.13倍。OA诱导处理后,BN10表达量也是先增加后降低,在12h达到最大值(为诱导前的2.5倍),72h下降至最低(仅为诱导前的25%)。研究结果显示,甲基茉莉酸和草酸均能诱导BN10的表达,推断BN10可能在油菜抗逆过程中具有一定的作用。
4.通过化学法(CaCl2法)将油菜BN5和BN10基因的RNAi载体和过表达载体,即pRNAi-BN5、pG4A-BN5、pRNAi-BN10和pG4A-BN10 4种质粒DNA成功转化农杆菌EHA105。通过除草剂筛选及PCR检测,分别获得含pRNAi-BN5、pG4A-BN5、pRNAi-BN10和pG4A-BN10质粒的T0代转基因拟南芥20株、10株、7株和10株。
Rapeseed (Brassica napus) is one of the most important oilseed crops in China. Virus disease is the main disease in oilseed rape. Turnip mosaic virus(TuMV), Cucumber mosaic virus(CMV), Youcai mosaic virus(YoMV) and Tobacco mosaic virus(TMV) are major virus that infecte oilseed rape. At present, the cultivation of resistant varieties is the best way to resist virus diseases. But we are lacking in highly resistant or immune varieties of rape and the molecular mechanism of resistance to virus is also lagging behind.
In this research, we selected two genes (named BN5 and BN10) form oilseed rape genome based on UniGene libraries and cDNA microarray induced by disease defense response signaling molecules on leaves. BN5 and BN10 genes were transformed into Arabidopsis thaliana by way of floral-dip mediated by Agrobacterium tumefaciens; The difference in expression of BN5 and BN10 genes in different tissues were detected by fluorescence quantitative PCR(Q-PCR). Viruses and chemical treatment were also analysed. The main results are as following:
1. The plants of Huaza No.6 which is a hypersensitive variety showd mosaic, mottle, blister spot, clear vein and shrinking symptoms infected by TuMV, CMV and YoMV. Majority of positive transgenic plants of pRNAi-BN5 were more susceptible to the viruses than the non-transgenic ones after inoculated with Cucumber mosaic virus (CMV), TuMV and YoMV viruses. Furthermore, transgenic plants were more susceptible to CMV than to YoMV and TuMV.
2. Gene expression profiles of transgenic plants transformed with BN5 RNA interference vector were detected by real-time quantitative PCR. The results showed that the expression level of BN5 gene was inhibited. The relative expression of BN5 gene in transgenic plants was only 22%-45% relative to the non-transgenic control. BN5 can be detected in roots, stems and leaves of transgenic plants and non-transgenic ones with the highest expression level in stems and the lowest in roots. TuMV, CMV and YoMV can induce up-regulated expression of BN5 gene in transgenic and non-transgenic plants although their degree is various. The expression of BN5 gene was highest and first reached the peak and then declined rapidly induced by CMV infection than by YoMV and TuMV. This result showed that BN5 gene was hypersensitive to CMV infection. BN5 gene may have important function in resistant to viruses.
3. BN10 can be detected in roots, stems and leaves in non-transgenic oilseed rape plants with the highest expression level in roots and the lowest in leaves. Methyl jasmonate (MeJA) and oxalic acid (OA) can induce up-regulated expression of BN10 gene in leaves analyzed by quantitative fluorescent PCR. The results suggested that BN10 gene expression up-regulated firstly and then decline induced by mathyl-jasmonate treated. BN10 had the highest expression level at 24h (2.58 times before the induction), downregulated to 72h (1.13 times before the induction). After oxalic acid induced, BN10 gene expression reached the maximum at 12h (2.5 times before the induction), dropped to the lowest at 72h (only 25% before the induction). These data suggested MeJA and OA could induce BN10 gene expression. It can be inferred tha BN10 may play a role in stress resistance pathway.
4. RNA interference vectors and plant over-expression vectors of BN5 and BN10 genes (namely pRNAi-BN5, pG4A-BN5, pRNAi-BN10 and pG4A-BN10) were transformed into Agrobacterium tumefaciens EHA105 by using CaCl2 treated method.20, 10,7 and 10 of TO generation of transgenic Arabidopsis thaliana plants of four vectors were obtained tested by herbicide screen and PCR test.
引文
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