农杆菌介导瞬时沉默小麦HCP1的研究

Transient Silencing of HCP1 Gene in Wheat Mediated by Agrobacterium

【目的】建立农杆菌介导的瞬时沉默小麦HCP1的方法,为简便、快速、高效地研究小麦基因功能提供一种可靠的技术支持。研究HCP1在叶锈菌侵染过程中对H2O2的清除作用,为阐明小麦抗叶锈菌侵染中信号转导机制奠定理论基础。【方法】采用农杆菌注射法,将携带有pe GFP载体的农杆菌注射到小麦叶片中,观察叶片中荧光强度及分布,分析外源质粒在小麦叶片中的表达情况。通过注射EHA105、LBA4404和C58C1 3种不同的农杆菌菌株,将HCP1的RNAi载体导入小麦叶片细胞中,利用半定量RT-PCR和实时荧光定量PCR检测HCP1瞬时沉默效率,并结合Rohringer染色分析,明确不同农杆菌对寄主叶片侵染能力的强弱,从而筛选出最适的试验菌株。在瞬时沉默的小麦叶片上,接种不亲和叶锈菌小种260,利用DAB染色,观察接种叶锈菌后叶片中H2O2的变化,分析HCP1在小麦与叶锈菌互作过程中的功能。【结果】叶片GFP荧光观察结果说明,在注射农杆菌48 h后GFP开始出现较高表达。RT-PCR和q RT-PCR结果表明,注射C58C1菌株的叶片12 h后,HCP1的表达量开始降低,并持续保持较低的表达量。注射EHA105菌株的叶片,在注射后72—96 h,HCP1的表达量迅速降低。而注射LBA4404菌株的叶片中HCP1的表达量基本没有变化。所以在3种不同农杆菌菌株介导的HCP1 RNAi转化中,沉默效率依次为:C58C1≥EHA105>LBA4404。注射不同农杆菌菌株叶片的Rohringer染色结果发现,EHA105侵染后引起的HR反应较其他两种菌株明显,说明EHA105对小麦叶片具有较高毒性,会引起叶片产生防卫反应,C58C1菌株在具有较高的沉默效率同时,对叶片的毒性较小,因此选取C58C1作为最适的试验菌株;沉默HCP1的小麦叶片接种叶锈菌小种260后,通过DAB染色发现,接种24 h和48 h后叶锈菌侵染诱发的小麦叶片H2O2的分布及积累量均多于对照组。【结论】建立了农杆菌介导的瞬时基因沉默体系,选用C58C1农杆菌菌株作为试验菌株,在处理后72 h可达到沉默目的基因的效果。确定HCP1参与了小麦与叶锈菌互作过程中H2O2的清除途径。

【Objective】Through transient silencing of the wheat HCP1 gene mediated by Agrobacterium, a simple, rapid, high efficient and reliable transgenic technique in wheat to identify the gene functions can be developed. The role of HCP1 gene in wheat in scavenging H2O2 that induced by inoculating Puccinia triticina was investigated using the transgenic plants overexpressing this target gene. The purpose of this study was to provide a theoretical basis for further understanding the signal transduction mechanism initiated by Puccinia triticina in wheat. 【Method】The Agrobacterium cells carrying the pe GFP vector were cocultured into the wheat leaves, the explants for wheat transformation. The intensity and distribution of the fluorescence signal in leaves mediated by Agrobacterium strains of EHA105, LBA4404 and C58C1 were detected and the expressions of exogenous gene in leaves were examined. The transient silencing efficiency of HCP1 mediated by RNAi in the transgenic plants was detected by semi-quantitative PCR and real-time quantitative PCR as well as the Rohringer staining analysis. RT-PCR and q RT-PCR analyses were also performed to determine the expression of HCP1 after 12 h inoculation in strain C58C1, and after 72-96 h coculture in strains EHA105 and LBA4404. The changes of H2O2 stained by DAB were observed and the function of HCP1 was investigated in the transgenic leaves with transient silencing of the HCP1 gene inoculated race 260, an incompatible P. triticina strain.【Result】The GFP fluorescence signals were strongly detected at 48 h after inoculation of Agrobacterium strains with the explants. Three Agrobacterium strains all efficiently mediated the HCP1-RNAi transformation, with the strongest silencing efficiency by strain C58C1, followed by strains EHA105 and LBA4404. Rohringer staining suggested that strain EHA105 had a high efficiency to mediate the RNAi effects of HCP1, indicating that this strain had a high toxicity to cause the defensive reaction in cells. By contrast, strain C58C1 had higher silencing efficiency and less toxicity than strain EHA105, indicating that C58C1 could be used as the Agrobacterium strain to mediate wheat transformation. DAB staining revealed that H2O2 accumulated largely in wheat leaves with the transient silencing HCP1, showing comparable rate with the inoculation progress of P. triticina race 260. Thus, the accumulation of H2O2 was shown more amount in the transgenic leaves than the control group at times of 24 h and 48 h after inoculation of the leaf rust race.【Conclusion】Strain C58C1 inoculated with the binary plasmids for 72 h could transform wheat leaves with high efficiency. Transgenic analysis confirmed that HCP1 gene is involved in scavenging H2O2 when wheat inoculated with P. triticina strain.