摘要
该研究结合蔷薇科数据库,经多序列比对、表达分析和验证,筛选了苹果和梨抗病反应Marker基因及其检测引物,并以腐烂病抗性资源杜梨和东北山荆子悬浮细胞为材料,经20%腐烂病菌(Vp)代谢物处理,采用实时荧光定量PCR(RT-qPCR)分析Marker基因对腐烂病信号响应的表达模式,为苹果、梨抗病反应的快速检测和杜梨抗腐烂病机理的系统研究奠定基础。结果显示:(1)经检索比对共筛选出水杨酸(SA)、茉莉酸(JA)、乙烯(ET)、系统获得型抗性(SAR)反应、病原相关分子模式激发的免疫反应(PTI)、植保素和防御相关等信号的15个Marker基因及其对应引物。(2)各处理cDNA浓度采用100 ng·μL^(-1)时Cq值均介于18~25,且PCR扩增效率高,表明所选的15个Marker基因的引物均可准确反映抗性反应的激活状况,可作为各自信号通路的Marker基因。(3)与对照相比,Vp代谢物处理后杜梨悬浮细胞中ROS的积累随处理时间的延长呈逐渐显著上升的趋势,并于处理6 h时,ROS信号值达到对照的3.2倍,表明Vp代谢物会激活体内的免疫反应(PTI),进而导致植物体内ROS的爆发。(4)RT-qPCR验证分析显示,Vp代谢物处理后,杜梨悬浮细胞中SA、JA、PTI、SAR、R基因、植保素、防御相关等信号通路的Marker基因都呈上调表达趋势,其中,SA、JA和SAR通路的Marker基因ChiV(Chitinase class V)、LOX1(Lipoxygenase 1)和PR4(Pathogenesis-Related 4)及防御相关基因PAL1(Phe Ammonia Lyase 1)的表达上调最为明显,最高分别可上升至对照的1718、691、6369和1072倍,表明杜梨受到Vp代谢物胁迫时,主要激活了植物体内的SA、JA、SAR和防御相关信号通路的基因,进而能够抵御病原的进一步入侵。研究发现,SA、JA、SAR和防御反应等信号参与了杜梨对腐烂病胁迫的响应,进而提高其抗病性。
In this study,combined with Rosaceae database,through multiple sequence alignment,expression analysis and validation,we screened the defense-related Marker genes and corresponding primers in the genome of apple(Malus domestica)and pear(Pyrus bretschneideri).We took the suspension cells of Duli and DongbeiShanjingzi as materials,treated with 20%metabolites of Valsa canker(Vp),and analyzed the expression patterns of marker genes in response to Vp signal by real-time fluorescent quantitative PCR(RT qPCR),which lays a foundation for the rapid detection of disease resistance reaction of apple and pear and systematic study of resistance mechanism to Vp of Duli.The results showed that:(1)a total of 15 Marker genes and corresponding primers for salicylic acid (SA), jasmonic acid (JA), ethylene (ET), systemic acquired resistance (SAR) response, pathogen-associated molecular pattern-primed immune re-sponse (PTI), phycocyanin and defense-related signals were screened by search and comparison. (2) The Cq values ranged from 18 to 25 at the cDNA concentration of 100 ng·μL^(-1) for each treatment, and the PCR amplification efficiency was high, which indicated that the primers of the 15 Marker genes could accu-rately reflect the activation of the resistance response and could be used as Marker genes for the respective signaling pathways. (3) Compared with control, the accumulation of ROS in suspension cells of Duli after Vp metabolite treatment showed a significant increase with the treatment time, and the value of ROS sig-nal reached 3.2 times of the control at 6 h, show that Vp metabolite activates the plant immune response (PTI), which induce the burst of ROS. (4) RT qPCR analysis showed that Marker genes of SA, JA, PTI, SAR, R-gene, phytogenesis and defense-related signaling pathways were up-regulated in Duli sus-pension cells after Vp metabolite treatment, among which, expression of SA, JA and SAR related genes ChiV (Chitinase class V), LOX1 (Lipoxygenase 1), PR4 (Pathogenesis-Related 4), and defense response related gene
作者
孙娥
闫文萍
余宏强
赵丹
朵虎
左存武
SUN E;YAN Wenping;YU Hongqiang;ZHAO Dan;DUO Hu;ZUO Cunwu*(College of Horticulture,Gansu Agricultural University,Lanzhou 730070,China;Forestry and Grassland Bureau,Linze County,Zhangye Gansu 734200,China)
出处
《西北植物学报》
CAS
CSCD
北大核心
2022年第9期1468-1476,共9页
Acta Botanica Boreali-Occidentalia Sinica
基金
国家自然科学基金(31860545)
甘肃省教育厅产业支撑引导项目。
关键词
腐烂病
Marker基因
植物免疫
差异表达
Valsa canker
Marker gene
plant immune response
differential expression
