摘要
为明确小麦组织蛋白酶B基因TaCTSB在小麦与条锈菌互作中的功能,采用PCR技术扩增获得TaCTSB基因的完整编码区序列;通过qRT-PCR技术检测TaCTSB基因的表达情况;利用农杆菌介导瞬时表达体系在烟草叶片上进行亚细胞定位,并验证其是否能够诱导细胞坏死;利用VIGS技术瞬时沉默TaCTSB,解析其对小麦与条锈菌互作的影响。结果表明,TaCTSB基因编码344个氨基酸,N端含有1~19 aa的信号肽,无跨膜结构;qRT-PCR结果显示,TaCTSB基因在小麦与条锈菌非亲和互作早期上调表达。瞬时表达分析发现,烟草叶片上瞬时表达TaCTSB基因能够诱导细胞坏死,且质壁分离后TaCTSB分泌到细胞外。在TaCTSB瞬时沉默植株上接种条锈菌毒性小种CYR31,产孢量变化不显著;而接种条锈菌无毒性小种CYR23,产孢量升高;组织细胞学观察发现,在小麦叶片中瞬时沉默TaCTSB,其活性氧面积和坏死面积显著下降,菌丝面积及菌丝长度均有所增加,表明沉默TaCTSB减弱了小麦对条锈菌的抗性。
In order to analyze the function of wheat cathepsin B gene TaCTSB in the interaction between wheat and Puccinia striiformis(Pst),we used PCR technology to amplify the full coding region sequence of TaCTSB gene.The expression of TaCTSB gene was determined by qRT-PCR technology,and the transient expression system mediated by agrobacterium was used to verify whether the TaCTSB can inhibit or induce cell necrosis in tobacco.TaCTSB gene was constructed into pCAMBIA1302 vector for transient expression of TaCTSB on Nicotiana benthamiana for subcellular localization.BSMV mediated VIGS technology was applied to silence TaCTSB during wheat-Pst interaction to assess the role of TaCTSB in wheat resistance to Pst.The results showed that TaCTSB gene encodes 344 amino acids,containing a N-terminal signal peptide(1-19 aa),and has no transmembrane structure;qRT-PCR results showed that TaCTSB gene is up-regulated in the early stage of wheat-Pst incompatible interaction.Transient expression of TaCTSB in N.benthamiana induced cell necrosis,and subcellular localization in tobacco found that TaCTSB was secreted out of the cell.Transient silencing of TaCTSB in wheat did not cause significant phenotype change in wheat infected by the virulent Pst race CYR31,by contrast silencing of TaCTSB repaired resistance of wheat to the avirulent Pst CYR23 with decreased reactive oxygen species accumulation and cell death occurrence,and increased infection hyphae length and infection area.These results suggested the positive role of TaCTSB in regulating wheat resistance to stripe rust fungus.
作者
蒋军
岳明星
於立刚
康振生
王晓杰
汤春蕾
JIANG Jun;YUE Mingxing;YU Ligang;KANG Zhensheng;WANG Xiaojie;TANG Chunlei(College of Plant Protection/Sate Key Laboratory of Crop Stress Biology for Arid Areas,Northwest A&F University,Yangling,Shaanxi 712100,China)
出处
《麦类作物学报》
CAS
CSCD
北大核心
2022年第7期781-789,共9页
Journal of Triticeae Crops
基金
陕西省科技创新团队项目(2018TD-004)。
