拟南芥酵母双杂交文库构建及ADPG1互作蛋白筛选

Construction of Yeast Two-Hybrid Library of Arabidopsis thaliana and Screening of ADPG1 Interacting Proteins

纤维素酶、半纤维素酶和果胶酶在植物果实发育和成熟过程中起着重要的作用。拟南芥开裂区多聚半乳糖醛酸酶1(ARABIDOPSIS DEHISCENCE ZONE POLYGALACTURONASE 1,ADPG1)是拟南芥角果开裂所必需的果胶酶,ADPG1在木质部的异位表达可以诱导拟南芥ccr1突变体病程相关(pathogenesis-related,PR)基因的表达。但ADPG1是如何促进ccr1释放激发子并诱导病害防御基因表达的尚不清楚。通过筛选其互作的蛋白,可进一步探究ADPG1参与ccr1突变体激发子释放和诱导防御基因PR1表达的作用机制。该文以拟南芥野生型和突变体ccr1为材料,通过提取总RNA,分离mRNA,合成双链cDNA,依次构建酵母双杂交初级和次级文库,构建诱饵载体pGBKT7-AtADPG1,进行自激活检测,与构建的次级文库共转化酵母感受态细胞,筛选到1个与ADPG1相互作用的候选蛋白AtGRP5,为进一步研究其参与植物诱导防御反应过程奠定了基础。

Cellulase,hemicellulase and pectinase play critical roles in the fruit development and ripening.ADPG1 (ARABIDOPSIS DEHISCENCE ZONE POLYGALACTURONASE 1) is an essential pectinase for silique dehiscence.In addition,the ectopic expression of ADPG1 in xylem can induce the expression of PR (pathogenesis-related) gene in Arabidopsis ccr1 mutant.However,the mechanism of?how ADPG1 promotes the release of elicitors in ccr1 mutant and induces the expression of defense-related gene is still unclear.To explore the role of ADPG1 involving in the release of elicitors and the induction of defense gene PR1 in ccr1 mutants,the interacting proteins with ADPG1 were screened.In this paper,the authors extracted the total RNA from wild-type and ccr1 Arabidopsis.Then,the extracted RNA was used to isolate mRNA and synthesize double-stranded cDNA.The primary and secondary libraries of yeast two-hybrid were constructed successively.The bait vector pGBKT7-AtADPG1 was constructed and tested for its self-activation.The pGBKT7-AtADPG1 was co-transformed into yeast competent cells with the secondary library,and a candidate protein named AtGRP5 that interacting with ADPG1 was identified.This study provided a basis for further study on the involvement of ADPG1 in plant defense response.