Genetic Transformation And Functional Analysis Of Cucumis Sativus Translationally Controlled Tumor Protein Genes CsTCTP1 And CsTCTP2

Cucumber(Cucumis sativus)is one of the main facility vegetables in China.It has been continuously affected by pathogenic bacteria such as powdery mildew [Sphaerotheca fuliginea(Schlecht)Poll.] and resulted a large-scale reduction in production in production.Previous studies in this laboratory have shown that translationally controlled tumor protein(TCTP)may negatively regulate cucumber resistance to powdery mildew.In this study,firstly,Cs TCTP1/Cs TCTP2 gene restorer line plants and overexpression line plants were obtained by Cs TCTP1 and Cs TCTP2 genes heterologously transformed into Arabidopsis thaliana.Secondly,the phenotypes of Arabidopsis thaliana in different lines were analyzed to investigate the effects of Cs TCTP1/Cs TCTP2 genes on growth and development of plants.At the same time,the roles of Cs TCTP1/Cs TCTP2 genes in the response of Arabidopsis to Golovinomyces cichoracearum infection were studied by inoculating with powdery mildew to different strains Arabidopsis,and the signaling pathway of Cs TCTP1/Cs TCTP2 genes involved in Arabidopsis’ s resistance to powdery mildew were preliminarily explored.This study will provide useful evidence for further research on the role and mechanism of cucumber Cs TCTP in the balanced regulation of plant growth and development and immune response.The main findings are as follows:(1)The constructed Cs TCTP1/Cs TCTP2 overexpression vector were heterologously transformed into Arabidopsis thaliana through Agrobacterium tumefaciens,and homozygous overexpression strains were obtained through Basta herbicide screening and molecular detection,and Cs TCTP1/Cs TCTP2 transformation was selected The attctp1 / attctp2 mutant restores Arabidopsis thaliana and provides material for further verification of the biological functions of the Cs TCTP1 and Cs TCTP2 genes.(2)The effects of Cs TCTP1 and Cs TCTP2 genes on the growth and development of Arabidopsis were analyzed by observing the growth phenotype and growth indicators of different strains of Arabidopsis.The results showed that Cs TCTP1 and Cs TCTP2 genes can promote the germination,primary root elongation,rosette diameter and plant height of Arabidopsis thaliana,shorten the growth cycle,but have no effect on fruit pods.(3)Using RT-q PCR technology,the expression analysis of related endogenous genes in different strains of Arabidopsis thaliana was carried out,and the signal pathways involved in the regulation of Cs TCTP1/Cs TCTP2 were initially explored.Analyzed to the expression changes of the genes related to the Sn RK1 and TOR signaling pathways in Arabidopsis thaliana.The results showed that in attctp1,attctp2 mutants and WT,KIN10 did not change significantly,and both restorer and overexpressed lines showed upregulation.Therefore,the Cs TCTP1/Cs TCTP2 gene has a positive regulatory effect on KIN10 gene expression.KIN11 was the least expressed in the attctp1 mutant,and there was no significant difference between the attctp2 mutant and WT.The expression of KIN11 was significantly up-regulated in the restored and overexpressed lines,and the Cs TCTP1 and Cs TCTP2 genes positively regulated the expression of KIN11.At the same time,in Cs TCTP1 transgenic lines,the expressions of At RABA4 a,At RABA4 b,At RABF1 and At RABF2 b genes are down-regulated,especially the expression of At RABA4 a and At RABF2 b genes is down-regulated.The expression levels of TOR signal pathway element gene tor and downstream response gene S6K1 showed a downward regulation trend.In Cs TCTP2 transgenic lines,the expressions of At RABA4 a,At RABA4 b,At RABF1 and At RABF2 b were up-regulated,and the levels of At RABA4 a,At RABF1 and At RABF2 b genes were up-regulated to a greater extent.The expression of tor and S6K1 was significantly up-regulated.(4)Different strains of Arabidopsis thaliana were inoculated with powdery mildew.Through phenotypic and disease index analysis,it was found that Cs TCTP1 and Cs TCTP2 transgenic Arabidopsis leaves had large disease spots and serious disease.According to the disease index survey,the disease index of the Cs TCTP1 and Cs TCTP2 transgenic Arabidopsis lines was significantly higher than that of other lines.Cs TCTP1 and Cs TCTP2 genes play a negative regulatory role in the interaction between Arabidopsis thaliana and powdery mildew.And through analysis of the expression levels of related signaling pathway genes and defense genes,the results show that the regulatory mechanism of Cs TCTP1 and Cs TCTP2 genes is very complex.Cs TCTP1 may participate in defense response to powdery mildew stress by regulating SA defense-related genes and ABA signaling pathways,while Cs TCTP2 is possible to regulate the defense response of pathogenic bacteria by mediating the TOR signaling pathway.