Regulation Of Potato Late Blight R Gene Expression By Different Promoters And Translation Regulators UORFs

Potato（Solanum tuberosum L.）is the third largest food crop in the world,and it is loved for its versatility as food and vegetables and abundant processed products.Potato late blight caused by the oomycete Phytophthora infestans is the most serious disease of potatoes.Resistance genes（R genes）play an important role in potato late blight resistance breeding practice,and the stack of multiple R genes is the most effective way to obtain broad spectrum and durable resistance.RB,R8 and Rpi-vnt1 are three broad spectrum and durable late blight resistance R genes of potato,which are expected to obtain strong broad spectrum and durable resistance by pyromiding three R genes by genetic engineering.Overexpression of disease R genes can improve resistance,but may cause damage to plant growth and development,especially the stack of multiple R genes may amplify the negative effects.Therefore,it is necessary to use appropriate aproches to regulate the expression of R genes in the process of pyramiding multiple R gens.In order to explore the appropriate regulatory ways of RB,R8 and Rpi-vnt1 genes in the process of pyromiding,different promoters and translation inhibition elements were used in this experiment to regulate the expression of R gene,and the effect of regulation was verified by transient expression of tobacco and stable genetic potatoes.The main results are as follows:1.The functional RB,R8 and Rpi-vnt1 genes were cloned by using the recognition principle of R protein and AVR protein and transient expression and P.infestans inoculation assay in N.benthamiana leaves.2.Transient expression experiments in N.benthamiana leaves revealed that overexpression of RB and Rpi-vnt1 genes caused cell death,while R8 gene did not,indicating that different R genes triggered different degrees of cell death.At the concentration for Agrobacterium that contains RB,R8 and Rpi-vnt1 genes alone did not cause cell death,co-injection of RB,R8 and Rpi-vnt1 activated reactive oxygen species burst and triggered rapid cell death in N.benthamiana leaves,suggesting that co-expression of multiple R genes caused much damage to plants.3.Semi-quantitative gene expression testof green fluorescent protein gene expression and Western blotting experiments demonstrated that the translational regulatory elements Atu ORFs _TBF1,Stu ORF_TBF1and Nbu ORF_TBF1from Arabidopsis thaliana,potato and N.benthamiana can inhibit e GFP expression at the translational level without affecting transcription.Stu ORF_TBF1and Nbu ORF_TBF1exhibited stronger inhibitory effects.4.The expression vectors controled by 35S promoter and pathogen-inducible promoter PVS3 promoter combined with different translational regulatory elements were constructed and expressed transiently in N.benthamiana leaves.The results showed that Atu ORFs _TBF1and Stu ORF_TBF1could inhibit cell death caused by overexpression of RB and Rpi-vnt1genes.This inhibitory effect did not affect the HR response induced by RB/IPIO and Rpi-vnt1/AVRvnt1 recognition,and Stu ORF_TBF1showed stronger inhibitory effect.5.RB transgenic potato lines regulated by different promoters and different translational regulatory elements were obtained.Late blight resistance evaliation showed that the resistance level of transgenic plants was positively correlated with the expression level of RB gene and protein.Both 35S::RB and PVS3::RB vector transgenic potatoes showed strong late blight resistance,and the 35S::RB transgenic potato was more resistant to late blight.35S::Atu-RB,35S::Stu-RB,PVS3::Atu-RB transgenic potatoes showed mediumresistance,but not stronger enough,indicating that translational regulatory elements play an role in inhibiting the traslation of RB protein.No obvious growth inhibit phenotype was observed in transgenic potato plants transformed by 35S::RB and PVS3::RB,indicating that the overexpression of RB gene did not cause obvious damages to plants.Based on the above studies,overexpression of R8 does not induce cell death,indicating it could be controlled by 35S promoter to increase expression level.While,overexpression of Rpi-vnt1 can easily induce cell death.No studies have shown that Rpi-vnt1 resistance is related to its expression level.Therefore,Rpi-vnt1 can be regulated by its own promoter.The resistance level of RB was positively correlated with its expression level,but overexpression of RB could trigger cell death in N.benthamiana leaves,pathogen-inducible PVS3 promoter may be a suitable promoter to drive RB gene expression in order to increase expression level while avoid negative effects.