Studies On The Physiological And Molecular Responsive Mechanism Of Glyphosate Phytotoxicity In Hevea Brasiliensis

Glyphosate is the most widely used herbicide in the word.Glyphosate spraying for weed control in rubber tree plantation will affect the growth of rubber tree,even lead to rubber trees die.However,the occurrence mechanism of glyphosate phytotoxicity in rubber tree is not clear.In order to study physiological and molecular response mechanism of rubber tree to glyphosate phytotoxicity,plant physiology,molecular biology,bioinformatics and transcriptome technical were used.The effect of glyphosate on physiological index in rubber tree were by means of plant physiological methods;Clond rubber tree HbEPSPS gene by RT-PCR and analyzed HbEPSPS structure with bioinformatics methods;Its expression in tissue,under treatment of glyphosate,powdery mildew,plant hormone and stress.by Q-RT-PCR method;Analyzed transcriptome of glyphosate spraying rubber tree leaves.The results are as follows:1,After leaf fall caused by spraying glyphosate of 170mg·L-1 in rubber tree,newborn young leaves showed longer and deformity phenotype,recovery leaves showed in leaves of next extension.Glyphosate caused chlorophyll content and soluble sugar contents declined,proline content,prolinecontents peroxidase(POD)and superoxide dismutase(SOD)activity increased,whilst water content did not show significant difference.2,The accumulation of shikimic acid in glyphosate spraying rubber was significantly difference before and after the spraying.Shikimic acid accumulation rise in half yellow half green phenotype rubber tree leaves,while shikimic acid content fall in new deform leaves and recovery leaves.Auxin and GA3 contents-did-not showed significant difference in leaves before and after spraying glyphosate,whilst Abscisic acid and Zeatin contents showed significant difference in leaves before and after spraying glyphosate.Abscisic acid and Zeatin contents fall in half yellow half green phenotype rubber tree leaves,while rise in in new deform leaves and recovery leaves.3,Rubber tree HbEPSPS gene cDNA sequence is 1891 bp long,contains a 1572 bp ORF,encoding 523 amino acids,containing a characteristic EPSPS family domain,have not transmembrane domain and signal peptide.Phylogenetic analysis showed that the HbEPSPS protein sequences showed highest identity with jatropha curcas EPSPS.According to the Q-RT-PCR analysis,HbEPSPS gene expression is highest in flower,followed by the bark and leaves,the lowest expressed in latex.After spraying glyphosate,the HbEPSPS expression rise in half yellow half green phenotype rubber tree leaves,while showed no difference in new deform leaves and recovery leaves.Powdery mildew and mechanical wounding can significantly reduce HbEPSPS expression.Drought,hormones,and H2O2 treatment induced HbEPSPS expression.4,Using the Illumina Hiseq 4000 sequencing platform,rubber tree leaves spraying glyphosate 0d,1d,2d and 3d were used for the transcriptome sequencing.It produced 18 Gb of data,after filtering,found 83486 All-Unigenes.After annotation and comparison of All-Unigenes with NR,NT,GO,COG,KEGG and Interpro database,predicted 59608 CDS,2389 genes encoding transcription factors,prediction of 9846 SSR markers loci and SNPS.Three groups of leaves sample Id vs Od,2d vs 0d and.3d vs 0d were found 16342,19100,and 20613.differentially expressed genes,respectively.Screening of differentially expressed genes of 46 responses to glyphosate,these genes are associated with protein synthesis,pigment and transcription factors,also found some unknown gene function.Using Q-RT-PCR technology to validation transcriptome data results,they are same.Conclusions were drawn based on above results:1,Glyphosate phytotoxicity caused leaves deform in rubber tree,the chloroplast structure change,photosynthesis inhibition,affect photosynthesis,the metabolism of active oxygen and osmotic regulation substances as well as other physiological processes.2,Glyphosate phytotoxicity causes shikimic acid pathway blocked and changed abscisic acid and zeatin contents,3,HbEPSPS was-related to glyphosate,hormone:drought,powdery mildew;and-stress reactions such as mechanical wounding.4,The transcriptome data screening glyphosate phytotoxicity related genes in rubber tree were genes in protein synthesis,pigment,and transcription factors.This study provide theoretical basis for elucidate the glyphosate phytotoxicity mechanism and molecular marker assisted glyphosate resistant breeding program in rubber tree.