Glyphosate inhibits the enzyme 5-enolpyruvylshikimate-3-phosphate synthase(EPSPS)and overexpression of the EPSPS gene is one of the molecular mechanisms conferring glyphosate resistance in weeds.A regulatory sequence ...Glyphosate inhibits the enzyme 5-enolpyruvylshikimate-3-phosphate synthase(EPSPS)and overexpression of the EPSPS gene is one of the molecular mechanisms conferring glyphosate resistance in weeds.A regulatory sequence of EPSPS gene was isolated previously,and an alteration in its 5´-untranslated region(UTR)pyrimidine(Py)-rich stretch element is involved in the regulation of EPSPS expression in glyphosate-resistant(GR)Eleusine indica.However,the transcription factors involved in this regulatory sequence remain to be elucidated.In this study,we investigated the regulatory network of EPSPS overexpression associated genes in a GR E.indica population by RNA-seq.The differentially expressed transcript analyses revealed that glyphosate treatment caused an increase in the expression of 2752 unigenes and a decrease in the expression of 4025 unigenes in the GR E.indica,compared to the glyphosatesusceptible(GS)E.indica.Among them,1373 unigenes were identified to be co-expressed with the EPSPS gene in GR E.indica.GO and KEGG pathway analyses showed that the up-regulated unigenes were mainly enriched in chloroplasts and associated with the shikimate biosynthesis pathway,chlorophy II and peroxisome metabolism processes.Notably,the expression of a Shikimate kinase which catalyzed the conversion of Shikimate to Shikimate 3-phosphate(S3P,a substrate of EPSPS),was also up-regulated.Eight transcription factors were identified as likely to be involved in the regulation of the EPSPS expression,and three of them(ARF2,ARF8 and BPC6)showed more binding sites because of a(CT)n insertion of the 5´-UTR Py-rich stretch element in GR.However,the yeast one-hybrid assay illustrated that ARF8 and BPC6 could bind to the 5´-UTR Py-rich stretch element of wild type EPSPS,but could not bind to the mutated form.Our data suggests that the transcriptional regulation of EPSPS expression is complex and was significantly altered in GR E.indica.These discoveries provide new references for further study of the EPSPS overexpression mechanism that endows glyphosate resistance.展开更多
基金This study was supported by the National Natural Science Foundation of China(31871984)the Guangdong Provincial National Natural Science Foundation,China(2017B030311006)+1 种基金the Department of Science and Technology of Guangdong Province,China(2019B121201003)the special fund for Scientific Innovation Strategy-Construction of High Level Academy of Agriculture Science,China(202105TD,R2020PYJX005).
文摘Glyphosate inhibits the enzyme 5-enolpyruvylshikimate-3-phosphate synthase(EPSPS)and overexpression of the EPSPS gene is one of the molecular mechanisms conferring glyphosate resistance in weeds.A regulatory sequence of EPSPS gene was isolated previously,and an alteration in its 5´-untranslated region(UTR)pyrimidine(Py)-rich stretch element is involved in the regulation of EPSPS expression in glyphosate-resistant(GR)Eleusine indica.However,the transcription factors involved in this regulatory sequence remain to be elucidated.In this study,we investigated the regulatory network of EPSPS overexpression associated genes in a GR E.indica population by RNA-seq.The differentially expressed transcript analyses revealed that glyphosate treatment caused an increase in the expression of 2752 unigenes and a decrease in the expression of 4025 unigenes in the GR E.indica,compared to the glyphosatesusceptible(GS)E.indica.Among them,1373 unigenes were identified to be co-expressed with the EPSPS gene in GR E.indica.GO and KEGG pathway analyses showed that the up-regulated unigenes were mainly enriched in chloroplasts and associated with the shikimate biosynthesis pathway,chlorophy II and peroxisome metabolism processes.Notably,the expression of a Shikimate kinase which catalyzed the conversion of Shikimate to Shikimate 3-phosphate(S3P,a substrate of EPSPS),was also up-regulated.Eight transcription factors were identified as likely to be involved in the regulation of the EPSPS expression,and three of them(ARF2,ARF8 and BPC6)showed more binding sites because of a(CT)n insertion of the 5´-UTR Py-rich stretch element in GR.However,the yeast one-hybrid assay illustrated that ARF8 and BPC6 could bind to the 5´-UTR Py-rich stretch element of wild type EPSPS,but could not bind to the mutated form.Our data suggests that the transcriptional regulation of EPSPS expression is complex and was significantly altered in GR E.indica.These discoveries provide new references for further study of the EPSPS overexpression mechanism that endows glyphosate resistance.
