Transcriptomic analysis reveals the transcription factors involved in regulating the expression of EPSPS gene, which confers glyphosate resistance of goosegrass (Eleusine indica)

doi:10.1016/S2095-3119(21)63682-1

摘要/Abstract

摘要：

草甘膦靶标酶是5-烯醇式丙酮酸莽草酸-3-磷酸合酶(EPSPS)，EPSPS基因的过表达是杂草抗草甘膦的分子机制之一。比较抗型和敏感型牛筋草中EPSPS基因调控序列，发现抗型牛筋草EPSPS基因5’端非翻译区5’-UTR Py-rich stretch元件发生突变，且该突变与提高抗草甘膦牛筋草EPSPS基因转录水平相关。然而，与该元件及整个EPSPS基因启动子序列结合的关键转录因子尚未可知。为进一步探究抗草甘膦牛筋草中EPSPS基因的转录调控机制，本研究利用RNA-seq技术分析了抗草甘膦牛筋草EPSPS过表达相关的基因调控网络，筛选出相关转录因子；通过酵母单杂交技术体外验证相关转录因子与5’-UTR Py-rich stretch元件的结合情况。转录本差异表达分析显示，与草甘膦敏感型(GS)牛筋草相比，抗草甘膦牛筋草在草甘膦处理后有2752个unigenes的表达增加，4025个unigenes的表达减少。其中，鉴定出1373个unigenes与EPSPS基因共表达。GO和KEGG通路分析表明，上调的unigenes主要富集于叶绿体，并与莽草酸生物合成通路、叶绿素II和过氧化物酶体代谢过程相关。值得注意的是，催化莽草酸转化为莽草酸3-磷酸(S3P, EPSPS的底物)的莽草酸激酶的表达也上调。筛选到8个转录因子可能参与EPSPS表达，其中预测三个转录因子(ARF2, ARF8和BPC6)能与5 ' utr Py-rich元件结合。酵母单杂交实验表明ARF8和BPC6能与野生型5’-UTR Py-rich stretch元件结合，但不再能与其突变体结合。本研究数据表明，与草甘膦敏感型牛筋草相比，抗草甘膦牛筋草中EPSPS的表达转录调控机制发生了显著的变化。首次筛选分析出与EPSPS基因表达调控相关的转录因子，可为进一步研究基于EPSPS过表达的牛筋草抗草甘膦机理提供了新的参考。

Abstract:

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 2 752 unigenes and a decrease in the expression of 4 025 unigenes in the GR E. indica, compared to the glyphosate-susceptible (GS) E. indica. Among them, 1 373 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.

Key words: transcriptomic , EPSPS , 5´-UTR Py-rich stretch element , transcription factor , glyphosate resistance

. [J]. Journal of Integrative Agriculture, 2021, 20(8): 2180-2194.

ZHANG Chun, YU Chao-jie, ZHANG Tai-jie, GUO Wen-lei, TIAN Xing-shan. Transcriptomic analysis reveals the transcription factors involved in regulating the expression of EPSPS gene, which confers glyphosate resistance of goosegrass (Eleusine indica)[J]. Journal of Integrative Agriculture, 2021, 20(8): 2180-2194.

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