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 glyphosate-susceptible (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.

草甘膦抑制酶 5-enolpyruvylshikimate-3-phosphate 合酶 (EPSPS)，EPSPS基因的过度表达是杂草产生草甘膦抗性的分子机制之一。先前分离出EPSPS基因的调控序列，并且其富含 5´ 非翻译区 (UTR) 嘧啶 (Py) 的拉伸元件的改变参与了抗草甘膦 (GR) Eleusine indica中EPSPS表达的调控。然而，参与该调控序列的转录因子仍有待阐明。在这项研究中，我们研究了G E. indica中EPSPS过表达相关基因的调控网络通过 RNA-seq 进行人口统计。差异表达的转录物分析显示，与草甘膦敏感 (GS) E. indica相比，草甘膦处理导致 G E. indica中 2752 个 unigenes 的表达增加和 4025 个 unigenes 的表达减少。其中，鉴定出1373个unigenes与GR E. indica中的EPSPS基因共表达。GO 和 KEGG 途径分析表明，上调的 unigenes 主要富集在叶绿体中，并与莽草酸生物合成途径、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被上调。八种转录因子被确定为可能参与EPSPS表达的调节，其中三个（ARF2、ARF8和BPC6）显示出更多的结合位点，因为 (CT) _n插入了富含 5´-UTR Py 的GR 中的拉伸元素。然而，酵母单杂交试验表明ARF8和BPC6可以与野生型EPSPS的富含 5´-UTR Py 的拉伸元件结合，但不能与突变形式结合。我们的数据表明EPSPS表达的转录调控是复杂的，并且在 G E. indica 中发生了显着改变。这些发现为进一步研究赋予草甘膦抗性的EPSPS过表达机制提供了新的参考。