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。

Glyphosate has been used worldwide for nearly 40 years, and 30 types of resistant weeds have been reported. Glyphosate is mass-produced and widely used in China, but few studies and reports on glyphosate-resistant weeds and resistance mechanisms exist. Previous studies found a goosegrass species with high glyphosate resistance from orchards in South China and its glyphosate resistant mechanism was described in this study. The cDNA of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS, EC 2.5.1.19), the target enzyme of glyphosate, was cloned from the glyphosate-resistant and -susceptible goosegrass, respectively, and referred as EPSPS-R and EPSPS-S. The Pro106 residue was known to be involved in the glyphosate resistance in most goosegrass populations. However, sequence analysis did not find the mutation at the Pro106 residue in the R biotype EPSPS amino acid sequence. The residue 133 and 382 was mutated in the R biotype EPSPS amino acid sequence instead, but it did not affect the EPSPS-S and EPSPS-R genes sensitivities to glyphosate. RT-PCR and Western blot analyses suggested that EPSPS mRNA and protein are mainly present in the shoot tissues both in the R and S goosegrass biotypes. The EPSPS-R rapidly responds to the glyphosate in R-biotype goosegrass and the induced expression was detected at 12 h post glyphosate treatment. The mRNA and protein expression of EPSPS-R increased constantly as the increasing concentration of glyphosate. However, the expression of the EPSPS-S was not induced significantly by glyphosate in the S goosegrass biotype. Quantification of real-time PCR results showed that the copy number of the EPSPS in R-biotype goosegrass was 4.7 times higher than that in the S goosegrass biotype. All the results implied that EPSPS gene amplification might mainly caused the glyphosate resistance of a goosegrass population collected from orchards in South China.

The rapid detection of glyphosate resistance in goosegrass (Eleusine indica) will enhance our ability to respond to new resistant populations of this major weed. Chlorophyll fluorescence (Fluo) and P700 (reaction center chlorophyll of photosystem I) absorbance were analyzed in one biotype of goosegrass that is resistant to glyphosate and in another that remains sensitive to the herbicide. Both biotypes were treated with a foliar spray of glyphosate. Differences in photosystem II maximum quantum yield (Fv/Fm), effective photochemical quantum yield (Y(II)), and non-photochemical quenching (NPQ) between the biotypes increased over time. Values for Fv/Fm and Y(II) differed between the two biotypes 24 h after treatment (HAT). Differentiated activities and energy dissipation processes of photosystem II (PSII) and energy dissipation processes of photosystem I (PSI) were manifested in the two biotypes 24 HAT with 20 mmol L–1 glyphosate. Differentiated energy dissipation processes of PSI were still apparent 24 HAT with 200 mmol L–1 glyphosate. These results indicate that the Fluo parameters related to PSII activity and energy dissipation and the P700 parameters related to energy dissipation are suitable indicators that enable rapid detection of glyphosate resistance in goosegrass.