基于EPSPS基因复增的抗草甘膦牛筋草与敏感生物型间适合度差异性机理

Eleusine indica is one of the most troublesome weeds heavily infesting corn and many other crops in China. In the previous work, we found that the amplification of EPSPS gene, coding the target enzyme of glyphosate, could result in glyphosate resistance in Eleusine indica. What’s more, there were significant differences in phenotypic characteristics and some photosynthetic parameters between the resistant and susceptible biotypes. However, little is known about the fitness variation and its mechanism between the resistant and susceptible biotypes of Eleusine indica. Therefore, using the resistant and susceptible biotypes, with homogeneous genetic background, as materials, this application project aims to (1) investigate the seed dormancy and germination characteristics and their biological characteristic parameters under different competitive environments (non-competition, intraspecific competition and corn co-cultured with Eleusine indica) and stress intensities (glyphosate treatments, temperature treatments and soil moisture treatments); (2) detect the response differences of the resistant and susceptible biotypes in PSⅡfunctions and absorbed light allocation under the aforementioned stress conditions by combining chlorophyll fluorescence and leaf gas exchange measurement; (3) explore the spatial and temporal expression changes of those differentially accumulated protein (basing on the scientific data of isobaric tags for relative and absolute quantitation from our research team) genes under the aforementioned stress conditions. The anticipated results will reveal the fitness cost of glyphosate-resistant Eleusine indica conferred by EPSPS gene amplification, and its mechanism will be illustrated preliminarily. This research will shed light on the green management of glyphosate-resistant weed populations.

牛筋草是严重威胁我国玉米等多种作物安全生产的恶性杂草之一。前期研究发现靶标酶EPSPS基因复增导致牛筋草对草甘膦产生抗性，且抗性、敏感生物型间的生物学表型特征和光合作用相关参数存在显著差异，但牛筋草抗性生物型与敏感生物型之间的适合度差异及其机理，仍知之甚少。本申请项目拟利用遗传背景一致的抗性及敏感生物型牛筋草为研究材料，研究两者的种子休眠、萌发特性及在不同生境（非竞争、种内竞争、杂草-玉米共生）和胁迫强度（草甘膦处理、温度处理、水分处理）下生物学特征参数的差异；将叶绿素荧光和气体交换测定相结合，研究两种生物型牛筋草光系统Ⅱ功能和光能分配对不同胁迫的响应差异；并基于蛋白质组学科学数据，探寻不同梯度胁迫下两种生物型牛筋草差异表达蛋白基因表达量时空变化特性。预期结果可揭示基于EPSPS基因复增的抗草甘膦牛筋草适合度代价，并初步阐明其机理，为绿色治理抗草甘膦杂草种群提供依据。

牛筋草是一种恶性禾本科杂草，草甘膦是防治牛筋草最常用的除草剂之一，近年来我国不同地区牛筋草种群对草甘膦抗性发展迅速。前期研究发现牛筋草EPSPS基因复增是其对草甘膦产生抗性的重要机理之一，但抗性（R）生物型与敏感（S）生物型间的适合度差异仍不清楚。本研究以遗传背景一致的牛筋草R生物型与S生物型为材料，设置非竞争、种内竞争和种间竞争等不同生态环境，全面解析了两生物型在种子萌发、营养生长和生殖生长等生活史周期内的适合度差异；并设置不同梯度草甘膦、温度、水分等常见胁迫因素，分析了两生物型对不同胁迫的响应差异，对造成差异的原因进行了分析。主要研究结果如下：R和S生物型的种子在收获后0~60 d的休眠特性存在显著差异。R和S生物型在20℃~35℃下的萌发率无显著差异；在18/12℃和15℃处理下，R生物型萌发率显著高于S生物型；在高温处理下，两生物型均出现“致死萌发”现象；渗透势−0.4 MPa至−0.6 MPa时，R生物型的萌发率显著高于S生物型。非竞争条件下，两生物型的经典生长分析和组合生长分析相关参数未表现出显著差异。不同竞争条件下，R和S生物型的株高、穗数、初级分枝长、种子量、千粒重等表现出显著差异，但繁殖成效无显著差异。在草甘膦50~1600 g/ha下，R生物型相对S生物型的适合度逐渐升高。R生物型对干旱胁迫的适应能力弱于S生物型，表现出一定的适合度代价。与高温（30/25℃）相比，两生物型在低温下（20/15℃）对草甘膦敏感性提高，R生物型相对S生物型的抗性倍数由高温时的8.9倍下降至低温时的3.1倍。低温下，两生物型的莽草酸积累量增加，叶绿素含量减少，Fv/Fm值下降。LC-MS/MS结果表明低温下草甘膦吸收量和转运量均减少，qPCR结果表明低温下EPSPS基因表达量降低。本研究发现对草甘膦抗性牛筋草的绿色防控和草甘膦的科学使用具有指导意义。