亚精胺参与牛筋草抗性调控的机理研究

Resistant weeds have become a serious threat to the management of farmland and the agricultural production, which has become a serious problem in the world. The bi-pyridine herbicides resistant biotype and susceptible biotypes of Goosegrass was used as materials in this project, through the application of spermidine and paraquat, the biological phenotypes of seedlings was observed, and the chlorophyll fluorescence parameters were determined. The content of endogenous spermidine, spermine and putrescine were analyzed by using HPLC to analysis its competitive mechanism. Combined with previous high pass sequencing results, the genes associated with the transporter gene were screened, the internal control was designed and gene was cloned. In order to provide a theoretical basis for the mechanism of bi-pyridine herbicides resistance in Goosegrass of spermidine, the gene expression of different biotypes of Goosegrass was analyzed by using PCR high-throughput fluorescence quantitative. The project will reveal the resistance mechanisms of Goosegrass for Goosegrass control and bi-pyridine herbicides long-term efficient application which will provide an important theoretical significance and application value.

抗药性杂草对农田杂草治理和农业生产构成严重威胁，成为备受全球关注的严重问题。本项目以联吡啶类除草剂抗性生物型和敏感生物型牛筋草为试验材料，通过喷施亚精胺和联吡啶类除草剂处理，观测牛筋草幼苗的生物学表型，测定其叶绿素荧光参数变化，利用高效液相技术分析内源亚精胺、精胺、腐胺的含量，分析亚精胺与联吡啶类除草剂的竞争关系，结合高通测序获得的基因，筛选亚精胺和转运体相关基因，设计内参并克隆基因，运用荧光定量PCR技术进行不同生物型牛筋草的基因表达差异性分析，从而为阐明亚精胺参与牛筋草联吡啶类除草剂抗性机理提供理论依据。本项目揭示牛筋草的抗药性机制，对抗性牛筋草的防除以及联吡啶类除草剂的长期高效应用都有重要的理论意义和应用价值。

抗药性杂草对农田杂草治理和农业生产均构成了严重威胁，成为备受全球关注的严重问题。为此，项目组主要开展了牛筋草幼苗生物学效应分析牛筋草植株体内除草剂含量测定、牛筋草幼苗生理生化机理研究、牛筋草抗性调控的机制研究和亚精胺及转运体基因的表达分析。项目组筛选出MZ04牛筋草种群对百草枯抗药性水平最低，QY05牛筋草种群是MZ04的12.94倍，同时，筛选出亚精胺的最佳浓度为1 mmol•L-1。利用高效液相色技术，建立了一种测定植物组织中百草枯含量的方法。施用1 mmol•L-1亚精胺可促进百草枯胁迫下敏感生物型牛筋草幼苗的生长发育，提高叶片的叶绿素含量和叶绿素荧光参数，提高牛筋草幼苗叶片中可溶性蛋白含量，增强牛筋草幼苗的渗透调节能力，同时能够增加抗氧化系统的保护能力，降低丙二醛的含量和细胞膜透性，提高细胞膜的稳定性。利用Illumina HiSeq平台测序，获得1,088,102,646个原始读数。移除低质量区域及接头后，总共获得了1,019,929,478个高质量读数，组装得到218,515个Transcript，69,786个Unigene。其中Transcript的平均长度为1,696.82 bp, Unigene的平均长度为1,109.42 bp。25,190条Unigene注释到GO三大功能分类中；18,457个Unigene被注释上KOG分类。与对照相比，百草枯和亚精胺+百草枯处理两种生物型牛筋草引起基因表达量显著上调，但在敏感生物型中表达量尤为突出。与百草枯处理相比，亚精胺+百草枯可以显著降低基因的表达量，其中敏感生物型中表达量极低。经转录组测序获得目标基因，从中筛选出4个亚精胺及转运体候选基因，利用PCR技术和HPLC技术，揭示亚精胺和转运体基因的其功能。说明亚精胺及其转运体参与了牛筋草对百草枯的抗性机制，为进一步研究牛筋草对百草枯的抗性机制提供了重要依据。