三七北方根结线虫flps基因靶向防治的研究

Root-knot nematode is an important factor causing continuous cropping obstacle of medicinal plants, and its prevention is the hot and hard issue at present. In this study, Meloidogyne hapla, common disease and causing heavy yield losses in the Panax notoginseng cultivation, serves as the object of research. And we screen the target genes for root-knot nematode control and confirm the mechanism of lauric acid (an active substance) controlling root-knot nematode by regulation of target genes in this study. First, flps genes encoding FMRFamide-like peptides act as a point of penetration. We confirm that flps are the key genes for regulating the nematode chemotaxis, parasitism and reproduction by RNAi technology, and ascertain the target genes for the M. hapla control. Secondly, lauric acid controls root-knot nematodes by regulating nematode chemotaxis and decreasing nematode parasitism and reproduction in the basis of chemotaxis experiments and pot experiments. These results state the mechanism and effects of lauric acid controlling root-knot nematode. High-through sequencing analysis reveals that lauric acid mediates the soil micro-ecology based on the regulation of the diversity, composition and relative abundance of nematode community in the notoginseng rhiosphere soil. The data offer materials and basis for the exploitation of nematicide. This study provides new idea and strategies for the control of notoginseng root-knot nematodes, and then contributes to sustainable development of medicinal plants and environmental protection.

根结线虫病害是造成药用植物连作障碍的重要因子之一，其防治是当前研究的热点及难点。本项目以三七栽培中普遍发生且造成重大损失的北方根结线虫病害为研究对象，筛选防治该类病害的靶基因并阐述活性物质-月桂酸通过调控靶基因进而防治线虫病害的机制。首先，以编码FMRFamide-like酰胺多肽的flps基因为切入点，采用RNAi技术验证flps是调控线虫趋向性迁移和寄生致病及繁殖的关键基因，确定防治线虫病害的靶基因。其次，通过趋向性试验及盆栽试验验证月桂酸通过调控线虫趋向性迁移，降低线虫寄生致病和繁殖能力，进而防治三七根结线虫病害，阐述月桂酸防治线虫病害的效果及机制；并通过高通量测序技术分析月桂酸对三七根际线虫群落多样性、组成及丰度的影响，阐述其对根际微生态的调控作用，为杀线剂的开发提供材料及依据。本研究为三七根结线虫病害的防治提供新思路和途径，进而促进药用植物的可持续发展和环境保护。

本项目以三七栽培中普遍发生且造成重大损失的北方根结线虫病害为研究对象，筛选防治该类病害的靶基因并阐述活性物质-月桂酸通过调控靶基因进而防治线虫病害的机制。研究内容主要包括：采用RNAi技术验证flps是调控线虫趋向性迁移和寄生致病及繁殖的关键基因，确定防治线虫病害的靶基因。通过趋向性试验及盆栽试验验证月桂酸通过调控线虫趋向性迁移，降低线虫寄生致病和繁殖能力，阐述月桂酸防治该类病害的效果及机制。.结果表明，1）Mh-flp-12基因在而龄幼虫期表达量最高，其次为寄生期，卵的表达量最低。2）Mh-flp-12调控线虫的趋向性迁移，与对照相比，Mh-flp-12 dsRNA处理后的J2s，其趋向性指数下降达到50%以上，基因表达下降70%以上。3）Mh-flp-12调控线虫的侵染能力。与对照相比，Mh-flp-12敲出之后处理的三七幼苗的根结数量显著下降46.8%；而根结内线虫Mh-flp-12 基因的表达量显著下降63.7%。4）Mh-flp-12敲出之后，线虫的繁殖能力下降，幼虫的死亡数增加，Mh-flp-12基因是调控线虫繁殖的关键基因。5）不同浓度的月桂酸对线虫的趋向性影响不同，0.5-1.0 mmol 浓度范围的月桂酸对线虫表现为吸引，趋向性指数为0.16~0.27，随着浓度的增加（2.0-4.0 mmol），月桂酸对线虫表现为排斥，趋向性指数为-0.11~-0.27。6）月桂酸依赖浓度降低线虫的侵染及繁殖。与对照相比（0 mmol），2.0 -4.0 mmol月桂酸显著减少了根结（56.3%-59.7%）的数量，降低寄生状态下Mh-flp-12基因表达,表达量降低了47.2%-60.4%；与对照相比，1.0 -4.0 mmol月桂酸显著减少了卵块数量（40.4%-65.8%）的数量。.本研究揭示Mh-flp-12基因是调控北方根结线虫趋向性迁移和寄生致病及繁殖的关键基因，确定月桂酸依赖浓度调控根结线虫趋向性、侵染及繁殖，解析月桂酸防治线虫病害的机制。该结果为北方根结线虫病害的生物学防治提供有效靶基因，为杀线剂的开发和使用提供材料及依据。