实验海洋生物学重点实验室知识库

根结线虫是一类重要的土传性植物病原线虫，广泛分布于世界各地。根结线虫寄主范围十分广泛，其二龄幼虫能进入植物根部形成根结，破坏根系功能，导致植物对水分和养分的吸收困难，严重影响植物的正常生长和发育，危害的植物种类超过3000多种，每年造成农产品的产量损失为10%-30%，严重时甚至绝收，已经成为我国农业生产的重要威胁之一。根结线虫病害发生于隐蔽的地下，易通过水土、植物残体等传播，难以根除，且易与其他细菌、真菌、病毒等形成复合侵染，导致防治难度大。现有的杀线虫剂品种少且大多数存在毒性高、对非靶标生物和环境危害严重等缺点。因此，研制开发新型高效低毒的环保型杀线虫剂是当务之急。

我国海洋生物资源十分丰富，海洋生物中含有许多陆地生物所没有的、具有特殊功能的活性物质，以海洋活性物质开发新型绿色农药，具有十分广阔的应用前景。

本课题以海洋源活性物质壳寡糖为原料，通过亚结构拼接原理，接枝不同活性基团，设计、合成了氨基脲类壳寡糖衍生物、胍类壳寡糖衍生物和马来酰化类壳寡糖衍生物。通过红外光谱、核磁共振氢谱、核磁共振碳谱和元素分析等表征手段对这些衍生物进行了结构表征。并以南方根结线虫为受试对象，对这三类壳寡糖衍生物的体外杀线活性进行了测试和评价。得到的主要结果如下：

1. 壳寡糖与1，2-二溴乙烷、水合肼通过两步反应制备了肼乙基壳寡糖衍生物，再以其为底物，借助碳酸二甲酯通过两次氨解反应，将肼乙基壳寡糖与三种二胺类物质相结合，得到了三种氨基脲类壳寡糖衍生物。对其进行的体外杀线活性测试的结果表明，氨基脲类壳寡糖衍生物对于根结线虫二龄幼虫的杀灭活性相对于壳寡糖有一定程度的提高。其中邻氨苯基脲胺乙基壳寡糖衍生物在浓度为4 mg/mL时对二龄幼虫的72 h校正致死率为27.13%，其72 h 半数致死浓度（LC₅₀）为6.888 mg/mL，而壳寡糖的LC₅₀(72 h) 为13.821 mg/mL。

2. 通过尿素与硫酸二甲酯的反应制备了甲基化尿素，将其与壳寡糖以及壳寡糖的两种多氨基衍生物进行反应，制备了三种胍类壳寡糖衍生物。对其进行的体外杀线活性测试的结果表明：胍类壳寡糖衍生物对二龄幼虫的杀灭活性相对于壳寡糖也有所提高。其中胍基化肼乙基壳寡糖衍生物在浓度为4 mg/mL时对二龄线虫的72 h校正致死率为58.34%，其LC₅₀(72 h) 为3.421 mg/mL。

3. 壳寡糖与马来酸酐直接反应，得到壳寡糖的马来酰化产物，并通过增加壳寡糖分子中的氨基接枝位点，进一步得到了两种多马来酰基的壳寡糖衍生物。对这三种壳寡糖衍生物进行体外杀线活性测试，结果表明马来酰化类壳寡糖衍生物相对于壳寡糖的体外杀线活性有了显著提高。其中马来酰化聚胺乙基壳寡糖衍生物在浓度为1 mg/mL时对二龄幼虫的杀灭活性达到100%，其LC₅₀(72 h)为0.072 mg/mL，明显低于壳寡糖，并且接近阳性对照噻唑磷。同时，马来酰化类壳寡糖衍生物对南方根结线虫卵的孵化也有很强的抑制作用，其中马来酰化聚胺乙基壳寡糖衍生物在浓度为0.2 mg/mL时即能完全抑制南方根结线虫卵的孵化。

本论文通过不同接枝方法制备了三类新型壳寡糖衍生物，所得衍生物相对于壳寡糖的杀线活性有不同程度的提高，并以马来酰化类壳寡糖衍生物的活性最强，具有较好的应用潜力。以壳寡糖为原料进行修饰改性制备杀线虫活性衍生物，不仅拓宽了壳寡糖的应用领域，也对新型杀线虫剂的开发具有一定的借鉴意义。

Root knot nematode is an important kind of soil borne plant pathogenic nematode，which is widely distributed all over the world. Root knot nematodes have a quite broad host range, their second-stage juveniles (J2) could penetrate plant roots and get inside to form root knots, which would damage the function of root system, lead to the difficulties in the absorption of water and nutrients, and seriously affect the normal growth and development of plants. Root knot nematodes parasitize more than 3000 species of plant, thereby causing yield losses rate of agricultural products is 10%-30% annually, or even total crop failure in serious cases, which has become one of the most important threats to agricultural production in our country. Root knot nematode disease occurs underground and is easily transmitted through soil, water and plant residues, which is difficult to eradicate and easily forms complex infection with other bacteria, fungi and viruses, leading to great difficulties in treatment. There are limited varieties of nematocides and most of them have some disadvantages such as high toxicity, serious harm to non-target organisms and environments. Therefore, it is urgent to develop new environmentally friendly nematocides with high efficiency and low toxicity.

China is rich in marine biological resources. There are many active substances from marine organisms with special functions that are not found in terrestrial organisms. The development of new green pesticides with marine active substances has a very broad application prospect.

In this study, marine active substance chitooligosaccharide (COS) was used as a raw material, and different active groups were grafted onto COS backbones, respectively, through substructural splicing methods, thus, the aminourea chitooligosaccharide derivatives (AUCOS), guanidinylated chitooligosaccharide derivatives and maleated chitooligosaccharide derivatives were designed and synthesized. The structures of these derivatives were characterized by infrared spectrum (FTIR), ¹H nuclear magnetic resonance (¹H NMR), ¹³C nuclear magnetic resonance (¹³C NMR) and elemental analysis. Meloidogyne incognita (M. incognita) was used to test the nematocidal activities of abovementioned COS derivatives in vitro. The main results are as follows:

1. COS reacted with 1, 2-dibromoethane and hydrazine hydrate successively to form the hydrazino-ethyl chitooligosaccharide (HECOS), and then HECOS was used as the substrate to combine with three diamines by two ammonolysis reactions with dimethyl carbonate to prepare three AUCOSs. The results of nematocidal activities of the AUCOSs against the second-stage juveniles (J2) of M. incognita in vitro showed that the AUCOSs had higher nematicidal activities in vitro compared with chitooligosaccharide. The corrected lethality rate of the o-aminophenyl urea aminoethyl chitooligosaccharide derivative (AUCOS3) against the J2s was 27.13% at the concentration of 4 mg/mL, and its lethal concentration of 50% (LC₅₀) at 72 h was 6.888 mg/mL, while the LC₅₀(72 h) of COS was 13.821 mg/mL.

2. Methylated urea was prepared by the reaction of urea and dimethyl sulfate, and three guanidinylated COS derivatives were prepared by the reactions of methylated urea with COS and two kinds of multi-amino COS derivatives, respectively. The nematicidal activities of the guanidinylated COS derivatives against the J2s in vitro were also better in comparasion with the original chitooligosaccharide. The corrected lethality rate of the guanidinylated hydrazino-ethyl chitooligosaccharide derivatives (GHECOS) in 72 h at the concentration of 4 mg/mL against the second-stage juveniles was 58.34%, and its LC₅₀(72 h) was calculated as 3.421 mg/mL.

3. COS reacted directly with maleic anhydride to obtain the maleated products of chitooligosaccharide, and two kinds of multi-maleyl COS derivatives were further obtained after introduing more amino graft sites onto COS backbones and reacting with maleic anhydride. The results of nematicidal activities tests showed that the maleated COS derivatives showed significantly improvement compared with COS on nematocidal activities. The corrected lethality rate of the maleated polyaminoethyl chitooligosaccharide derivative (MPACOS) against the second-stage juveniles of M. incognita in 72 h at the concentration of 1 mg/mL reached 100%, its LC₅₀(72 h) was 0.072 mg/mL, this value was significantly lower than the LC₅₀(72 h) of COS, and close to the positive control lythidathion’s. In addition, the maleated COS derivatives also had strong inhibitory effects on the incubation of M. incognita eggs and the maleated polyaminoethyl chitooligosaccharide could completely inhibit the incubation of M. incognita eggs at the concentration of 0.2 mg/mL.

In this paper, three new types of chitooligosaccharide derivatives were prepared through different grafting methods, and the obtained derivatives showed different degrees of improvement on lethal activities against M. incognita compared with COS, and the maleated COS derivatives had better utilization potentiality which had the highest nematocidal activities. The preparation of active nematocidal derivatives by modifying the raw COS not only broadens the application fields of COS, but also has certain reference significance for the development of new green nematocides.