黄单胞菌L4毒素抑制杂草的作用机理研究
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摘要
本文对黄单胞菌L4毒素的理化性质包括稳定性、脂溶性、pH对活性影响、分子量大小进行了初探，在此基础上在细胞水平上对黄单胞菌毒素14抑制目标杂草的作用机理进行了较为深入的研究，同时研究了毒素抑制作用的条件、构建了施药模式。利用发酵得到的细菌毒素进行抑制和杀伤杂草试验国内外报道较多，但细菌除草剂作用机理的深入研究报道甚少，因此本研究为黄单胞菌L4毒素作为细菌除草剂的开发应用提供基础，对根细菌和植物交互作用的研究提供理论依据，同时对环境保护和现代农业可持续发展具有重要的意义。
黄单胞菌L4经种子培养24h、摇瓶发酵培养72h、离心得含毒素的上清液，乙酸乙酯萃取试验和透析试验表明该毒素是脂溶性小分子化合物，热稳定性好，pH4时活性最高。助剂是毒素发挥活性作用的重要因素，助剂选择试验表明吐温、氨硅、JFC和十二烷基磺酸钠助剂在低浓度时有助于毒素抑制作用，其中1％吐温较好。
在温室条件下，黄单胞菌14感染目标杂草后，采用考马斯亮蓝法测定杂草叶子可溶性蛋白质含量、紫外分光法测定核酸合成、电导率法测定细胞膜透性、叶绿素荧光法测定光能转化效率、愈创木酚法测定过氧化物酶活性和硫代巴比妥酸法测定丙二醛含量的变化，并分别利用透射电镜法和扫描电镜法观察了杂草叶子感染毒素前后的细胞膜超微结构、细胞膜表皮结构的图象，同时采用开放系统测定呼吸速率改变及徒手切片法统计根尖有丝分裂指数，实验结果表明：毒素处理3h后叶绿体，线粒体结构遭受严重破坏；处理2h后呼吸速率加强106％、过氧化物酶活性增加50％，但丙二醛含量没有显著变化；最终导致呼吸旺盛，叶面气孔关闭，蒸腾作用的热量无法散发，使叶片坏死萎蔫。因此黄单胞菌L4毒素的作用靶位是杂草叶片的线粒体，通过破坏线粒体内膜结构阻遏氧化磷酸化偶联，从而使ATP能量代谢不能正常进行。
The chemical and physical characters of Xanthomonas L4 phytotoxin, including its heat stability, liposolubility, molecular weight and effect of pH on weed control activity, then its action mechanism on the cell level was explored, and the inhibitory condition of Xanthomonas L4 phytotoxin was also further studied. Researches on bacterial herbicide are mostly limited to weed control test with phytotoxin from bacterial fermentation broth. However, mechanism of weed control was reported scarcely in the home and abroad. So works reported in this research will provide foundation for developing Xanihomonas L4 phytotoxin as a new kind of bacterical herbicide.
The tests of ethyl acetate extraction and dialyzation from Xanihomonas L4 fermentation broth demonstrated the L4 phytotoxin was very liposoluble compound and belonging to micromolecular, further experiments indicated it was very stable under high temperature and phytotoxin have highest bioactivity at pH4. The experiment of selecting adjvant showed that Tween-80, JFC, SDS, Silicone was suitable for Xanthomonas L4 phytotoxin at lower concentration and 1% Tween-80 was best adjvant.
After Amaranthus retro/lex (redroot amarant) were infected by Xanthomonas L4 phytotoxin in greenhouse, the variation of soluble protein, DNA, peroxidase (POD) activity, malon dialdehyde (MDA), photosynthetic conversion efficiency, cell memebrane permeability and respiratory ratio of leaves were dertemined with Coomassie Brilliant Blue, UV spectroscopy, guaiacol, thiobarbituric acid, PAM201-pulse modulation fluorometer, conductometry and LI-6400 Portable Photosynthetic System, and the change of cell ultrastructure and epidermis of leaves were also inspected with transmission electron microscope(TEM) and scanning electron microscope(SEM). The experiment results showed respiratory ratio and POD activity increased 106 % and 50 % Respectively after treat 2h, photosynthetic conversion
efficiency, MDA and soluble protein was not inhibited. Ultrastructre observation
revealed that the thylakoids of chloroplast and cristae of mitochondria swelled after 2h treating with the poison; however after 3h treating, cell membrane was disrupted,
chloroplast was disintegrated and mitochondria was vesiculated, finally respiratory ratio was inhanced, at last stoma was closed, heat of transpiration couldn't be emitted and leaf began to wilt. So the Xanthomonas L4 phytotoxin's target is mitochondria, which disrupted ATP synthesis metabolic process by blocking process of oxidation coupling with phosphorylation.
黄单胞菌L4经种子培养24h、摇瓶发酵培养72h、离心得含毒素的上清液，乙酸乙酯萃取试验和透析试验表明该毒素是脂溶性小分子化合物，热稳定性好，pH4时活性最高。助剂是毒素发挥活性作用的重要因素，助剂选择试验表明吐温、氨硅、JFC和十二烷基磺酸钠助剂在低浓度时有助于毒素抑制作用，其中1％吐温较好。
在温室条件下，黄单胞菌14感染目标杂草后，采用考马斯亮蓝法测定杂草叶子可溶性蛋白质含量、紫外分光法测定核酸合成、电导率法测定细胞膜透性、叶绿素荧光法测定光能转化效率、愈创木酚法测定过氧化物酶活性和硫代巴比妥酸法测定丙二醛含量的变化，并分别利用透射电镜法和扫描电镜法观察了杂草叶子感染毒素前后的细胞膜超微结构、细胞膜表皮结构的图象，同时采用开放系统测定呼吸速率改变及徒手切片法统计根尖有丝分裂指数，实验结果表明：毒素处理3h后叶绿体，线粒体结构遭受严重破坏；处理2h后呼吸速率加强106％、过氧化物酶活性增加50％，但丙二醛含量没有显著变化；最终导致呼吸旺盛，叶面气孔关闭，蒸腾作用的热量无法散发，使叶片坏死萎蔫。因此黄单胞菌L4毒素的作用靶位是杂草叶片的线粒体，通过破坏线粒体内膜结构阻遏氧化磷酸化偶联，从而使ATP能量代谢不能正常进行。
The chemical and physical characters of Xanthomonas L4 phytotoxin, including its heat stability, liposolubility, molecular weight and effect of pH on weed control activity, then its action mechanism on the cell level was explored, and the inhibitory condition of Xanthomonas L4 phytotoxin was also further studied. Researches on bacterial herbicide are mostly limited to weed control test with phytotoxin from bacterial fermentation broth. However, mechanism of weed control was reported scarcely in the home and abroad. So works reported in this research will provide foundation for developing Xanihomonas L4 phytotoxin as a new kind of bacterical herbicide.
The tests of ethyl acetate extraction and dialyzation from Xanihomonas L4 fermentation broth demonstrated the L4 phytotoxin was very liposoluble compound and belonging to micromolecular, further experiments indicated it was very stable under high temperature and phytotoxin have highest bioactivity at pH4. The experiment of selecting adjvant showed that Tween-80, JFC, SDS, Silicone was suitable for Xanthomonas L4 phytotoxin at lower concentration and 1% Tween-80 was best adjvant.
After Amaranthus retro/lex (redroot amarant) were infected by Xanthomonas L4 phytotoxin in greenhouse, the variation of soluble protein, DNA, peroxidase (POD) activity, malon dialdehyde (MDA), photosynthetic conversion efficiency, cell memebrane permeability and respiratory ratio of leaves were dertemined with Coomassie Brilliant Blue, UV spectroscopy, guaiacol, thiobarbituric acid, PAM201-pulse modulation fluorometer, conductometry and LI-6400 Portable Photosynthetic System, and the change of cell ultrastructure and epidermis of leaves were also inspected with transmission electron microscope(TEM) and scanning electron microscope(SEM). The experiment results showed respiratory ratio and POD activity increased 106 % and 50 % Respectively after treat 2h, photosynthetic conversion
efficiency, MDA and soluble protein was not inhibited. Ultrastructre observation
revealed that the thylakoids of chloroplast and cristae of mitochondria swelled after 2h treating with the poison; however after 3h treating, cell membrane was disrupted,
chloroplast was disintegrated and mitochondria was vesiculated, finally respiratory ratio was inhanced, at last stoma was closed, heat of transpiration couldn't be emitted and leaf began to wilt. So the Xanthomonas L4 phytotoxin's target is mitochondria, which disrupted ATP synthesis metabolic process by blocking process of oxidation coupling with phosphorylation.
引文
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