溴氰菊酯对秀丽隐杆线虫的毒性效应

发布时间：2018-06-18 04:49

本文选题：溴氰菊酯 + 秀丽隐杆线虫　；参考：《湖南农业大学》2015年硕士论文

【摘要】：溴氰菊酯是常见的一种人工合成的拟除虫菊酯,具有药效的高效性,结构的稳定性、作用对象的广泛性等特征,其毒性主要有急性毒性、亚慢性毒性、致畸、致突变性,中毒者主要表现为痉挛、流涎、呕吐、四肢不调等症状。拟除虫菊酯类杀虫剂对昆虫等生物的主要作用靶标为电控门钠离子通道。在前工作中我们发现溴氰菊酯对秀丽隐杆线虫有较高的杀虫活性,但由于秀丽隐杆线虫不具备钠离子通道,溴氰菊酯对秀丽隐杆线虫的作用机制如何?是否作用于钙离子通道?为此,本研究溴氰菊酯对秀丽隐杆线虫的毒性效应,并通过实时荧光定量PCR技术检测了溴氰菊酯对秀丽隐杆线虫编码钙离子通道α1亚基egl-19、cca-1、unc-2的表达量的影响。本研究首先研究探讨了不同浓度溴氰菊酯暴露下秀丽隐杆线虫的毒性效应。将L4期秀丽隐杆线虫于5、0.5和0.05mg·L-1溴氰菊酯中暴露24h,以溶剂和K-m溶液为对照,分别在体式显微镜下观察其后代数目、怀卵数、排卵速率、头部摆动频率、身体弯曲频率及菌落接触率,结果发现溴氰菊酯暴露24h后,其后代数目、怀卵数及30min排卵的速度均有降低,其头部的摆动频率和身体的弯曲频率与对照组相比明显下降,并且与溴氰菊酯的浓度有密切关系,5mg·L-1暴露组与对照组相比秀丽隐杆线虫与大肠杆菌菌落接触率明显下降。本研究还通过运用荧光定量PCR技术,研究了50、5、0.5和0.05mg·L-1溴氰菊酯处理秀丽隐杆线虫3、6、12、24h,然后测试其编码钙离子通道的α1亚基egl-19、cca-1、 unc-2相对表达量的差异,结果表明不同浓度溴氰菊酯暴露秀丽隐杆线虫后,可以导致3个基因表达量的不同程度的改变,其中egl-19在暴露12h时,浓度为0.05 mg·L-1上调幅度最大,为12.5倍；cca-1在暴露24 h后,浓度为50mg·L-1时,表达量最高,达13.6倍；unc-2在暴露24h后,浓度为0.05mg·L-1时,表达量最高,达8.27倍。综上所述,本研究通过溴氰菊酯对秀丽隐杆线虫的毒性效应研究,证明了溴氰菊酯对秀丽隐杆线虫可造成后代数目减少、怀卵数减少及排卵速率降低的生殖中毒症状,还可导致头部摆动及身体弯曲频率降低的运动障碍和觅食缺陷,并且推测这些缺陷可能与编码钙离子通道的α1亚基egl-19、cca-1、unc-2有一定的关系。本研究从生理和分子水平初步研究了溴氰菊酯对秀丽隐杆线虫的作用位点,为进一步探究拟除虫菊酯对秀丽隐杆线虫具体靶标位点和研发新型杀线虫药剂提供一定的科学依据。
[Abstract]:Deltamethrin is a kind of synthetic pyrethroid, which has the characteristics of high efficacy, stability of structure and extensive action object. Its toxicity mainly includes acute toxicity, subchronic toxicity, teratogenicity, mutagenicity, etc. The main manifestations of the poisoning were spasms, salivation, vomiting, limb disorders and other symptoms. The main target of pyrethroid insecticides on insects and other organisms is electronic gate sodium channel. In the previous work, we found that deltamethrin has higher insecticidal activity against Rhizoma elegans, but what is the action mechanism of deltamethrin on nematode because it does not have sodium channel? Does it act on calcium channels? Therefore, the toxic effect of deltamethrin on cryptospira elegans was studied, and the effect of deltamethrin on the expression of 伪 1 subunit egl-19cca-1 unc-2 was detected by real-time quantitative PCR. In this study, the toxic effects of different concentrations of deltamethrin on cryptospira elegans were studied. The L4 nematodes were exposed to 50.5 and 0.05mg L-1 deltamethrin for 24 h. The number of offspring, the number of eggs, ovulation rate and the frequency of head swinging were observed under asana microscope, respectively, using solvent and K-m solution as control. Body bending frequency and colony contact rate. The results showed that the number of offspring, the number of pregnant eggs and the speed of 30min ovulation were all decreased after deltamethrin exposure for 24 hours, and the frequency of head swinging and bending of body were obviously decreased compared with the control group. There was a close relationship between the concentration of deltamethrin and the exposure rate of Escherichia coli in 5 mg L-1 exposure group, compared with the control group, the contact rate between C. elegans and Escherichia coli colonies was significantly decreased. In this study, fluorescence quantitative PCR technique was used to study the relative expression of 伪 1 subunit egl-19 cca-1, a 伪 1 subunit encoding calcium channel, treated with 0.05mg L-1 deltamethrin for 24 h. The results showed that different concentrations of deltamethrin could lead to the changes of three gene expression levels in different degree after exposure to Deltamethrin. After 12h exposure, the concentration of egl-19 was 0.05mg L-1, and the up-regulation was 12.5-fold after exposure to Ca-1 for 24 h. When the concentration of 50mg L-1 was the highest, the expression of 13.6 times of unc-2 was the highest, and that of 0.05mg L-1 was 8.27 times. To sum up, the toxic effect of deltamethrin on nematode elegans was studied, which proved that deltamethrin could cause reproductive poisoning symptoms of reducing the number of offspring, reducing the number of pregnant eggs and decreasing the ovulation rate. It is also suggested that these defects may be related to the 伪 1 subunit egl-19cca-1 unc-2 which encodes calcium channel. In this study, the action sites of deltamethrin on Cryptoderma elegans were preliminarily studied at physiological and molecular levels, which provided a scientific basis for further exploring the specific target sites of pyrethroids against Cryptoderma elegans and for the development of new nematicidal agents.
【学位授予单位】：湖南农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R114

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