Caenorhabditis elegans对重金属离子Cd~（2+）、Pb~（2+）和Zn~（2+）的反应以及利用RNAi研究几种影响其寿命的基因
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摘要
本文研究了秀丽小杆线虫Caenorhabdits elegans对重金属Cd~(2+)、Zn~(2+)、Pb2+的反应以及利用RNAi对影响C. elegans寿命的一些因素进行了研究。
首先研究了三种重金属对秀丽小杆线虫的毒害作用。结果表明:24h时,三种重金属的毒性为Pb~(2+)>Cd~(2+)>Zn~(2+),半致死浓度分别为41774μg·L~(-1)、65945μg·L~(-1)、113323μg·L~(-1);48h时,三种重金属的毒性为Pb~(2+)>Cd~(2+)>Zn~(2+),半致死浓度分别为31567μg·L~(-1)、38679μg·L~(-1)、63423μg·L~(-1);72h时,三种重金属的毒性为Cd~(2+)≈Pb~(2+)>Zn~(2+),半致死浓度分别为24669μg·L~(-1)、24946μg·L~(-1)、40668μg·L~(-1)。三种重金属的毒性中Cd~(2+)随时间变化的速度最快,Pb~(2+)随时间变化的速度最慢;Pb~(2+)随浓度的变化最快,Zn~(2+)随浓度变化最慢。
重金属浓度对C. elegans联合作用的影响的实验表明:当Cd~(2+)和Zn~(2+)共同作用于线虫时,在低浓度时,呈现出简单的相加作用,高浓度时,呈现出拮抗作用;Pb~(2+)和Zn~(2+)共同作用于线虫时,四个浓度都呈现出简单的相加作用;当Cd2+和Pb~(2+)共同作用于线虫时,低浓度呈现出简单的相加效应,高浓度呈现出协同作用。
用AI指数法分析C. elegans暴露重金属的时间对重金属联合作用的影响,结果表明:当Cd~(2+)和Zn~(2+)以1:2的质量比混合时,在24h、48h和72h,其相加指数AI均小于0,联合作用表现为拮抗作用;当Pb~(2+)和Zn~(2+)以1:2的质量比混合时,在24h、48h和72h,其相加指数AI均小于0,联合作用表现为拮抗作用;当Cd~(2+)和Pb~(2+)以1:1的质量比混合时,在24h,其相加指数大于0,联合作用表现为协同作用;在48h和72h,其相加指数均小于0,联合作用表现为拮抗作用。
利用转基因线虫PC72,发展了一种可用于生物监测的高灵敏度的生物监测器。实验结果表明:在三种重金属的毒害下,染色率都呈现出随致死率变化而变化的特点,延长毒害时间能增加其染色率,空白对照即便染色时间延长也未曾出现明显的染色率增加。利用计分法评估线虫的染色效果,得分也和染色率呈现出相关变化。与致死率相比,用染色评估水体毒性的方法更加快捷、灵敏。
转基因线虫PC72染色的方法用于检验厦门海域海水水质取得了比较好的效果,检测结果显示染色率与致死率的变化趋势相同,染色率与致死率相关,但利用染色率检测能够缩短检测时间,提高监测灵敏度。
其次利用RNAi法研究了几种影响C. elegans寿命的基因。构建了四个RNA干扰载体:R148.3a、Cnfκb (C. elegans nfκb like)、sqv-4、helicase。将四种食物喂食线虫,研究其对线虫寿命的影响。
R148.3a (RNAi)菌喂食的线虫呈现出产卵数目下降,寿命缩短的特点,并且会使N2、daf-2(e1370)、daf-16(mu86)突变体的免疫力下降。
Cnfκb (RNAi)菌喂食N2线虫并不能使其寿命发生明显变化,但却会使其免疫力下降,易于被金黄色葡萄球菌感染。同时Cnfκb缺失使免疫力下降的作用依赖于daf-16,当daf-16突变时,Cnfκb不能使daf-16突变体的免疫力进一步下降。helicase(RNAi)菌喂食的线虫,产卵数目较对照N2下降,寿命缩短了2.9天。同时helicase功能的缺失会使线虫幼虫部分致死。
sqv-4(RNAi)菌喂食的线虫,产卵数目大幅度下降,并有很强的胚胎致死能力,所产的卵大多数无法孵化成幼虫。同时sqv-4干扰后,线虫的寿命会缩短约1.5天。
lin-28(n1119)突变体寿命相对于N2延长了8%,lin-4(e912)突变体寿命相对于N2缩短了47%,let-7(n2853)寿命相对N2缩短了36%。然而双突变体lin-28(n719); lin-4(e912)的寿命相对于lin-28(n1119)无明显改变,说明lin-28位于lin-4下游。
Caenorhabditis elegans was used in this paper to study the factors affecting lifespan.
We studied the toxic effects of three heavy metals Cd~(2+), Pb~(2+) and Zn~(2+) on C. elegans. The results indicated that: after C. elegans exposed to the three heavy metals 24hs, the toxicity of three heavy metals was Pb~(2+)> Cd~(2+)>Zn~(2+), LC 50 of three heavy metals was 41774μg·L~(-1), 65945μg·L~(-1) and 113323μg·L~(-1) respectively; after exposed to the three heavy metals 48hs, the toxicity of the three metals was: Pb~(2+)>Cd~(2+)>Zn~(2+), LC 50 of three heavy metals was 31567μg·L~(-1),38679μg·L~(-1) and 63423μg·L~(-1) respectively; after exposed to the three heavy metals 72hs, the toxicity of the three metals was Cd~(2+)≈Pb~(2+)>Zn~(2+), LC50 of three heavy metals was 24669μg·L~(-1), 24946μg·L~(-1) and 40668μg·L~(-1) respectively. In the three metals, the toxicity of Cd~(2+) changed most with the time and the toxicity of the Pb~(2+) changed least with the time. The toxicity of Pb~(2+) changed most with the concentration and the toxicity of Zn~(2+) changed least with the concentration.
The experiments of concentration effects in combination of two metals presented that: the toxic effect of Cd~(2+) and Zn~(2+) in combination was additional in low concentration and was antagonistic in high concentration; the toxic effect of Pb~(2+) and Zn~(2+) in combination was additional in all concentration in our experiment; the toxic effect of Cd~(2+) and Pb~(2+) was additional in low concentration and synergistic in high concentration.
Using additive index method to know the time effects in the combination, we found that: when Cd~(2+) and Zn~(2+) were mixed in 1: 2 in weight, the AI of 24h, 48h and 72 h were minus and the combination effects were antagonistic; when Pb~(2+) and Zn~(2+) were mixed in 1: 2 in weight, the AI of 24h, 48h and 72 were minus and the combination effects were antagonistic; when Cd~(2+) and Pb~(2+) were mixed in 1: 1 in weight, the AI of 24h was positive and the combination effect was synergistic but the AI of 48h and 72 h were minus and the combination effects were antagonistic.
Transgenic worm PC72 was used as a biomonitor for the heavy metal contamination which has a high sensitivity. Results indicated: in all metals, the staining ratio changed with the lethal ratio and prolonging the processing time could enhance the staining ratio. We used score method to estimate the effect and found that score changed with the staining ratio. Compared with lethality method, staining method was more quick and sensitive.
The staining method also got a good result in the practice of monitoring the pollution of sea water in Xiamen. The staining ratio and the lethal ratio had correlation. The staining method could shorten the experiment time and enhance the sensitivity.
We used RNAi to study the genes which could affect the lifespan of C. elegans. Four RNAi vectors: R148.3a, Cnfkb (C. elegans nfkb homologus), sqv-4 and helicase were constructed.
The result of R148.3a RNAi showed that lost function of R148.3a could reduce the egg number, shorten the lifespan and decrease the immunity in N2, daf-2(e1370) and daf-16(mu86) mutants.
Cnfkb was a homologus of human nfκb light polypeptide gene in C.elegans. The similarity of this gene to nfκb was 31. 09%. Feeding N2 with Cnfκb RNAi bacteria could not change the lifespan but decreased N2 immunity, and the worm easy infected with S. aureus. The ability of cnfkb reduced the immunity depended on daf-16. Lost function of Cnfkb by RNAi couldn’t reduce the lifespan of daf-16(mu86) mutant further.
Nematodes fed on helicase (RNAi) bacteria laid less eggs and the lifespan of the nematode was 2.9 days shorter than normal nematode. Lost function of helicase by RNAi also made some larvae die at early stage.
When the nematodes were fed on sqv-4 (RNAi) bacteria, the number of eggs nematodes laid was reduced dramaticly. Lost function of sqv-4 the worm has the high embryo lethality. 96. 7 % of the eggs couldn’t become larva and died in the early stage. At same time the lifespan of the sqv-4 mutant by RNAi was 1.5 day shorter than N2.
The lifespan of lin-28(n1119) mutant was 8% longer than N2. The lifespan of lin-4(e912) mutant was 47% shorter than N2. The lifespan of let-7(n2853) mutant is 36% shorter than N2. The double mutant lin-28(n719);lin-4(e912) mutant lived no longer than lin-28(n1119) mutant. This implies that lin-28 works downstream of lin-4.
首先研究了三种重金属对秀丽小杆线虫的毒害作用。结果表明:24h时,三种重金属的毒性为Pb~(2+)>Cd~(2+)>Zn~(2+),半致死浓度分别为41774μg·L~(-1)、65945μg·L~(-1)、113323μg·L~(-1);48h时,三种重金属的毒性为Pb~(2+)>Cd~(2+)>Zn~(2+),半致死浓度分别为31567μg·L~(-1)、38679μg·L~(-1)、63423μg·L~(-1);72h时,三种重金属的毒性为Cd~(2+)≈Pb~(2+)>Zn~(2+),半致死浓度分别为24669μg·L~(-1)、24946μg·L~(-1)、40668μg·L~(-1)。三种重金属的毒性中Cd~(2+)随时间变化的速度最快,Pb~(2+)随时间变化的速度最慢;Pb~(2+)随浓度的变化最快,Zn~(2+)随浓度变化最慢。
重金属浓度对C. elegans联合作用的影响的实验表明:当Cd~(2+)和Zn~(2+)共同作用于线虫时,在低浓度时,呈现出简单的相加作用,高浓度时,呈现出拮抗作用;Pb~(2+)和Zn~(2+)共同作用于线虫时,四个浓度都呈现出简单的相加作用;当Cd2+和Pb~(2+)共同作用于线虫时,低浓度呈现出简单的相加效应,高浓度呈现出协同作用。
用AI指数法分析C. elegans暴露重金属的时间对重金属联合作用的影响,结果表明:当Cd~(2+)和Zn~(2+)以1:2的质量比混合时,在24h、48h和72h,其相加指数AI均小于0,联合作用表现为拮抗作用;当Pb~(2+)和Zn~(2+)以1:2的质量比混合时,在24h、48h和72h,其相加指数AI均小于0,联合作用表现为拮抗作用;当Cd~(2+)和Pb~(2+)以1:1的质量比混合时,在24h,其相加指数大于0,联合作用表现为协同作用;在48h和72h,其相加指数均小于0,联合作用表现为拮抗作用。
利用转基因线虫PC72,发展了一种可用于生物监测的高灵敏度的生物监测器。实验结果表明:在三种重金属的毒害下,染色率都呈现出随致死率变化而变化的特点,延长毒害时间能增加其染色率,空白对照即便染色时间延长也未曾出现明显的染色率增加。利用计分法评估线虫的染色效果,得分也和染色率呈现出相关变化。与致死率相比,用染色评估水体毒性的方法更加快捷、灵敏。
转基因线虫PC72染色的方法用于检验厦门海域海水水质取得了比较好的效果,检测结果显示染色率与致死率的变化趋势相同,染色率与致死率相关,但利用染色率检测能够缩短检测时间,提高监测灵敏度。
其次利用RNAi法研究了几种影响C. elegans寿命的基因。构建了四个RNA干扰载体:R148.3a、Cnfκb (C. elegans nfκb like)、sqv-4、helicase。将四种食物喂食线虫,研究其对线虫寿命的影响。
R148.3a (RNAi)菌喂食的线虫呈现出产卵数目下降,寿命缩短的特点,并且会使N2、daf-2(e1370)、daf-16(mu86)突变体的免疫力下降。
Cnfκb (RNAi)菌喂食N2线虫并不能使其寿命发生明显变化,但却会使其免疫力下降,易于被金黄色葡萄球菌感染。同时Cnfκb缺失使免疫力下降的作用依赖于daf-16,当daf-16突变时,Cnfκb不能使daf-16突变体的免疫力进一步下降。helicase(RNAi)菌喂食的线虫,产卵数目较对照N2下降,寿命缩短了2.9天。同时helicase功能的缺失会使线虫幼虫部分致死。
sqv-4(RNAi)菌喂食的线虫,产卵数目大幅度下降,并有很强的胚胎致死能力,所产的卵大多数无法孵化成幼虫。同时sqv-4干扰后,线虫的寿命会缩短约1.5天。
lin-28(n1119)突变体寿命相对于N2延长了8%,lin-4(e912)突变体寿命相对于N2缩短了47%,let-7(n2853)寿命相对N2缩短了36%。然而双突变体lin-28(n719); lin-4(e912)的寿命相对于lin-28(n1119)无明显改变,说明lin-28位于lin-4下游。
Caenorhabditis elegans was used in this paper to study the factors affecting lifespan.
We studied the toxic effects of three heavy metals Cd~(2+), Pb~(2+) and Zn~(2+) on C. elegans. The results indicated that: after C. elegans exposed to the three heavy metals 24hs, the toxicity of three heavy metals was Pb~(2+)> Cd~(2+)>Zn~(2+), LC 50 of three heavy metals was 41774μg·L~(-1), 65945μg·L~(-1) and 113323μg·L~(-1) respectively; after exposed to the three heavy metals 48hs, the toxicity of the three metals was: Pb~(2+)>Cd~(2+)>Zn~(2+), LC 50 of three heavy metals was 31567μg·L~(-1),38679μg·L~(-1) and 63423μg·L~(-1) respectively; after exposed to the three heavy metals 72hs, the toxicity of the three metals was Cd~(2+)≈Pb~(2+)>Zn~(2+), LC50 of three heavy metals was 24669μg·L~(-1), 24946μg·L~(-1) and 40668μg·L~(-1) respectively. In the three metals, the toxicity of Cd~(2+) changed most with the time and the toxicity of the Pb~(2+) changed least with the time. The toxicity of Pb~(2+) changed most with the concentration and the toxicity of Zn~(2+) changed least with the concentration.
The experiments of concentration effects in combination of two metals presented that: the toxic effect of Cd~(2+) and Zn~(2+) in combination was additional in low concentration and was antagonistic in high concentration; the toxic effect of Pb~(2+) and Zn~(2+) in combination was additional in all concentration in our experiment; the toxic effect of Cd~(2+) and Pb~(2+) was additional in low concentration and synergistic in high concentration.
Using additive index method to know the time effects in the combination, we found that: when Cd~(2+) and Zn~(2+) were mixed in 1: 2 in weight, the AI of 24h, 48h and 72 h were minus and the combination effects were antagonistic; when Pb~(2+) and Zn~(2+) were mixed in 1: 2 in weight, the AI of 24h, 48h and 72 were minus and the combination effects were antagonistic; when Cd~(2+) and Pb~(2+) were mixed in 1: 1 in weight, the AI of 24h was positive and the combination effect was synergistic but the AI of 48h and 72 h were minus and the combination effects were antagonistic.
Transgenic worm PC72 was used as a biomonitor for the heavy metal contamination which has a high sensitivity. Results indicated: in all metals, the staining ratio changed with the lethal ratio and prolonging the processing time could enhance the staining ratio. We used score method to estimate the effect and found that score changed with the staining ratio. Compared with lethality method, staining method was more quick and sensitive.
The staining method also got a good result in the practice of monitoring the pollution of sea water in Xiamen. The staining ratio and the lethal ratio had correlation. The staining method could shorten the experiment time and enhance the sensitivity.
We used RNAi to study the genes which could affect the lifespan of C. elegans. Four RNAi vectors: R148.3a, Cnfkb (C. elegans nfkb homologus), sqv-4 and helicase were constructed.
The result of R148.3a RNAi showed that lost function of R148.3a could reduce the egg number, shorten the lifespan and decrease the immunity in N2, daf-2(e1370) and daf-16(mu86) mutants.
Cnfkb was a homologus of human nfκb light polypeptide gene in C.elegans. The similarity of this gene to nfκb was 31. 09%. Feeding N2 with Cnfκb RNAi bacteria could not change the lifespan but decreased N2 immunity, and the worm easy infected with S. aureus. The ability of cnfkb reduced the immunity depended on daf-16. Lost function of Cnfkb by RNAi couldn’t reduce the lifespan of daf-16(mu86) mutant further.
Nematodes fed on helicase (RNAi) bacteria laid less eggs and the lifespan of the nematode was 2.9 days shorter than normal nematode. Lost function of helicase by RNAi also made some larvae die at early stage.
When the nematodes were fed on sqv-4 (RNAi) bacteria, the number of eggs nematodes laid was reduced dramaticly. Lost function of sqv-4 the worm has the high embryo lethality. 96. 7 % of the eggs couldn’t become larva and died in the early stage. At same time the lifespan of the sqv-4 mutant by RNAi was 1.5 day shorter than N2.
The lifespan of lin-28(n1119) mutant was 8% longer than N2. The lifespan of lin-4(e912) mutant was 47% shorter than N2. The lifespan of let-7(n2853) mutant is 36% shorter than N2. The double mutant lin-28(n719);lin-4(e912) mutant lived no longer than lin-28(n1119) mutant. This implies that lin-28 works downstream of lin-4.
引文
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