【作者】 李艳梅；

【导师】 徐世清； 袁红霞； 司马杨虎；

【作者基本信息】 苏州大学 ， 发育生物学， 2011， 硕士

【摘要】 铅是一种对人类和小鼠等哺乳类动物以及水生动物存在显著不良影响的重金属,但铅对无脊椎动物、特别是对昆虫的影响鲜有研究报道。zonghe特异蛋白基因只在家蚕是鳞翅目模式昆虫,也是一种应用广泛的实验动物。本实验以幼虫期全龄添食Pb（NO3）2对家蚕进行铅暴露,调查了Pb²⁺在家蚕体内组织的蓄积及其对家蚕生长和生殖发育的影响,重点调查了Pb²⁺暴露后家蚕组织的抗氧化相关基因和生殖发育相关基因的表达和酶活性的变化。主要获得了以下结果:1.铅胁迫家蚕的Pb²⁺蓄积有明显组织和性别差异原子吸收光谱法（GFAAS）测定结果显示,铅暴露后家蚕5龄幼虫的消化吸收组织中肠和马氏管,以及性腺组织中,Pb²⁺含量随着添食Pb（NO3）2浓度的提高而增加,有明显的蓄积效应;蚕粪中Pb²⁺含量也随着添食Pb（NO3）2浓度的提高显著增加,且具有浓度梯度效应。说明铅胁迫家蚕幼虫具有较强的排泄解毒能力,同时铅离子能够在幼虫生殖腺蓄积。在相同暴露浓度时,家蚕5龄雌性幼虫的中肠内Pb²⁺含量高于雄性,但两性中肠内Pb²⁺含量都随着暴露浓度的提高而显著增加。两性5龄幼虫马氏管中的Pb²⁺含量与消化管中接近,但随暴露浓度的提高呈现先升高后降低的趋势,40mg/kg暴露浓度下,雄性幼虫马氏管中Pb²⁺的含量显著高于雌性,提示雄性通过马氏管排泄血液中Pb²⁺能力比雌雄幼虫高。5龄幼虫生殖腺中的Pb²⁺的蓄积量显著低于消化管和马氏管,饲料中Pb²⁺浓度为40-160mg/kg时,雌性生殖腺中Pb²⁺含量均高于雄性,暗示雌性生殖腺可能受到Pb²⁺的影响更大。2.家蚕脂肪体、中肠和生殖腺中抗氧化解毒酶活性及相关基因表达对铅胁迫敏感昆虫受重金属胁迫后,抗氧化解毒酶系成员有通过联合作用来清除铅离子胁迫产生的自由基危害的作用。本实验在10-160mg/kg铅暴露后,家蚕5龄6 d幼虫体内的SOD活性,在脂肪体和中肠组织中上升,而在生殖腺中下降;CAT活性在脂肪体中上升,在中肠和生殖腺中下降。在实验浓度范围内,Sod基因的表达水平在脂肪体、中肠和生殖腺内均出现下降趋势,而Cat基因在3个调查组织中都有不同程度的上调表达,但这2个基因表达水平的变化与各自蛋白酶活性的变化不同步,显示体内SOD和CAT酶活性的升高,不完全依赖基因转录与蛋白质合成。推测铅离子胁迫后,家蚕幼虫脂肪体具有上升抗氧化酶活性解毒;而生殖腺的抗氧化酶活性下降,有氧化受损的可能。谷胱甘肽S-转移酶（GST）和谷胱甘肽过氧化物酶（GSH-Px）是生物体内重要的解毒酶。正常家蚕5龄6 d幼虫生殖腺中的GST酶活性分别是脂肪体和中肠中的20.0倍、34.6倍,GSH-Px酶活性分别是脂肪体和中肠中的46.6倍和57.5倍。幼虫食下含Pb²⁺ 10 mg/kg以上的人工饲料时,生殖腺中2种解毒酶的活性成倍下降;饲料含Pb²⁺ 20-80 mg/kg时,生殖腺中的Gstd1和Gsh-Px基因mRNA转录水平也显著下调,且雄性比雌性更加显著。幼虫食下含Pb²⁺ 10-160 mg/kg的人工饲料时,脂肪体中2种解毒酶活性显著升高,Pb²⁺对2种酶的影响浓度分别为10-160 mg/kg和20-80 mg/kg,但在20-80 mg/kg Pb²⁺范围内,Gsh-Px和Gstd1基因mRNA的转录却受到抑制,并且雌性幼虫脂肪体中的GSH-Px酶活性及基因mRNA转录水平、雄性幼虫脂肪体中的GST酶活性及其基因mRNA转录水平对Pb²⁺暴露更加敏感。幼虫食下含Pb²⁺ 20 mg/kg以上的人工饲料时,中肠GST酶活性显著上升,而GSH-Px酶活性变化较小,性别间差异不明显;Gstd1基因mRNA转录水平有上调趋势,Gsh-Px基因mRNA转录水平则受到显著抑制。结果显示,家蚕幼虫生殖腺虽然有比脂肪体和中肠更强的GST酶和GSH-Px活性,但对Pb²⁺的毒害敏感,雄性比雌性受影响更大,生殖腺通过抗氧化防御系统抵御Pb²⁺毒害的作用很弱。3.铅胁迫对家蚕卵黄蛋白原基因和卵特异蛋白基因表达有显著影响家蚕卵黄蛋白原基因Vg与卵特异蛋白基因Esp是家蚕的2个生殖发育相关的重要基因。EST电子表达谱结果显示,家蚕Vg只在吐丝期和蛹期脂肪体与卵巢组织中表达,家蚕Esp只在蛹期卵巢中表达。为了解重金属铅是否存在环境雌激素效应,本文实验调查了铅暴露后,Vg和Esp基因在其特异时空的表达谱。结果表明,在20-160 mg/kg Pb²⁺暴露浓度范围内,家蚕5龄雌性幼虫脂肪体Vg基因的表达量较未接触铅离子的对照蚕呈下降趋势;20 mg/kg Pb²⁺低暴露浓度下,家蚕雄性5龄幼虫脂肪体中Vg基因的表达量为对照的1.15倍,出现了Vg基因的微弱诱导表达,但Pb²⁺暴露浓度提高到40-160 mg/kg时,雄蚕脂肪体中Vg基因的表达量比对照显著降低。幼虫期正常两性家蚕生殖腺中都有Vg基因的微弱表达,其中雌性的表达水平显著高于对照。在20 mg/kg组雄性生殖腺中Vg基因的表达水平较对照显著升高,和幼虫脂肪体中的结果一致,暗示该铅暴露浓度有诱导雄性Vg基因表达的环境雌激素效应可能。家蚕蛹期脂肪体中Vg基因的转录活性显著高于幼虫期,而且蛹期有显著的性别差异,雄蛹脂肪体中Vg基因的表达量仅为雌性的3.59%。20-160 mg/kg Pb²⁺暴露后,家蚕雄蛹脂肪体中Vg基因转录水平显著下调（P<0.01）,而雌蛹脂肪体中Vg基因的表达量显著上调（P<0.01）,但其中20 mg/kg组显著下降（P<0.01）。蛹期生殖腺中Vg基因的表达量性别差异小于幼虫期;铅离子暴露后,3d龄蚕蛹卵巢中Vg表达水平显著下降,而睾丸中Vg表达水平在低水平波动,但160 mg/kg浓度组出现显著下调。家蚕幼虫期与蛹期Vg基因的转录受铅离子暴露影响方式可能不同。家蚕蛹期生殖腺中Esp基因mRNA水平调查结果显示,实验浓度范围内铅离子胁迫,造成了雌性生殖腺中Esp基因表达的显著下调,但没有诱导雄性家蚕生殖腺中Esp基因表达。更多还原

【Abstract】 Bombyx mori is the model insect of lepioloptera and it is a widely used experimental animals. Research on heavy metals like lead on mamals （such as human and mouse ）and aquatic organisms is common, but relatively little in invertebrates. Research on the insect was less. In our experiment, we used Pb（NO3）2 as the resource of Pb²⁺ to treat Bombyx mori during the whole laval stage. We investgested the accumulation of Pb²⁺ in tissues of silkworm, the basic physical and reproductive development-related indicators and the effects of lead exposure on the expression level of antioxidant genes and the reproductive development related genes and activity of antioxidant enzymes in tissues of Bombyx mori. The major results are as follows:1.When forced by lead（Pb）, a heavy metal, the accumulations of Pb²⁺ differs apparently in different tissues of silkworms. A different rule appeared between males and females.In the midgut, gonad and malpighian tube of Bombyx mori, the content of Pb²⁺ increased with improved concentration of Pb（NO3）2 in food which was measured by GFAAS, showing an obvious Pb²⁺ accumulation effect. The content of Pb²⁺ in excrements of silkworms also increased apparently with an effect of concentration gradient. This showed that,the larva of Bombyx mori had a strong excreting ability to detoxification and there were accumulations of Pb²⁺ in gonads.The content of Pb²⁺ in midgut of both sexes obviously increased with the improved exposion concentration of Pb²⁺ , presenting an accordance trend. However, when in the same exposion concentration of Pb²⁺ , the content of Pb²⁺ in midgut of females was higher than males.The content of Pb²⁺ in malpighian tube was close to that in alimentary canal. Meanwhile, with the improved exposion concentration of Pb²⁺, the content of Pb²⁺ in both sexes showed a thend of rising at first and then decreasing. Under the condition of 40 mg/kg exposion concentration, the malpighian of male larvas had a higher excretion ability of Pb²⁺ than that of females.The accumulated amount of Pb²⁺ in gonad was obviously lower than that in alimentary canal and malpighian tube. When the concentration of Pb²⁺ in forage was 40-160 mg/kg, the content of Pb²⁺ in female gonad was higher than males, suggesting that female gonads were influenced greater by Pb²⁺ probably.2.The antioxidant detoxifying enzymes activity in fat body, midgut and gonad of Bombyx mori and expressions of relative genes were sensitivity to Pb²⁺ forcing.After forced by Pb²⁺ ,a heavy metal , silkworms would clear the radical created by Pb²⁺ by koinonia of antioxidant detoxifying enzymes like SOD、CAT、GST and GSH-Px in order to reduce the harm to organism, which was also a flexible response to overcoming harmful environment and preventing toxicosis.When dealed with heavy metals, SOD and CAT in Bombyx mori’s fat body and midgut have poison excited and poison inhibit effect, but the ranges were different. When exposed in 10-160 mg/kg of lead, the activity of SOD in both fat body and midgut of Bombyx mori’s 5 instar larvaes would significantly rise, however, they were inhibited in gonades. Moreover, the active of CAT in fat body would significantly rise in fatbody under 10-160 mg/kg lead exposure,while the active was inhibited in midgut and gonades.Detected the mRNA expression level of Sod and Cat, we found that the Sod expression levels in fatbody、midgut and gonads of Bombyx mori’s 5 instar larvaes were all decreasing. Differently, the expression of Cat were up-regulated in the three tissues showing that rised of enzyme active were not completely rely on the gene transcription and the synthesis of proteins. There were asynchronism between the changes of enzyme activities of CAT and SOD and mRNA expression level changes of relative genes. It suggested that after lead stress the fatbody of silkworm could rise the enzyme active to antidote the stress; But the enzyme active was depressed in gonads suggesting that it was easier to be harmed.Glutathione S-transferase （GST） and glutathione peroxidase （GSH-Px） are important detoxifying enzymes in vivo. In order to study the influencing mechanism of heavy metal lead on lepidopterous insect, we fed the Bombyx mori with artificial diet added with Pb（NO3）2 for Pb²⁺ exposure. Enzyme activity assay and real-time PCR were employed to investigate the activity of enzymes and the mRNA transcription level of related genes in tissues of day 6 larvae of the 5th instar. In normal day 6 larvae of the 5th instar, GST enzyme activity in gonad was 20.0 and 34.6 times of that in fat body and midgut respectively, and GSH-Px enzyme activity was 46.6 and 57.5 times of that in fat body and midgut respectively. After the larvae fed on artificial diet containing 10 mg/kg or higher Pb²⁺, activity of the two enzymes in gonad decreased by many times. When the artificial diet contained 20-80 mg/kg Pb²⁺, mRNA transcriptional levels of Gstd1 gene and Gsh-Px gene were also significantly down-regulated, with more significant down-regulation in males over females. After the larvae fed on artificial diet containing 10-160 mg/kg Pb²⁺, the activity of GST and GSH-Px in fat body increased significantly. Effective Pb²⁺ concentrations to these two enzymes could be divided into two ranges: 10-160 mg/kg and 20-80 mg/kg, respectively. However, within the range of 20-80 mg/kg Pb²⁺, mRNA transcriptions of Gsh-Px and Gstd1 were inhibited. Moreover, GSH-Px activity and mRNA level of Gsh-Px in fat body of female larvae, and GST activity and mRNA level of Gstd1 in fat body of male larvae were more sensitive to Pb²⁺ exposure. After 20 mg/kg or more Pb²⁺ exposure, GST activity in the midgut increased dramatically, but GSH-Px activity only changed slightly and there was no significant difference between male and female larvae. mRNA transcriptional level of Gstd1 gene had a trend of up-regulation, while that of Gsh-Px was significantly down-regulated. The results suggested that, although silkworm larvae have higher enzyme activity of GST and GSH-Px in gonads than in midgut and fat body, they are sensitive to toxicity of Pb²⁺. Nevertheless, male larvae are more sensitive than female ones, and the antioxidant defense system in gonads is quite weak in resisting toxicity from Pb²⁺.3.Lead stress had a significant impact on the expression of Bombyx mori vitellogenin gene and egg specific protein gene.Vitellogenin gene and egg specific protein gene were two key genes of Bombyx mori’s reproductive development. EST electric expression profiles showed that Vg gene only expressed in fat body and ovary on both spinning stage and pupa stage of silkworm, and specific protein gene Esp only expressed in testis on pupa stage.In order to investgate if lead have the enviromental estrogen effect, the paper study the specific expression profiles of Vg and Esp. The results indicated that expression of Vg gene trended to decrease than normal with disposals of Pb²⁺ concentrations within 20-160 mg/kg in the fat body of 5L female silkworm. Meanwhile, the expression of all disposed groups dropped dramatically except that of 20 mg/kg concentration group in male silkworm which turned out to be up significantly and it was as 1.15 times as that of control group in male. It can be driven that faint induction of Vg gene expression took place in male fat body in the concentration of 20 mg/kg, though it was still in a relatively low level compared with that of female fat body. When the lead exposure concentration rised to 40-160 mg/kg, expression of Vg gene in fatbody of males trended to decrease remarkably than normal. In gonads of female and male Bombyx mori there were little expression of Vg, but higer in females than males. In testis of silkworms under 20 mg/kg lead exposure, the Vg expression level was rised absolutely, which was coincident with the result of fatbody of male larves suggesting that the concentration of lead had the enviromental estrogen effect of induction expression of Vg in males.The expression of Vg gene in fat body on pupa stage of normal silkworm was tremendously higher than that of larva fat body and had a sex difference. The expression of Vg gene in all disposed groups rose drastic （P<0.01） in comparison to the control except the group exposed to the concentration of 20 mg/kg which decreased drastic （P<0.01）. The expression of Vg gene in male fat body on pupa stage of normal silkworm was also lower than that of female fat body, which was tantamount to 3.59% of the female control. The mRNA expression level of Vg gene in all disposed male fat body on larva stage dropped dramatically within the Pb²⁺ concentration of 20 mg/kg-160 mg/kg compared with that of the control. Therefore, a disparity existed in mRNA expression of Vg gene in fat body between pupal and larva stages. The difference between sexes of pupa stage was less than the stage of larva; After lead exposure, Vg expression in ovary of 3d pupa depressed significantly while fluctuation at low levels intesis. Therefore, a disparity existed in influence pattern of Vg gene between pupal and larva stages.Investigation on the mRNA expression level of Esp gene in genital gland on pupal stage showed that Pb²⁺ concentrations used in this study made expression of Esp gene down in females, while Esp gene expression wasn’t induced in males. Therefore, Esp gene is different to Vg gene, thus Esp gene can not be used as the indicators of environmentally endocrine disruptors.更多还原