BmNPV胁迫下抗性与非抗性家蚕血液中金属络合物的差异分析
发布时间：2018-09-14 17:56
【摘要】：蚕桑业在中国已有五千多年历史,是中国乃至亚洲一项重要的经济产业。家蚕核型多角体病毒(BmNPV)感染带来的的家蚕核型多角体病毒病是养蚕过程中比较常见的一类传染性蚕病,感染的家蚕产丝量严重下降甚至死亡。科学家已经筛选并培育出多种对家蚕核型多角体病毒具有抗性的家蚕品种,并投入到蚕桑生产中。目前,已有许多关于家蚕与BmNPV感染、抗性的研究,主要研究方向为家蚕基因组学和家蚕蛋白质组学,也有学者将转基因技术应用到家蚕抗BmNPV品种的培育中。但还未有报道从家蚕金属蛋白组学方面探讨家蚕对BmNPV的抗性机理。金属蛋白组学是蛋白质组学的重要组成部分,它主要研究与蛋白质结合的金属离子的功能以及其在生物体内的分布定位。对金属组学的研究通常需要特定的分离检测方法,以完成金属蛋白的定性定量分析。我们选取了BmNPV的高抗家蚕品种及其非抗性家蚕品种,通过桑叶将病毒喂食给家蚕,利用高效液相色谱和电感耦合等离子体质谱,分离和检测抗性、非抗性家蚕及喂食病毒后家蚕血液的差异金属络合物,试图从金属配合物方面为家蚕抗BmNPV的研究提供一个新的突破口,同时,为家蚕体内金属蛋白的分析研究提供更加简便有效的分离收集方法。我们的研究结果如下:1.喂食病毒后的抗性和非抗性品种家蚕,CuMnZnRb四种元素含量和连续六天的变化趋势基本不变。非抗性家蚕血液中K、Ca、Mg、Fe和Ni五种元素在喂食BmNPV后的第三天的含量和趋势出现明显差异,抗性家蚕的K、Ca、Mg、Fe和Ni五种元素在喂食BmNPV后第五天差异明显。2.利用筛选出的体积排阻色谱分离条件,运用体积排阻色谱法(SEC)对四组家蚕血淋巴进行了初步的分离检测。结果表明,在喂食病毒后的第三天,未感染病毒的抗性和非抗性家蚕的SEC谱图的8min峰差异明显。抗性家蚕感染BmNPV后SEC图谱中8min的其中一个峰检测不到。在喂食病毒后第五天,感染BmNPV后的抗性家蚕的SEC图谱的8min峰发生明显改变。非抗性家蚕感染病毒后保留时间10~12min的其中两个峰检测不到。3.比较分析四组家蚕血淋巴的SEC-ICP-MS检测图,发现添食BmNPV后的第三天,与未感染BmNPV的非抗性家蚕相比,感染病毒后的非抗性家蚕血淋巴的SEC-ICP-MS图谱的11min峰几乎检测不到。喂食BmNPV后的第五天,抗性、非抗性家蚕及其感染BmNPV后血淋巴的Mn、Zn和Cu元素的SEC-ICP-MS检测图都出现明显差异。4.利用葡聚糖G100凝胶柱对体积排阻色谱分离结果中的8min组分进行了重点分离并收集分离组分。收集的分离组分的SEC检测结果在0~30min只出现一个8min谱峰,分离组分的SEC-ICP-MS检测结果和家蚕血液检测结果基本一致。并且证明运用半制备的葡聚糖凝胶柱分离收集8min组分的分离方法比传统的HPLC-馏分收集器收集的馏分浓度高,不需要再次对馏分进行合并浓缩,为家蚕金属蛋白的研究提供更加简便有效的分离收集方法。
[Abstract]:Silkworm industry has a history of more than 5000 years in China, and is an important economic industry in China and even Asia. Silkworm nuclear polyhedrosis virus (BmNPV) infection is one of the most common infectious silkworm diseases in silkworm rearing. The silkworm silk production of infected silkworms has declined seriously and even died. A variety of silkworm varieties resistant to Bombyx mori nuclear polyhedrosis virus have been developed and put into silkworm production. At present, there are many studies on silkworm and BmNPV infection and resistance. The main research directions are silkworm genomics and silkworm proteomics. Some scholars have applied transgenic technology to the breeding of silkworm varieties resistant to BmNPV. Metalloproteinomics is an important part of proteomics. It mainly studies the function of metal ions bound to proteins and their distribution in organisms. Metalloproteinomics studies usually require specific separation. We selected BmNPV resistant silkworm varieties and their non-resistant silkworm varieties, fed the virus to the silkworm through mulberry leaves, separated and detected the resistant, non-resistant silkworm and the different gold in the blood of the silkworm after feeding the virus by high performance liquid chromatography and inductively coupled plasma mass spectrometry. The results are as follows: 1. Four elements of resistant and non-resistant silkworms, namely, Cu? Mn? Zn? Rb, are contained in the virus. The content and trend of K, Ca, Mg, Fe and Ni in the blood of non-resistant silkworms were significantly different on the third day after feeding BmNPV, and the content and trend of K, Ca, Mg, Fe and Ni in the blood of resistant silkworms were significantly different on the fifth day after feeding BmNPV. 2. Using the screened conditions of volume exclusion chromatography, the content and trend of these five elements were significantly different. Four groups of silkworm hemolymph were isolated and detected by volume exclusion chromatography (SEC). The results showed that there was a significant difference in the 8-minute peak of SEC spectra between non-infected and non-infected silkworms on the third day after feeding the virus. One of the peaks in SEC spectra of 8-minute after BmNPV infection was not detected in resistant silkworms. Five days later, the SEC peaks of resistant silkworms infected with BmNPV changed significantly. Two of the peaks remained for 10-12 minutes after infection could not be detected. 3. The SEC-ICP-MS patterns of hemolymph of four groups of silkworms were compared and analyzed. It was found that on the third day after feeding BmNPV, the SEC peaks of resistant silkworms infected with BmNPV were lower than those of non-resistant silkworms infected with BmNPV. The 11min peak of the SEC-ICP-MS pattern of the non resistant silkworm hemolymph was almost impossible to detect. After fifth days of feeding BmNPV, there was a significant difference in the SEC-ICP-MS detection map of the Mn, Zn and Cu elements of the hemolymph after the resistance, non resistant silkworm and BmNPV infection. The components were separated and collected. The SEC test results of the isolated components showed only one 8min peak in 0~30min. The SEC-ICP-MS test results of the separation components were basically the same as those of the silkworm blood test. It was proved that the separation method of 8min components separated by the half prepared Sephadex gel column was better than that of the traditional H. The concentration of fractions collected by PLC-fraction collector is high, so it is not necessary to concentrate the fractions again, which provides a more convenient and effective method for the separation and collection of silkworm metalloproteins.
【学位授予单位】：江苏科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S884.51
本文编号：2243447
[Abstract]:Silkworm industry has a history of more than 5000 years in China, and is an important economic industry in China and even Asia. Silkworm nuclear polyhedrosis virus (BmNPV) infection is one of the most common infectious silkworm diseases in silkworm rearing. The silkworm silk production of infected silkworms has declined seriously and even died. A variety of silkworm varieties resistant to Bombyx mori nuclear polyhedrosis virus have been developed and put into silkworm production. At present, there are many studies on silkworm and BmNPV infection and resistance. The main research directions are silkworm genomics and silkworm proteomics. Some scholars have applied transgenic technology to the breeding of silkworm varieties resistant to BmNPV. Metalloproteinomics is an important part of proteomics. It mainly studies the function of metal ions bound to proteins and their distribution in organisms. Metalloproteinomics studies usually require specific separation. We selected BmNPV resistant silkworm varieties and their non-resistant silkworm varieties, fed the virus to the silkworm through mulberry leaves, separated and detected the resistant, non-resistant silkworm and the different gold in the blood of the silkworm after feeding the virus by high performance liquid chromatography and inductively coupled plasma mass spectrometry. The results are as follows: 1. Four elements of resistant and non-resistant silkworms, namely, Cu? Mn? Zn? Rb, are contained in the virus. The content and trend of K, Ca, Mg, Fe and Ni in the blood of non-resistant silkworms were significantly different on the third day after feeding BmNPV, and the content and trend of K, Ca, Mg, Fe and Ni in the blood of resistant silkworms were significantly different on the fifth day after feeding BmNPV. 2. Using the screened conditions of volume exclusion chromatography, the content and trend of these five elements were significantly different. Four groups of silkworm hemolymph were isolated and detected by volume exclusion chromatography (SEC). The results showed that there was a significant difference in the 8-minute peak of SEC spectra between non-infected and non-infected silkworms on the third day after feeding the virus. One of the peaks in SEC spectra of 8-minute after BmNPV infection was not detected in resistant silkworms. Five days later, the SEC peaks of resistant silkworms infected with BmNPV changed significantly. Two of the peaks remained for 10-12 minutes after infection could not be detected. 3. The SEC-ICP-MS patterns of hemolymph of four groups of silkworms were compared and analyzed. It was found that on the third day after feeding BmNPV, the SEC peaks of resistant silkworms infected with BmNPV were lower than those of non-resistant silkworms infected with BmNPV. The 11min peak of the SEC-ICP-MS pattern of the non resistant silkworm hemolymph was almost impossible to detect. After fifth days of feeding BmNPV, there was a significant difference in the SEC-ICP-MS detection map of the Mn, Zn and Cu elements of the hemolymph after the resistance, non resistant silkworm and BmNPV infection. The components were separated and collected. The SEC test results of the isolated components showed only one 8min peak in 0~30min. The SEC-ICP-MS test results of the separation components were basically the same as those of the silkworm blood test. It was proved that the separation method of 8min components separated by the half prepared Sephadex gel column was better than that of the traditional H. The concentration of fractions collected by PLC-fraction collector is high, so it is not necessary to concentrate the fractions again, which provides a more convenient and effective method for the separation and collection of silkworm metalloproteins.
【学位授予单位】：江苏科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S884.51
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