BmNPV脅迫下抗性與非抗性家蠶血液中金屬絡(luò)合物的差異分析
發(fā)布時(shí)間：2018-09-14 17:56
【摘要】：蠶桑業(yè)在中國(guó)已有五千多年歷史,是中國(guó)乃至亞洲一項(xiàng)重要的經(jīng)濟(jì)產(chǎn)業(yè)。家蠶核型多角體病毒(BmNPV)感染帶來的的家蠶核型多角體病毒病是養(yǎng)蠶過程中比較常見的一類傳染性蠶病,感染的家蠶產(chǎn)絲量嚴(yán)重下降甚至死亡�？茖W(xué)家已經(jīng)篩選并培育出多種對(duì)家蠶核型多角體病毒具有抗性的家蠶品種,并投入到蠶桑生產(chǎn)中。目前,已有許多關(guān)于家蠶與BmNPV感染、抗性的研究,主要研究方向?yàn)榧倚Q基因組學(xué)和家蠶蛋白質(zhì)組學(xué),也有學(xué)者將轉(zhuǎn)基因技術(shù)應(yīng)用到家蠶抗BmNPV品種的培育中。但還未有報(bào)道從家蠶金屬蛋白組學(xué)方面探討家蠶對(duì)BmNPV的抗性機(jī)理。金屬蛋白組學(xué)是蛋白質(zhì)組學(xué)的重要組成部分,它主要研究與蛋白質(zhì)結(jié)合的金屬離子的功能以及其在生物體內(nèi)的分布定位。對(duì)金屬組學(xué)的研究通常需要特定的分離檢測(cè)方法,以完成金屬蛋白的定性定量分析。我們選取了BmNPV的高抗家蠶品種及其非抗性家蠶品種,通過桑葉將病毒喂食給家蠶,利用高效液相色譜和電感耦合等離子體質(zhì)譜,分離和檢測(cè)抗性、非抗性家蠶及喂食病毒后家蠶血液的差異金屬絡(luò)合物,試圖從金屬配合物方面為家蠶抗BmNPV的研究提供一個(gè)新的突破口,同時(shí),為家蠶體內(nèi)金屬蛋白的分析研究提供更加簡(jiǎn)便有效的分離收集方法。我們的研究結(jié)果如下:1.喂食病毒后的抗性和非抗性品種家蠶,CuMnZnRb四種元素含量和連續(xù)六天的變化趨勢(shì)基本不變。非抗性家蠶血液中K、Ca、Mg、Fe和Ni五種元素在喂食BmNPV后的第三天的含量和趨勢(shì)出現(xiàn)明顯差異,抗性家蠶的K、Ca、Mg、Fe和Ni五種元素在喂食BmNPV后第五天差異明顯。2.利用篩選出的體積排阻色譜分離條件,運(yùn)用體積排阻色譜法(SEC)對(duì)四組家蠶血淋巴進(jìn)行了初步的分離檢測(cè)。結(jié)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蠶的SEC譜圖的8min峰差異明顯�？剐约倚Q感染BmNPV后SEC圖譜中8min的其中一個(gè)峰檢測(cè)不到。在喂食病毒后第五天,感染BmNPV后的抗性家蠶的SEC圖譜的8min峰發(fā)生明顯改變。非抗性家蠶感染病毒后保留時(shí)間10~12min的其中兩個(gè)峰檢測(cè)不到。3.比較分析四組家蠶血淋巴的SEC-ICP-MS檢測(cè)圖,發(fā)現(xiàn)添食BmNPV后的第三天,與未感染BmNPV的非抗性家蠶相比,感染病毒后的非抗性家蠶血淋巴的SEC-ICP-MS圖譜的11min峰幾乎檢測(cè)不到。喂食BmNPV后的第五天,抗性、非抗性家蠶及其感染BmNPV后血淋巴的Mn、Zn和Cu元素的SEC-ICP-MS檢測(cè)圖都出現(xiàn)明顯差異。4.利用葡聚糖G100凝膠柱對(duì)體積排阻色譜分離結(jié)果中的8min組分進(jìn)行了重點(diǎn)分離并收集分離組分。收集的分離組分的SEC檢測(cè)結(jié)果在0~30min只出現(xiàn)一個(gè)8min譜峰,分離組分的SEC-ICP-MS檢測(cè)結(jié)果和家蠶血液檢測(cè)結(jié)果基本一致。并且證明運(yùn)用半制備的葡聚糖凝膠柱分離收集8min組分的分離方法比傳統(tǒng)的HPLC-餾分收集器收集的餾分濃度高,不需要再次對(duì)餾分進(jìn)行合并濃縮,為家蠶金屬蛋白的研究提供更加簡(jiǎn)便有效的分離收集方法。
[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.
【學(xué)位授予單位】：江蘇科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S884.51
本文編號(hào)：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.
【學(xué)位授予單位】：江蘇科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S884.51
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