苹斑芫菁Mylabris calida Palla斑蝥素合成期体内可溶性蛋白的比较蛋白组学分析
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摘要
斑蝥素(Cantharidin, C10H12O4)是鞘翅目(Coleoptera)芫菁科(Meloidae)昆虫分泌的萜类防御物质,化学名:六氢-3a,7a-二甲基-4,7-环氧异苯并呋喃-1,3-二酮。研究发现它有杀虫作用,但资源问题无法解决。目前斑蝥素的生产采用人工提取和化学合成两种方法,两者都存在明显的局限性,无法实现斑蝥素的大规模生产,生物合成是解决这一问题的有效途径。但由于芫菁生理系统和斑蝥素产生过程的复杂性,到目前为止人们对斑蝥素的合成机理还知之甚少。以前的研究主要集中于斑蝥素含量检测、斑蝥素衍生物合成及毒理等方面,有关斑蝥素生物合成方面的研究少有报道,体内合成是一个极其复杂的过程,是资源昆虫研究中的热点问题之一。
蛋白质组学是在蛋白水平大规模研究基因表达的产物,直接研究蛋白的表达水平和相关蛋白的活性状态。芫菁雄虫羽化后0～30天无论在形态还是斑蝥素合成量方面都发生了显著变化。因此本研究利用SDS-PAGE、双向电泳和质谱相结合的蛋白质组学研究方法对不同时期的苹斑芫菁Mylabris calida雄虫的蛋白表达进行了比较研究,以进一步研究这个复杂的生物学过程,同时也为阐释斑蝥素合成机理提供信息。在综述蛋白质组学的研究现状和进展,包括蛋白质组学研究中的关键技术—2DE、生物质谱以及生物信息学的进展和现状,概述了斑蝥素的研究进展和有待进行研究的内容的基础上,提出了本课题工作的研究方向和目的。
本研究主要获得如下结果:
1.确定了研究芫菁蛋白质组学的技术条件。本试验采用不同的方法对芫菁可溶性蛋白进行了双向电泳研究,从样品提取液、等点聚焦条件、第二向聚焦条件、试验中常见的问题等因素对芫菁双向电泳研究体系进行了全面的分析和优化。结果发现,对于芫菁可溶性蛋白,采用尿素裂解液液氮粉碎高速离心法比采用咪唑缓冲液组织匀浆法更为适合,蛋白损失少,图谱均匀清晰。因此本试验经过各种条件优化确定了一套适合于芫菁可溶性蛋白样品分析的双向电泳研究方法,利用这种方法所获得的双向电泳图谱中的蛋白点分辨率和重复性更高。在建立蛋白点肽质量指纹图谱分析体系时,确定了胶内消化过程中的胰酶用量,挖点大小、基质辅助激光解吸飞行时间质谱的参数选择等试验条件,构建了芫菁质谱分析研究平台。
2.斑蝥素合成期芫菁可溶性蛋白样品的SDS-PAGE电泳分析。通过观察斑蝥素合成增长期的蛋白质表达变化,探讨相关合成酶的分布范围。利用蛋白质凝胶电泳技术,对苹斑芫菁在羽化后0～25天的体内可溶性蛋白进行了分析。经蛋白质电泳图谱分析发现,有3条蛋白带的表达量在芫菁雄虫中明显大于雌虫,这些蛋白的分子量分别为:80KD,45KD,30 KD;其中分子量约为80 KD,45KD的蛋白带在芫菁雄虫羽化后第2天出现,其浓度不断的增加。30KD的蛋白带在芫菁雄虫羽化后1小时出现,主要作用是调控成虫的生长和变态发育。推测蛋白组份45KD中的某种或几种关联蛋白质在芫菁雄虫羽化后斑蝥素合成增长期发挥了重要作用。
3.斑蝥素合成期的蛋白质双向电泳及质谱鉴定。前人研究发现芫菁大量合成斑蝥素发生在羽化后,通过对这一时期的芫菁体内可溶性蛋白研究,探索斑蝥素合成相关酶蛋白的种类和合成机理。采用蛋白质双向电泳和飞行质谱技术,分析了合成前期、大量合成早期和大量合成末期的蛋白质组成的变化。研究发现,合成前期蛋白质具有区别于其它时期蛋白质的明显特征:蛋白质大多分布在PI值4-7、分子量20-80KD的区域,且分布不均匀,主要集中在偏酸性一端,20～45KD蛋白质差异明显。芫菁羽化后1小时的蛋白质点数目为471个,羽化后第2天增加到569个,羽化后第4天蛋白点数目增加到645个;随着芫菁进入斑蝥素大量合成期,蛋白点数目明显增加,大量合成早期和合成末期蛋白点数为827个和999个,比合成准备期增长了25%以上。结果显示:三个时期蛋白质组成的变化趋势刚好与芫菁合成斑蝥素含量显著峰值变化相吻合,表明芫菁体内斑蝥素的合成是多种蛋白参与的复杂过程,这些蛋白分子量范围在20～45 kD。利用MALDI-TOF MS对明显差异蛋白进行鉴定时,发现这些蛋白质分别是Actin-87E isoform 1(肌动蛋白87E亚型1)、putative secreted protein(分泌蛋白)、LD04994p(肌动蛋白,LD04994p)、GM19279(肌动蛋白,GM19279)、cytoplasmic actin(胞质型肌动蛋白)、molybdenum cofactor biosynthesis protein C(钼辅因子合成蛋白C)、ABC transporter ATP-binding protein(ATP结合ABC转运子蛋白)、pheromone binding protein 1(信息素结合蛋白1)、ATP-dependent DNA ligase(ATP依赖DNA连接酶)、zinc-containing alcohol dehydrogenase superfamily protein (含锌乙醇脱氢酶)。
Cantharidin (C10H12O4, hexahydro-3aα, 7aα-dimethyl-4β, 7β-epoxyisobenzofuran-1, 3-diketone) is secreted defensive substance found in meloid insects. It is used as a medicine and also is found of have insecticidal effect in plant protection. However, artificial extraction and chemical synthesis are only ways to produce small amount of cantharidin, and neither can achieve large-scale cantharidin production. Biosynthesis is an effective way to solve this problem but because of the complexity of the physical system and the sythesis process of cantharidin. we know little about the mechanism of cantharidin synthesis. Previous studies mainly focused on cantharidin content detection, cantharidin derivatives synthesis and toxicology, few studies reported in vivo synthesis of cantharidin. cantharidin synthesis is an extremely complex process.
Proteomics is a large-scale study of the gene expression at the protein level, which ultimately provides direct measurement of protein expression levels and insight into the activity state of all relevant proteins. Male blister beetle undergo significant changes in morphology and cantharidin content during the 0-30 days after eclosion. Changes of soluble protein composition at the stage of cantharidin synthesis were analysed by SDS-PAGE , two-dimensional gel electrophoresis and mass spectrometry in order to study this complex biological processes, explain the mechanism of cantharidin synthesis.
Outlining the progress of the cantharidin study and the problem to be carried out, I give a proteomics overview of the research, including key techniqs in proteomics research -2DE,mass spectrometry and bioinformatics. Research topics and objectives are proposed for this study.
The main results are as follows:
1. Establishment of technical conditions for blister beetle proteomics research
The experimental conditions of using two-dimensional gel electrophoresis in this study was optimized by different experimental conditions, including different sample processing, focusing conditions, the SDS-PAGE focus condition, the experiment factors. The results showed that, for the blister beetle soluble protein, using liquid nitrogen urea lysate with high-speed centrifugation is more suitable than the imidazole buffer method for blister beetle tissue, providing a pattern with less loss and more clearity. Therefore, we established two-dimensional gel electrophoresis analysis methods suitable to soluble protein samples of blister beetle through optimizing experimental conditions and the resolution and repeatability of a two-dimensional gel electrophoresis pattern of protein spots is higher with the methods. Experimenting with in-gel digestion, trypsin digestion, size of excised gel, the parameters for matrix-assisted laser desorption time-of-flight mass spectrometry were analyzed and optimum parameters were choosen for experiments. We have built the MS research platform with reference of the silkworm protein MALDI-TOF MS research platform.
2. This study observed the change of protein expression during the rapid growth period of cantharidin synthesis to probe the distribution of related synthesis enzymes. Using protein gel electrophoresis technology,soluable proteins of Mylabris calida Palla from 0 to 25 days after eclosion were analyzed. On protein electrophoresis patterns of Mylabris calida, 3 protein expression bands were found significantly higher in males than females, and their molecular weights are about 80 kD, 45 kD and 30 kD. The bands of 80 kD, 45 kD in males appeared during the first 2 days after emergence and their concentration content increased continously. The protein band of 30 kD expressed at 1 h after eclosion playing a major role in the growth and metamorphosis of adult development. It was presumed that one or several related proteins of 45 kD protein group played an important role in the synthesis of cantharidin.
3. The study on soluble protein after eclosion that was the stage of synthesis cantharidin in Mylabris calida Palla would help to clarify the synthesis mechanism of cantharidin and involved enzymes of the synthesis. The changes of soluble protein composition during the pre-stage, early stage and advanced stage of cantharidin synthesis in Mylabris calida Palla were analysed by SDS-PAGE and two-dimensional electrophoresis. The SDS-PAGE results showed that there were significant differences at different stages during 1h to 25d after eclosion for expression of 80 kD, 45 kD and 30 kD proteins. The characteristic of the pre-stage of synthesis was that most proteins were 20-80 KD, distributed at PI 4-7 and located in the acidic side, especially for 20 kD to 45kD proteins. There were 471 protein spots in the 2D map at one hour after eclosion, while there were 569 spots the second day and 645 spots the fourth day after eclosion. During the period of cantharidin synthesis, the amount of protein spots increased, up to 827 spots and 999 spots at the early stage and advanced stages of cantharidin synthesis, respectively. The protein spots were increased a kind of 25% compared to pre-stage. The results revealed that the developing trend of proteins produced during the three periods was consistent with the course of cantharidin synthesis, indicating that synthesis of cantharidin is a complex process involved a variety of proteins with a molecular weight were range from 20 kD to 45 kD. The obviously different spots were identified as Actin-87E isoform 1, putative secreted protein, LD04994p, GM19279, cytoplasmic actin, molybdenum cofactor biosynthesis protein C, ABC transporter ATP-binding protein, pheromone binding protein 1, ATP-dependent DNA ligase, zinc-containing alcohol dehydrogenase superfamily protein .
蛋白质组学是在蛋白水平大规模研究基因表达的产物,直接研究蛋白的表达水平和相关蛋白的活性状态。芫菁雄虫羽化后0～30天无论在形态还是斑蝥素合成量方面都发生了显著变化。因此本研究利用SDS-PAGE、双向电泳和质谱相结合的蛋白质组学研究方法对不同时期的苹斑芫菁Mylabris calida雄虫的蛋白表达进行了比较研究,以进一步研究这个复杂的生物学过程,同时也为阐释斑蝥素合成机理提供信息。在综述蛋白质组学的研究现状和进展,包括蛋白质组学研究中的关键技术—2DE、生物质谱以及生物信息学的进展和现状,概述了斑蝥素的研究进展和有待进行研究的内容的基础上,提出了本课题工作的研究方向和目的。
本研究主要获得如下结果:
1.确定了研究芫菁蛋白质组学的技术条件。本试验采用不同的方法对芫菁可溶性蛋白进行了双向电泳研究,从样品提取液、等点聚焦条件、第二向聚焦条件、试验中常见的问题等因素对芫菁双向电泳研究体系进行了全面的分析和优化。结果发现,对于芫菁可溶性蛋白,采用尿素裂解液液氮粉碎高速离心法比采用咪唑缓冲液组织匀浆法更为适合,蛋白损失少,图谱均匀清晰。因此本试验经过各种条件优化确定了一套适合于芫菁可溶性蛋白样品分析的双向电泳研究方法,利用这种方法所获得的双向电泳图谱中的蛋白点分辨率和重复性更高。在建立蛋白点肽质量指纹图谱分析体系时,确定了胶内消化过程中的胰酶用量,挖点大小、基质辅助激光解吸飞行时间质谱的参数选择等试验条件,构建了芫菁质谱分析研究平台。
2.斑蝥素合成期芫菁可溶性蛋白样品的SDS-PAGE电泳分析。通过观察斑蝥素合成增长期的蛋白质表达变化,探讨相关合成酶的分布范围。利用蛋白质凝胶电泳技术,对苹斑芫菁在羽化后0～25天的体内可溶性蛋白进行了分析。经蛋白质电泳图谱分析发现,有3条蛋白带的表达量在芫菁雄虫中明显大于雌虫,这些蛋白的分子量分别为:80KD,45KD,30 KD;其中分子量约为80 KD,45KD的蛋白带在芫菁雄虫羽化后第2天出现,其浓度不断的增加。30KD的蛋白带在芫菁雄虫羽化后1小时出现,主要作用是调控成虫的生长和变态发育。推测蛋白组份45KD中的某种或几种关联蛋白质在芫菁雄虫羽化后斑蝥素合成增长期发挥了重要作用。
3.斑蝥素合成期的蛋白质双向电泳及质谱鉴定。前人研究发现芫菁大量合成斑蝥素发生在羽化后,通过对这一时期的芫菁体内可溶性蛋白研究,探索斑蝥素合成相关酶蛋白的种类和合成机理。采用蛋白质双向电泳和飞行质谱技术,分析了合成前期、大量合成早期和大量合成末期的蛋白质组成的变化。研究发现,合成前期蛋白质具有区别于其它时期蛋白质的明显特征:蛋白质大多分布在PI值4-7、分子量20-80KD的区域,且分布不均匀,主要集中在偏酸性一端,20～45KD蛋白质差异明显。芫菁羽化后1小时的蛋白质点数目为471个,羽化后第2天增加到569个,羽化后第4天蛋白点数目增加到645个;随着芫菁进入斑蝥素大量合成期,蛋白点数目明显增加,大量合成早期和合成末期蛋白点数为827个和999个,比合成准备期增长了25%以上。结果显示:三个时期蛋白质组成的变化趋势刚好与芫菁合成斑蝥素含量显著峰值变化相吻合,表明芫菁体内斑蝥素的合成是多种蛋白参与的复杂过程,这些蛋白分子量范围在20～45 kD。利用MALDI-TOF MS对明显差异蛋白进行鉴定时,发现这些蛋白质分别是Actin-87E isoform 1(肌动蛋白87E亚型1)、putative secreted protein(分泌蛋白)、LD04994p(肌动蛋白,LD04994p)、GM19279(肌动蛋白,GM19279)、cytoplasmic actin(胞质型肌动蛋白)、molybdenum cofactor biosynthesis protein C(钼辅因子合成蛋白C)、ABC transporter ATP-binding protein(ATP结合ABC转运子蛋白)、pheromone binding protein 1(信息素结合蛋白1)、ATP-dependent DNA ligase(ATP依赖DNA连接酶)、zinc-containing alcohol dehydrogenase superfamily protein (含锌乙醇脱氢酶)。
Cantharidin (C10H12O4, hexahydro-3aα, 7aα-dimethyl-4β, 7β-epoxyisobenzofuran-1, 3-diketone) is secreted defensive substance found in meloid insects. It is used as a medicine and also is found of have insecticidal effect in plant protection. However, artificial extraction and chemical synthesis are only ways to produce small amount of cantharidin, and neither can achieve large-scale cantharidin production. Biosynthesis is an effective way to solve this problem but because of the complexity of the physical system and the sythesis process of cantharidin. we know little about the mechanism of cantharidin synthesis. Previous studies mainly focused on cantharidin content detection, cantharidin derivatives synthesis and toxicology, few studies reported in vivo synthesis of cantharidin. cantharidin synthesis is an extremely complex process.
Proteomics is a large-scale study of the gene expression at the protein level, which ultimately provides direct measurement of protein expression levels and insight into the activity state of all relevant proteins. Male blister beetle undergo significant changes in morphology and cantharidin content during the 0-30 days after eclosion. Changes of soluble protein composition at the stage of cantharidin synthesis were analysed by SDS-PAGE , two-dimensional gel electrophoresis and mass spectrometry in order to study this complex biological processes, explain the mechanism of cantharidin synthesis.
Outlining the progress of the cantharidin study and the problem to be carried out, I give a proteomics overview of the research, including key techniqs in proteomics research -2DE,mass spectrometry and bioinformatics. Research topics and objectives are proposed for this study.
The main results are as follows:
1. Establishment of technical conditions for blister beetle proteomics research
The experimental conditions of using two-dimensional gel electrophoresis in this study was optimized by different experimental conditions, including different sample processing, focusing conditions, the SDS-PAGE focus condition, the experiment factors. The results showed that, for the blister beetle soluble protein, using liquid nitrogen urea lysate with high-speed centrifugation is more suitable than the imidazole buffer method for blister beetle tissue, providing a pattern with less loss and more clearity. Therefore, we established two-dimensional gel electrophoresis analysis methods suitable to soluble protein samples of blister beetle through optimizing experimental conditions and the resolution and repeatability of a two-dimensional gel electrophoresis pattern of protein spots is higher with the methods. Experimenting with in-gel digestion, trypsin digestion, size of excised gel, the parameters for matrix-assisted laser desorption time-of-flight mass spectrometry were analyzed and optimum parameters were choosen for experiments. We have built the MS research platform with reference of the silkworm protein MALDI-TOF MS research platform.
2. This study observed the change of protein expression during the rapid growth period of cantharidin synthesis to probe the distribution of related synthesis enzymes. Using protein gel electrophoresis technology,soluable proteins of Mylabris calida Palla from 0 to 25 days after eclosion were analyzed. On protein electrophoresis patterns of Mylabris calida, 3 protein expression bands were found significantly higher in males than females, and their molecular weights are about 80 kD, 45 kD and 30 kD. The bands of 80 kD, 45 kD in males appeared during the first 2 days after emergence and their concentration content increased continously. The protein band of 30 kD expressed at 1 h after eclosion playing a major role in the growth and metamorphosis of adult development. It was presumed that one or several related proteins of 45 kD protein group played an important role in the synthesis of cantharidin.
3. The study on soluble protein after eclosion that was the stage of synthesis cantharidin in Mylabris calida Palla would help to clarify the synthesis mechanism of cantharidin and involved enzymes of the synthesis. The changes of soluble protein composition during the pre-stage, early stage and advanced stage of cantharidin synthesis in Mylabris calida Palla were analysed by SDS-PAGE and two-dimensional electrophoresis. The SDS-PAGE results showed that there were significant differences at different stages during 1h to 25d after eclosion for expression of 80 kD, 45 kD and 30 kD proteins. The characteristic of the pre-stage of synthesis was that most proteins were 20-80 KD, distributed at PI 4-7 and located in the acidic side, especially for 20 kD to 45kD proteins. There were 471 protein spots in the 2D map at one hour after eclosion, while there were 569 spots the second day and 645 spots the fourth day after eclosion. During the period of cantharidin synthesis, the amount of protein spots increased, up to 827 spots and 999 spots at the early stage and advanced stages of cantharidin synthesis, respectively. The protein spots were increased a kind of 25% compared to pre-stage. The results revealed that the developing trend of proteins produced during the three periods was consistent with the course of cantharidin synthesis, indicating that synthesis of cantharidin is a complex process involved a variety of proteins with a molecular weight were range from 20 kD to 45 kD. The obviously different spots were identified as Actin-87E isoform 1, putative secreted protein, LD04994p, GM19279, cytoplasmic actin, molybdenum cofactor biosynthesis protein C, ABC transporter ATP-binding protein, pheromone binding protein 1, ATP-dependent DNA ligase, zinc-containing alcohol dehydrogenase superfamily protein .
引文
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