【作者】 覃吕高；

【导师】 陈克平；

【作者基本信息】 江苏大学 ， 食品营养与安全， 2012， 博士

【摘要】 家蚕是一种重要的经济昆虫,桑蚕业无论在古代还是现代,在中国农业中都占有重要地位。引起蚕病的毒病主要有核型多角体病毒(Nuclear polyhedrosis virus, NPV)、质型多角体病毒和浓核病毒。其中NPV引起的脓病每年给桑蚕业造成的损失巨大。不仅如此,食物中的病毒也对食品安全造成严重威胁。作为NPV的天然宿主,家蚕与病毒的相互作用关系一直是众多研究者关心的问题。在自然界中,有少数家蚕品系具有天然的抗病毒能力。尽管已经有多种蛋白或基因被报道有抵抗NPV的作用,但到今天为止,科学界还未确定哪种蛋白或基因决定了家蚕的抗NPV性状,更不清楚家蚕抗NPV的详细机制。另外,在雌雄家蚕之间有一些差异性状,如雄蚕比雌蚕更健壮、不同龄期雄蚕与雌蚕的生长速率不一样等,这其中的机制至今还不很清楚。如果能了解宿主抵抗病毒的机制,将会大大推动蚕业生产给,降低病毒给食品安全性带来的风险。本研究用蛋白质组学技术鉴定家蚕中的抗病毒相关蛋白。用抗病毒品系NB、感病毒品系306和它们的杂交F1代来组配成正交组和反交组,比较各品系家蚕间的差异蛋白,希望能找出与抗病毒或感病毒相关的蛋白。另外,还用二维电泳技术分离雄蚕与雌蚕中肠的总蛋白,希望能找到与不同性别家蚕性状差异相关的蛋白,为家蚕育种和蚕业生产提供理论基础。研究的主要结果如下：1.对抗性亲本NB、感性亲本306及F1代经口接种病毒后,死亡率的分析显示,F1代与NB抗性亲本都没有出现家蚕个体死亡,对病毒表现出同样的抵抗能力,而感性亲本306全部感染病毒死亡。表明家蚕抗病毒性状的遗传方式是显性遗传。2.将各品系家蚕中肠的总蛋白提取出来后,用二维电泳分离总蛋白白并比较各品系家蚕的蛋白表达谱。发现在正交组(NB(?),306(?),NBx306)中,有七个蛋白只在抗性品系中表达,包括pc21g12150, protein o-fucosyltransferase 2, protein LST8 homolog, caspase-1, hemolymph protein, serine protease, glucose phosphate dehydrogenase。有三个蛋白只在感性品系中表达,包括heat shock protein 90, phosphodiesterase subunit alpha和adenosine kinase。在反交组(306(?),NB(?),306xNB)中,有六个蛋白只在抗性品系中表达,包括caspase-1, carboxylesterase, serine protease, hypothetical protein, antennal binding protein,还有一个未成功鉴定的蛋白。有两个蛋白只在感性品系中表达,包括arginine kinase和wall-associated receptor kinase-like 4。将正交组和反交组的结果结合起来看,发现有两个蛋白：半胱天冬酶-1(caspase-1)和丝氨酸蛋白酶(serine protease)无论在正交组还是反交组中,都只在抗性品系中表达,在感性品系中不表达。3.将正交组和反交组中所有的抗性或感性品系特异表达蛋白做基因聚类分析和KEGG pathway分析,然后统计这些差异蛋白主要参与的生物途径和分子功能。对各个差异蛋白的功能进行分析,发现半胱天冬酶-1、丝氨酸蛋白酶和羧酸酯酶有可能参与家蚕的抗病毒过程,而热休克蛋白90可能是导致家蚕对NPV病毒敏感的因素。4.半胱天冬酶-1、丝氨酸蛋白酶在抗性亲本NB、F1代(正交组和反交组)中都检测到表达,而在感性亲本306中检测不到表达,表明它们可能在家蚕的抗病毒过程中发挥关键作用。因此,用Western blot对它们在抗性和感性品系中的差异表达进行验证。实验结果再次显示这两个蛋白只有抗性品系中能检测到表达,在感性品系中检测不到,与二维电泳结果一致。5.在五龄第二天,将NB家蚕分成不同的性别组,提取中肠组织,用蛋白质组学方法比较不同性别家蚕蛋白质表达谱的差异。发现有5个蛋白只在雄蚕中表达,包括predicted protein, muscle glycogen phosphorylase, isocitrate dehydrogenase, uridine 5’-monophosphate synthase, vacuolar ATPase B subunit。5个蛋白只在雌蚕中表达,包括vacuolar ATP synthase catalytic subunit A, coA-substrate-specific enzyme activase, efl alpha-like factor isoform 1, hypothetical protein,还有一个未成功鉴定的蛋白。8个蛋白在雄蚕中的表达量更高,包括cone cGMP-specific 3’,5’-cyclic phosphodiesterase subunit alpha’, vacuolar ATP synthase subunit B, thiol peroxiredoxin, H+ transporting ATP synthase subunit d, predicted protein, actin-depolymerizing factor 1, mRNA transport regulator 3,还有一个未成功鉴定的蛋白。25个蛋白在雌蚕中的表达量更高,包括heat shock protein 90, wall-associated receptor kinase-like 4等。将鉴定到的差异蛋白做基因聚类和KEGG pathway分析,统计了雄蚕表达水平高和雌蚕表达水平高的蛋白主要参与的生物途径和分子功能,分析这些途径和功能与不同性别家蚕性状差异的关系。从差异蛋白中选取六个重要的蛋白,包括5’-尿苷酸合成酶,巯基过氧化还原酶,H+ ATP合成酶D亚基,mRNA转运调节因子3,丝氨酸蛋白酶抑制剂-2,磷酸甘油酸变位酶,用荧光定量PCR验证这六个蛋白在不同性别家蚕章的差异表达。实验结果与蛋白质组实验结果一致,即5’-尿苷酸合成酶只在雄蚕中检测到表达,巯基过氧化还原酶,H+ ATP合成酶D亚基和mRNA转运调节因子3在雄蚕中的表达量比雌蚕中的高,丝氨酸蛋白酶抑制剂-2,磷酸甘油酸变位酶在雌蚕中的表达量比雄蚕中的高。更多还原

【Abstract】 Silkworm is one of important economic insects. Whether in ancient or modern agriculture, sericulture occupies an important position in China. Silkworm diseases are mainly caused by nuclear polyhedrosis virus (Nuclear polyhedrosis virus, NPV), cytoplasmic polyhedrosis virus and densonucleosis virus, among these the nucleopolyhedorosis caused by NPV causes most great loss. In addition, virus which exist in food also bring threaten to food safety. As the natural host of NPV, the interaction of silkworm and virus attracts a lot of attention from all over the world. There have some silkworm strains that have natural existing anti virus ability. Though some genes or proteins were reported capable to anti-virus, the conclusions needed further conformation, and the detail mechanisms that these genes and proteins resist against NPV also needed more researches. On the other hand, there are some different between male and female silkworms, such as male silkworm is stronger than female, the growth rate between different silkworm sex is different, and the mechanisms result in these differences still unclear to day. Understanding the host resistance mechanisms to the virus will greatly promote sericulture as well as reduce the risk of the virus to food security.In this study, proteomics technology was used to identify anti-viral proteins in silkworm. NPV-resistant strain NB, susceptible strain 306 and their F1 hybrid were used for construct direct cross group and reciprocal cross group, and analyzed which proteins were related with silkworm resistance to NPV. In addition, in order to find proteins that related to the difference between different genders, two-dimensional electrophoresis was used for separate total protein of midgut from male and female silkworm, this can provide a theoretical basis for silkworm breeding and sericulture. The main findings are as follows: 1. When administered NPV virus per os to NPV-resistant parent NB, susceptible parent 306 and their F1 hybrid, the result of mortality analysis showed that resistant parent NB and F1 hybrid were alive, they showed the same resistance to NPV. However, susceptible parent 306 died after NPV infection. The result suggested that the inheritance mode of silkworm antiviral traits is dominant inheritance.2. After extracted midgut total protein from various silkworm strains, two-dimensional electrophoresis was used for separate the total protein, and protein expression profiling of these silkworm strains were analyzed. In direct cross group (NB♀,306(?),NB×306), seven proteins expressed exclusively in resistant strains, including pc21g12150, protein o-fucosyltransferase 2, protein LST8 homolog, caspase-1, hemolymph protein, serine protease, glucose phosphate dehydrogenase. Three proteins expressed only in susceptible strain, including heat shock protein 90, phosphodiesterase subunit alpha, adenosine kinase. In reciprocal cross group (306♀, NB(?),306xNB), six proteins expressed exclusively in the resistant strains, including caspase-1, carboxylesterase, serine protease, hypothetical protein, antennal binding protein, as well as an unidentified protein. Two proteins expressed only in susceptible strain, including arginine kinase and wall-associated receptor kinase-like 4. Combined the results form direct cross group and reciprocal cross group, it was found that two proteins, caspase-1 and serine protease expressed only in resistant strains but not in susceptible strain.3. Resistant or susceptible strain-specific proteins in two cross groups were subject to GO and KEGG pathway analysis, and the biological processed and molecular functions of these proteins were analyzed. It was found that caspases-1, serine protease and carboxylesterase may be involved in the process of silkworm resist to NPV, whereas heat shock protein 90 maybe a factor made silkworm susceptible to NPV.4. Because caspase-1 and serine protease were showed specific in resistant parent and F1 hybrid (D-F1 and R-F1), and couldn’t be detected in susceptible parent, suggesting that they may play a key role in silkworm antivirus. Thus Western blot was employed to confirm the different expression of the two proteins in resistant and susceptible strains. The results show that these two proteins could be detected only in resistant strains, and could not be detected in susceptible strains, consistent with result in two-dimensional electrophoresis.5. In the second day of fifth instar, NB silkworm was divided into two groups according to different genders, and proteomic method was used for compare the protein profile of midgut from two silkworm sexes. Finally, it was found that five proteins expressed only in male silkworm, including predicted protein, muscle glycogen phosphorylase, isocitrate dehydrogenase, uridine 5’-monophosphate synthase, vacuolar ATPase B subunit. Five proteins only expressed in female, including vacuolar ATP synthase catalytic subunit A, coA-substrate-specific enzyme activase, efl alpha-like factor isoform 1, hypothetical protein, as well as an unidentified protein.8 proteins expressed higher in male silkworm, including cone cGMP-specific 3’,5’-cyclic phosphodiesterase subunit alpha’, vacuolar ATP synthase subunit B, thiol peroxiredoxin, H+ transporting ATP synthase subunit d, predicted protein, actin-depolymerizing factor 1, mRNA transport regulator 3, as well as an unidentified protein.25 proteins expressed higher in female, including heat shock protein 90, wall-associated receptor kinase-like 4, et al. GO and KEGG pathway analysis was also employed to analyzed the different proteins, for proteins higher in male or female silkworm, their biological processed and molecular functions was analyzed respectively. Finally, the relationship of these processes (or functions) and special traits in two silkworm sexes were also explored. Six proteins were selected for confirm their different expression, including uridine 5’-monophosphate synthase, thiol peroxiredoxin, H+ transporting ATP synthase subunit d, mRNA transport regulator 3, serpin-2 and phosphoglyceromutase. qRT-PCR was used to detected their expression level in different silkworm sexes. The result was similar with that in 2-DE, which showed that uridine 5’-monophosphate synthase could only be detected in male silkworm, thiol peroxiredoxin, H+ transporting ATP synthase subunit d, mRNA transport regulator 3 expressed higher in male silkworm, whereas serpin-2 and phosphoglyceromutase expressed higher in female silkworm.更多还原