Proteomics Analysis Of Anti Nuclear Polyhedrosis Virus Related Proteins And Male-female Difference In Silkworm, Bombyx Mori

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.