| As a kind of important economic insect, silkworm(Bombyx mori) play a very important role in the production of human life and cultural history. Silkworm is an oligophagous insect, and mulberry leaf is its only natural feed, which severely restricts the development of the sericulture industry. Artificial diet feeding silkworm will become an important innovation and inevitable trend in the future sericulture industry, but the technology of artificial diet feeding silkworm in our country can't meet the practical demand. The bad feeding habits to the artificial diet of most silkworm in our country become the main obstacle that prevent the development of the technology, so increasing the feeding habits and adaptability of the silkworm to the artificial diet become an important research subject. Previous studies have done a lot of research in the feeding habits of silkworm in terms of physiology and genetics, and achieved fruitful results, but the molecular mechanism of trophic differences is unclear and the related genes have not been found.We used the silkworm strains with obviously different feeding habits as the experimental material in this study, and analyzed the silkworm feeding habits related molecular markers using Simple Sequence Repeats(SSR). Real-time PCR was employed to detect the expression of the c GMP-dependent protein kinase(PKG) in different strains of silkworm. And pharmacological activation of the PKG was used to verify the function of pkg1. The main results are as follows:1. We used 3 pairs of silkworm strains with different feeding habits as the materials, and employed SSR-PCR using 152 pairs of SSR primers, and then selected 18 polymorphic SSR markers which mainly located on the chromosome 3rd, 7th, 9th, 10 th, 12 th, 13 th, 14 th, 16 th, 17 th, 19 th, 20 th and 21 st. There are 3 polymorphic SSR marks on the chromosome 20 th, and many special bands amplified from the parents with high feeding ability. It indicated that the genes related foraging artificial diet may locate on the chromosome 20 th. The SSR marker S2009 was the special mark of the silkworm with high feeding ability. Further more, it indicated that the genes related foraging artificial diet may locate near the SSR marker S2009 of the chromosome 20 th. The SSR marker S1902 also amplified many special bands from the parents with high feeding ability, which indicated that the genes related foraging artificial diet may also locate near the SSR marker S1902 of the chromosome 19 th. The SSR marker S0911 and S1603 amplified many special bands from the parents with low feeding ability, which indicated that the no-feeding genes may locate near the SSR marker S0911 of the chromosome 9th and the SSR marker S1603 of the chromosome 16 th. Therefore, we speculate that the silkworm feeding-related traits may be controlled by multiple genes and belong to quantitative traits. And the foraging gene of different silkworm strains may be different to some extent, and their inheritance pattern will be different as well.2. We determined the expression pattern of pkg1 in the 3rd instar larvae head of Bombyx mori strains with different feeding habits. The expression level of pkg1 in the head of Bombyx mori larvae strains with high feeding habit was significantly higher than that in B. mori strains with low feeding habit. The pkg1 gene expression in various larval tissues of Youshi-1 larvae at 96 h after the 5th instar indicated that the pkg1 gene expressed in the head, midgut, fat body, malpighian tubules and sericterium, and the gene performs the highest express level in the fat body, the malpighian tubules followed, and the gene performs the lowest express level in the head and the midgut.3. We hungered the 4th instar larvae of Bombyx mori strains with different feeding habits for 12 hours, and then we found that the expression level of pkg1 in the head of Bombyx mori larvae strains increased significantly. We carried out the experiment of mulberry leaf, artificial diet without mulberry leaf powder and repellent stimulus after the 4th instar B. mori larvae strains with different feeding habits hungered for 12 hours. The expression differences of pkg1 reduced between B. mori larvae strains with high feeding habit and B. mori larvae strains with low feeding habit after mulberry leaf stimulated. In contrast, The expression differences of pkg1 increased between B. mori larvae strains with high feeding habit and B. mori larvae strains with low feeding habit after artificial diet without mulberry leaf powder stimulated, and the expression level of pkg1 was reduced significantly after artificial diet stimulated. The repellent stimulus had a little influence on the pkg1 expression. The results suggest that pkg1 may be closely related to the feeding habits of the silkworm, and the up-regulated expression of this gene may play the role of promoting the silkworm to eat artificial diet, but the specific mechanism remains to be further research.4. We fed the high feeding strain of Jingsong B larvae at 96 h after the 4th instar with 8-Br-c GMP at different consentration. The results showed that the artificial diet acceptance became worse significantly as the consentration of 8-Br-c GMP incressing. We injected the Youshi-1 larvae at 24 h after the 5th instar with 8-Br-c GMP at different consentration. As a result, when the injection consentrations are 500 ?M, 1000 ?M and 5000 ?M, the artificial diet acceptance became worse significantly as the consentration of 8-Br-c GMP increasing, and the silkworm was poisoned at the injection consentration of 5000 ?M, and then the silkworm returned normal after 48 hours of injection. We injected the Youshi-1 larvae at 24 h after the 5th instar with c GMP at different consentration. The results showed that there are no significant changes of the feeding habits to the artificial diet of the silkworm. So the relationship between pkg1 gene and the feeding habits of silkworm need other methods to verify. |
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