The Analysis Of Cloning TrpA1 Gene And Identification Of The Related Genes About Diapause In Silkworm, Bombyx Mori

Insects are the animals with the largest population and the most widely distribution in the world, which is attributed to its amazing capacity of adaptive behaviors. Diapause is the most important physiological property for insects to adapt to environment. Diapause is a physiological state induced by environment in the specific developmental stage to avoid unfavorable circumstance. It is different from dormancy that if insects entered diapause, it will not resume to development even the suitable environment coming. However, the suitable environment is beneficial to maintain diapause state. For insects, diapause has the significant means, on one hand, diapause enables insects survive the unfavourable environmental conditions and makes their life cycle to consistent with their feeding; on the other hand, once diapause terminates, they are in the same developmental level which can improve their mating opportunities and then help the population to survive and multiply. Hence, diapause plays an important role in survive, reproduction and evolution of insects.Bombyx mori is an economically important insect and excellent model organism. Its diapause phenomenon is always the research hotspot. Research on the diapause mechanism of silkworm can not only provide a theoretical basis for the study of insect diapause; but it can also contribute to sericulture to expand production and give full play to its economic traits. At the same time, a clear understanding on the mechanism of diapause can also be applied to pest control, which is of great significance for agricultural production and environmental protection. Based on different diapause characteristic owned by different silkworm strains, this study researches the mechanism how the Bm Trp A1 gene affect the diapause of bivoltine silkworm. Then, the Digital Gene Expression Profiling(DGE) sequence was performed to screen differentially expression gene about silkworm diapause and to verify their function. The main results in this study are as follow:1. Bm Trp A1 expression analysis during embryonic development in different voltinism strains of silkwormTo research whether expression disparity of Bm Trp A1 exists in different silkworm strains with the voltinism variance, we investigated Bm Trp A1 gene expression patterns in embryonic development in different voltinism strains of silkworm by quantitative PCR. It was found that, Bm Trp A1 expressed consistently under 25 ℃ and 15 ℃ incubation during the whole embryonic development in univoltine and multivoltine strains; However, in bivoltine strains, it has a higher expression under 25℃ incubation condition than under 15 ℃ incubation condition during the whole embryonic development. In addition, the expression reached the peak in embryonic reversal and head pigmentation stage which are sensitive to temperature under 15℃ incubation condition. These results may suggest that Bm Trp A1 gene involved in the regulation of eggs diapause in bivoltine silkworm, but not in univoltine and multivoltine strain.2. The cloning and bioinformatics analysis of Bm Trp A1To find the reason why BmTrpA1 has effect on egg diapause of silkworm, we cloned the Bm Trp A1 gene and used bioinformatic to analyze the Bm Trp A1 protein. The cloning results showed that only 27 single-base nucleotide mutations were detected in the open reading frame which led three amino acids mutation; Bioinformatic analysis indicated that the Bm Trp A1 protein has the same structure in different voltinism strains. We speculated that the reason why different voltinism strains have different diapause phenomenon is the upstream regulatory sequence of Bm Trp A1, but not the Bm Trp A1 itself.3. The analysis of the upstream sequence of Bm TrpA1 in different voltinism strains21 strains, which could be classified to three voltinism groups, were used to perform alignment analysis of the upstream 5Kb sequence of Bm Trp A1 by analysis on the genome re-sequencing result. And it showed that the single nucleotide polymorphism is concentrated on the upstream(scaf23 : 3596000-3597000). These variation points provided theoretical basis for further researches on diapause regulation of Bm Trp A1 in bivoltine strain.4. DGE sequencing and data analysis of embryonic diapause in bivoltine silkwormFor further analysis on the molecular regulation of embryonic diapause, we used the embryo which developed to head pigmentation stage under different incubation temperature(25 ℃ \15 ℃) to structure two data base: 25-Hp and 15-HP. Then we performed the Digital Gene Expression Profiling(DGE) sequence and analyzed the sequencing data. The analysis results, including the mapping of two Clean Reads to genome, sequencing saturation analysis of two sequencing samples and randomness assessment of two sequencing samples, suggested a high creditability of the sequencing data. Meanwhile, to confirm the reality of DEGs, 19 genes in KEGG Pathway about diapause were selected for q RT-PCR analysis. Relative expression level of 16 genes showed a significant change in the same direction between the q RT-PCR and DGE sequence methodologies. This results illustrated a substantial conformity between q RT-PCR and DGE sequence data,and indicated it can be used for the subsequent analysis.5. The identification and functional verification of diapause genes in silkwormWe used the embryo which developed to head pigmentation stage under different incubation temperature to perform Digital Gene Expression Profiling sequence. Based on the research results of insects diapause, Gene Ontology and KEGG Pathway analysis, we screened period、timeless and takeout genes which may involve in silkworm diapause.In Dazao, the used bivoltine strain, period and takeout genes expressed significantly higher under 25℃ incubation than 15℃, while they expressed highly but equal under the two condition in J106 and Nis, the used univoltine strain and multivoltine strain respectively. Based on the above findings, we further speculated that period and takeout genes may participate in the regulation of silkworm diapause. To confirm the speculation, RNAi was used to further verify the function of period and takeout genes. We firstly synthesized the period-ds RNA and takeout-ds RNA and injected them into eggs of the bivoltine strain(Dazao) collceted within 2h after oviposition. These injected eggs were incubated under 25℃ and fed to oviposite. The result showed that the offspring eggs of the period-ds RNA and takeout-ds RNA injected individuals, still entered into diapause. Diapause is a complicated regulatory process. Therefore, genome editing to knock down period and takeout genes may be need to validate their functions in diapause and to confirm whether period and takeout genes involved in the regulation of bivoltine silkworm diapause.