The Study On The Effects Of 20E And JH On Inducing The Expression Of The Silk Protein Genes In Silkworm,Bombyx Mori

Ecdysone(20E) and juvenile hormone(JH) coordinately regulates growth of insects.The process of Lepidoptera silk synthesis and secretion is regulated by the hormone. 20 E exerts its effects through a heterodimer protein composed of an ecdysone receptor(Ec R)and ultraspiracle(USP). JHIII can be bound to USP protein specificly, causing a conformational change in the receptor molecule, which will be helpful to promote the dimerization.Silkworm is an important economic insect that produces silk material. However, its role in regulation the expression of silk genes remains obscure. To study the expression and regulation of 20 E and JH on silk protein genes, this study examined the expression of Ser-1,Fib-H, Fib-L and P25 in silk gland using real-time quantitative PCR. In order to identify the Ec REs of silk protein, this study detected the interaction of domain protein complex(Ec R-B1D/USPD) and predicted Ec REs taking the method of isothermal titration calorimetry(ITC) technology, respectively, and further analysis of the 20E-Ec R/USP-Ec R regulation model. The main research results are as follows:1. The expression characteristics of Ser-1 in silkworm middle silk grands exposed to hormone in vitro.In order to investigate the expression characteristics of Ser-1 in middle silk grands induced by hormone. In the present study we cultured the silk gland with different hormone. The results showed that the expression of Ser-1 were suppressed by 20 E or JH,however, co-treatment with 20 E and JH significantly up regulated the expression level of Ser-1 at 72 h. The results indicated that it was more favorable for the expression of silk protein genes with the joint action 20 E and JH.2. The expression characteristics of Fib-H, Fib-L and P25 in silkworm posterior silk grands exposed to the hormone in vitro.2.1 Effect of hormone on the expression of Fib-HIn order to study the expression of Fib-H after induced by hormone, this study we cultured the posterior silk gland with different hormone. The results showed that the expression of Fib-H were suppressed by 20 E or JH, and were significantly upregulated by co-treatment with 20 E and JH at 72 h. In a medium containing 20 E or JH, first training time for 24 h and then transferred to a medium containing 20 E and JH had no significant difference on the expression of Fib-H.2.2 Effect of hormone on the expression of Fib-LIn order to study the expression of Fib-L after induced with hormone, this study we cultured the posterior silk gland with different hormone. The results showed that 0.1 μg/m L concentrations of 20 E had no obvious effect on the expression of Fib-L, in contrast, 0.5μg/m L and 2.5 μg/m L 20 E promoted it. The treatment with different concentrations of JH inhibited the expression of Fib-L.In addition, the treatment with 0.5 μg/m L 20 E and 0.5 ng/m L JH inhibited the expression of Fib-L; the treatment group with 0.5 μg/m L 20 E and 5 ng/m L JH and with 0.1μg/m L 20 E and 5 ng/m L JH group both improved the expression of Fib-L, the latter had a better induction effect. In a medium containing 20 E, first training time for 24 h and then transferred to a medium containing 20 E and JH, this treatment enhanced the expression of Fib-L; In a medium containing JH, first training time for 24 h and then transferred to a medium containing 20 E and JH, this treatment inhibited the expression of Fib-L. 20 E and JH which priority treatment had no significant difference on the expression of Fib-L.2.3 Effect of hormone on the expression of P25In order to study the expression of P25 after induced with hormone, this study we cultured the posterior silk gland with different hormone.The results showed that the expression of P25 were up regulated by 20 E and suppressed by JH.In addition, the treatment with 0.5 μg/m L 20 E and 0.5 ng/m L JH inhibited the expression of P25; The treatment with 0.5 μg/m L 20 E and 5 ng/m L JH or with 0.1 μg/m L20 E and 5 ng/m L JH both promoted the expression of P25,the latter had a better induction effect. In a medium containing 20 E or JH, first training time for 24 h and then transferred to a medium containing 20 E and JH, had no significant difference on the expression of P25.3. Co-expression of ecdysone receptor domain protein Bm Ec R-B1 D and ultraspiracle domain protein Bm USPDWe cloned USPD and ligated into p ET-28 a vector resulting in recombinant expression vectors of p ET-28a-USPD. The recombinant plasmids p ET-28a-USPD were transformed into E.coli BL21 and then Ec R-p ET21b-MDX12 recombinant plasmids were transformed into E.coli BL21 which contain p ET-28a-USPD, induced the expression of protein by SDS and western blot. The purification of target proteins was carried out through Ni-NTA affinity chromatography column. We obtained the Bm Ec R-B1D/USPD complex and the purity of this protein was above 95 %.4. Isothermal titration calorimetric study on binding interactions between Bm Ec R-B1D/USPD protein with Ec REs of silk protein genesUnder in vitro conditions, we used ITC to obtain a series of thermodynamic parameters of the binding interaction between Bm Ec R-B1D/USPD protein and Ec REs of silk protein, including the affinity constant(K), reaction heat(ΔH) and entropy(ΔS). The results indicated that Ec R-B1D/USPD could be bound to Ec REs predicted. The Ec REs predicted were the core sequence of the Ec REs which were conserved in the core sequence.5. 20E-Ec R/USP-gene model analysisTaking the method of itc, we found that 20 E can be combined with Ec R-B1D/USPD protein, Ec R-B1D/USPD protein can be combined with Ec RE, and 20E-Ec R/USP complex can be combined with Ec RE.This study illustrated the effect of 20 E and JH on the expression of Ser-1, Fib-H,Fib-L, P25 at the level of transcription and verify the Ec REs of silk protein genes by ITC,these study provides data support for the hormonal regulation of silk protein genes and provides new insights into the mechanism for improving the yield of silk.