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

Ecdysone(20E)and juvenile hormone(JH)can regulate the growth and development of insects.The growth and development of silkworm glands and the synthesis of silk protein are also regulated by 20 E and JH.In order to study the regulation effect of 20 E and JH on expression of silk protein genes,we analyzed the difference of the genes' expression level of trimolters of silkworm “sanguang” and tetramolters of silkworm “haoyue”.We cultured the silkworm gland in vitro and analyzed the expression of silk protein genes which induced by hormones.And we analyzed the important regions and factors related to hormone induction in the 5' flanking sequence of silk fibroin genes.The results are as follows:1 The differences in the expression of silk protein genes between trimolters of silkworm “sanguang” and tetramolters of silkworm “haoyue”1.1 The expression trends of ser-1 gene in trimolters of silkworm “sanguang” and tetramolters of silkworm “haoyue” were first rapid increase and then decreased.The ser-1 gene of trimolters of silkworm “sanguang” 4th instar highest raised 3.0 times;The ser-1 gene of tetramolters of silkworm “haoyue” 4th instar highest raised 1.5 times and highest raised 2.8 times in 5th instar.Among them,the fold increase of trimolter “sanguang” 4th instar was the highest.1.2 The expression of silk fibroin genes in trimolters of silkworm “sanguang” and tetramolters of silkworm “haoyue” showed a tendency to rise straight from newly molted silkworms.In trimolters of silkworm “sanguang” 4th instar,the expression levels of fib-H,fib-L and p25 genes were highest up to 2.8,2.9 and 5.2 times,respectively;In tetramolters of silkworm “haoyue” 4th instar,the expression levels of fib-H,fib-L and p25 were up-regulated at 1.4,1.4 and 2.2 times,respectively;in the 5th instar the highest amount were raised 4.5,6.2,24.3 times.Among them,tetramolters of silkworm “haoyue” 5th instar raised the highest of times,followed by trimolters of silkworm “sanguang” 4th instar.1.3 Whether trimolters of silkworm “sanguang” 4th instar or tetramolters of silkworm“haoyue” 4th instar and 5th instar,the expression levels of silk fibroin genes were: p25,fib-H,fib-L.Increased expression of multiple fwere: p25,fib-L,fib-H.2 Expression of silk protein genes in early stage(0-24h)after hormones treated silk glands in vitro2.1 20 E treatment on the early effects of silk protein genes expression20E treatment alone,medium20 E group on ser-1 gene expression level and the most significantly promote at 12 h;different concentrations of 20 E treatment inhibited fib-H gene expression levels;for fib-L gene expression,high 20 E treatment has a role in promoting.The expression of p25 gene up-regulated at different concentrations of 20 E,and the effect of medium and high concentration was significant.2.2 JH treatment on the early effects of silk protein gene? s expressionJH? alone treament mainly inhibited the expression of silk protein genes.The expression of ser-1 gene was up regulated by low concentration of JH ?treatment.The expression of fib-H,fib-L and p25 gene was inhibited by different concentrations of JH?.2.3 20 E and JH co?-treatment on the early effects of silk protein genes expressionUnder normal circumstances,20 E and JH? co-treatment were more effective than single hormone treatment in favor of silk protein genes expression,but the appropriate concentration ratio to promote the role were more obvious.The expression of ser-1 and p25 genes was promoted by medium concentration of 20 E and JH ?treatment group.The medium concentration of 20 E and low concentration of JH ? group to promote ser-1 and fib-H genes expression.However,in some special cases,when JH ?is too low,co-treatment not only does not promote the expression of silk protein genes,but seriously inhibited fib-L and p25 genes expression.3 Functional analysis of silk fibroin genes promoter after hormone inductionThe results showed that in Bmfib-H and Bmfib-L full length 5' flanking areas(Bmfib-H total length-1164~+36 bp;Bmfib-L total length-1061~+15 bp)and different short pieces,20 E,JH? treatment alone or 20 E and JH ?co-treatment were all inhibited the activity of Bmfib-H and Bmfib-L promoter fragments,and there were not found the activity up-regulated fragments.20E,JH? treatment alone or 20 E and JH? co-treatment all significantly inhibited the activity of Bmp25 promoter-1342~-1032 bp fragment,but significantly up-regulated the activity of-743~-662 bp fragment,indicating that Bmp25 promoter-1342~-1032 bp had suppression regions which associated with 20 E or JH?;While the region of-743~-662 bp had sequences or binding factors that promoted the induction of hormones,and further mutations showed that GA-BF and HNF-1A transcription binding sites were necessary for the hormones to promote the Bmp25 promoter-743~+38 bp region activity.In conclusion,in this study,we analyzed the expression of silk protein genes of trimolters of silkworm“sanguang” and tetramolters of silkworm“haoyue”,found that the expression of silk protein genes were up-regulated in the low level 20 E and JH?;in the middle and late stages,the expression of ser-1 gene decreased with the 20 E and JH ?titers,then the expression level of silk fibroin genes continued to increase to the highest level,indicating that in the silk body,the effect of 20 E and JH on silk protein genes expression is more complex,needed to be studied in the future;The expression of silk protein genes in silk gland was induced by hormones.It was found that 20 E and JH? co-treatment were more beneficial to the expression of silk protein genes than hormone alone,but the concentration of JH? in co-treatment group was too low to inhibit fib-L and p25 genes expression.In addition,by analyzing the activity of p25 promoter induced by hormone,it was found that GA-BF and HNF-1A transcriptional binding sites were necessary for hormone-promoted Bmp25 promoter-743~+38 bp region activity.These results provide theoretical references for hormonal regulation of silk protein genes in future researchers.