| As a very common substance with extremely low content in the biological world,hormone is quite important for the regulation of life development.Belonging to sterol series,estrogen is one kind of the female sex hormone,which includes estradiol,estrone and estriol.17-beta-estradiol（E2）,as a representative of vertebrate estrogen and with highest activity has been widely and deeply researched.The biological function of E2 was meditated by its receptor（ER）in vertebrate.The typical mechanism is that E2 binds to and actives estrogen receptor,then the receptor dimerizate,following combine with estrogen response element located at the target gene promoter to regulate its transcription activity,and then has an influence on the vertebrate’s growth and development,multiply and metabolism.In fact,since sex hormone had been identified in higher animals,some researchers had reported that higher animals’ steroid sex hormone exist in some organization or individual extraction from invertebrates,such as insects,and have some physical function.Silkworm,Bombyx mori as one kind of significant economic insects is the model organism about Lepidoptera.So far,there were some reports about silk worm’s E2,but it was limited compared to wide research about vertebrate E2.It was identified that silkworm’s ovary contains some content of E2 and have the ability to metabolism E2.The extractive from silkworm pupa can induce vertebrate to generate some estrogenic effects: mouse with ovary removed was treated by extractive from silkworm pupa,can recovery and mitigate uterine weight decrease,increase E2 levels in its blood.And E2 has some effect on the physical function of silkworm’s silk gland,fat body and other tissue.When Drosophila melanogaster genomic sequencing was finished in 2003,however,the ER gene can’t be found in their database,in other words,Insect has no the estrogen receptor gene.So it wasn’t clearly how the E2 plays its function in silkworm.The typical function in vertebral ovipara of E2 is to induce the expression of vitellogenin gene through estrogen receptor.As invertebrate,Silkworm has vitellogenin protein belonging to ovipara animals.The estrogen receptor does not exist in insects,while estrogen related receptors（ERR）exist in some insect species.ERR in vertebrate can participate in the estrogen signal pathway.This research is mainly aimed at identifying E2’s function in silkworm.Though detecting the content of E2 in different development stage of silkworm’s hemolymph analyze the silkworm’s E2 origin and using Bm Vg as a biomarker study the function and signal path way of the E2 in silkworm.The mainly results are as follows: 1.Silkworms have the ability to synthesis E2Previous studies have been reported that the ovary of the silkworm contain E2.This paper has detected E2 in the blood of silkworm through HPLC,and the concentration is 145.35 pg/ml.The silkworm is an open blood circulation system,and the E2 need to be transported by blood to play its function.So we speculated that the E2 has physiological function in silkworm.Although many insects,including silkworm,have detected E2,there was no unified vision about the source of detected E2 at the moment.The mainstream view think about E2 in insect is from food.Using ELISA to detect E2 content from wandering to the second day of pupal in haemolymph of silkworm which have stopped feeding,we has found that the content of E2 in male and female is slightly different but both have fluctuation at this stage.There is a peak at about 36 h after wandering,then began to decline to the previous level,and finally began to rise again after pupation.Because the silkworm has not eaten in detection period,which suggests that the E2 in silkworm is not derived from food and silkworm has the ability to synthesize E2.2.The transcriptional regulation of E2 to BmVgRT-PCR and q RT-PCR have found that Bm Vg is female specific and high level expression and also was detected weakly expression in the male.And the expression trend in the male is consistent with the female: it began to highly express from the second day of mounting,reached a peak before pupation,and then began to decline.q RT-PCR showed the maximum expression values of female BmVg is 130 times higher than that of males.5nmol/g ecdysteroids（20E）can up-regulated the expression of Bm Vg in fat body of female and male silkworm and increased the content of vitellin of female silkworm’s egg,eventually led to the increase of weight of silkworm eggs.The up-regulation expression by 20 E in the female silkworm could be inhibited by 20 E receptor inhibitors（RH-5992）.Ovaries and the equal 20E（5nmol/g）amounts of E2 can induce the expression of Bm Vg in male silkworm.And ovarian primordial can develop into the egg in the haemolymph of male silkworm.By SDS-PAGE of the total protein of their eggs,there is no obvious difference between the species of its visible protein compared with the eggs development in female silkworm using silver nitrate staining.Besides,it can be detected the existence of vitellin in the eggs that developed in the male silkworm.5nmol/g of E2 decreased the transcription of BmVg in the fat body of female silkworm.In the female silkworm with ovary removed,the BmVg accumulated in haemolymph and the Bm Vg transcription in the fat body was up regulated.BmVg protein that purification from 2 or 3 day of the pupation of female silkworm haemolymph was injected into the haemolymph of the silkworm.But there is no significant difference in the expression of BmVg gene in the fat body compared to the control.But BmVg protein that early（after wandering 36h）appeared in silkworm blood will be degraded.Only when the endogenous BmVg is present,the BmVg protein which was injected can be absorbed by the ovary,and finally increased the content of vitellin in eggs.This may be related to the extent of ovarian development.In addition,feeding on E2 and 20 E can both promote the mounting of silkworm,but the male silkworm is more sensitive to 20 E.2nmol/g and 5nmol/g of 20 E can make the male silkworm mount 24 h earlier than normal,while 10nmol/g of 20 E can only advance 12 h.And 2nmol/g,5nmol/g and 10nmol/g of E2 and 20 E can make the female silkworm wandering 12 h earlier than normal.3.The element in Bm Vg promoter which lead promoter to response E2 and inhibit its activityThe 798 bp of BmVg promoter,which was reported that can make the foreign gene show the same expression mode of endogenous BmVg in silkworm,was scanned on the JASPAR web site,and then got 11 elements that were similar to the estrogen responsive elements of vertebrates’ promoters.Combined with the existing literature reports and information analysis,we selected two nucleic acid sequences on the Bm Vg promoter ——ERRE like 1 and ERRE like 2,which might enable the Bm Vg promoter to respond to E2.Through truncating（remove ERRE like 1）and mutating（ERRE like 2）Bm Vg promoter constructed the luciferase report vector that can drive the luciferase expression in BmE cells.Luciferase enzyme activity found that these two elements of Bm Vg promoter in BmE cells can inhibit the activity of BmVg promoter and treatment with 10-6M E2 can further inhibit the activity of it.Additionally,the inhibition effect of ERRE like 1 was more obvious than that of ERRE like 2.EMSA experiments showed that there were transcription factors extracted in the nuclear protein of BmE cells and the fatbody of female silkworm specifically bind to the elements.4.Molecular characterization of BmERR in silkwormTwo spliceosome sequences of Bm ERR were gained by PCR technology based on the transcriptome resequencing data of silkworm ovaries.Compared with the short-ERR,the long-ERR is 21 bp longer,and it is the main form of ERR in silkworm ovary.It has the typical structure of ERR protein family.The full-length cDNA is 1296 bp and encode 431 amino acid.Its initiation codon is ATG and the termination codon is TAA.Phylogenetic analysis showed that BmERR had higher consistency compared with other ERR amino acid sequences in insects.But ERR in silkworm,Bombyx mori,is the most ancient,compared with other reported insect ERR sequences.5.BmERR can bind to the motif responsed to E2 in BmVg promoterActive BmERR protein by prokaryotic expression and purification incubation with ERRE-like 1,BmERR was able to detect a clear,specific lag in the membrane though EMSA,while incubation with ERRE-like 2 show no lag.Which proved that the ERRE-like 1 could be specifically combined with BmERR,but ERRE-like 2 was not associated with BmERR in vitro.The transcription factor that binded to ERRE-like 2 in the nucleoprotein of BmE cells and the female silkworm fat body is not BmERR.Therefore,ERRE like 1 was named as ERRE.The introduction of the BmERR antibody can reduce the amount of hysteresis band after the incubation between nuclear protein and ERRE probe in the BmE cells and fat body,which illustrated that BmERR was existed in the nuclear protein of the BmE cells and fat body that binding to ERRE.And through the CHIP experiment,we further prove that BmERR can bind to the ERRE on the promoter of BmVg.6.Overexpression BmERR inhibit the activity of BmVg promoterBmERR co-overexpression with Bm Vg promoter containing ERRE-like 1 element drive the expression of fluorescein in BmE cell lines.Double fluorescence experimental results showed that the presence of Bm ERR in BmE cells can significantly decrease the activity of Bm Vg promoter.Furthermore,ERRE-like 1 element was introduced into the A4 promoter by PCR.Co-overexpression with BmERR could reduce the expression of red fluorescent protein.In addition,10-6M estrogen could aggravate the inhibition of BmERR on the promoter of ERRE-like 1 component.7.The expression of BmERR is induced by E2BmE cell lines also expressed Bm ERR,10-6M E2 can upregulate the expression of Bm ERR in BmE cells.RT-PCR testing the expression of Bm ERR and Bm Vg in female silkworm fat body show that the expression of these two genes showed opposite trends.When 5nmol / g E2 was injected into female silkworm at wandering 48 h,the expression of Bm ERR in the fat body raised 12 h later,while the Bm Vg gene expression level was significantly reduced compared with the control 24 h after in the fat body.The Bm ERR expression has no significant differences in gonads of silkworm,but there is a peak of expression before and after pupation,and have very similar trend compared with Bm Vg express pattern in fat body.In summary,based on all the results of this study,silkworm also possesses E2 and has a certain ability to synthesize it.Like with vertebrae oviparity Vg,BmVg expression may also be subjected to the regulation of E2,the difference is E2 inhibit Bm Vg expression in female silkworm.Further study reveal that the inhibition of E2 to Bm Vg expression in female silkworm fat body is effected by inducing transcription factor Bm ERR bind to the regulatory element（ERRE like 1-5’ATCTAGGGTCACAG3 ’）of BmVg promoter.8.BmERR can regulate the expression of BmVg through participating in the pathway of BmEcRBmEcR and BmUsp can’t bind to the ERRE on the promoter of Bm Vg individually,but can combine with the ERRE when they exist at the same time,which explains that BmERR make the function through participating in the signal pathway of BmEcR.Although E2 plays its biological function by ER in the vertebrates,there is no E2 in the insects.Microscale thermophoresis（MST）preliminary confirmed that E2 can combine with EcR in the silkworm,so we speculate that EcR plays as a “receptor” of E2 and mediates E2 to regulate the physiological process in the silkworm.In conclusion,E2 was also present in the silkworm and the silkworms have the ability to synthesis E2.Like Vg in the vertebral oviparity,the expression of BmVg is also regulated by E2.But the difference is that E2 inhibits the transcription of BmVg in the female silkworms’ fat body.The further analysis suggests that E2 inhibits the transcription of BmVg in the female silkworms’ fat body through inducing the transcription factor BmERR bind to the ERRE（5’ ATCTAGGGTCACAG3’）of the BmVg promoter.The ERR in the vertebrate works by taking part in the pathway of ER,but there is no ER in the silkworm.So E2 regards EcR as a “receptor” to induce the expression of BmERR,and then BmERR regulates the expression of BmVg through participating in the pathway of EcR. |
