Identification Of MBF2 Family And Its Member MBF2-1 Involved In The Regulation Of The Transcription Of Silk Fibroin Heavy Chain

The silkworm,Bombyx mori,as an important economic insect,is cultivated for silk production.The silk-producing organ,which is highly specialized,is known as the silk gland,and it can be divided into anterior(ASG),middle(MSG),and posterior(PSG)silk gland regions.and the silk is composed of silk fibroin and sericin,which are synthesized in the posterior silk gland and middle silk gland,respectively.In general,the fibroin which is the major silk protein component consists of the fibroin heavy-chain(fibH),fibroin-light-chain(fib L),and P25 proteins.The yield of silk closely depends upon the developmental stage of the silkworm.Silkworms only spin a large amount of silk proteins at the end of the last instar phase to make a cocoon.However,silkworms also secrete a very small amount of silk proteins during other larval instar stages,except during each molting stage.These silk proteins are synthesized under strict distinct temporal-and spatialspecificities that are regulated at the transcriptional level.The identification and characterization of transcription factors involved in the regulation of fibroin genes represents a key step in understanding the mechanism of silk protein synthesis.Although many efforts have been made,the mechanism is not clear.Multiprotein bridge factor 2(MBF2)was separated from silk glands and identified as a mediator of transcription factor.Liu et al(2000)analyzed the expression pattern of MBF2 and found that its m RNA transcripts could not be detected in the fat body or trachea,although MBF2 could generally be detected in the silk gland.Additionally,it was expressed in a temporally and spatially specific manner.These findings suggest that MBF2 may be involved in the regulation of silk protein synthesis.Based on the information platform of the silkworm genome database,Bioinformatics analysis of MBF2 and the research about its function in the regulation of fibH research was carried out,and the main results are as follows: 1.Identification and expression analysis of MBF2 family genes from the silkworm Bombyx mori and their function analysisTo identify of MBF2 family member genes in the silkworm genome,MBF2 protein sequence was screened against the NCBI non-redundant(nr)and silkworm databases to identify homologous genes.ULtimately,nine genes were identified and named MBF2-1,-2,-3,-4,-5,-6,-7,-8,-9,respectively.Bioinformatics analysis showed that there were transcripted in silkworm and the gene duplication event was happened in the process of evolution.The analysis of their amino acid sequences showed that their predicted protein lengths ranged from 110 to 118 amino acids,and all MBF2 family members contain a signal peptide,which implies that these proteins might be secreted.Furthermore,multiple protein sequence alignments showed 41% amino acid sequence identity among the MBF2 family members,which suggested that these sequences are highly homologous.Phylogenetic tree showes the MBF2 family gene is inesct-specific,and MBF2-1,MBF2-2,MBF2-3,and MBF2-7 grouped together,and were related to MBF2 protein from Danau plexippus and Samia cynthia.However,the other members display high similarity to ?REPAT proteins,which means that the MBF2 could be involved in the immune response of silkworm.Tissue distribution analysis showed that these genes were expressed in a tissuespecific manner.MBF2-1 was highly expressed in malpighian tubule,MBF2-5 was specifically expressed in the midgut,MBF2-2,MBF2-3,MBF2-4,MBF2-6,and MBF2-8 were expressed highly in the haemolymph,MBF2-9 was detected in the fat body and malpighian tubules,while MBF2-7 was highly in the fatbody,testies,and ovary.Developmental profile analysis showed that the family genes were Stagespecific and highly expressed in the different periods.MBF2-4,MBF2-6,MBF2-8,and MBF2-9 exhibited a high expression level during the wandering and pupation stage.In contrast,MBF2-2,MBF2-3,and MBF2-5 were lowly expressed during the pupation and the moth stage,but highly expressed during the larval stage.We also found MBF2-1 exhibited a sustained high-level of expression in the 5th instar and the wandering stage of silkworm,while MBF2-7,different with the genes we mentioned above,was detected during the whole growth and development.It is known that the larval stage is the key stage of feeding to obtain the energy,and the pupa is the period of the adult organs organogenesis and formation.What's more,silk gland,midgut,haemolymph,and fat body are important tissues that are involved in material and energy metabolism.We speculate that these genes could play a vital role in the metabolism and development of silkworm.Analysis of the expression patterns of nine MBF2 family members after Bacillus bombysepticus infection showed that these genes clearly exhibited differentially induced expression patterns.Among them,MBF2-4,MBF2-7,and MBF2-9 were significantly up-regulated in the head 1 h after silkworm larvae were injected with Bb,but then declined to a lower level compared with controls 3 h after injection.The MBF2-8 gene was up-regulated in response to Bb at the 12 h time point and then recovered to normal level.The expression of MBF2-2 and MBF2-3 showed different patterns,as they were down-regulated 3 h after Bb injection and then recovered to normal levels.These findings suggest that they were involved in response to Bb.Furthermore,we found the MBF2 family genes were modulated by starvation and the expression of these genes recovered upon re-feeding.MBF2-1,-4 and-8 showed a similar pattern of variation as they decreased significantly upon starvation,but increased again after re-feeding the insect.By contrast,some genes were up-regulated by starvation and down-regulated by re-feeding,such as MBF2-2,-3,-6 and-7.However,the expression of MBF2-5 and-9 were also affected,but compared with other genes,relatively slow change.These findings suggest that clear connection between these genes and nutrient metabolism.2.Prokaryotic expression and analysis of expression pattern of MBF2Prokaryotic expression vector of MBF2 was constructed and the recombinant protein was purified to prepare the polyclonal antibody for the subsequent expriments.q PCR analysis of different sections of silk gland showed that MBF2 transcription occurs only in the posterior silk gland.However,Western blotting analysis showed that MBF2 protein existed in the anterior and middle silk gland regions,except posterior silk gland and Malpighian tubules.The immunohistochemistry analysis of silk showed that MBF2 protein existed highly in the outer layer of a cocoon.These results suggest MBF2 is a secretory protein,which is consistent with the previous prediction results.The MBF2-8 had been proved to be involved in the immune response in Bombyx mori,so the MBF2 in cocoon may be involved against foreign pathogens invasion,which laid the foundation for the further study of the immune function of MBF2,which means it may participate in the regulation of fibH.An analysis of temporal expression patterns by PCR and western blot showed that the expression pattern of MBF2 was opposite of the fibH gene,as its expression was high during the fourth molting stage,decreased gradually during the fifth instar feeding stage,and disappeared at the end of the fifth instar phase,which means it may participate in the regulation of fibH.3.MBF2 is involved in the regulation of fbi HTo gain insights into the potential roles of MBF2 in the silkworm,dual luciferase reporter assays were performed and fibH promoter activity decreased when MBF2 was over-expressed in vitro,which suggests it participate in the regulation of fibH.However,previous studies showed that MBF2—a multiprotein bridge factor—could not directly bind to DNA,and the DNA binding domain was not predicted in the above study.Therefore MBF2 should be combined with other transcription factors to play the regulatory role.Subcellular localization was assessed by expressing Bmdimm and MBF2 in Bm E cells and showed that MBF2 expression overlapped with Bmdimm in the nucleus.So MBF2 was predicted to interact with Bmdimm.To verify the aforementioned interactions,a Far-western blot assay was used with purified Bmdimm and SUMO-MBF2 fusion protein and showed that they could interact with each other in vitro.BIFC assay was simultaneously performed and the result was in good agreement with the results of vitro experiment.These data demonstrated that MBF2 protein interacted with Bmdimm protein in the nucleus.Interestingly,Bmdimm has been proved to be involved in regulation of the fibH gene and worked as a positive regulator,but the fibH promoter activity was downregulated when MBF2 and Bmdimm were co-expressed.Together,these findings suggest MBF2 inhibits the fibH promoter activity,as well as the Bmdimm-mediated activation of the fibH promoter.4.FTZ-F1 is involved in the regulation of fbiH MBF2 was identified as a co-activator of Bm FTZ-F1 in vitro transcription assay,but whether they interact with each other directly is still unknown.First,Prokaryotic expression vector of Bmftz-f1 was constructed and the recombinant protein was purified to prepare the polyclonal antibody for the subsequent expriments.Tissue distribution analysis by qPCR showed that Bmftz-f1 transcripts were detected in all of the tissues on day 3 of the fifth instar,and western blot analysis showed that Bm FTZ-F1 was detected in all of the tissues tested,but it was highly expressed in the silk gland and Malpighian tubules.Developmental profile analysis showed that Bm FTZ-F1 was highly expressed in the posterior silk gland during the fourth molting stage and the beginning of the fifth instar,and then expression decreased gradually until it disappeared at the end of the fifth instar and the wandering stage.Bm FTZ-F1 exhibited opposite expression patterns with fibH,but same with MBF2.Furthermore,Subcellular localization,BiFC,Co-IP,and Farwestern blot assays were performed and revealed that Bm FTZ-F1 could interact with MBF2 in vivo and vitro.These finding show that Bm FTZ-F1 might be involved in regulation of fibH via its interaction with MBF2.A Basic Local Alignment Search Tool(BLAST)search of the fibH promoter using the FTZ-F1 recognition sequence was performed,and found that its potential binding sites were located-389~-397 bp and-652~-659 bp upstream of the transcription initiation site.EMSA and ChIP analysis identified an FTZ-F1 response element-389~-397 bp upstream of the transcription initiation site of the fibH promoter.Furthermore,these results were confirmed by truncating or mutating the fibH promoter.The fibH promoter activity increased when we truncated the FTZ-F1 response element,and the same result was obtained by mutating the FTZ-F1 response element.Additionally,the promoter activity no longer decreased when FTZ-F1 was over-expressed.Together,these results clearly demonstrate that the fibH(-389--397)region contains an FTZ-F1 response element,and that Bm FTZ-F1 exerts a negative effect on fibH transcription by binding to this element.We have shown that MBF2 and FTZ-F1 can down regulate fibH promoter activity seprately,when MBF2 and FTZ-F1 were co-expressed;fibH promoter activity was down-regulated.These results suggest that they were involved in the regulation by interact with each other.In addition,through the analysis of the expression level in Dazao and 872 by qPCR showed that the expression level MBF2 and Bmftz-f1 in 872 is much lower than that in Dazao.5.The effect of 20 E on fibH regulationTo date,studies of the transcriptional regulation of fibH by 20 E in the silkworm have been rarely reported.To determine how 20 E regulates the expression of fibH,dual luciferase reporter assays were performed and reveal that 20 E could regulate the fibH in a dose dependent manner.The question how 20 E regulate the fibH was raised;Bmftz-f1 in the PSG could be induced by 20 E in vivo and in vitro.And the MBF2 showed the same express pattern with Bm FTZ-F1;therefore MBF2 may be controlled by 20 E.To test this hypothesis,a prediction of potential regulatory elements in the MBF2 promoter region revealed several possible transcription factorbinding elements,including E74 A,Br-C,and Ec R sequences.Hormone treatments experiment showed that the MBF2 expression was induced in PSG in vivo and vitro,and the expression of MBF2 and fibH have a relationship of restricting each other.Together,these findings indicate that MBF2 is involved in regulating fibH expression,and that this process is modulated by 20 E in B.mori.Additionally,MBF2 could interact with Bm FTZ-F1.Together,these findings suggest that the regulation of fibH by 20 E is mediated by MBF2 and Bm FTZ-F1.Interestingly,the response element of Bm FTZ-F1 is close to that of Bmdimm.Furthermore,they both could interact with MBF2 directly.This finding prompted us to investigate the possibility that Bm FTZ-F1 interacts with Bmdimm.A Far-western assay using purified Bm FTZ-F1 and Bmdimm were performed and reveal that Bm FTZ-F1 directly binds to Bmdimm in vitro.To confirm whether Bm FTZ-F1 and Bmdimm interact in vivo,A Bi FC assay was conducted and reveal the same result with the Far-western assay.Dual luciferase reporter assays were performed to detect the effects of Bm FTZ-F1 and Bmdimm on the fibH promoter when they are coexpressed.Promoter activity increased significantly when only Bmdimm was overexpressed.The result is consistent with a previous study.However,compared with the over-expression of Bmdimm alone,fibH promoter activity did not increase when Bmdimm and Bm FTZ-F1 were co-expressed.These findings suggest that Bm FTZF1 binds to Bmdimm and counteracts the effect of Bmdimm on fibH expression.In summation,the expression of FTZ-F1 and MBF2 induced by 20 E,and then they can interact with Bmdimm and inhibit its effect on the regulation of fibH to achieve the low amount of fibH expression or no expression.