The BmSuc1 gene,which encodes a novel animal-typeβ-fructofuranosidase(EC 3.2.1.26),was first cloned and identified in silkworm(Bombyx mori).As an essential sucrase,the activity of BmSUC1 is unaffected by alkaloidal s...The BmSuc1 gene,which encodes a novel animal-typeβ-fructofuranosidase(EC 3.2.1.26),was first cloned and identified in silkworm(Bombyx mori).As an essential sucrase,the activity of BmSUC1 is unaffected by alkaloidal sugar mimics in mulberry leaves.This enzyme may also directly regulate the degree of sucrose hydrolysis in the silkworm midgut.In addition,BmSUC1 is involved in the synthesis of sericin 1 in the silk gland tissue.However,the mechanism underlying the regulation of BmSuc1 transcription remains unclear.In this study,we analyzed the BmSuc1 promoter activity using a dual-luciferase reporter assay and identified 4 regions that are critical for transcriptional activation.The gene encoding a predicted transcription factor(TATA-box-binding protein;BmTBP)capable of binding to the core promoter regions was cloned.A quantitative real-time polymerase chain reaction analysis indicated the gene was highly expressed in the midgut.Downregulating BmTBP expression via RNA interference decreased the expression of BmSuc1 at the transcript and protein levels.An electrophoretic mobility shift analysis and chromatin immunoprecipitation indicated that BmTBP can bind to the TATA-box cis-regulatory element in the BmSuc1 promoter.Furthermore,a bioinformatics-based analysis and a far-western blot revealed the interaction between BmTBP and another transcription factor(BmTfIIA-S).The luciferase reporter gene assay results confirmed that the BmTBP–BmTfIIA-S complex increases the BmSuc1 promoter activity.Considered together,these findings suggest that BmTBP regulates BmSuc1 expression through its interaction with BmTfIIA-S.展开更多
基金supported by National Natural Science Foundation of China(Grant No.31872423)the International Science&Technology Cooperation Plan of Anhui Province(Grant No.2022h11020020)the Innovative Research Groups of Anhui Agricultural University(Grant No.ANRC2019032)to Y.Meng.
文摘The BmSuc1 gene,which encodes a novel animal-typeβ-fructofuranosidase(EC 3.2.1.26),was first cloned and identified in silkworm(Bombyx mori).As an essential sucrase,the activity of BmSUC1 is unaffected by alkaloidal sugar mimics in mulberry leaves.This enzyme may also directly regulate the degree of sucrose hydrolysis in the silkworm midgut.In addition,BmSUC1 is involved in the synthesis of sericin 1 in the silk gland tissue.However,the mechanism underlying the regulation of BmSuc1 transcription remains unclear.In this study,we analyzed the BmSuc1 promoter activity using a dual-luciferase reporter assay and identified 4 regions that are critical for transcriptional activation.The gene encoding a predicted transcription factor(TATA-box-binding protein;BmTBP)capable of binding to the core promoter regions was cloned.A quantitative real-time polymerase chain reaction analysis indicated the gene was highly expressed in the midgut.Downregulating BmTBP expression via RNA interference decreased the expression of BmSuc1 at the transcript and protein levels.An electrophoretic mobility shift analysis and chromatin immunoprecipitation indicated that BmTBP can bind to the TATA-box cis-regulatory element in the BmSuc1 promoter.Furthermore,a bioinformatics-based analysis and a far-western blot revealed the interaction between BmTBP and another transcription factor(BmTfIIA-S).The luciferase reporter gene assay results confirmed that the BmTBP–BmTfIIA-S complex increases the BmSuc1 promoter activity.Considered together,these findings suggest that BmTBP regulates BmSuc1 expression through its interaction with BmTfIIA-S.
