Identification Of Hypoxia-responsive Genes'transcription Factors And The Regulatory Mechanisms In Callosobruchus Maculatus

Oxygen is essential for organisms including insects.It is an efficient method to control cowpea bruchid(Callosobruchus maculatus)in the stored beans by lowering the oxygen content in the airtight containers.However,cowpea bruchid,especially the 4^thinstar larvae,shows a strong tolerance to hypoxia stress.Under 2%oxygen,the cowpea bruchid stops feeding and growth.Once resume to normoxia,it returns to normal development.Previous studies have mainly focused on the biology aspects,a limited researches were conducted to uncover the molecular mechanisms in respond to hypoxia stress in cowpea bruchid.In this study,we obtained the hypoxia-responsive genes and analyzed the common cis-regulatory elements(CREs)among the hypoxia-responsive genes'promoter regions to identify transcription factors regulating the expression of the differentially expressed genes under hypoxia.In addition,we studied the molecular mechanism of transcription factors regulating the expression of relevant genes in respond to hypoxia stress.The results are of great significance to reveal the tolerance mechanisms in respond to hypoxia stress and the research provides a new method to study gene transcriptional regulation.The main results are as follows:(1)CmFKH activates the transcriptional expression of hypoxia-responsive gene CmPFKFB3We successfully cloned and obtained the Cm PFKFB3 promoter sequence.Through part-by-part promoter activity verification and EMSA experiments,we found the transcription factor binding site regulating the expression of Cm PFKFB3was located at-520?-461 bp(P8).CREs analysis indicated P8 contains 3 CREs including FKH,KR,HRE.The EMSA competition binding experiments showed that FKH plays an important role in the transcriptional expression of Cm PFKFB3.The FKH binding protein CmFKH was successfully cloned form the midguts of cowpea bruchid.CmFKH encodes 432 amino acids with a highly conserved DNA binding domain.We also cloned the KR binding protein Cm KLF,which encodes 239amino acids with a highly conserved Cys2/His2 zinc finger.Overexpression of CmFKH in Drosophila S2 cells significantly enhanced LUC activity and hypoxia-treated S2 cells that overexpressed CmFKH further increased LUC activity.However,S2 cells overexpressing Cm KLF,Cm HIF1,and CmFKH with point mutations in the DNA binding domain did not exhibit any changes in LUC activities,respectively.In vitro synthesized CmFKH could specifically bind to CRE FKH.Under hypoxia stress,the m RNA expression of CmFHK firstly increased and then decreased,while the expression of Cm PFKFB3 m RNA continued to display its upward trajectory.The results demonstrated that CmFKH could interact with FKH CRE in Cm PFKFB3 promoter region to activate the expression of Cm PFKFB3 under hypoxia stress.(2)CREs AREB6,CDX2,CEBP and CHR function analysis in the transcription of hypoxia responsive genesHypoxia resulted in 602 differentially expressed genes(DEGs),of which 408genes were upregulated and 194 genes were downregulated.After DEGs annotation,GO clustering and KEGG pathway analysis,the DEGs were mainly clustered in the pathways related to the energy metabolism processes,indicating that hypoxia stress has a great impact on the metabolism processes in cowpea bruchid.We selected 46DEGs with translation initiation sites and having GO annotation or KEGG pathways.Among these genes,37 DGEs'promoter sequences were obtained after aligning with genomic database.Using Common TFs software,19 and 18 common CREs were obtained from the up-and down-regulated genes'promoter regions,respectively.Among which,CREs DFD,HOAX3,BARX2,BTN,SLOU,SMARCA3 and DRI were commonly shared by both the hypoxia-induced and repressed genes.After transcription factor binding sites analysis,some of the common CREs,including AREB6,CEBP,CDX2,ABDB,CHR,SMARCA3,were labeled with?-³²P and incubated with nuclear extracts.The results showed these?-³²P labeled probes exhibited different binding patterns with midgut nuclear extracts from normoxic and hypoxia-treated 4^thinstar larvae and the competition assays confirmed the binding specificities.Further luciferase activity assays showed that CREs ABDB and SMARCA3 did not have any functions in the promoter regions of hypoxia responsive genes Cm HSP60,Cm ENOPH,respectively.While AREB6,CDX2,CEBP and CHR played important roles in regulating the expression of CmScylla/CmLPCAT,CmL-Gal DH,Cm GPCPD1 and Cm E63-1/Cm PKA,respectively.(3)CmZFH function analysis in hypoxia responsive genes CmScylla and CmLPCAT transcriptional expressionThe AREB6 binding protein CmZFH was successfully cloned form the midguts of cowpea bruchid.CmZFH encodes 961 amino acids.Its N-terminal,C-terminal zinc-finger clusters and homeodomain in the middle are highly conserved.Overexpression of CmZFH in Drosophila S2 cells could significantly activate the expression of CmScylla and CmLPCAT,while such activation effects could not be observed when AREB6 was removed from the promoters of CmScylla and CmLPCAT,respectively.The resulted suggested CmZFH could activate the expression of CmScylla and CmLPCAT through interacting with AREB6 on their promoter regions,respectively.Prokaryotic expression of CmZFH protein could specifically bind to AREB6.CmZFH lacking the N-terminal zinc finger cluster could not bind to AREB6,nor could it activate the transcription of CmScylla.However,CmZFH without N-terminal zinc-finger cluster or homeodomain could bind to CRE AREB6 and activate the transcription of CmScylla as well.Hypoxia activated the m RNA expression of CmZFH,CmScylla,and CmLPCAT in cowpea bruchid.CmZFH was induced a little earlier than CmScylla and CmLPCAT,demonstrating the regulatory effect of CmZFH on CmScylla and CmLPCAT expression.Feeding ds CmZFH successfully silenced CmZFH,leading to a decrease in upward trend of CmScylla,CmLPCAT under hypoxia stress.In summary,when cowpea bruchid was challenged by oxygen deprivation,CmZFH could interact with AREB6 CRE on the promoter regions of CmScylla,CmLPCAT to activate CmScylla,CmLPCAT expression,respectively.The N-terminus zinc-finger cluster of CmZFH plays a vital role in the CmScylla activation.