Identification Of BHLH Transcription Factor Family Members In Bombyx Mori And In Silico Cloning Of Their Genes

Basic helix-loop-helix transcription factors form a super family of eukaryotic proteins.Its members play crucial roles in the regulation of organismal growth and development.The bHLH motif is composed of approximately 60 amino acids.It has one basic region,which is capable of binding DNA,and a HLH(Helix 1-Loop-Helix 2) structure,in which the loop region varies in different bHLH proteins.Since the discovery of the first bHLH protein in 1989,nearly 1000 bHLH sequences have been identified in various organisms.Animal bHLHs have been classified into 45 families according to their varied roles in regulating gene expression.Besides,they are divided into 6 groups based on their target DNA elements and their own structural characteristics.Group A has 22 families,They mainly regulate neurogenesis, myogenesis and mesoderm formation.Group B has 12 families.They mainly regulate cell proliferation and differentiation,sterol metabolism and adipocyte formation,and glucose-responsive gene expression.Group C has 7 families.They are responsible for the regulation of midline and tracheal development,circadian rhythms,and for the activation of gene transcription in response to environmental toxins.Group D has only 1 family.It forms inactive heterodimers with group A bHLH proteins.Group E has 2 families,which regulate embryonic segmentation,somitogenesis and organogenesis etc. Group F also has 1 family.It regulates head development and formation of olfactory sensory neurons etc.So far,the total number of animals of which bHLH family members have been identified is 15.The members identified in each species are 118 in human,102 in mouse, 84 in puffer fish,78 in lancelet,68 in sea anemone,63 in Lottia gigantea,64 in Capitella sp.I,59 in fruit fly,57 in Daphnia pulex,50 in sea urchin,50 in red flour beetle,46 in sea squirt,39 in nematode,33 in Hydra magnipapillata and 16 in sponge.Silkworm(Bombyx mori)is an important model insect of Lepidoptera.Silkworm genome sequencing project has been finished in October 2003.However,there has been no report on how many bHLH family members existing in the genome.This study employed bioinformatic methods to conduct studies on "Identification of bHLH transcription factor family members in Bombyx mori and in silico cloning of their genes".Its objectives includes to find out the number of bHLH transcription factor family members in silkworm and to explore gene structures that code for the bHLH transcription factors.The major results obtained are as follows:1.The silkworm genome has regions that code for 52 bHLH transcription factors. This figure is comparable with those in other insects.Twelve of the silkworm bHLH motifs have introns.One of them has two coding regions,locating on Ctg038409 and Ctg063456 respectively.2.All 52 identified silkworm bHLH transcription factors have been assigned into their corresponding groups and families.It was found that group A has 21 silkworm bHLH members.They are BmASCal,BmASCa2,BmASCa3,BmASCa4,BmMyoD, BmE12/E47,BmNgn,BmAtonal,BmMist,BmBeta3,BmNet,BmDeliah,BmMesp, BmTwist,BmParaxis,BmMyoRa,BmHand,BmPTFa,BmPTFb,BmSCL and BmNSCL.Group B has 10 members.They are BmSRC,BmMyc,BmMnt,BmMax, BmUSF,BmMITF,BmSREBP,BmAP4,BmMLX and BmTF4.Group C has 12 members.They are BmClock1,BmClock2,BmClock3,BmARNT,BmBma11, BmBma12,BmAHR1,BmAHR2,BmAHR3,BmSim,BmTrh and BmHIF.Group D has one member,BmEmc.Group E has 7 members.They are Hey1,BmHey2, BmH/E(spl)1,BmH/E(spl)2,BmH/E(spl)3,BmH/E(spl)4 and BmH/E(spl)5.Group F has one member,BmCOE.Coding regions for ASCb,NeuroD,Oligo,MyoRb,Figαand Mad families were not found in silkworm genome.3.Structural information of 11 silkworm bHLH genes was obtained through in silico cloning.They are BmASCa4,BmAtonal,BmBeta3,BmMesp,BmTwist, BmParaxis,BmMyoRa,BmMyc,BmUSF,BmEmc and BmH/E(spl)4.Partial structural information of 5 silkworm bHLH genes was also obtained.They are BmMLX,BmTF4, BmClock1,BmARNT and BmHIF.4.Functions of 5 BmbHLH proteins have been deduced,among which BmASCa4 is encoded by l'sc gene and promotes formation of neural precursers;BmAtonal is encoded by ato gene and controls the formation of chordotonal organs and photoreceptors;BmTwist plays roles in controlling differentiation of mesodermal lineage and inhibiting functions of transcription factor MyoD;BmMyoRa inhibits the formation of muscle cells;and BmEmc exerts negative control through forming heterodimers that lack DNA-binding ability with other bHLH proteins.5.Emc gene structures have also been explored in human,rat,mouse,red flour beetle,fruit fly,Aedes aegypti,Anopheles gambie str.PEST,honeybee,chicken, Xenopus,zebrafish and sea urchin.Based on their structures,it seems that,as the evolutionary level of the organisms heightens,exon and intron lengths of the Emc genes shorten gradually,tending to support the introns-early theory.The achievements of this study accrued fundamental knowledge for future studies on regulating mechanisms of silkworm growth and development.They can also facilitate further studies on expression patterns of the bHLH genes and on comparison of bHLH gene structures and functions between different species.