Glycerol-3-phosphate dehydrogenase (GPDH) is a soluble cytosolic NAD-dependent enzyme present in eukaryotic organisms and has some important functions,such as energy transfer.Bombyx mori is a central model animal to research metamorphosis and energy metabolism.In order to determine the role of GPDH in B.mori,we cloned a complete gene of BmGpd based on protein (lethal (2) k05713) sequence of Drosophila melanogaster from NCBI (GenBank accession number:NP_725495.1),which is 27 983 bp in full length and contains 16 exons and 15 introns (GenBank accession number is EF154335).The two complete mRNA transcripts from B.mori strain C108 are 3 456 bp and 2 979 bp in full length (GenBank accession numbers are GQ865685 and GQ865686,respectively),both including a 2 166 bp ORF that encodes 721 amino acids (Mw 82.1 kD,pI 6.43).BmGPD protein includes several membrane-spanning regions and a signal peptide sequence with a length of 20 aa.The results of phylogenetic analysis and motif search revealed that BmGPD belonged to glycerol-3-phosphate dehydrogenase (GPDH) family,which was not reported in B.mori and could be abundantly inducible expression in Escherichia coll.Semi-quantitative RT-PCR analysis indicated that a high-level of BmGpd mRNA occurred in tissues (organs) of the midgut,silk gland,gonad and fat body,and it occurred at the middle stage of the 5th instar larva;however,a low level in the blood,as well as on day 3 of the pupal stage,and a lower level occurred during the diapause.The result of microarray-based gene expression profile showed that the expression abundance of BmGpd was very high in head and integument,while low in fat body and Malpighian tubule on day 3 of the 5th instar larva,and there were no obvious differences between the male and female strains.Expression profile of ESTs indicated that the expression abundance was the highest in the midgut on day 2 of the 4th instar larva.RNAi result suggested that there are strong metabolic compensation pathways to make up for the silence of BmGpd gene.This study provides a basis for further investigating the expression regulation of GPDH and its relationship with metamorphosis and energy metabolism in B.mori.

Bombyx mori is the model insect of lepioloptera with diapausing during its egg stage.Diapause inducements are due to temperature and photoperiod in the incubation process of parental eggs.to investigate the relationship of diapause inducements and temperature and photoperiod,we design this experiment to search the affect of temperature and photoperiod on the gene sod and cat.When eggs were incubated in 25 LL,their sod mRNA would keep at a high level,but the transcription level was rapidly down-regulated at EAT1 800℃.h and 3 600℃.h.The expression of sod gene in 20 LD was significantly higher than in 15 DD,this phenomenon was more obvious when the embryonic nervous had fully grown in the later incubation stage.The expression of cat gene had no obvious differences when eggs were incubated in 25 LL,20 LD or 15 DD but it was notable that the mRNA level of cat all increased in the late incubation stage,which was in accordance with the fact that the eggs were more sensitive to diapause-inducing incubation temperature and photoperiod of this time.After artificial hatching treatment with hydrochloric acid at 24h after oviposition,the sod mRNA in diapause-terminated eggs (DTE) was much higher than diapause-destined eggs (DDE).From 24 to 192hours after oviposition,eggs at progeny went into the diapause stage,the transcription level of sod subsequently decreased.After we examined the common effect of illumination and temperature on the sod gene of progeny eggs,it was observed that the sod mRNA increased under constant darkness at 15℃.The cat mRNA level of DTE was lower than DDE at first after oviposition,but then the mRNA increased,which indicated that the expression of cat might be inhibited by hydrochloric acid at the early activation.Then we investigated the common effect of illumination and temperature on the cat gene of progeny eggs and found that the expression level of cat in 20 LD was lower than in 25 LL and 15 DD.

Glycerol-3-phosphate dehydrogenase (GPDH) is a soluble cytosolic NAD-dependent enzyme present in eukaryotic organisms and has some important functions,such as energy transfer.Bombyx mori is a central model animal to research metamorphosis and energy metabolism.In order to determine the role of GPDH in B.mori,we cloned a complete gene of BmGpd based on protein (lethal (2) k05713) sequence of Drosophila melanogaster from NCBI (GenBank accession number:NP_725495.1),which is 27 983 bp in full length and contains 16 exons and 15 introns (GenBank accession number is EF154335).The two complete mRNA transcripts from B.mori strain C108 are 3 456 bp and 2 979 bp in full length (GenBank accession numbers are GQ865685 and GQ865686,respectively),both including a 2 166 bp ORF that encodes 721 amino acids (Mw 82.1 kD,pI 6.43).BmGPD protein includes several membrane-spanning regions and a signal peptide sequence with a length of 20 aa.The results of phylogenetic analysis and motif search revealed that BmGPD belonged to glycerol-3-phosphate dehydrogenase (GPDH) family,which was not reported in B.mori and could be abundantly inducible expression in Escherichia coli.Semi-quantitative RT-PCR analysis indicated that a high-level of BmGpd mRNA occurred in tissues (organs) of the midgut,silk gland,gonad and fat body,and it occurred at the middle stage of the 5th instar larva;however,a low level in the blood,as well as on day 3 of the pupal stage,and a lower level occurred during the diapause.The result of microarray-based gene expression profile showed that the expression abundance of BmGpd was very high in head and integument,while low in fat body and Malpighian tubule on day 3 of the 5th instar larva,and there were no obvious differences between the male and female strains.Expression profile of ESTs indicated that the expression abundance was the highest in the midgut on day 2 of the 4th instar larva.RNAi result suggested that there are strong metabolic compensation pathways to make up for the silence of BmGpd gene.This study provides a basis for further investigating the expression regulation of GPDH and its relationship with metamorphosis and energy metabolism in B.mori.

Pyruvate dehydrogenase (PDH) is the former enzyme in pyruvate dehydrogenase complex and participates in generating acetyl coenzyme A,the initiator of tricarboxylic acid (TCA) cycle.It also plays a decisive role in the distribution of nutrition composition.By means of in silico cloning,RT-PCR and rapid amplification of cDNA ends (RACE) technology,a full-length cDNA which was similar to Drosophila melanogaster lethal(1) G0344 gene with pyruvate dehydrogenase function was cloned from silkworm (Bombyx mori) and designated as Bm-l(1) .It is 1 630 bp long,contains a complete ORF of 1 200 bp,186 bp of 5'-UTR and 207 bp of 3'-UTR.Bm-l(1) gene contains 8 exons and 7 introns.It encodes 399 amino acids,with predicted molecular mass of 43.93 kD and isoelectric point of 8.07.The deduced amino acids showed that an E1-dh domain was located between the 69th and the 365th amino acid residues.This structural domain is uniquely owned by thiamine pyrophosphate dependent dehydrogenases.Protein seconda-ry structure prediction showed that 28.8% of the protein is composed of α helix and 12.0% of β sheet.Multiple sequence alignment with Clustal W program revealed that Bm-l(1) coded protein has over 63% sequence identity with PDHs from Tribolium castaneum and other insect species.Nevertheless,their conservative regions are in high consensus.Bm-l(1) mRNA had high transcriptional levels during the whole egg,larval,and pupal stages,and in emerging adults,head,silk gland,gonad,fat body,midgut and hemolymph of 3-day-old larvae of the 5th instar,and the difference between tissues was relatively low.

An Expressed Sequence Tag (EST) is a short sub-sequence of a transcribed cDNA sequence. ESTs represent gene expression and give good clues for gene expression analysis. Based on EST data obtained from NCBI, an EST analysis package was developed (apEST). This tool was programmed for electronic expression, protein annotation and Gene Ontology (GO) category analysis in Bombyx mori (L.) (Lepidoptera: Bombycidae). A total of 245,761 ESTs (as of 01 July 2009) were searched and downloaded in FASTA format, from which information for tissue type, development stage, sex and strain were extracted, classified and summed by running apEST. Then, corresponding distribution profiles were formed after redundant parts had been removed. Gene expression profiles for one tissue of different developmental stages and from one development stage of the different tissues were attained. A housekeeping gene and tissue-and-stage-specific genes were selected by running apEST, contrasting with two other online analysis approaches, microarray-based gene expression profile on SilkDB (BmMDB) and EST profile on NCBI. A spatio-temporal expression profile of catalase run by apEST was then presented as a three-dimensional graph for the intuitive visualization of patterns. A total of 37 query genes confirmed from microarray data and RT-PCR experiments were selected as queries to test apEST. The results had great conformity among three approaches. Nevertheless, there were minor differences between apEST and BmMDB because of the unique items investigated. Therefore, complementary analysis was proposed. Application of apEST also led to the acquisition of corresponding protein annotations for EST datasets and eventually for their functions. The results were presented according to statistical information on protein annotation and Gene Ontology (GO) category. These all verified the reliability of apEST and the operability of this platform. The apEST can also be applied in other species by modifying some parameters and serves as a model for gene expression study for Lepidoptera.