Expression Analysis And Characterization Of Sorbitol Dehydrogenase Gene From The Silkworm, Bombyx Mori

Sorbitol dehydrogenase (SDH) is the key enzyme to convert sorbitol to glycogen.In the silkworm, Bombyx mori there are three SDH genes, BmSDH-1, BmSDH-2a andBmSDH-2b. The variation of sorbitol content in the silkworm eggs shows closelyparallel relationship with the onset, duration and termination of diapause procedure.Up to date, a large number of researches have been done focusing on the experssionand activity of BmSDH in the eggs. There is little research on the other developmentstages, tissues and promoter activity in the silkworm.In this thesis, based on the bioinformatics analysis of BmSDH-1, BmSDH-2a andBmSDH-2b in gene structure, homologies and protein structure, we analyzed thetemporal and spatial expression of three BmSDH genes by using semi-RT-PCR, andcompared the effects of two different incubation on BmSDH expression,25℃in12hlightness/12h darkness and17.5℃in darkness. Further more, two heterogeneousBmSDH-2a promoter fragments were cloned and characterized.1. Bioinformatic analysis of BmSDH genesBmSDH-1,-2a and-2b genes are all located in the silkworm chromosome21.BmSDH-1gene has a full-length of6.035kb nucleotides composed of8exons and7introns; BmSDH-2a gene has a full-length of10.75kb nucleotides composed of6exons and5introns; and BmSDH-2b gene has a full-length of10.15kb nucleotidescomposed of8exons and7introns. The primary protein structure analysis showedthat the similarity among BmSDH-1and BmSDH-2a, BmSDH-2b were49.7%, thesimilarity between BmSDH-2a and BmSDH-2b was96.4%. We constructed aphylogenetic tree with MEGA4.0software based on the homologous protein aminoacid sequences of BmSDH-1,-2a,-2b and other13species including insects,anphibiens, mammalians and human. In this tree, BmSDH-1formed an independentbranch; while BmSDH-2a and-2b ware in another sub-branch, they showed a closegenetic relationship with Apis aegypti, Bombus impatiens and Harpegnathos saltator.2. Analysis on expression of BmSDH genes and effects of incubation conditionsduring embryo stage on their expressionAccording to the principle that diapause ability of bivoltine silkworm strainsprogeny is regulated by the incubation conditions during embryo stage of parent eggs,i.e. when parent eggs are incubated under25℃their progeny eggs are in diapause, when parent eggs are incubated under20℃or lower temperature in darkness theirprogeny are non-diapause. Diapause terminated hibernating eggs (in single batch) ofQiufeng, a bivoltine silkworm strain were used. Each egg batch was divided into twosemi-batches. One group was incubated at17.5℃in darkness (Lower temperaturein Darkness, LD) and the other at25℃in lightness (Normal temperature andLightness, NL), respectively. By using semi-quantitative RT-PCR, we analyzed theBmSDH genes transcript levels in different development stages of the feamalesilkworm and tissues. The results showed that BmSDH-1, BmSDH-2a and BmSDH-2bwere expressed at various developmental stages tested in bivoltine silkworm strainQiufeng. In the fifth instar of larval stage3d, BmSDH-1in NL group has a higherexpression in the fat body, the BmSDH-2a and BmSDH-2b transcript levels in blood,fat body and ovary of NL incubated were higher than those of LD incubated. In thepupal stage3d, BmSDH-2a and BmSDH-2b transcription levels in blood and ovary ofNL incubated were lower than those of LD incubated. In the progeny eggs from6h to24h post ovulation, BmSDH-1transcript level was very low and almost withoutchange. While BmSDH-2a and BmSDH-2b transcript levels gradually increased, butthere was no visible difference between LD and NL incubation.3. Cloning and characterization of BmSDH-2a gene promoterAccording to the nucleotide sequence of BmSDH-2a gene promoter region, twoupstream and one downstream primer were designed. And two heterogeneousBmSDH-2a promoter fragments of674bp and355bp were cloned by PCR usinggenomic DNA of posterior silkgland from the fifth instar larvae as template,respectively. Online bioinformatic analysis predicted that674bp promoter fragmentcontains the core promoter elements such as TATA box and GC box, and somepotential embryo development related transcription factor binding sites includingGATA-1, HSF1, CREB, CdxA, cap and Oct-1, the posterior four element weremultiple appeared. A luciferase reporter plasmid using pGL3.0basic vector driven byheterogenous BmSDH-2a promoter fragments, BmSDH-2aP-674andBmSDH-2aP-355were constructed, respectively. BmN cells were transfected withone of the reporter plasmids and cells transfected with pGL3.0-luc were served as thepositive control. Cells were harvested72post transfection and the luciferase activitieswere tested on LuminoMeter20/20. The results revealed that both two promoterfragments showed transcription activities. But the promoter activity of674bp is morehigher, which is2.4folds of that of355bp one, implying there is(are) one or several positive regulation elements in the region of-674nt to-355nt.These results provide new experimental data for elucidating the molecularmechanism of silkworm diapause.