Characterization Of Sorbitol Dehydrogenase Promotor From The Silkworm, Bombyx Mori

Sorbitol dehydrogenase(SDH) is a key enzyme for sorbitol converted to glycogen. The changes of sorbitol content in silkworm eggs have a closely parallel relationship with diapause start, duration and stop of egg stage. There are three SDH genes in silkworm, respectively BmSDH-1, BmSDH-2a and BmSDH-2b. Although there are many related research reports on silkworm sorbitol dehydrogenase, they mainly focus on research on protease expression, and there are little research refereence on these three characteristics of SDH gene promoter. To explore how BmSDH is involved in the regulation molecular mechanism of bivoltine silkworm, this paper will do research on silkworm bivoltine variety "Qiufeng" 3 SDH gene promoter of the same length, BmSDH-2a promoters of different length and promoter activity on BmSDH-2adealed with hormone.The results are listed as bellow:1.Activity of Bombyx mori sorbitol dehydrogenase mainly comes from the expression of BmSDH-2aBased on the nucleotide sequences of Bombyx mori three sorbitol dehydrogenase gene, specific primers are designed for amplifing the three promoters, bivoltine(bvt) varieties Qiufeng genomic DNA as the template.We use pGL3.0 basic plasmid as initial vector to construct three luciferase reporter gene(luc) expression vector driven by BmSDH promoter fragments, respectively pGL3-BmSDH-1-bvt-1074, pGL3-BmSDH-2a-bvt-1082 and pGL3- BmSDH-2b-bvt-1175.These recombinant plasmids would be transfected into BmN cells to detect the activity of BmSDH promoter fragment by analyse luciferase expression levels. The result shows that, for the same promoter length, BmSDH-2a promoter activity was significantly higher than that of BmSDH-2b(9.7 times) and BmSDH-1(21.0 times).As the result shows, it implied the BmSDH activity mainly comes from the expression of BmSDH-2a.2. Negative regulatory element exists between-355 ~1082 bp of BmSDH-2a gene promoterTo further investigate the characteristics of BmSDH-2a gene promoter, We cloned Bivoltine varieties BmSDH-2a 674 bp and 355 bp promoter fragment, respectively, and constructed reporter plasmid pGL3-BmSDH-2a-bvt-674 and pGL3-BmSDH-2a-bvt-355 controlled by BmSDH-2a promoter fragments with different lengths.After then, the control group, pGL3-BmSDH-2a-bvt-1082, was added in the experiment that the two recombinant plasmids were transfected into BmN cells.The results showed that: BmSDH-2a of 355 bp promoter activity was significantly higher than that of the 674(1.3 times) bp and 1082 bp(3.3 times) promoter, which indicates that there could be negative regulatory element existing between-355 bp and-674 bp and or(or)-674 bp and-1082 bp.3. BmSDH-2a gene promoter activities changes when dealed with different insect hormonesThis experiment also performed in vitro cell transfection using the 1082 bp BmSDH-2a gene promoter which were processed by the diapause hormone, juvenile hormone and ecdysone.The results showed that: When using different concentrations of diapause hormone for processing, BmSDH-2a gene promoter activity did not change significantly, compared with no diapause hormone. This described that diapause hormone did not directly regulate the expression of BmSDH.When juvenile hormone concentration were 2, 4, 6 ?g/mL, promoter activity of BmSDH-2a was significantly decreased, respectively, 0.67, 0.64 and 0.67 times, compared to juvenile hormone concentration with 0:00.When the concentration of juvenile hormone was 1 ?g/mL, the promoter activity of BmSDH was not significant, indicating a high concentration of JH inhibited the expression of BmSDH.When molting hormone concentration was 1 ?g/mL, promoter activity of BmSDH-2a was significantly reduced, 0.63 times with the concentration of molting hormone being 0:00.However, when dealed with lower concentration of 2, 4, 6 ?g/mL, promoter activity was significantly reduced, respectively, 0.22, 0.17, 0.16 times with the concentration of molting hormone being 0:00, which indicated high concentrations of ecdysone also inhibited the expression of BmSDH.These results above accumulated experimental datafor the study of the function of BmSDH.Meanwhile, it also helps to clarify the molecular mechanisms of diapause.