Functional Analysis Of Sorbitol Dehydrogenase Gene And Ecdysteroidogenic Shadow In The Silkworm,Bombyx Mori

The silkworm,Bombyx mori is an important economic and lepidopteran model insect.Diapause and developmental metamorphosis are important characteristics of the silkworm.As we all know the study of its molecular mechanism is a hotspot and a difficult point in the field of sericulture science,while it has not been fully elucidated so far.Bombyx mori is a typical embryonic diapause insect,which can be divided into univoltine,bivoltine,and multivoltine according to the number of generations that occur in one year under natural conditions.According to the number of larval stages of molting,it can be divided into trimolter,four-moulter and pentamolter.To further investigate the characteristics of the sorbitol dehydrogenase gene and the role of the ecdysteroidogenic pathway Shadow gene in diapause and developmental metastasis,we take the bivoltine strain Qiufeng as marerial to research the function of these genes by using 5’RACE,qRT-PCR and CRISPR/Cas9technques.The main results were as follows.1.Determination of transcription initiation site and promoter characteristics of the sorbitol dehydrogenase genes in Bombyx mori.To clarify the transcriptional characteristics of sorbitol dehydrogenase genes BmSDH-1,BmSDH-2a and BmSDH-2b in B.mori.The transcription initiation sites of three BmSDH genes were determined by 5’RACE technique;The 2 kb upstream and 200 bp downstream sequences of the transcription initiation site were analyzed using online web such as NNPP,JASPAR,Alibaba2.1,etc.The RACE results show that the transcription initiation sites of BmSDH-1,BmSDH-2a and BmSDH-2b are located at 41,41 and 40 bp upstream of translation initiation sites,respectively.It is predicted that the core promoter region and the transcription factor binding site are mostly concentrated in the near 1 kb sequence region;there are no TATA boxes at 25-30 bp upstream of the transcription start sites of these three genes,indicating that they are different from the typical eukaryotes’promoter structure.2.Promoter properties of BmSDH-1,-2a,-2b and regulation of hormone on BmSDH-2a in vitro.The promoters of three BmSDH genes about 1 kb length and BmSDH-2a in different length were cloned by PCR.Then report plasmids with a fruit fly luciferase gene driven by BmSDH promoters in different length,pGL3-BmSDH-P-luc,were constructed.Using dual luciferase detection system,the promoter activity of BmSDH was detected by co-transfecting the BmN cells with pGL3-BmSDH-P-luc and pRL-CMV which contains a renin-luciferase reporter gene,and the effects of hormones on the promoter activity of BmSDH-2a was detected by adding juvenile hormone analogue（JHA）,or ecdysone hormone（20-E）,or diapause hormone（DH）to culture media in the gradient concentrations.The promoter activity of BmSDH-2a was significantly higher than those of BmSDH-1 and BmSDH-2b.For the BmSDH-2a gene,the promoter activity of the 355 bp fragment was significantly higher than those of the 674 bp and 1 117 bp fragments.In BmN cells,the activity of 1 117 bp promoter of BmSDH-2a increased with the increase of DH concentration;however,when the DH concentration was higher than 100 ng/mL,the promoter activity was decreased to some degree but maintained at a high level.In JHA treated Bm N cells,the promoter activity was decreased gradually as the hormone concentration increased.In 20-E treated BmN cells,the promoter activity significantly increased when 0.1 ng/mL of 20-E was applied,and then decreased gradually with the increase of hormone concentration.The results showed that a certain concentration of 20-E can significantly enhance the promoter activity of BmSDH-2a.3.Expression profile of several genes on ecdysteroidogenic pathway related to diapause in pupal stage of Bombyx mori bivoltine strain.Studies have shown that Ecdysone is involved in regulation of embryonic diapause in the silkworm,B.mori.However,its mechanism still remains unclear.To explore the role of ecdysteroidogenic pathway genes in diapause process of bivoltine B.mori,the eggs of"Qiufeng",a bivoltine strain,were used as the study materials and arranged into diapause eggs producers（DEPs）and non-diapause eggs producers（NDEPs）,respectively.The differential expression of ecdysteroidogenic pathway genes between two groups was analysed during the early pupal stage.The results showed that Neverland,Spook and Cyp18a1 were up-regulated in DEPs group.There was no significant difference in the expression levels of Phanton and Shade genes between the two groups.The Disembodied and Shadow genes were up-regulated in the NDEPs group.The expression of Shadow was significantly increased in the NDEPs in day-3 pupae and reached the peak simultaneously,indicating that Shadow was in coincidence with diapause process.To validate this hypothesis,a repression of Shadow by RNA interference was performed in day-2 pupae of NDEPs.The expression of Shadow was downregulated by RNAi,andβFtz-F1,a downstream gene of ecdysteroidogenic pathway was also decreased.Furthermore,the genes encoding the kynurenine-synthetase were upregulated in the ovary,and Brown,AdenoK which link Shadow to the kynurenine-synthase gene were also upregulated in the fat body.The progeny eggs appeared a light purple colour at 48 h after oviposition,revealing a certain tendency to diapause.We speculate that inhibition of Shadow upregulates 3-hydroxy-kynurenine synthesis by increasing the expression of Brown and AdenoK.In addition,Shadow was cloned,and expressed in E.coli for further functional study of Shadow protein.4.Functional characterization of Shadow in Bombyx mori by CRISPR/Cas9 mediated genome editing.The Shadow gene was knocked out using the CRISPR/Cas9 gene editing system and microinjection technology to further study the function of the Shadow gene.First,the cleavage-active gRNA target site was screened in BmN cells,then the non-diapause eggs produced in the NDEP group were microinjected with a mixture of cleavage-active gRNA and Cas9 protein.After hatching,the silkworms were routinely raised to mating.The generation 1 was produced,and the generation 0 was extracted from the genome to detect the mutation;the result showed that a chimera was detected in the generation 0,and the mutant homozygote was selected in generation 2.The mutant individuals develop normally in the1^stt instar and molt on time.At the beginning of the 2ⁿdd instar,the silkworms were lustrous,and they remained unstained for 6-8 days.They could not enter the 3^rdd instar normally.Finally,the energy was exhausted and died.The mutant sequence showed that 42 bases were deleted in exon 3,and the transcription level was analyzed.It was found that the expression of Shadow at the mRNA level was significantly lower than control group,presumably due to the mutant.The low expression of the Shadow gene resulted in these mutants exhibiting a state of insomnia at the 2ⁿdd instar.The addition of 20-E could make the mutation entered the 3^rdinstar,indicating that the lack of 20-E synthesis in the silkworm is the reason for the emergence of 2ⁿdd insomnia.Experiments show that the Shadow gene is an important gene in the ecdysone synthesis pathway.Through the analysis above,we determined the transcription initiation site of BmSDH-1,-2a,-2b genes in Bombyx mori,and the promoter activity of BmSDH-2a was influenced by 20-E;The accumulation of ecdysone was found on the third day of the pupal stage in the NDEP group.The inhibition of Shadow gene can up-regulate the3-hydroxy-kynurenine synthesis pathway in silkworm,suggesting that Shadow gene may be involved in the regulation of silkworm diapause.Finally,the CRISPR/Cas9 system was successfully used to knock out the BmShadow gene in the silkworm"Qiufeng"variety and screen out the mutant homozygote.These results provided experimental evidences for further elucidation of the regulation mechanism of the diapause preparation period of silkworm.