Modification Of BmVg Promoter And Development Of Fat Body Bioreactor In The Pupal Stage Of Silkworm

Silkworm is an important economic insect.Its economic benefits can be divided into main value-silk and added value-silkworm pupa.A large number of silkworm pupae are produced due to silk reeling every year,and the economic value of silkworm pupa is less excavated.Therefore,it is particularly important to improve the added value of silkworm.Among them,the use of silkworm pupae to develop bioreactor has become an important mean.The use of silkworm to develop pupa bioreactor has become an important mean.Silkworm bioreactor refers to the transfer of exogenous genes into silkworm body,using silkworm as host to express exogenous protein.The selection of suitable promoters is crucial for the development of bioreactors.Studies have shown that the maximum content of Vitellogenin(Vg)protein accounted for 40% of the total protein of female silkworm pupa.Bm Vg begins to be expressed on day 2 after wandering,and continues to be expressed throughout the pupa stage.Vg expression is gender-,tissue-and stage-specific,and satisfied the conditions of developing bioreactor.Our research team found that the Bm Vg promoter with a length of 1.0K bp is more active than the previously reported Bm Vg promoter,and has the characteristics of endogenous Bm Vg,however,the expression of exogenous genes driven by the Bm Vg promoter with a length of 1.0K bp was much lower than that of endogenous Bm Vg.It has also been reported that increasing the enhancement element can improve the expression of target genes.Therefore,this study hopes to explore the main reasons that affect the gender-specific expression of silkworm vitellogenin and the effect of enhancer elements on Bm Vg promoter driven target gene expression,which will lay the foundation for the application of Bm Vg promoter and the construction of pupa fat body bioreactor.The main research results obtained are as follows:1.The effect of DSX CRE on Bm Vg promoter activityThere are reports that female and male DSX transcription factors can regulate Bm Vg expression by binding to DSX cis-regulatory elements(CRE)on the Bm Vg promoter.First of all,this study cloned the Bm Vg P1.0K promoter of mutant DSX CRE(Bm Vg P1.0K-DSXm)by using the Bm Vg promoter with a length of 1.0K bp.The double luciferase activity experiment showed that the activity of the Bm Vg promoter with a length of 1.0K after the mutation of DSX CRE was significantly reduced.Then,a transgenic vector for Bm Vg P1.0K-DSXm to drive the expression of red fluorescent protein(Ds Red)was constructed.through microinjection of silkworm eggs and screening of transgenic positive individuals,a transgenic strain of mutant DSX CRE was obtained.RT-PCR and q PCR results showed that after mutation of DSX CRE,the expression of exogenous Ds Red and endogenous Bm Vg in the wandering fat body of males was not significantly change,while the expression of exogenous Ds Red and endogenous Bm Vg in the wandering fat body of females was significantly decreased.These results indicate that the low expression of Bm Vg in males is not because male DSX binds to the DSX CRE of Bm Vg promoter to inhibit Bm Vg expression.Subsequently,this study cloned the Bm Vg P1.0K promoter of triple repeat DSX CRE(Bm Vg P1.0K-3DSX)by using the Bm Vg promoter with a length of 1.0K bp.The double luciferase activity experiment showed that the activity of the Bm Vg promoter with a length of 1.0K after the triple repeat of DSX CRE was significantly increased.and then a transgenic vector for Bm Vg P1.0K-3DSX to drive Ds Red expression was constructed.Through microinjection of silkworm eggs and screening of transgenic positive individuals,a transgenic strain with triple repeat DSX CRE was obtained,RT-PCR and q PCR results showed that after triple repeat of DSX CRE,the expression of exogenous Ds Red in the wandering fat body was extremely significantly increased,and the expression of endogenous Bm Vg was significantly reduced.The above results indicate that after triple repeat DSX CRE,the Bm Vg P1.0K promoter activity increased and the ability to drive target gene expression increased.This suggests that the DSX of male splicing also up-regulates Bm Vg expression,but the up-regulation is not as high as the DSX of female splicing.2.The effect of enhancer elements on Bm Vg P1.0K driven foreign protein expressionAccording to literature reports,the ability of the Gal4/UAS system to drive the expression of foreign proteins can be improved by adding enhancement elements.Inspired by this,two GAL4 transgenic strains driven by Bm Vg P1.0K without translation enhancement elements(GP)and adding translation enhancement elements(ISGP)were constructed.Meanwhile,in order to improve the start-up efficiency,this study increased the copy number of UAS components in the UAS system according to the literature report,the transgenic strain of 20×UAS elements fused with the Ds Red was constructed.The two GAL4 transgenic strains obtained were hybridized with UAS transgenic strain to detect the expression of exogenous Ds Red in the fat body of the hybrid strain.The results showed that red fluorescence was observed in the female fat bodies of two hybrid lines on the second day of pupa.The results of q PCR and Western bloting also showed that the expression of exogenous Ds Red in the fat body of the female hybrid line containing the enhancement elements was significantly higher than that in the hybrid line without the enhancement element.However,no red fluorescence was observed in the male fat body of the hybrid lines without enhancement elements,while the red fluorescence was observed in the male fat body of the hybrid line containing enhancement elements,and the fluorescence intensity is similar to that of female hybrid strain without reinforcement elements.The results of q PCR and Western bloting also showed that the expression of exogenous Ds Red in the fat body of the male hybrid line containing the enhancement elements was significantly higher than that in the hybrid line without the enhancement element.This indicates that the enhancement elements can significantly promote the Bm Vg P1.0K driven exogenous protein,suggesting that the differential expression of Bm Vg P1.0K driven exogenous protein in male and female individuals can be significantly improved by adding the enhancement element.