Expression Analysis Of Promoters Of Five Silk Protein Genes In Transgenic Silkworms

The silkworm(Bombyx mori)is an important silk-producing insect that has been domesticated for over 5,000 years.The silk gland is a specialized organ of B.mori that synthesizes and secretes silk proteins.This organ is usually divided into three parts according to their morphology and function,including the anterior silk gland,middle silk gland and posterior silk gland.The middle silk gland and posterior silk gland are synthesizes and secretes sericin(approximately 25%)and fibroin(approximately 75%),respectively,two main components of silk.As the biological basis of the silk industry,the silk gland determines the yield and quality of the silk.Silk fibroin is composed of three subunits,fibroin heavy chain(fibH),silk fibroin light chain(fib L)and glycoprotein P25,which are assembled in a 6: 6: 1 ratio to form a basic structural unit.The components of sericin are more complicated and contains at least sericin 1(Ser1),sericin 2(Ser2),sericin 3(Ser3)proteins.According to early studies,the regulation of silk protein synthesis mainly occurs at the transcription level,that is,the signals of hormones is transmitted firstly to the transcription factors,then the transcription factors transmit the signals directly to the fibroin and sericin encoding genes(mainly acting in the promoter region,but also in the intron region)to turn on or turn off the silk protein genes in a spatiotemporal mannar,and finally guide efficient synthesis of silk proteins.This regulatory process is very complicated.In the past decades,researchers have focused on the regulation of silk protein synthesis and much progress have been achieved,however,the detailed regulatory mechanism has not yet been fully clarified.Promoters are core functional elements for the control of gene expression.Studying on the expression and regulation characteristics of the promoters of fibroin and sericin-encoding genes would help to reveal the molecular mechanism of the regulation of silk protein synthesis.In this paper,we selected the promoters(fibH,fibL,Ser1,Ser2,and Ser3)of five major silk protein genes to generate transgenic silkworms by using the modified GAL4/UAS binary expression tool,and investigated the spatiotemporal expression characteristics of the five promoters at individual level.The main results are as follows:1.Generation of GAL4/UAS transgenic silkworms(1)Based on the original GAL4/UAS system commonly used to analyze gene function in B.mori,we modified both the GAL4 and UAS constructs and constructed GAL4 expression vectors under the control of the fibH,fibL,Ser1,Ser2,Ser3 promoters and the UAS expression vector harboringthe reporter gene EGFP(enhanced green fluorescent protein).(2)After microinjecting the plasmid DNA of GAL4 and UAS constructs into the early embryos of the multiple silkworm strain Nistari,the corresponding basic transgenic lines were obtained,named as fibH-GAL4(HG4),fibL-GAL4(LG4),Ser1-GAL4(S1G4),Ser2-GAL4(S2G4),Ser3-GAL4(S3G4)and UAS-EGFP(UEGFP),respectively.(3)The GAL4 transgenic lines HG4,LG4,S1G4,S2G4,S3G4 were respectively crossed with the UEGFP,and the offspring harboring the GAL4/UAS were selected by dual fluorescence screening,named HG4/UEGFP,LG4/UEGFP,S1G4/UEGFP,S2G4/ UEGFP and S3G4/UEGFP,respectively.Molecular detection confirmed that all GAL4/UAS transgenic lines were successfully established and can be used for further analysis.2.Spatiotemporal expression characteristics of promoters of fibroin genes(1)The expression characteristics of the fibH promoter.First,main tissues of the HG4/UEGFP at day-6 fifth-instar larvae were examined,the result showed that EGFP was specifically expressed in the posterior silk gland of the transgenic silkworm.Further,the posterior silk glands of transgenic silkworms developed at day-8 embryo until the third day of pupae were investigated.The results showed that EGFP was clearly expressed since day-8 embryo,and EGFP expression continued until day-3 of pupae,andthe expression level of day-6 fifth-instar larvae was the highest.The results of molecular detection were consistent with that of the fluorescence observation.(2)Expression characteristics of the fibL promoter.Tissue and stage detection of LG4/UEGFP revealed that the spatiotemporal expression of EGFP driven by the fibL promoter was highly similar to that of the fibH promoter,EGFP was specifically expressed in the posterior silk gland of the transgenic silkworm;EGFP was expressed since day-8 embryo until the third day of pupae,and the peak of expression was day-6 fifth-instar larvae.The results of molecular detection were consistent with that of the fluorescence observation.Interestingly,degradation of the posterior silk glands of both the HG4/UEGFP and LG4/UEGFP was delayed about one day compared to the posterior silk gland of wild-type silkworm(WT).We speculated that it is related to the GAL4/UAS system,especially the high expression of GAL4,but it needs to be further confirmed.In addition,previous studies showed that the fibH and fibL genes expressed at larval instars,but not at molting stages.Our studies revealedthat EGFP driven by the promoter of either fibH or fibL genes was also expressed at four molting stages,suggesting that these two promoters are still active during molting stages or they are not completely inhibited by in vivo hormone signals.This phenomenon is worthy of further investigation.3.Spatiotemporal expression characteristics of promoters of sericin genes(1)Expression characteristics of the Ser1 promoter.Detection of EGFP expression in main tissues of day-6 fifth-instar larvae of S1G4/UEGFP showed that EGFP was only expressed in the middle and posterior regions of the middle silk gland.Further testing confirmed that EGFP was expressed continuously in the middle and posterior regions of the middle silk gland since the third day of fifth-instar larvae until day-2 prepupa,and the peak expression appeared at day-6 fifth-instar larvae.The results of molecular detection were consistent with that of the fluorescence observation.(2)Expression characteristics of the Ser2 promoter.Detection of main tissues of day-6 fifth-instar larvae of S2G4/UEGFP showed that EGFP was only expressed at the fore-end of the anterior region of the middle silk gland.Further detection revealed that EGFP was expressed at the anterior region of the middle silk gland during day-1 four-instar larvae to the fourth molting stage.During day-1 fifth-instar larvae to day-2 prepupa,EGFP was expressed in the anterior region of the middle silk gland.These results reveal for the first time the spatiotemporal expression characteristics of the Ser2 promoter at the individual level.(3)Expression characteristics of the Ser3 promoter.Detection of main tissues of day-6 fifth-instar larvae of S3G4/UEGFP showed that EGFP was only expressed at the rear-end of the anterior region of the middle silk gland.Subsequent detection on the middle silk gland developed from day-1 fifth-instar larvae to day-4 of pupal stage revealed that EGFP was expressed at the rear-end of the anterior region of the middle silk gland since day-6 fifth-instar larvae,and expressed continuously until day-2 prepupa.The results of molecular detection were consistent with that of the fluorescence observationTaken together,the above results indicated that the EGFP driven by the Ser1,Ser2 and Ser3 promoters are specifically expressed in the middle silk gland,but the regions and stages of EGFP expression are significantly different,suggesting that the three promoters have different spatiotemporal expression patterns,which reflects that the regulatory mechanism of expression of sericin genes as well as sericin protein synthesis are more complicated anddeserves further study.In summary,this study demonstrated the spatiotemporal expression characteristics of promoters of five major silk protein-encoding genes fibH,fibL,Ser1,Ser2,and Ser3,by analyzing the expression patterns of EGFP driven by them,and provides some theoretical references and transgenic materials for further clarification of the regulatory mechanism of silk protein synthesis.