| The regulation promoter region of LSP and HSC70-4 are cloned from genomic DNA from B. mori and B. mandarina. A series of luciferase reporter plasmids, driven by LSP and HSC70-4 promoters, are constructed, respectively. Via the transient expression system in BmN cells or silkworm transfected by lipofectin, the functional and characterestic analysis of the promoter are investigated. This will be helpful to further elucidate the regulation mechanism of LSP and HSC70-4 expression and to apply of the promoters in stable transformation cell system or transgenic silkworm.1. The Cloned BmandLSP and BmLSP 5'FR and Functional analysis of BmLSP promoterThe Cloned BmandLSP and BmLSP 5'FR, consisting of the first intron, the first exon, the core promoter region and 5'-upstream region, harbor the classic TATA box, sequences commonly found in insect genes that were specifically transcribed in fat body and the deduced ERE. The 5'-upstream region contains the homologous sequence with the first intron of Fib-L (S) and the inactive mariner like element (MLE). Using PCR and restriction endonuclease methods, a series of luciferase reporter plasmids, driven by different length of BmLSP promoters, are constructed. Via the transient expression system in BmN cells, the effects of the regulation elements and foreign insect hormones on the BmLSP promoter activity are investigated. The results show that the BmLSP promoter activity is enhanced by the intron with 5.4-5.8 folds, and by S with 4.42 folds, suggesting that the intron and S harbor the enhancer-like element. However, MLE in 5'-upstream region presents a negative effect on promoter activity. The effects of juvenile hormone analogue (JHA) on the BmLSP promoter activity appear the typical dose-dependent manner, that is, low concentration treatments increase the BmLSP promoter activity and high concentration treatments decrease it. Meanwhile, insect ecdysone (MH) treatment present no significant effect2. The Cloned BmandHSC70-4 and BmHSC70-4 5'FR, and Characterestic and functional analysis of BmandHSC70-4 and BmHSC70-4 promotersThe Cloned BmandHSC70-4 and BmHSC70-4 5'FR, consisting of the partial first exon, the central promoter region and 5'-upstream region, harbor six GATAs, two CAATs and two HSFs, but no TATA and GC box. Under normal condition, transcriptional activities of BmandHSC70-4 or BmHSC70-4 promoter are high in BmN, Sf21 cells and the 5lh instar larvae. By heat-shock treatment at 37癈 for 2hr, the enhancements of BmandHSC70-4 or BmHSC70-4 promoter activity are achieved in BmN cells, sf21 cells and 4lh instar silkworm larvae, respectively. But heat-shock treatment for 5th instar silkworm larvae lead to a drastically decrease of the promoters' activity. In 5th instar silkworm larvae, JHA treatments have no significant effect, and injection of low level of MH (1-3 V g per os) can increase BmHSC70-4 promoter activity, while high level of MH (5 M g per os) presents no effect. Transcriptional activity of promoter is changed with the different developmental stages and reaches the highest level at the wandering stage in 5" instar. From the above results obtained from in vitro and in vivo, we can deduceIIIthat BmandHSC70-4 or BmHSC70-4 promoter is both constitutive and inducible promoter, suggesting that its potential application in cell stable expression system or transgenic Bombyx mori.3. BmNPV hr3 enhancing BmHSC70-4 and BmandHSC70-4 promoter transcriptional activityBmNPV hr3, derived from B. mori Nucleopolyhedrovirus, functions as replication initiation site in viral DNA and enhancer for several promoters' transcription. Here, hr3 also can increase the transcriptional activiy of the BmHSC70-4 and BmandHSC70-4 promoter by 16-18 folds in BmN cells and 190.57-242.19 folds in 5th instar larvae. The ArJ-mediated enhancement is orientation-independent.In the 5th instar larvae, MH treatments enhance the transcriptional activity of the promoter combination. The maximum stimulating effect, by 5ug MH per os treatment, is achieved about 17.54 times, compared with the cont... |
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