Transcription activator IE1
A technology of transcription activators and terminators, which is applied in genetic engineering, plant gene improvement, biochemical equipment and methods, etc., can solve complex problems such as the reduction of exogenous gene expression, and achieve the effect of improving efficiency and expression level
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Embodiment 1
[0035] Example 1 Transcription activator IE1
[0036] Bombyx mori baculovirus (BmNPV) transcriptional activator IE1 Gene cloning: Isolate and purify the BmNPV genome (Chongqing strain, CQ strain) from the Chongqing local silkworm Dazao strain infected with baculovirus as a template. No. GI: 438817 IE1 Gene sequence Design an upstream primer containing a BamH I site F: 5' CGCggatccATGACGCAAATTAATTTTAA 3' and a downstream primer containing a Not I restriction site R: 5' ATTTgcggccgcTTAATTAAATTCAA 3', using PCR amplification, the amplification conditions are: 94 Pre-denaturation at °C for 4 minutes, denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 90 seconds, a total of 30 cycles; the final extension at 72°C for 10 minutes, and storage at 4°C. The virus IE1 gene was connected to the sequencing carrier for sequencing identification, which contained the nucleotide sequence shown in SEQ ID NO:1, and its amino acid sequence was show...
Embodiment 2
[0037] Example 2 Enhancer Hr3
[0038] Cloning of the homologous repetitive sequence Hr3 of the Bombyx mori baculovirus (BmNPV): The BmNPV genome (Chongqing strain, CQ strain) was isolated and purified from the Dazao strain of Bombyx mori infected with baculovirus as a template. Virus T3 strain (BmNPV T3 strain) GenBank accession number: hr3 homologous repeat sequence of L24902 Design an upstream primer containing an Nco I site F: 5'CATGccatggAAAAAGAAGCCGTGCCCA 3', and a downstream primer containing an Nco I site R : 5'CATGccatggCAGCGTCGTGAAAAGAGG 3', amplified by PCR, the amplification conditions are: 94°C pre-denaturation for 4 minutes, 94°C denaturation for 30 seconds, 54°C annealing for 30 seconds, 72°C extension for 45 seconds, a total of 30 cycles; the final 72°C Extended for 10 min and stored at 4°C, the baculovirus homologous repeat sequence ( hr3 ) was connected to a sequencing vector for sequencing identification, and its nucleotide sequence is shown in SEQ ID NO:...
Embodiment 3
[0039] Example 3 Construction of binary hybrid transgenic sericin 1 expression vector containing hr3 enhancer and IE1
[0040] Using the genome of cultivar Dazao as a template, primers specific to the promoter region of sericin I gene (sericin1) were designed according to the silkworm 9-fold genome database: Pser1-F: 5'CAAAgtcgacGAAAACAGCACACACTAC 3', Pser1-R: 5'AAAAggatccAGACCCGATGATAAGACG 3', Pser1signal peptide- R: 5'AAAAggatccTGTATCTCGATTGCCGGGGTGGTGACCGAAGGCTTTTACGCTgagcgcagccaacgcaatc' 3, two sericin I (sericin1) promoters were obtained by PCR amplification, one of which did not contain a signal peptide for intracellular expression, named Pser1 ( figure 1 ), its nucleotide sequence is shown in SEQ ID NO:5, and a signal peptide is secreted and expressed, named Pser1sp ( figure 2 ), whose nucleotide sequence is shown in SEQ ID NO:4, and cloned into T vector for sequencing identification.
[0041] The transgenic backbone vectors pBac[3xp3EGFPaf] (Carsten Horn. et al 20...
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