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Acyrthosiphon pisim CYP6A13 promoter and activity analysis

A pea aphid and promoter technology, applied in the field of DNA sequence, can solve the problem of the lack of a pea aphid promoter and achieve the effect of stable expression

Inactive Publication Date: 2014-05-28
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no promoter of the pea aphid that can be applied to the study of eukaryotic expression.

Method used

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  • Acyrthosiphon pisim CYP6A13 promoter and activity analysis
  • Acyrthosiphon pisim CYP6A13 promoter and activity analysis
  • Acyrthosiphon pisim CYP6A13 promoter and activity analysis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1: Cloning of the CYP6A13 promoter of the pea aphid

[0025] The promoter of the pea aphid P450 gene CYP6A13 (the promoter sequence includes the DNA nucleotide sequence of the -1bp to -821bp region relative to the transcription start site of SEQ ID NO: 1), in the 5' region of the pea aphid CYP6A13 gene identified in the sequence.

[0026] The pea aphid P450 gene CYP6A13 is registered in NCBI GenBank (accession number: HM009309), and the sequence listing shows the DNA sequence of the promoter and 5' untranslated region of the above gene of the present invention. In the list of promoter sequences in this document, the base of the transcription initiation site is indicated by +1, and ATG is marked in red. And use the promoter analysis website to analyze the core elements of the promoter. Promoter analysis website http: / / www.gene-regulation.com / pub / programs / alibaba2 / index.html.

[0027] Taking pea aphid genomic DNA ( figure 1 ) as a template, using a pair of spe...

Embodiment 2

[0028] Example 2: Construction of the pea aphid CYP6A13 promoter expression vector pGL3-CYP6A13(-1322 / +229)

[0029] The pisa aphid CYP6A13 promoter and 5' untranslated region (see sequence listing) cloned in Example 1 were inserted into the pGL3 vector, thereby constructing the pGL3-CYP6A13(-821 / +349) vector.

[0030] More specifically, the promoter sequence was amplified using primers (5-ACGCGGTCGTTCGCTAGATTCAACAGGGT-3, 5-CTCGAGAATCGGCTTGATGTAGGGCAC-3) and cloned into pGL3 vector MluI and XhoI sites to construct pGL3-CYP6A13(-1322 / +229 ), used to drive the expression of Luc+, identified by PCR ( Figure 4 ) and enzyme digestion identification ( Figure 5 ) to obtain the recombinant plasmid of the promoter and the vector.

Embodiment 3

[0031] Example 3: Identification of the activity of the CYP6A13 promoter of the pea aphid of the present invention

[0032] Sf9 cells were seeded in 24-well plates (4×10 5 cells / well), the pGL3-CYP6CY3(-2230 / +71) construct (2μg / well) and the control reporter gene plasmid phRL-TK (Promega; 0.2 μg / well),. After 48 hours, cells were harvested and the resulting lysates were used to measure luciferase activity (Promega). Such as Figure 6 As shown, the cells transfected with pGL3-CYP6A13(-821 / +349) had very high luciferase activity.

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Abstract

The invention discloses an acyrthosiphon pisim CYP6A13 promoter and activity analysis, and belongs to the technical field of biological engineering. The invention provides an obtained acyrthosiphon pisim CYP6A13 promoter sequence which is represented by SEQ ID NO: 1; the regions from -1bp to -821bp of the SEQ ID NO: 1 represent a DNA sequence of the acyrthosiphon pisim CYP6A13 promoter; the regions from +1bp to +349bp of the SEQ ID NO: 1 represent a DNA sequence of a 5' non-translating region of a gene CYP6A13 of acyrthosiphon pisim P450. The acyrthosiphon pisim CYP6A13 promoter can be used for high-efficiency expression of a eukaryotic gene and is applied to a research on acquisition of low-abundance genes, so that high-level and stable expression can be obtained; an active significance is realized for the research on a target function.

Description

technical field [0001] The invention belongs to the technical field of bioengineering and relates to a DNA sequence which can be used as a promoter to regulate gene expression. In particular, it relates to a promoter sequence derived from the P450 gene CYP6A13 of the pea aphid and highly expressed in cells. Background technique [0002] The pea aphid (Acyrthosiphon pisum) is an important piercing-sucking pest that damages crops through feeding and virus transmission. Pea aphid P450 gene CYP6A13 (Genebank No. XP001948488) and peach aphid P450 gene CYP6CY3 (Genebank No. HM009309) are highly homologous, so it is inferred that the gene has similar functions. Puinean et al. (2010) showed that the overexpression of CYP6CY3 in green peach aphid resulted in a high level of resistance to imidacloprid, which also showed that the promoter of CYP6CY3 gene in green peach aphid most likely has a high induction activity. It can be inferred that the promoter of CYP6A13 of P450 gene of pea...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/79
Inventor 尚庆利潘怡欧杨晨席景会杨巽辛雪成
Owner JILIN UNIV
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