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Myzus persicae resistance associated CYP6CY3 promoter and activity analysis

A technology related to imidacloprid and resistance of peach aphid, applied in the field of DNA sequence and inducible promoter sequence, can solve the problem of lack of promoter of peach aphid

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 peach aphid promoter that can be applied to eukaryotic expression research

Method used

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  • Myzus persicae resistance associated CYP6CY3 promoter and activity analysis
  • Myzus persicae resistance associated CYP6CY3 promoter and activity analysis
  • Myzus persicae resistance associated CYP6CY3 promoter and activity analysis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1: Cloning of Peach Aphid CYP6CY3 Promoter

[0025] Chromosome walking primers GSP1 (5-GTCCTCATCTGGAACAGTCCTCCGTAT-3) and GSP2 (5-CAGTCGGTGGTTGACGATGATAGATTC-3) were designed according to the CYP6CY3 mRNA sequence of peach aphid (Genbank No. HM009309). Genomic DNA of green peach aphid ( figure 1 ), and digested with straight cutting enzymes AfeI, EcoRV-HF, PvuII, SmaI, PmeI, StuI, purified DNA and ligated adapter primers. PCR amplification was carried out according to the designed specific primers, and the amplification was carried out according to the Genome walker (Clontech) PCR method. The target fragment was amplified and analyzed by 1% agarose gel electrophoresis, and a band with a length of about 2300bp was amplified ( figure 2 ), and recycle. The recovered fragment was ligated with the pGEM-T vector to obtain a recombinant plasmid, which was extracted and verified by enzyme digestion ( image 3 ), and sequenced.

[0026] Green peach aphid CYP6CY3 pr...

Embodiment 2

[0029] Example 2: Construction of the expression vector pGL3-CYP6CY3(-2230 / +71) of peach aphid CYP6CY3 promoter

[0030] The promoter and 5' untranslated region (see sequence listing) of peach aphid CYP6CY3 cloned in Example 1 were inserted into the pGL3 vector, thereby constructing the pGL3-CYP6CY3(-2230 / +71) vector.

[0031] More specifically, the promoter sequence was amplified using primers (5-ACGCGTATTTATGACTATAACGAGG-3, 5-CTCGAGCAGTCGGTGGTTGACGATGAT-3) and cloned into pGL3 vector MluI and XhoI sites to construct pGL3-CYP6CY3(-2230 / +71 ), 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

[0032] Embodiment 3: Identification of the activity of the promoter of CYP6CY3 of peach aphid according to the present invention

[0033] Sf9 cells were seeded in 24-well plates (4×10 5 cells / well), the pGL3-CYP6CY3 (-2230 / +71) construct (2 μg / well) and the control reporter gene plasmid phRL-TK (Promega; 0.2 μg) were transiently co-transfected with CellfectinII reagent (Invitrogen; 2 μL / well). / hole),. After 48 hours, cells were harvested and the resulting lysates were used to measure luciferase activity (Promega). Such as Figure 6 As shown, pGL3-CYP6CY3 (-2230 / +71) transfected cells have very high luciferase activity.

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Abstract

The invention discloses a myzus persicae resistance associated CYP6CY3 promoter and activity analysis, and belongs to the technical field of biological engineering. The sequence of the promoter is represented by SEQ ID NO: 1; the regions from -1bp to -2230bp of the SEQ ID NO: 1 represent a DNA sequence of the myzus persicae resistance relevant CYP6CY3 promoter; the regions from +1bp to +71bp of the SEQ ID NO: 1 represent a DNA sequence of a 5' non-translating region of a gene CYP6CY3 of myzus persicae P450. The myzus persicae resistance associated CYP6CY3 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. It specifically relates to an inducible promoter sequence derived from P450 gene CYP6CY3 of peach aphid and highly expressed in cells. Background technique [0002] Green peach aphid is a worldwide agricultural pest that harms crops through feeding and virus transmission. Imidacloprid is a neonicotinoid insecticide that can effectively control green peach aphid, but field populations have developed different levels of resistance to imidacloprid. Puinean et al. (2010) showed that the overexpression of CYP6CY3 in green peach aphid resulted in a high level of resistance to imidacloprid. This may be an important physiological function of green peach aphid using imidacloprid as a chemosensory signal to initiate its tolerance mechanism and respond to imidacloprid stress. This also shows that the promoter...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/85
Inventor 尚庆利杨晨潘怡欧席景会毕锐杨巽辛雪成
Owner JILIN UNIV
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