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Specific expression and activity analysis of ace2 promoter in central nervous system of cotton aphid

A central nervous system, specific technology, applied in the field of DNA sequence, can solve problems such as no, and achieve the effect of specific and stable expression

Inactive Publication Date: 2015-08-12
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, no Ace2 promoter has been isolated from cotton aphid

Method used

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  • Specific expression and activity analysis of ace2 promoter in central nervous system of cotton aphid
  • Specific expression and activity analysis of ace2 promoter in central nervous system of cotton aphid
  • Specific expression and activity analysis of ace2 promoter in central nervous system of cotton aphid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1: Cloning of the cotton aphid Ace2 promoter

[0025] Chromosome walking primers GSP1 (5-CGCTTCTCCGTATATTCTGGCTCTTG-3) and GSP2 (5-GGCGTAATGAAAGTCCATAAGTTGAAGC-3) were designed according to the Ace2 mRNA sequence of cotton aphid (GenbankNo.AF502082). Extraction of cotton aphid genomic DNA ( figure 1 ), 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 Genomewalker (Clontech) PCR method. The target fragment was amplified and analyzed by 1% agarose gel electrophoresis, and a band with a length of 1378bp 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] Cotton aphid Ace2 promoter (the DNA...

Embodiment 2

[0029] Example 2: Construction of the cotton aphid Ace2 promoter expression vector pGL3-Ace2(-1284 / +94)

[0030] The Ace2 promoter and 5' untranslated region of Aphid gossypii cloned in Example 1 (see the sequence listing) were inserted into the pGL3 vector, thereby constructing the pGL3-Ace2(-1284 / +94) vector.

[0031] More specifically, the promoter sequence was amplified using primers (5-GGTACCCTGTACTTGTTTTGATCGA-3, 5-CTCGAGGGCGTAATGAAAGTCCATA-3) and cloned into pGL3 vector KpnI and XhoI sites to construct pGL3-Ace2(-1284 / +94 ), 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: Identify the activity of Ace2 promoter of cotton aphid according to the present invention

[0033] Sf9 cells were seeded in 24-well plates (4×105 cells / well), and pGL3-Ace2(-1284 / +94) construct (2 μg / well) and control were transiently co-transfected with Cellfectin II reagent (Invitrogen; 2 μL / well) Reporter 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 Sf9 cells (Spodoptera frugiperda ovary cells) transfected with pGL3-Ace2(-1284 / +94) have no luciferase activity, which indicates that it has a highly specific expression property of the central nervous system.

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Abstract

The invention discloses an aphis gossypii nervous centralis specificity expression Ace2 promoter and activity analysis, belongs to the technical field of bioengineering, and provides a sequence of the aphis gossypii nervous centralis specificity expression Ace2 promoter. The sequence of the promoter is represented by SEQ ID NO:1; the regions from -1bp and -1284bp of the sequence SEQ ID NO:1 represent a DNA sequence of the aphis gossypii nervous centralis specificity expression Ace2 promoter; the regions from +1bp to +30bp of the sequence SEQ ID NO:1 represent a DNA sequence of a 5' untranslated region of a gene Ace2 of aphis gossypii AChE. The aphis gossypii nervous centralis specificity expression Ace2 promoter can be used for high-efficiency expression of eukaryotic genes and applied to target gene obtaining research, so that high-level and stable specificity expression is obtained; the aphis gossypii nervous centralis specificity expression Ace2 promoter has an active significance for target function research.

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 a promoter sequence derived from the cotton aphid acetylcholinesterase (AChE) gene Ace2 and highly specifically expressed in cells. Background technique [0002] Cotton aphid (Aphisgossypii) is an important worldwide agricultural pest, which harms crops through feeding and virus transmission. Acetylcholinesterase is the main target of carbamate and organophosphate insecticides. It is highly and specifically expressed in the central nervous system of Ace2 cotton aphid. The targets of the two classes of insecticides are insensitive. Compared with the Ace1 gene of cotton aphid, the expression level of Ace2 was significantly higher than that of Ace1. This indicated that the promoter of Ace2 gene of cotton aphid probably had higher activity. So far, there is no Ace2 promoter ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/85
Inventor 尚庆利潘怡欧杨晨席景会毕锐杨巽辛雪成
Owner JILIN UNIV
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