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Aphis gossypii gossypol-induced CYP6J1 promoter and activity analysis

A technology of CYP6J1 and cotton aphid gossypol, which is applied in the field of bioengineering and can solve problems such as lack of cotton aphid promoter

Inactive Publication Date: 2014-06-04
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no cotton aphid promoter that can be applied to eukaryotic expression research

Method used

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  • Aphis gossypii gossypol-induced CYP6J1 promoter and activity analysis
  • Aphis gossypii gossypol-induced CYP6J1 promoter and activity analysis
  • Aphis gossypii gossypol-induced CYP6J1 promoter and activity analysis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1: Cloning of the cotton aphid CYP6J1 promoter

[0025] Chromosome walking primers GSP1 (5-TCATCAAATACTATTCCACGGTTGGGA-3) and GSP2 (5-GCTTTTGAAACTGATAGAAACCGACGAAC-3) were designed according to the CYP6J1 mRNA sequence of cotton aphid (Genbank No. JN989967). 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 1202bp 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 CYP6J1 promoter...

Embodiment 2

[0030] Example 2: Construction of the cotton aphid CYP6J1 promoter expression vector pGL3-CYP6J1(-848 / +297)

[0031] The cotton aphid CYP6J1 promoter and 5' untranslated region (see sequence listing) cloned in Example 1 were inserted into the pGL3 vector, thereby constructing the pGL3-CYP6J1(-848 / +297) vector.

[0032] More specifically, the promoter sequence was amplified using primers (5-ACGCGTGACTTCTGCCTCAAAGAAG-3, 5-CTCGAGTTTAGTTACCTCCTTAGTAT-3) and cloned into the pGL3 vector MluI and XhoI sites to construct pGL3-CYP6J1(-848 / +297 ), 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

[0033] Example 3: Identification of the activity of the cotton aphid CYP6J1 promoter of the present invention

[0034] Sf9 cells were seeded in 24-well plates (4×105 cells / well), and were transiently co-transfected with pGL3-CYP6CY3(-2230 / +71) construct (2 μg / well) and control 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 cells transfected with pGL3-CYP6J1(-848 / +297) have very high luciferase activity.

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Abstract

An aphis gossypii gossypol-induced CYP6J1 promoter and activity analysis belong to field of bioengineering technology. The invention provides an obtained aphis gossypii gossypol-induced CYP6J1 promoter sequence as shown in SEQ ID NO:1, wherein the region from -1bp to -848bp of the SEQ ID NO:1 is the DNA sequence of the aphis gossypii gossypol-induced CYP6J1 promoter, and the region from +1bp to +297bp of the SEQ ID NO:1 is the DNA sequence of the 5' untranslated region of the aphis gossypii P450 gene CYP6J1. The promoter of the invention can be used for high level expression of eukaryotic genes, and for obtaining low-abundance genes for research so as to realize high-level and stable expression of the genes, and has positive significance on 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. Specifically, it relates to an inducible promoter sequence derived from cotton aphid P450 gene CYP6J1 and highly expressed in cells. Background technique [0002] Cotton aphid (Aphis gossypii) is an important worldwide agricultural pest, which harms crops through feeding and virus transmission. Gossypol is a terpene compound unique to cotton plants, and it is also a type of secondary substance that was first discovered in cotton and has the most obvious anti-aphid effect. The previous research results showed that the expression of cytochrome P450 gene CPY6J1 of cotton aphid could be induced by high dose of gossypol. This may be an important physiological function of the gossypol aphids use gossypol as a chemosensory signal to initiate their defense mechanism and respond to gossypol stress. This als...

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毕锐辛雪成
Owner JILIN UNIV
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