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Analysis of ofcyp promoter and activity of dinbu-inducible corn borer

An inducible, corn borer technology, applied in the field of bioengineering, can solve the problems of no inducible high activity promoter, high inducible activity, no

Inactive Publication Date: 2015-09-30
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At the same time, this also shows that the promoter of the OfCYP gene in the corn borer is likely to have a high inducible activity. So far, there is no inducible high-activity promoter isolated from the corn borer
At present, there is no dinbu-inducible promoter of the corn borer that can be applied to the study of eukaryotic expression.

Method used

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  • Analysis of ofcyp promoter and activity of dinbu-inducible corn borer
  • Analysis of ofcyp promoter and activity of dinbu-inducible corn borer
  • Analysis of ofcyp promoter and activity of dinbu-inducible corn borer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1: Cloning of the OfCYP promoter of the corn borer

[0025] Chromosomal walking primers GSP1 (5-CGACAAATGTCCTGTGAGACTTCTCCTAC-3) and GSP2 (5-CTTTTTCTTCCAGTAGTTGTGGTTTTTCG-3) were designed according to the mRNA sequence of P450 gene OfCYP of the corn borer P450 (Genbank No. EU807990). Genomic DNA was extracted from the corn borer ( 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 661bp 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. ...

Embodiment 2

[0029] Embodiment 2: Construction of the corn borer OfCYP promoter expression vector pGL3-OfCYP (-393 / +268)

[0030] The corn borer OfCYP promoter and 5' untranslated region (see sequence listing) cloned in Example 1 were inserted into the pGL3 vector to construct the pGL3-OfCYP(-393 / +268) vector.

[0031] More specifically, the promoter sequence was amplified using primers (5-ACGCGTGTGAAGCCCAGTAAAAGCT-3, 5-CTCGAGCTTTTTTTCTCCAGTAGTTGTG-3) and cloned into pGL3 vector MluI and XhoI sites to construct pGL3-OfCYP(-393 / +268 ), 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 the corn borer OfCYP promoter described in the present invention

[0033] Sf9 cells were seeded in 24-well plates (4×105 cells / well), and pGL3-OfCYP(-393 / +268) 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, pGL3-OfCYP (-393 / +268) transfected cells have very high luciferase activity.

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Abstract

A DIMBOA induction type corn borer OfCYP promoter and activity analysis belong to the field of bioengineering technology. The invention provides an obtained DIMBOA induction type corn borer OfCYP promoter sequence as shown in SEQ ID NO:1, wherein the region from -1bp to -393bp of the SEQ ID NO:1 are the DNA sequence of the DIMBOA induction type corn borer OfCYP promoter, and the region from +1bp to +268bp of the SEQ ID NO:1 are the DNA sequence of the 5' non-translation region of the corn borer P450 gene OfCYP. The promoter of the invention can be used for high-efficiency expression of eukaryotic genes, can be used for research of obtaining low-abundance genes so as to obtain high-level and stable expression, and has active meaning 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. It specifically relates to an inducible promoter sequence derived from the corn borer P450 gene (Genebank No. EU807990) and highly expressed in cells. Background technique [0002] The corn borer is an important worldwide agricultural pest. The larvae of the rice heart leaf stage eat the mesophyll or eat the unexpanded heart leaves, resulting in "mosaic leaves". , The moth hole is easy to fold. At the ear stage, the ears and tender grains are eaten by moth, causing grain defects and mildew, and the quality decreases. During the growth and development of corn, many secondary biomasses are produced, some of which have obvious defense functions against pests and affect the behavior, growth and development and reproduction of pests. Dingbu is the most important one among hydroxamic acids. Some people c...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/79
Inventor 尚庆利潘怡欧杨晨席景会杨巽毕锐
Owner JILIN UNIV