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Vector for verifying enhancer and application of vector

A carrier and sub-function technology, applied in the field of plant genetic engineering, can solve problems such as the lack of substantial experimental evidence

Inactive Publication Date: 2019-04-09
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although it can be judged that certain fragments may function as enhancers or enhance gene expression through sequence comparison combined with gene expression, there is a lack of substantial experimental evidence, so additional targeted experiments are needed to verify

Method used

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  • Vector for verifying enhancer and application of vector
  • Vector for verifying enhancer and application of vector
  • Vector for verifying enhancer and application of vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Construction of the p3301-mini35S-GUS vector of the present invention

[0039] (1) Use the specific primer sequence (sequence shown below) to pCMABIA3301 plasmid DNA (provided by the Rapeseed Research Laboratory of Huazhong Agricultural University, for details, see figure 1 Amplify with the sequence table SEQ ID NO: 1) to obtain the PCR product of the full-length 2126bp fragment of the GUS reporter gene. The DNA sequences used for the amplification primers are as follows: Among them, the sequences marked in bold are protected bases, and the sequences marked underlined are restriction sites.

[0040] (2) KOD-plus standard reaction system (purchased from TOYOBO company) was used for gene amplification. The 50μl PCR reaction system included: 10ng plasmid DNA template, 5μl 10×PCR buffer for KOD-plus, 5μl dNTPs (2mM), 2μl MgSO 4 (25mM), 3μl PCR primers (forward and reverse primers 1.5μl each, the concentration is 10μM) and 1μl KOD-plus-(1U / μl), supplemented...

Embodiment 2

[0056] Example 2: Application and evaluation of the enhancer vector p3301-mini35S-GUS constructed by the present invention

[0057] (1) In the applicant's previous research, it was found that relative to the promoter of BnaA10.CO, a 537bp (sequence shown in SEQ ID NO: 14) and 2.0kb fragment were inserted at 335bp and 553bp upstream of the BnaC9.CO promoter, respectively (sequence shown in SEQ ID NO: 16) (see Figure 5 in A panel). At the same time, based on the transcriptome sequencing results of leaves of spring Brassica napus (Spring Rapeseed for short) Westar (a commonly used variety material, originally from Canada’s spring Brassica napus variety) (transcriptome sequencing library provided by Wuhan Jinosek Technology Co., Ltd. Construction and bioinformatics analysis), it was found that the expression level of BnaC9.CO gene was significantly higher than that of BnaA10.CO gene (see Figure 5 in panel B). Therefore, it was speculated that the two fragments inserted in the...

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Abstract

The invention belongs to the technical field of plant gene engineering and particularly relates to a vector for verifying an enhancer and the application of the vector. The vector p3301-mini35S-GUS for verifying the enhancer comprises a GUS (beta-glucuronidase gene) report gene and a CaMV mini35S promoter sequence, and the vector has a nucleotide sequence of SEQ ID NO:11 as shown in the specification. The vector provided by the invention can be adopted to verify potential or candidate enhancer fragments in a gene promoter, study the action of a target fragment in enhancing gene expression, andfurthermore analyze function differences of promoters, can be further applied to analysis on gene functions, and particularly can be applied to study and development on equipotential gene function differences of plants.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and in particular relates to a carrier for verifying an enhancer and its application. The enhancer vector p3301-mini35S-GUS contains GUS (beta-glucuronidase gene) reporter gene and CaMVmini35S promoter, and the nucleotide sequence of the vector p3301-mini35S-GUS of the present invention is as shown in the sequence table SEQ ID NO: 11. The vector prepared by the present invention can be used to verify the potential or candidate enhancer fragments in gene promoters, and explore the effect of the target fragments on enhancing gene expression, so as to analyze the functional differences of the promoters, and further be used for the analysis of gene functions, especially It is applied to the study of functional differences of alleles in plants. Background technique [0002] Enhancers (enhanccr) are a class of cis-regulatory elements (Cis-Regulatory Elements, CREs), which regulate c...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12Q1/6895A01H5/00A01H6/82A01H6/20
CPCC12N15/8222C12N2830/001C12Q1/6895
Inventor 王晶郭涛易丽聪李格田彦勇万明
Owner HUAZHONG AGRI UNIV
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