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Plant expression vector containing green fluorescence protein reporter gene, and constructing method and application of plant expression vector

A plant expression vector and green fluorescent protein technology, applied in the field of plant genetic engineering, can solve problems such as difficulty in identification of transgenic plants, and achieve the effect of expanding the scope of application

Inactive Publication Date: 2014-03-12
SHENYANG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The technical problem to be solved in the present invention is: to construct a plant expression vector for easy and rapid identification of transgenic plants, and to solve the problem of difficult identification of transgenic plants

Method used

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  • Plant expression vector containing green fluorescence protein reporter gene, and constructing method and application of plant expression vector
  • Plant expression vector containing green fluorescence protein reporter gene, and constructing method and application of plant expression vector
  • Plant expression vector containing green fluorescence protein reporter gene, and constructing method and application of plant expression vector

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Experimental program
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Embodiment 1

[0029] Embodiment 1 Construction of a plant expression vector containing green fluorescent protein reporter gene

[0030] 1. Obtaining the full-length sequence of GFP gene

[0031] 1. Design primers for full-length sequence amplification of GFP gene

[0032] Obtain the full-length sequence of the pCXGFP-P vector on NCBI, and design primers for amplifying the TA cloning site according to the sequence of the pCXGFP-P vector, introduce an EcoR I restriction site at the 5' end, and introduce a SacI restriction site at the 3' end. The length of the amplified fragment is about 700bp. After design, the upstream primer for the full-length amplification of the GFP gene sequence is shown in SEQ ID NO1, and the downstream primer for the full-length amplification of the GFP gene sequence is shown in SEQ ID NO2.

[0033] Upstream primer for full-length amplification of GFP gene sequence: 5'-C GAATTC ATGGGTAAAGGAGAAGAA-3'

[0034] Downstream primer for full-length amplification of GFP ...

Embodiment 2

[0056] Example 2 Construction of pRI101-GFP plant expression vector containing Hawthorn NAC domain gene

[0057] 1. Acquisition of Hawthorn NAC domain gene

[0058] Take 0.1-0.2 g of hawthorn fruit, and extract total RNA according to conventional methods.

[0059] 2. Design the amplification primers for the full length of the hawthorn NAC domain gene sequence

[0060] The RNA extracted from hawthorn fruit samples was sent to the company for high-throughput transcriptome sequencing. Refer to the various parameters of the transcriptome sequencing results, such as gene annotation, gene expression difference and other data, to find out the NAC transcription factor gene. According to the ID number of the relevant gene, the relevant sequencing reads are searched in the database, and the sequenced reads are de novo sequenced and assembled in order, and finally assembled into a continuous long sequence, and then compared with the model plant for analysis (BLAST) , to determine the ...

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Abstract

The invention discloses a constructing method of a plant expression vector containing a green fluorescence protein reporter gene. The constructing method comprises the following steps: using a pRI-AN-101 vector as a skeleton vector, amplifying the GFP full-length gene sequence on a pCXGFP-P vector by a PCR technology, inserting the GFP full-length gene forward sequence into the pRI-AN-101 vector to obtain a recombinant vector pRI-AN-101-GFP, performing site-specific mutagenesis on NdeI recognition sites in the GFP full-length gene sequence to cause the NdeI to be used for double digestion when the plant expression vector is constructed, and finally obtaining the plant expression vector containing the GFP gene, namely pRI101-GFP. The green fluorescence protein reporter gene contained in the vector can identify transgenic plants quickly and conveniently.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, in particular to a plant expression vector suitable for genetic transformation of dicotyledonous plants. Background technique [0002] Plant expression vectors are an important part of plant genetic engineering research. It is an important medium for carrying the target gene into the host cell for amplification and expression. Through plant expression vectors, exogenous target genes can enter host cells and be highly expressed, laying the foundation for elucidating the functions of new genes and improving plant traits by using genetic resources. Therefore, obtaining a stable and efficient plant expression vector is an important prerequisite for genetic transformation. [0003] After the exogenous gene is introduced into the plant, the identification of the transgenic plant is a key step. A commonly used method for identifying transgenic plants is to perform PCR on transgenic ...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/65C12N15/66
Inventor 张志宏韩国粉田义代红艳马跃从佩华李贺刘月学李晓明
Owner SHENYANG AGRI UNIV
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