A plant pollen-specific promoter pchf32 and its application

A technology of PCHF32 and plant pollen, which is applied in the field of plant pollen-specific promoter PCHF32 and its application, and can solve the problems of few reports

Active Publication Date: 2016-09-28
HAINAN BOLIAN RICE GENE TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are few reports on rice pollen-specific promoters

Method used

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  • A plant pollen-specific promoter pchf32 and its application
  • A plant pollen-specific promoter pchf32 and its application
  • A plant pollen-specific promoter pchf32 and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Acquisition of rice pollen-specific promoter PCHF32

[0040] 1. Extraction of rice genomic DNA

[0041] Rice genomic DNA was extracted according to the method instructions of the Plant DNA Isolation Kit (Chengdu Fuji Biotechnology Co., Ltd.). The genome was derived from fresh leaves of rice Nipponbare variety. The extracted genomic DNA was aliquoted and stored at -20°C for later use.

[0042] 2. Design PCR PCHF32 primers

[0043] Primers were designed using the Gibson Assembly method, and the amplified product was inserted into the NcoI and HindIII restriction sites of the 1300GUSplus vector. The primers for amplifying the PCHF32 gene are shown in the sequences SEQ ID NO:2 and SEQ ID NO:3. Among them, about 15 nucleotide sequences at the 5' ends of the upstream and downstream primers were repeated with the corresponding connection positions of the vector to facilitate Gibson Assembly connection.

[0044] PCR reaction system (50μ1):

[0045] DNA template...

Embodiment 2

[0053] Example 2: Construction of the recombinant expression vector 1300gus-PCHF32 of the promoter PCHF32

[0054] See the build process figure 1 , Insert the amplified product of Example 1 into the 1300GUSplus vector NcoI and HindIII restriction sites.

[0055] Primers SEQ ID NO: 2 and SEQ ID NO: 3 amplify the PCR product and recover a product of about 2000 bp by 1% agarose gel electrophoresis. The vector 1300GUSplus was digested with NcoI and HindIII, and the linearized vector was recovered.

[0056] The 2X ligation kit connects the promoter PCHF32 to 1300GUSplus, the 10ul system is as follows:

[0057] 2.5 μl PCHF32 PCR product (50ng)

[0058] 2.5μ1 enzyme-cut carrier (100ng)

[0059] 5μ1 Ligation Mix

[0060] Ligation procedure: 50°C for 60 minutes.

[0061] Transformation: Take 5 μl of the ligation product above to transform Escherichia coli competent cells by electroporation. Select positive clones for sequencing. Named 1300gus-PCHF32. The 1300GUSplus vector cont...

Embodiment 3

[0062] Example 3: Construction of recombinant expression vector DX2181-PCHF32 containing PCHF32 promoter sequence

[0063] refer to figure 2 The construction process of this method is to construct the recombinant cloning vector pGEM-PCHF32 containing the PCHF32 promoter sequence first, and then construct the recombinant expression vector DX2181-PCHF32 containing the PCHF32 promoter sequence based on it.

[0064] 1. Construction of recombinant cloning vector pGEM-PCHF32 containing PCHF32 promoter sequence

[0065] With reference to the method of embodiment 1, the primer pair shown in sequence SEQ ID NO:2 and SEQ ID NO:3 is amplified with sequence such as the primer pair shown in SEQ ID NO:5 and SEQ ID NO:6 The obtained amplified product was connected to the pGEM-T vector, and the operation steps were carried out according to the instructions of the pGEM-T vector produced by Promega Company to obtain the recombinant cloning vector pGEM-PCHF32. Transform Escherichia coli compete...

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Abstract

The invention provides a plant pollen specific promoter PCHF32 and an application thereof, and belongs to the technical field of gene engineering. The invention further provides an expression vector containing the plant pollen specific promoter PCHF32, a gene expression box and a vector containing the gene expression box. The invention further provides a primer pair of amplifying the pollen specific promoter PCHF32. The pollen specific promoter PCHF32 is a rice endogenous gene, an exogenous gene can be driven to be accurately and specifically expressed in pollen in rice gene engineering, and a novel method for driving the exogenous gene to be specifically expressed in the pollen is provided.

Description

technical field [0001] The invention belongs to the field of plant molecular biology, and specifically relates to a plant pollen-specific promoter PCHF32 and its application. Background technique [0002] In the study of gene structure and function, the regulation of transcription level is a key step, and the initiation of gene transcription is the result of the joint action of a series of trans-acting factors and transcription sites. A gene encoding a protein contains a promoter and a coding region. The promoter is generally located upstream of the coding region and is the binding site for trans-acting factors. There are two main regions: the core promoter region and the transcriptional regulatory region. The core promoter is the shortest promoter fragment at the initiation of transcription, generally 40 nt, including the core promoter element, which is a DNA sequence recognized and bound by RNA polymerase families I, II and III. The transcriptional regulatory region is lo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/82C12N5/10C12N15/11A01H5/00
Inventor 黄培劲吴永忠张维安保光龙湍李新鹏
Owner HAINAN BOLIAN RICE GENE TECH CO LTD
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