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Corn callus specific promoter and application

A technology of callus and promoter, applied in application, recombinant DNA technology, angiosperms/flowering plants, etc., can solve problems such as energy waste, increased cell burden, and accumulation of metabolites, so as to avoid unnecessary waste and increase area The effect of high expression amount and sensitivity

Inactive Publication Date: 2013-10-02
ANHUI AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The expression of exogenous genes is continuous in time and space. In genetic engineering, highly active constitutive promoters are often used to drive the expression of exogenous promoters in plants, because of their constant RNA and protein expression, no time and space Specificity, not induced by external environmental factors, etc., but this expression often leads to the accumulation of intracellular metabolites, increasing the burden on cells and causing energy waste

Method used

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  • Corn callus specific promoter and application
  • Corn callus specific promoter and application
  • Corn callus specific promoter and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1. Maize callus specific promoter LEG clone

[0019] According to the full-length sequence of the LEG promoter published on the NCBI website, design primers for PCR amplification of this fragment, and add Hin dIII (AAGCTT) restriction site, downstream add Nco I (CCATGG) restriction site

[0020] The primer sequences are as follows:

[0021] Primer 1 (upstream primer): 5'- CCCAAGCTT AACTGCTGGGAGATGGAACT-3'

[0022] Primer 2 (downstream primer): 5'- CATGCCATGG CTTTGCTTTGCCGTGATAGG-3'

[0023] Using the extracted corn B73 genomic DNA (extracted by Quanshijin Plant Genome Kit) as a template, PCR amplification was performed with primers 1 and 2, and the PCR reaction system was:

[0024] 10×PCR buffer 5 μL dNTP (10mM) 4 μL Primer 1 2 μL Primer 2 2 μL DNA template 1 μL Taq enzyme 0.2 μL wxya 2 o to 50 μL

[0025] The PCR reaction conditions were: pre-denaturation: 94°C 6min; denaturation: 94°C 45s; ...

Embodiment 2

[0027] Example 2. LEG Establishment of gene promoter expression vector

[0028] Connected to the pEASY T1 Cloning Vector LEG Fragment (small fragment) with CaMV35S promoter (large fragment) on Agrobacterium binary vector pCAMBIA1301 with HindIII and Nco Carry out double digestion with enzyme I, in a water bath at 37°C for 3 hours, and digest 30 μL of the system as follows:

[0029] large (small) fragment 22 μL BSA 3μL 10×buffer K 3μL Hind III 1μL NCOI 1 μL total capacity 30μL

[0030] Ligate the above size fragments with T4DNA ligase, and ligate at 25°C for 2~12h. The ligation system is as follows:

[0031] large fragment 1 μL small fragment 7 μL 10×ligase buffer 1 μL T 4 DNA ligase 1 μL

[0032] Take 3~5 μL of the constructed vector pCAM–p LEGPlasmids were gently injected into 200 μL EHA105 Agrobacterium competent cells, ice bathed for 5 minutes, liquid nitrogen quick-frozen for 1 m...

Embodiment 3

[0033] Example 3. Agrobacterium-mediated transformation of rice

[0034] Agrobacterium tumefaciens (Agrobacterium a mediated) Agrobacterium-mediated method will contain pCAM–p LEG Agrobacterium expressing the vector was introduced into rice.

[0035] 3.1 Obtaining callus from mature rice embryos

[0036] Before the experiment, the mature seeds used in the experiment were dried under strong sunlight for 3-4 hours and then shelled. First wash with sterilized water until clear, then soak in 75% ethanol for 5 minutes, then use sterilized water, hypochlorous acid and Tween prepared sterilized water (specific ratio: sterilized water: hypochlorous acid = 1:1, Total volume: Tween=1mL:1ul) Soak for 30min, shake continuously during the period, so as to sterilize the surface, then rinse with sterile water until the water becomes clear, then put the mature seeds on sterile filter paper to absorb the water After that, they were placed on the callus induction medium and cultured at 26...

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Abstract

The invention relates to a corn callus specific promoter, and an application of the specific promoter for constructing an expression carrier. The specific promoter enables specific expression in paddy rice callus. According to the invention, a segment of promoter sequence with length of 1910bp is cloned in corn B73 inbred line, the sequence is shown as SEQIDNO: 1 in a sequence table. The promoter of the present invention expresses only in the callus, thereby expression products of the target gene is accumulated in a certain space, the expression amount of the callus parts can be increased, and the corn callus specific promoter has important application value for directionally improving the corn quality.

Description

technical field [0001] The invention relates to a plant promoter and its application, in particular to the cloning, function verification and analysis of a maize callus-specific promoter and its application in cultivating safe transgenic plants. Background technique [0002] Although the genetic information contained in different types of cells is the same, the proteins they express are quite different, which is the result of differential expression of different genes, and the most important element that controls the expression of these genes in different time and space is the activation son. The so-called promoter is a DNA sequence located upstream of the 5' end of the transcription start point, which can correctly guide RNA polymerase II to bind to the template. The study of the promoter sequence found that its basic structural features: a TATA-box rich in AT bases located upstream of the transcription start point determines the correct initiation of transcription. The TA...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/82A01H5/00
Inventor 项艳董庆程备久江海洋朱苏文马庆
Owner ANHUI AGRICULTURAL UNIVERSITY
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