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A kind of low temperature inducible promoter and its application

A low-temperature induction and promoter technology, applied in the field of non-coding nucleic acid, can solve the problem of few inducible promoters

Inactive Publication Date: 2016-10-26
YANGTZE UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, so far, there are still few inducible promoters that can be used in transgenic research

Method used

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  • A kind of low temperature inducible promoter and its application
  • A kind of low temperature inducible promoter and its application
  • A kind of low temperature inducible promoter and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Embodiment 1: The low temperature inducible promoter of the present invention pCD1 separation

[0023] 1.1 Preparation of sweet buckwheat genomic DNA

[0024] The sweet buckwheat seedlings with the culture substrate are 8000LUX under the light condition, and the temperature is 25 o C conditions for 3 weeks. Then, the light conditions were changed to 8000LUX, and then the DNA was extracted using the new plant genomic DNA rapid extraction kit (catalogue number DN15) of Beijing Aidelai Biotechnology Co., Ltd.

[0025] 1.2 Promoter pCD1 Cloning and sequence analysis of

[0026] Using the DNA extracted above as a template, the upstream promoter sequence of the FeDREB1 gene was amplified by PCR. The amplification primers are as follows, a HindⅢ restriction site is introduced into the upstream primer, and a BamhⅠ restriction site is introduced into the downstream primer:

[0027] Upstream primer: 5'- CCCAAGCTTATAGACCTAGTCAGGATCA -3'

[0028] Downstream primer: 5'- GCG...

Embodiment 2

[0042] Embodiment 2: promoter pCD1 Construction of Plant Transient Expression Vector

[0043] Using Bam HI and Hind III Endonuclease digestion of the recombinant cloning vector (T+ pCD1 )and pC0390::GUS vector, and then the promoter pCD1 and pC0390::GUS The GUS reporter gene of the vector was connected to construct a plant transient expression vector ( figure 2 , image 3 ). The constructed plant transient expression vector was named as pCD1::GUS . pC0390::GUS carrier and pC35S:: GUS The carrier was provided by Dr. Xu Weirong of Ningxia University, and the specific construction process can be found in literature (Xu et al., 2010, Planta, 231:475–487). Using the electric shock method, the constructed pCD1::GUS carrier, pC0390::GUS vehicle (negative control) and pC35S:: GUS The vector (positive control) was respectively introduced into the Agrobacterium tumefaciens competent strain GV3101 for future use.

Embodiment 3

[0044] Example 3: Identification of promoters under low temperature conditions pCD1 active

[0045] 3.1

[0046] 3.1.1 Tobacco culture and transient transformation

[0047] After being treated at 4°C for 1 week, the seeds of Nicotiana benthamiana were sown in the sterilized substrate of the nutrient bowl, covered with plastic wrap, grown in the seedling hardening room for 3 weeks, and then transferred to the artificial intelligence climate incubator after emergence. Tobacco plants grown for 5 weeks in an incubator were used for Agrobacterium-mediated transient transformation assays. The specific transformation method is as follows: ① Inoculate 20 μl of Agrobacterium containing promoter and GUS fusion construct in 20 mL of fresh LB liquid medium, add rifampicin 50 mg L -1 , Kan 50mg L -1 , cultured overnight on a constant temperature shaker at 28°C at 240rpm; ②Transfer the overnight cultured Agrobacterium to a 50mL sterilized centrifuge tube, centrifuge at room temperature...

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Abstract

The invention discloses a low-temperature inducible promoter and application thereof, belongs to the field of genetic engineering, and in particular relates to non-coded nucleic acid for adjusting the genetic expression in DNA recombination techniques. The nucleotide sequence of the promoter is as shown in SEQIDNO:1. By adopting the promoter disclosed by the invention, experimental and technical support can be provided for research and development of plant biological reactors. Vaccines, antibodies and other medical protein can be expressed by utilizing the low-temperature inducible promoter and taking plants as the biological reactors, the conventional agriculture scope is broken through and the medical field is achieved.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a non-coding nucleic acid for regulating gene expression in DNA recombination technology. Background technique [0002] In the study of plant genetic engineering, it has become an important method to express foreign target genes in receptors to improve the resistance of crops to adversity stresses such as drought, low temperature, salinity, and pests and diseases, or to improve crop quality and crop agronomic traits. An important means of crop breeding at present. The regulation of gene expression in eukaryotes includes pre-transcriptional regulation, transcriptional regulation, post-transcriptional regulation, translational regulation and post-translational regulation. Among them, the regulation of transcription level is coordinated by cis-acting elements and transcription factors, which is the key link of expression regulation. Therefore, the isolation and resea...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/82A01H5/00
Inventor 方正武刘志雄王彩丽李晓方
Owner YANGTZE UNIVERSITY
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