Stress inducible promoter, stress inducible promoter plant expression vector and expression method of induced target gene

A plant expression vector and target gene technology, applied in the field of stress-inducible promoters, can solve problems such as affecting plant physiological balance, protein overexpression, affecting yield, etc., to avoid energy waste and possible damage, efficient start, and enhance resistance. sexual effect

Active Publication Date: 2018-04-06
JIANGHAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although constitutive promoters have the advantages of stability, high efficiency and broad spectrum when initiating the expression of exogenous genes, due to the efficient and continuous expression of target genes throughout

Method used

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  • Stress inducible promoter, stress inducible promoter plant expression vector and expression method of induced target gene
  • Stress inducible promoter, stress inducible promoter plant expression vector and expression method of induced target gene
  • Stress inducible promoter, stress inducible promoter plant expression vector and expression method of induced target gene

Examples

Experimental program
Comparison scheme
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Example Embodiment

[0024] Example 1

[0025] This example is about Brachypodium BdDREB-47 Cloning of gene promoters.

[0026] 1.1 Preparation of genomic DNA of Brachypodium

[0027] The leaves of normally growing Brachypodium bismuth were extracted by CTAB method, the concentration and quality of the extracted DNA were detected by ultra-trace nucleic acid protein analyzer Q5000, and the DNA concentration was diluted to 100ng / μl for use.

[0028] 1.2 Cloning of promoters

[0029] Using the above-extracted Brachypodium edodes DNA as a template, using the primers of SEQ ID NO: 2 and SEQ ID NO: 3, PCR amplification was performed to obtain the sequence of SEQ ID NO: 1.

[0030] The PCR reaction system is as follows:

[0031] reagent

[0032] PCR reaction program: pre-denaturation at 98°C for 5 min; denaturation at 98°C for 30s, annealing at 55°C for 20s (annealing temperature increased by 0.5°C for each cycle), extension at 72°C for 90s, and amplification for 20 cycles; denaturation at...

Example Embodiment

[0037] Embodiment two

[0038] This example is about the construction of a stress-inducible promoter (pBdDREB-47) plant expression vector.

[0039] use Eco RI and Hin dⅢ (purchased from NEB Company) double-digested the recombinant plasmid pBdDREB-47-T and the vector pCAMBIA1381-GUS, see figure 2 , respectively recovered the target fragment (1388bp) and the pCAMBIA1381-GUS vector fragment, connected them with T4 DNA ligase (purchased from NEB Company), transferred them into E. coli competent cells Trans-T1 by heat shock method, and picked a single clone for positive See results after testing image 3 , amplify and extract the plasmid, and further digest and identify the positive plasmid ( EcoR I and Hin dⅢ double enzyme digestion) see the results Figure 4 , and the sequencing results are correct, indicating that the plant recombinant expression vector pC1381-pBdDREB-47-GUS was successfully constructed, and the pC1381-pBdDREB-47-GUS recombinant plasmid can be further...

Example Embodiment

[0041] Embodiment Three

[0042] This example is about transforming tobacco with pC1381-pBdDREB-47-GUS recombinant plasmid through Agrobacterium-mediated method.

[0043] 3.1 Preparation of transgenic recipient material

[0044] Seeds of wild-type tobacco were sown in flower pots and placed in a 25-degree cultivation room (12h light / 12h dark), covered with plastic wrap to keep moisture, and the plastic wrap was gradually removed after the seedlings emerged. Two weeks later, the plants were transplanted, and when the tobacco was 2 months old, the leaves could be used for transgenic experiments.

[0045] 3.2 Disinfection and pre-cultivation of tobacco leaves

[0046] Tobacco leaves with good growth conditions were selected, and the tender leaves were cut off with scissors, and the tobacco leaves were soaked and sterilized with 75% ethanol for 50 seconds on the ultra-clean workbench. Then soak and sterilize with 2.5% sodium hypochlorite solution for 10 minutes, shake continuou...

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Abstract

The invention relates to a stress inducible promoter sequence which comprises a DNA nucleotide sequence of 1388bp of SEQ ID NO: 1 and belongs to the technical field of plant genetic engineering. The invention also provides a stress inducible promoter plant expression vector and a method of expressing a target gene of a plant at low temperature, drought and salt induction. The target gene replacedby a GUS gene is inserted into the plant expression vector comprising the promoter. The recombinant vector is imported into the target plant; and SEQ ID NO:2 and SEQ ID NO:3 are PCR primers which separately comprise ECoRI and HindIII digestion sites. The primers are suitable for amplifying the DNA sequence of SEQ ID NO: 1. The stress inducible promoter sequence provided by the invention can be used for efficient expression of a foreign gene under cold start, drought and salt stress, and is suitable for researches on plant genetic breeding and improvement.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a stress-inducible promoter, a stress-inducible promoter expression vector and a method for inducing the expression of a target gene. Background technique [0002] Cold damage, drought and soil salinization are important disasters that limit agricultural production and threaten food security. In particular, the cold wave in spring and autumn and drought in the critical period of crop growth can lead to large-scale damage to crops and serious yield reduction. Therefore, in addition to strengthening the research on defense technology against low temperature, drought and salt damage, through plant genetic engineering technology, stress resistance genes can be expressed in crops to improve the resistance of crops to adversity stress, reduce low temperature, drought and stress. The serious damage caused by high salt to crops has become an important means of crop breeding. ...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/82
CPCC07K14/415C12N15/8222
Inventor 李丽丽陈利红李甜甜高利芬周俊飞彭海
Owner JIANGHAN UNIVERSITY
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