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A technology of promoters and DNA molecules, applied in the field of inducible promoters, can solve the problems of toxicity, increase the metabolic burden of plants, unfavorable normal growth of plants, etc., and achieve the effect of good application prospects.
Inactive Publication Date: 2011-05-25
INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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In most cases, people do not want exogenous genes to be widely expressed in the entire plant and throughout the growth period of transgenic plants, because on the one hand, it will increase the metabolic burden of the plants; Conducive to the normal g
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Embodiment 1
[0050] Example 1, Discovery of PRO-TaFbox2 (promoter of TaFbox2) sequence
[0051] 1. Discovery of PRO-TaFbox2 sequence
[0052] 1. Screening of BAC mixed pool
[0053] (1) After mixing the strains stored in the BAC mixing tank, take 5ul and inoculate them into LB medium, culture at 220rpm on a shaker overnight at 37°C, extract plasmid DNA by alkaline lysis, and design primers according to the open reading frame of TaFbox gene for amplification , carry out next step screening to the positive mixing pool.
[0054] (2) After diluting the preserved bacterial solution of the positive BAC mixed pool, spread it on the surface of LB solid medium, culture overnight at 37°C, and then pick a single clone 5 times the number of clones in the mixed pool and inoculate it in a 384-well plate. Cultivate overnight at 37°C, and store the culture medium frozen.
[0055] (3) All single clones in each 384-well plate were used to prepare a mixed pool, inoculated in LB medium and cultured overnig...
Embodiment 2
[0069] Embodiment 2, the effect verification of promoter
[0070] 1. Acquisition of promoter (PRO-TaFbox2) DNA
[0071] Design a pair of primers as follows:
[0072] Primers were designed according to the nucleotide sequence of PRO-TaFbox2, plus restriction sites SalI and EcoRI, the primer sequences are as follows:
[0073] Primer F: 5'- GTC GAC GACTGCGAGTTGCTG-3';
[0074] Primer R: 5'- GAATTC GACAAGTTGAGAAGGAA-3'.
[0075] The gDNA of Chinese spring wheat was extracted as a template, and the above primers were used for PCR amplification to obtain the target gene.
[0076] The PCR reaction system is shown in Table 2.
[0077] Table 2 PCR reaction system
[0078]
[0079] Reaction program: 94°C for 5min, (94°C for 30sec; 55°C for 30sec; 72°C for 1min) x 32, 72°C for 10min.
[0080] The blunt end plus A reaction system is shown in Table 3.
[0081] Table 3 plus A reaction system
[0082] Element
Volume (μl)
10×Buffer
2
Mg 2+
...
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Abstract
The invention discloses an induced promoter comprising the following DNA molecules of (1) or (2) or (3): (1) a DNA molecule shown in a sequence 1 in a sequence table; (2) a DNA molecule which is hybridized with the DNA sequence confined by (1) under the strict condition and has the function of the promoter; and (3) a DNA molecule which has more than 90% homology with the DNA sequence confined by (1) or (2) and has the function of the promoter. The invention discloses the promoter PRO-TaFbox2 sequence of the relevant haloduric gene TaFbox2 of wheat, and the overall length of the nucleotide sequence of the promoter is 3076bp. The 2kb zone of the promoter is analyzed and confirmed to contain a plurality of transcription element binding sites relevant to biological stress induction and non-biological stress induction, which shows that a downstream gene controlled by the promoter is bound to be expressed by the stress induction. The induced promoter has very good application prospect in the biological engineering by serving as the induced expression vector of a plant source.
Description
technical field [0001] The present invention relates to an inducible promoter. Background technique [0002] Among the multi-level levels of gene expression regulation, transcriptional regulation is a very cost-effective regulation method for plants. Various regulatory factors combine with promoters to turn on or turn off gene expression to achieve the purpose of regulation. Promoter is the most important regulation method in gene transcription. Because genes are controlled by different regulatory factors at different levels, this control mechanism not only determines the level of gene expression, but also determines the temporal and spatial order of gene expression. Therefore, it can be considered that Promoter determines the spatiotemporal specificity of gene expression to some extent, and plays a very important role in the transcription process. Promoters are important elements of genetic engineering expression vectors, and the study of promoters is of great significance...
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