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A tobacco and protein technology, applied in the field of tobacco genetic engineering, can solve the problems of producing harmful substances, affecting the grade and quality of tobacco, and poor appearance quality.
Active Publication Date: 2022-04-29
ZHENGZHOU TOBACCO RES INST OF CNTC
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Problems solved by technology
"Green-yellow smoke" not only has poor appearance, but also produces harmful substances when burned, which in turn affects the grade and quality of tobacco
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Embodiment 1
[0032] In this example, the cloning of the tobacco NtAUX / IAA1 gene and the construction of the silencing vector are briefly introduced as follows.
[0033] (1) Tobacco NtAUX / IAA1 gene cloning
[0034] According to the previous research on the tobacco genome and related AUX / IAA1 genes, the specific coding sequence was selected as the target fragment, and the primer sequences for PCR amplification were designed as follows:
[0035] NtAUX1-F: 5'-ACC GAATTC AAACTTTGCCTTTGCTTG- 3', (the "GAATTC" part of the sequence underlined is the EcoRI restriction site)
[0036] NtAUX1-R: 5'-ACC GGATCC TCCAGGGTACATCACCAG- 3'; (The partial sequence of "GGATCC" underlined is the BamHI restriction site);
[0037] The NtAUX / IAA1 gene was obtained by PCR amplification using the cDNA of the cultivated tobacco leaf K326 as a template;
[0038] The PCR amplification program was: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 55°C for 15 s, extension at 72°C for 30...
Embodiment 2
[0050] On the basis of Example 1, using Agrobacterium-mediated VIGS technology, the inventors further transformed the constructed recombinant TRV2-NtAUX1 vector into tobacco plants, and verified and analyzed the phenotypic changes of related plants. A brief introduction to the specific experimental process as follows.
[0051] (1) Transformation of Agrobacterium
[0052] It should be noted that, referring to the operation of Example 1 and the prior art, the inventor simultaneously prepared TRV2-GFP and TRV2-PDS recombinant vectors as controls, and the specific transformation process is as follows:
[0053] The positive cloning plasmids of TRV2-GFP (vector control), TRV2-PDS (VIGS efficiency control) and TRV2-NtAUX1 were transformed into Agrobacterium GV3101 competent cells by electric shock transformation respectively, using 50mg / L Kan and 50mg / L Kan and 50mg / L The YEB plate of L Rif was cultured and screened, and after inverting at 28°C for 2 days, the Agrobacterium carrying...
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Abstract
The invention belongs to the technical field of tobacco genetic engineering, and specifically relates to a patent application for tobacco AUX / IAA gene and its application. Tobacco AUX / IAA protein consists of 212 amino acids, of which the 83-211th amino acid is its conserved domain. The protein is related to the content of pigment substances in plant leaves, and after reducing the expression of the protein, the content of pigment substances in the leaves is significantly reduced. The coding gene of tobacco AUX / IAA protein includes 639 bases, and its specific nucleic acid fragment is the 170-583rd base. The invention provides a tobacco AUX / IAA gene through the research on the regulatory genes related to the tobacco pigment content. Further through the gene silencing technology, after silencing the gene, the content of pigments in tobacco has been significantly reduced. Using this characteristic can provide new research directions and references for the development of new varieties of tobacco plants.
Description
technical field [0001] The invention belongs to the technical field of tobacco genetic engineering, and specifically relates to tobacco AUX / IAA and its application patent application. Background technique [0002] As one of the five classic plant hormones, auxin plays an important role in regulating almost all growth and development processes of plants. Auxin signal transduction is mainly regulated by two types of transcription-related proteins, Aux / IAAs (AUXIN / INDOLE ACETIC ACIDs) proteins and auxin response factors (Auxin Response Factors, ARFs). ARF has a DNA-binding domain that can recognize and bind to auxin-responsive elements in the promoters of auxin-responsive genes, thereby activating the expression of auxin-responsive genes. Under the condition of no or low concentration of auxin in plants, Aux / IAA protein can bind to ARF and inhibit its transcriptional activity, making it unable to drive the expression of downstream target genes; when the concentration of auxin ...
Claims
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