Specific promoter ZFP6 of tobacco glandular hair and application of specific promoter
A specificity, promoter technology, applied in the field of bioengineering, can solve the problems of normal plant growth obstruction, metabolic balance disorder, etc., and achieve the effect of increasing transgene, improving aroma quality, and important theoretical significance
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Embodiment 1
[0019] Example 1 ZFP6 gene promoter fusion GUS reporter gene vector construction and tobacco genetic transformation
[0020] (1) The leaves of Arabidopsis thaliana were taken, and the genomic DNA was extracted by CTAB method.
[0021] (2) Design specific primers according to the upstream sequence of the Arabidopsis ZFP6 gene transcription initiation site on Genbank, and add SalⅠ and NotⅠ restriction enzyme sites to the 5‵ ends of the primers respectively. The sequences are as follows:
[0022] Primer 1: 5‵-AGTCGACTGCAACGTAAAAGGTCCTGA-3‵;
[0023] Primer 2: 5‵-AAGCGGCCGCCAAAGAGAGAGAGAGAGACAGATTT-3‵.
[0024] (3) PCR amplification was performed using the extracted DNA as a template.
[0025] high fidelity enzyme HS DNA Polymerase (TaKaRa Code: DR010A) system:
[0026] 5×PrimeSTAR Buffer (Mg 2+ plus) 10 μl,
[0027] dNTP Mixture (2.5mM each) 4μl,
[0028] Primer 1 (10 μM) 1 μl,
[0029] Primer 2 (10 μM) 1 μl,
[0030] Template DNA 1 μl,
[0031] HS DNA Polymerase 0.5...
Embodiment 2
[0049] The character observation of embodiment 2 transgenic tobacco
[0050] The epidermal hairs of tobacco are divided into two categories according to the presence or absence of secretory glands. One is the glandular hairs that can produce secretions, and the other is the protective hairs that do not have the ability to secrete. The glandular hairs are divided into long-stalked glandular hairs and short-stalked glands according to their shape. hair.
[0051] The transgenic tobacco obtained in Example 1 was subjected to histochemical staining (GUS staining) analysis, and the results were as follows: figure 1 As shown, after the 60-day-old tobacco leaves were stained with GUS, the promoters of the present invention (A, B) were specifically expressed in the glandular hairs of the tobacco leaves (the glandular hairs appeared blue), while the control (C, D ) has no color reaction, indicating that the promoter of the present invention is active in tobacco glandular trichomes and ...
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