Drought induced type promoter and application thereof
A drought-inducible, promoter technology, applied in the field of non-coding nucleic acids, can solve the problem of few inducible promoters
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Embodiment 1
[0021] Embodiment 1: The drought-inducible promoter of the present invention pCD4 separation
[0022] 1.1 Preparation of sweet buckwheat genomic DNA
[0023] 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.
[0024] 1.2 Promoter pCD4 Cloning and sequence analysis of
[0025] 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:
[0026] Upstream primer: 5'- CCCAAGCTTAAGGTTCTTTGCAAGTC -3'
[0027] Downstream primer: 5'- GCGGATCCGATCGA...
Embodiment 2
[0042] Embodiment 2: promoter pCD4 Construction of Plant Transient Expression Vector
[0043] Using Bam HI and Hind III Endonuclease digestion of the recombinant cloning vector (T+ pCD4 )and pC0390::GUS Carrier ( figure 2 , image 3 ), and then the promoter pCD4 and pC0390::GUS The GUS reporter gene of the vector is connected to construct a plant transient expression vector. The constructed plant transient expression vector was named as pCD4::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 pCD4::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.
[0044]
Embodiment 3
[0045] Example 3: Identification of promoters under drought conditions pCD4 active
[0046] 3.1
[0047] 3.1.1 Tobacco culture and transient transformation
[0048] 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 6000rpm...
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