A dreb1 transcription factor in cotton, its coding gene and its application
A technology of transcription factors and cotton, which is applied in the fields of application, genetic engineering, and plant genetic improvement, and can solve problems such as failing to meet the standards of industrialization
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Embodiment 1
[0020] Cotton SSH library construction under cold stress in embodiment 1:
[0021] The specific method is:
[0022] Using PCR-select from Clontech TM cDNA Subtraction Kit, construct a subtractive library by suppression subtractive hybridization method according to the method indicated in the product manual. During the experiment, the mRNA of cotton seedling leaves treated at low temperature for 6 hours was used as a sample (tester), and the mRNA of untreated cotton seedling leaves was used as a control (driver).
[0023] (1) Test materials:
[0024] Ji Mian 14 (National Cotton Medium-Term Bank, acquired by China Cotton Research Institute, uniform number: ZM-30270) was sown on sterilized vermiculite, cultivated at 25°C, photoperiod 16h / 8h, and watered 1 / 2 2MS medium (9.39mMKNO 3 , 0.625mM KH 2 PO 4 , 10.3mM NH 4 NO 3 , 0.75mM MgSO 4 , 1.5mM CaCl 2 , 50 μM KI, 100 μM H 3 BO 3 , 100 μM nSO 4 , 30 μM ZnSO 4 , 1 μM Na 2 MoO 4 , 0.1 μM CoCl 2 , 100 μM Na 2 EDTA, 1...
Embodiment 2
[0035] Cloning of embodiment 2GhCBF2 coding gene
[0036] In the unigene with homologous sequences in Example 1, the clone Gh-C072 sequence is shown in SEQ ID NO: 3:
[0037] 1 ACTTTTCTCA CAGCGGAGAT GGCAGCTCGT GCCCACGACG TAGCGGCCAT AGCTCTGAGG
[0038] 61 GGGAGGTCGG CTTGCCTGAA CTTCGCGGAC TCAGCTTGGA GGCTTCCGGT TCCGGCTTCT
[0039] 121 GCTGATCCCA AGGATATCCA GAAGGCGGCG GCAGAGGCGG CGGAGGCATT TAGACAACCG
[0040] 181 GCTGAGCAAT GGGACGGAAA CTCTGAAGCT AATGCAAAGG GAGATGAAAA CAAAGGGAGT
[0041] 241 TTGTGTGAGA ATGGGTTTTA TTTGGACGAG GAGGCGGTTT TTGGAACACA AAGTTATCTG
[0042] 301 GAAAACATGG CGGCAGGGAT GATGATGTCA CCCCCTCGTT GTGGGT
[0043] Sequence analysis showed that the encoded amino acid sequence of this sequence belonged to DREB1 class protein, and this protein was named GhCBF2 in this paper.
[0044] (1) Cloning of the full-length gene of GhCBF2
[0045] According to the obtained GhCBF2 gene fragment, two specific primers were designed as the 5' end-specific primers of 3'RACE:
[...
Embodiment 3
[0095] Embodiment 3GhCBF2 plant expression vector construction
[0096] The construction process of the plant expression vector rd29A-GhCBF2-2300 is shown in Figure 1.
[0097] The plant binary expression vector pCAMBIA2300 (purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd.) was selected as the plant expression vector, and the 35S promoter of the NP TII gene containing double enhancers was replaced with the Pnos promoter to reduce the expression of NPTII protein in plants. Express. The inducible promoter rd29A and Tnos were selected as the promoter and terminator of GhCBF2 gene. Specific steps are as follows:
[0098] Using SEQ ID NO: 11 and SEQ ID NO: 12, Pnos was amplified using the plant expression vector PBI121 (purchased from Beijing Huaxia Ocean Technology Co., Ltd.) as a template, and TaKaRa's PrimeSTAR HS DNA polymerase was used. 50 μl PCR reaction system: 10 μl 5×PS Buffer, 3 μl 2.5 mM dNTP, 1.0 μl PBI121, 1.0 μl PrimeSTAR, 10 μM primers SEQ ID NO: ...
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