Transcription factor of DREB class in cotton, its coding gene, and application
A transcription factor and coding technology, applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve the problem of the function of A-6 group members being analyzed
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Embodiment 1
[0043] Embodiment 1, the cloning of cotton DREB class transcription factor coding gene GhDBP2
[0044] The cDNA sequence of the cotton DREB transcription factor coding gene GhDBP2 was cloned by combining bioinformatics and conventional PCR. and DBF2 are involved in rab17 regulation through the drought-responsive element in an ABA-dependent pathway. Plant J. 2002, 30: 679-689) encoded amino acid sequence was searched with tblastn program in the cotton EST database (http: / / www.tigr. org / ), the result was a sequence (TC18189), which was spliced from several EST sequences from Upland cotton and Sea island cotton, with a translation start codon and a stop codon, indicating that the sequence contained the full-length open Reading frame (ORF), sequence alignment results show that the amino acid residue sequence encoded by this sequence has a high similarity with the amino acid residue sequence of ZmDBF1. In order to verify that the cDNA sequence also exists in upland cotton (Gossy...
Embodiment 2
[0045] Example 2, Sequence Analysis of GhDBP2 Protein and Homology Evolution Analysis of DNA Binding Domain
[0046] 1. Sequence analysis of GhDBP2 protein
[0047] Sequence analysis of the amino acid residue sequence of GhDBP2 revealed that the GhDBP2 protein contains an AP2 / ERF domain that is conserved among DREB subfamily transcription factors, namely amino acid residues 160-217 from the amino terminal (N terminal) (The 587th-760th base from the 5' end in SEQ ID №: 2 is its coding sequence), a total of 58 amino acids; there is an acidic region at its C-terminus, that is, from the 278th-350th amino-terminal Amino acid residues (bases 941-1159 from the 5' end in SEQ ID No. 2 are its coding sequence), the acidic region may function as a transcriptional activation region; behind the DNA binding region is the amphipathic nuclear localization Signal sequence (NLS), that is, amino acid residues at position 257-273 from the amino terminal (in SEQ ID No.: 2, bases at position 878-9...
Embodiment 3
[0052] Example 3, Analysis of the binding characteristics of GhDBP2 and DRE elements
[0053] 1. Preparation of GhDBP2 AP2 / ERF domain fusion protein with His-tag (6×His)
[0054] 1. Construction of GhDBP2 AP2 / ERF domain expression vector
[0055] Using GhDBP2 cDNA as a template, primer P1 (upstream primer): 5'-CG GGATCC ATGGTTAGCTTTCTTTGC-3' (the underlined base is the restriction endonuclease BamH I recognition site) and P2 (downstream primer): 5'-CG GAATTC Under the guidance of TCAAGCGTCAACAGAGGA-3' (the underlined base is the restriction endonuclease EcoRI recognition site), PCR amplified GhDBP2 AP2 / ERF with BamHI recognition site added at the 5' end and EcoR I recognition site added at the 3' end Domain coding sequence. After the reaction, the PCR amplification product was detected by 1.2% agarose gel electrophoresis, and the target fragment of about 300bp was recovered and purified, digested with restriction enzymes BamHI and EcoRI, and double-digested with the same ...
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