Eggplant chalcone synthase smchs1 protein and its encoding gene
A chalcone synthase and gene technology, which can be used in genetic engineering, plant gene improvement, enzymes and other directions, and can solve the problem of lack of eggplant CHS1 gene and its encoded protein.
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Embodiment 1
[0048] Embodiment 1, the cloning of eggplant SmCHS1 gene
[0049] 1. Acquisition of plant material
[0050] The plant material used in this experiment is the excellent eggplant germplasm resource A. spp. The phenotype of Lanshanhexian eggplant was purple stem, purple flower and purple fruit. The experimental materials were cultivated in artificial plastic greenhouses in the Aerospace Breeding Base of Pujiang Town, Minhang District, Shanghai. Seedlings are raised, grown and fruited under natural conditions. The peel of eggplant was collected for RNA and DNA extraction.
[0051] 2. Extraction of RNA and DNA
[0052] Total RNA was extracted by the TRIzol method (TRIzol was purchased from Sangon Bioengineering (Shanghai) Co., Ltd.). The integrity of RNA was identified by formaldehyde denaturing gel electrophoresis, and then the purity and concentration of RNA were determined on a spectrophotometer (Thermo Scientific NANODROP 1000 Spectrophotometer).
[0053] Total DNA was ...
Embodiment 2
[0063] Example 2, sequence information and homology analysis of eggplant CHS1 gene
[0064] The full-length CDS open reading frame sequence of the novel eggplant CHS1 gene of the present invention is 1170 bp, and the detailed sequence is shown in SEQ ID NO.1. According to the CDS open reading frame sequence, the amino acid sequence of eggplant CHS1 is deduced, with a total of 389 amino acid residues, a molecular weight of 42485.2 Daltons, and a theoretical isoelectric point (pI) of 7.04. For the detailed sequence, see the sequence shown in SEQ ID NO.3 . Genomic DNA of SmCHS1 includes 2 exons and 1 intron, and the detailed sequence is shown in SEQ ID NO.2.
[0065] The CDS open reading frame sequence of eggplant CHS1 and the amino acid sequence of its encoded protein were analyzed by BLAST program in Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDS translations+PDB+SwissProt+Superdate+PIR database Acid and protein homology search, it was found that it has 9...
Embodiment 3
[0066] Embodiment 3, promoter cloning and sequence analysis of eggplant CHS1 gene
[0067] Primers were designed using the Genome Walking Kit kit (Bao Bioengineering (Dalian) Co., Ltd.)
[0068] SP1: 5′-CCACTATGTCTTGCCTAGCATCAAGG-3′
[0069] SP2: 5′-CTCCTTGAGCTCAGTCTTATGTTCAC-3′
[0070] SP3: 5′-AGCACTCTGATCAACACAGTTAGAAGG-3′
[0071] Using DNA as a template, three rounds of PCR amplification were performed, and the amplified product was recovered and connected to the pMD-19T (Bao Biological Engineering (Dalian) Co., Ltd.) vector, transformed into E. coli DH5α, and screened positive by α complementation and colony PCR The clones were sent to Yingwei Jieji (Shanghai) Trading Co., Ltd. for sequencing. Sequencing results showed that the promoter of eggplant CHS1 gene was 1726bp. The sequence was submitted to PLACE and PlantCARE, two cis-element online prediction software for analysis, and it was found that it contained many light-responsive elements, including Box 4, Box I,...
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