Eggplant chalcone isomerase smchi protein and its coding gene
A technology of chalcone isomerase and eggplant, which can be used in isomerase, genetic engineering, plant genetic improvement and other directions, and can solve problems such as
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Embodiment 1
[0047] Embodiment 1, the cloning of eggplant SmCHI gene
[0048] 1. Acquisition of plant material
[0049] 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.
[0050] 2. Extraction of RNA and DNA
[0051] 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 NANODROP1000 Spectrophotometer).
[0052] Total DNA was extr...
Embodiment 2
[0062] Example 2. Sequence information and homology analysis of eggplant CHI gene
[0063] The full-length CDS open reading frame sequence of the novel eggplant CHI gene of the present invention is 705 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 CHI was deduced, with a total of 234 amino acid residues, a molecular weight of 25248.8 Daltons, and a theoretical isoelectric point (pI) of 5.37. For the detailed sequence, see the sequence shown in SEQ ID NO.3 . The genomic DNA of SmCHI includes 4 exons and 3 introns, and the detailed sequence is shown in SEQ ID NO.2.
[0064] The CDS open reading frame sequence of eggplant CHI 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 86....
Embodiment 3
[0065] Embodiment 3, promoter cloning and sequence analysis of eggplant CHI gene
[0066] Using the GenomeWalking Kit kit (Bao Bioengineering (Dalian) Co., Ltd.), design primers
[0067] SP1: 5′-CCATTTCGCAGCTAGAAACGGAATTG-3′
[0068] SP2: 5′-CTTCCTTCAATCTCCAAACCTCTACTCC-3′
[0069] SP3: 5′-GGTATTGTTGGAACCAACAGGCTTCAC-3′
[0070] 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 Clones were sent to Yingwei Jieji (Shanghai) Trading Co., Ltd. for sequencing. Sequencing results revealed that the promoter of eggplant CHI gene was 1718bp. 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, G-Box, etc...
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