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Eggplant chalcone isomerase SmCHI protein and coding gene thereof

A technology of chalcone isomerase and eggplant, applied in the direction of isomerase, genetic engineering, plant gene improvement, etc., can solve problems such as unexamined

Active Publication Date: 2015-07-01
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no relevant literature report on the CHI gene and its encoded protein in eggplant

Method used

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  • Eggplant chalcone isomerase SmCHI protein and coding gene thereof
  • Eggplant chalcone isomerase SmCHI protein and coding gene thereof
  • Eggplant chalcone isomerase SmCHI protein and coding gene thereof

Examples

Experimental program
Comparison scheme
Effect test

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 new 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 86.81...

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 Bioengineering (Dalian) Co., Ltd.) vector, transformed into Escherichia 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, et...

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Abstract

The invention discloses eggplant chalcone isomerase SmCHI protein and a coding gene thereof. The protein comprises (a) protein consisting of an amino acid sequence as shown in SEQ ID NO.3; or (b) protein derived from the amino acid sequence in (a), which is substituted, lost or added by one or more amino acids and has the activity of eggplant chalcone isomerase. The cDNA and gDNA of the coding gene respectively have base sequences as shown in SEQ ID NO.1 and SEQ ID NO.2; and the gene is expressed in roots, stems, leaves, flowers, peels and pulp, and has the expression quantity in peels remarkably higher than that in other tissues. Under stress of low temperature, the expression quantity of the gene reaches the maximal value in 48 hours, which is 3.65 times that of an unprocessed gene. The eggplant chalcone isomerase SmCHI protein and the coding gene thereof provide a theoretical basis for improving plant quality by utilizing the genetic engineering technology to obtain high oxidation resistance medicines or foods in the future, thereby having great application values.

Description

technical field [0001] The invention relates to a key enzyme and its encoding gene in the eggplant anthocyanin synthesis pathway, in particular to an eggplant chalcone isomerase SmCHI protein and its encoding gene, and belongs to the field of biotechnology. Background technique [0002] The human body will inevitably produce a large number of free radicals due to metabolic activities. As a strong reducing substance, anthocyanins can remove free radicals in the human body and delay the aging of the body. The synthetic pathway of anthocyanins is a part of the synthetic pathway of flavonoids, and it is also a very extensive and in-depth plant secondary metabolic pathway studied at present, which has been elaborated in the main model plants such as Arabidopsis and petunia. The biosynthesis of anthocyanins is mainly divided into three stages: the first stage is from phenylalanine to coumaryl-CoA, which is a common step in many secondary metabolisms; the second stage is from couma...

Claims

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Application Information

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IPC IPC(8): C12N9/90C12N15/61C12N15/11C12N15/113
CPCC12N9/90C12Y505/01006
Inventor 陈火英蒋明敏任丽高莉洁周腾夏
Owner SHANGHAI JIAO TONG UNIV
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