Dracaena cambodiana chalcone isomerase DcCHIL1, encoding gene thereof and application

A chalcone isomerase and gene technology, applied in the field of plant biology, can solve problems such as inability to increase the content of flavonoids, and achieve the effects of great economic value, broad application prospects, and yield improvement

Inactive Publication Date: 2017-09-22
INST OF TROPICAL BIOSCI & BIOTECH CHINESE ACADEMY OF TROPICAL AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The object of the present invention is to provide a D. hainanensis chalcone isomerase DcCHIL1 gene and D. hainanensis ch...

Method used

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  • Dracaena cambodiana chalcone isomerase DcCHIL1, encoding gene thereof and application
  • Dracaena cambodiana chalcone isomerase DcCHIL1, encoding gene thereof and application

Examples

Experimental program
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Effect test

Embodiment 1

[0029] Example 1 Cloning of Chalcone Isomerase Gene DcCHIL1 from Dracaena hainanensis

[0030] Through the analysis of the sequencing results of the stem transcriptome of Dracaena hainanensis in the early stage, the specific primers P1 forward primer 5'-ATGGGCTCTGAGATAGTGATGGTG-3'(SEQ ID NO:3) and P2 reverse primer 5'-GCAGCAGATAACATAGTCCCAAG-3'( SEQ ID NO: 4), the CDS sequence of DcCHIL1 gene was amplified by PCR using the first-strand cDNA of the stem of Dracaena hainan as a template, and the amplification conditions were: pre-denaturation at 94°C for 5min; denaturation at 94°C for 30sec, annealing at 55°C 30sec, 70°C extension for 1min, a total of 35 cycles; 70°C extension for 8min. The amplified PCR product was connected to the pMD18-T vector, transformed into Escherichia coli competent cells, positive clones were screened and sequenced, and the full-length coding sequence 642bp of the chalcone isomerase gene DcCHIL1 of Dracaena hainanensis was obtained.

[0031] The above...

Embodiment 2

[0032] Example 2 Sequence analysis of the chalcone isomerase gene DcCHIL1 from Dracaena hainanensis

[0033] The full-length open reading frame of the chalcone isomerase gene DcCHIL1 of Dracaena hainanensis is 642bp, and the detailed sequence is shown in SEQ ID NO:1. The amino acid sequence of DcCHIL1 was deduced according to the sequence of the open reading frame, with a total of 213 amino acid residues (see SEQ ID NO: 2 for the detailed sequence), a molecular weight of 23.8 kDa, and a theoretical isoelectric point of 4.97.

[0034] 31 different types of CHI sequences were selected from different plants such as Arabidopsis thaliana and corn (for details of plant species and CHI protein sequence numbers, see figure 1 ) as a template to analyze the phylogenetic status of DcCHIL1. Use MEGA7 software to perform multiple sequence alignment on these 32 CHI sequences, and construct a phylogenetic tree (such as figure 1 ). It can be clearly seen from the figure that these CHIs can...

Embodiment 3

[0035] Example 3 Construction of PET28a-DcCHIL1 recombinant expression vector

[0036]Using Premier 5 software to analyze the restriction endonuclease cutting sites of DcCHIL1 coding region of Dracaena hainanensis chalcone isomerase gene, the two restriction endonucleases used to construct the vector were determined to be EcoRI and SalI. The primers PE1: 5′-GCGAATTCATGGGCTCTGAGATAGTGATGGTG-3′ (forward primer, marked as the EcoRI restriction site) and PE2: 5′-GCGTCGACAGCAGCAGATAACATAGTCCC-3′( Reverse primer, marked as the SalI restriction site), and pMD18-DcCHIL1 (prepared in Example 1) was used as a template for PCR amplification. The amplification conditions were: 94°C pre-denaturation for 5min; 94°C denaturation for 30sec, 58°C Anneal for 30 sec, extend at 70°C for 1 min, total 35 cycles; extend at 70°C for 8 min. The amplified PCR product was connected to the pMD18-T vector to obtain the pMD18-E DcCHIL1 recombinant plasmid, and the correctness of the target sequence was co...

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Abstract

The invention discloses dracaena cambodiana chalcone isomerase DcCHIL1, an encoding gene thereof and an application. The nucleotide sequence of the gene comprises: (a) a nucleotide sequence as shown in SEQ ID NO: 1; or (b) a nucleotide sequence formed by substituting, deleting and/or increasing one or a plurality of proteins expressing the same function of the nucleotide sequence as shown in SEQ ID NO: 1; or (c) a nucleotide sequence with 90% or more homologous proteins expressing the same function of the nucleotide sequence (a) or (b). The amino acid sequence of the dracaena cambodiana chalcone isomerase DcCHIL1 is as shown in SEQ ID NO: 2, or is an amino acid sequence formed by substituting, deleting or increasing one or a plurality of amino acids of SEQ ID NO: 2 and has the same function. The dracaena cambodiana chalcone isomerase DcCHIL1 gene is obtained by cloning for the first time, the obtained DcCHIL1 proteins have chalcone isomerase activity, the gene is transferred into dracaena cambodiana by biotechnology, so that dracaena cambodiana flavone accumulation and the yield of dragon's blood can be improved, and the gene has a wide application prospect and great economic values.

Description

technical field [0001] The invention relates to the field of plant biotechnology, in particular to a chalcone isomerase DcCHIL1 from Dracaena hainanensis and its coding gene and application. Background technique [0002] Flavonoids are a class of small molecular secondary metabolites that are widely involved in biological processes such as plant flower color formation, UV protection, plant fertility, auxin transport, resistance to osmotic stress, and cell cycle regulation. In addition, flavonoids are also a kind of antioxidant with strong biological activity, which has medical value such as regulating the body's immunity, anti-oxidation, anti-aging and anti-virus. Chalcone isomerase (CHI) is a key rate-limiting enzyme in the biosynthetic pathway of flavonoids, which catalyzes the isomerization of chalcones to flavanones, which are then converted into various types of flavonoids. The CHI superfamily contains four types of proteins: type Ⅰ is commonly found in vascular plants...

Claims

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Application Information

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IPC IPC(8): C12N15/61C12N9/90C12N15/10C12N15/70C12N1/21C12N15/82A01H5/00C12R1/19
Inventor 朱家红赵婉梅文莉戴好富彭世清王辉
Owner INST OF TROPICAL BIOSCI & BIOTECH CHINESE ACADEMY OF TROPICAL AGRI SCI
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