Chalcone isomerase and coding gene, expression vector, host bacteria and application thereof

A technology of chalcone isomerase and coding gene, which is applied in the field of bioengineering, can solve problems such as limiting CHI regulation and improvement, limiting CHI evolution research, etc., and achieves good practical application value

Inactive Publication Date: 2018-07-06
GUIZHOU NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report on the cloning and functional research of CHI gene in Rubiales, which greatly limits the evolutionary research of CHI, and also limits the utilization of CHI in Rubiales and its anthocyanin biosynthesis. Regulation and improvement, from the molecular and genetic perspectives, there are no reports on chalcone isomerase genes of Rubiaceae plants, especially Japanese snakeroot

Method used

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  • Chalcone isomerase and coding gene, expression vector, host bacteria and application thereof
  • Chalcone isomerase and coding gene, expression vector, host bacteria and application thereof
  • Chalcone isomerase and coding gene, expression vector, host bacteria and application thereof

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Embodiment 1

[0040] This embodiment provides a chalcone isomerase, the amino acid sequence of the chalcone isomerase is shown in SEQ ID NO.1, the estimated protein molecular weight is 25.018kD, and the isoelectric point is 4.95.

[0041] This embodiment also provides a gene encoding chalcone isomerase, and the base sequence of the gene encoding chalcone isomerase is shown in SEQ ID NO.2.

[0042] The coding gene of chalcone isomerase is obtained by amplifying the cDNA of the CHI gene that controls the synthesis of anthocyanins in the Japanese snakeroot grass with primers designed according to the transcriptome sequencing results of the Japanese snakeroot grass petal tissue, and then amplified by PCR.

[0043] Compared with the chalcone isomerase CHI with known crystal structure of alfalfa, the results show that the CHI of snakeroot has a ubiquitous active site of chalcone isomerase, the results are as follows figure 1 As shown, CHI-MEDICAGO.PRO represents the amino acid sequence of alfalfa...

Embodiment 2

[0045] This example provides a study on the function of the chalcone isomerase gene, including further verification and determination through gene expression in vitro.

[0046] Construction of the recombinant plasmid: use the cDNA of the cloned full-length Japanese snakeroot chalcone isomerase OjCHI gene as a template, and perform PCR of the OjCHI open reading frame with primers containing BamH I and Hind III restriction sites respectively Amplify. The amplified DNA fragments were double-digested with two restriction enzymes, BamHI and HindIII, respectively, and ligated and cloned into the pET-32a vector. The thus constructed recombinant plasmid was named pET32-OjCHI, and introduced into the host cell Escherichia coli BL21(DE3) cells to prepare a large amount of soluble recombinant protein.

[0047] Prokaryotic expression of soluble recombinant chalcone isomerase protein;

[0048]The above-mentioned Escherichia coli was streak-inoculated in LB solid medium containing Amp, an...

Embodiment 3

[0052] Due to heterologous prokaryotic expression, it is easy to cause enzyme inactivation, mainly because the Japanese snakeroot chalcone isomerase belongs to the expression product of plant eukaryotic genes, and Escherichia coli belongs to the prokaryotic expression system. Different expression systems may be in the The folding process of the protein in the later stage is different, which leads to the inactivation of the protein, so the successful expression of the protein does not necessarily make the protein have the corresponding biological activity; therefore, the verification of the biological activity is required; in addition, due to the codon preference, it may also cause the protein to be inactivated. Expression failed, so sequence codon optimization can be performed based on the gene sequence of the cloned japonica chalcone isomerase provided by the present invention; so that the efficiency of prokaryotic expression is improved.

[0053] With naringenin chalcone as s...

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Abstract

The invention provides chalcone isomerase and a coding gene, an expression vector, host bacteria and application thereof and belongs to the technical field of biological engineering. The chalcone isomerase provided by the invention is obtained by expression of a chalcone isomerase coding gene obtained by cloning ophiorrhiza japonica for the first time, and the coding gene is connected to the expression vector and transferred to the host bacteria. When the chalcone isomerase coding gene is applied to regulation and control of plant anthocyanin or seed color, regulation and control effects are better, and actual application values are realized.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to chalcone isomerase and its coding gene, expression vector, host bacteria and application. Background technique [0002] Anthocyanin compound is a very important plant secondary metabolite, which not only imparts color to flowers, fruits, leaves and other tissues, but also has many important functions. It can protect plant cells from ultraviolet rays, resist pathogens and herbivores, promote the interaction between plants and microorganisms as a signal molecule, affect the growth and development of pollen, the generation of root nodules, and the transport of hormones in plants. At the same time, a large number of experiments have proved that there is also a close relationship between anthocyanins and human health. It has biological activities such as anti-oxidation, anti-virus, and anti-cell proliferation, and has been used to treat diseases such as arteriosclerosis and car...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/90C12N15/61C12N15/82A01H5/00A01H5/10A01H6/20A01H6/76
CPCC12N9/90C12N15/825C12Y505/01006
Inventor 孙威
Owner GUIZHOU NORMAL UNIVERSITY
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