Asparagus Chalcone Isomerase Gene, Its Encoded Protein and Its Application

A chalcone isomerase and gene technology, applied in the field of plant genetic engineering, can solve problems such as unclear protein sequence, and achieve the effects of great economic value, promotion of growth and broad application prospects

Active Publication Date: 2019-06-04
VEGETABLE & FLOWER INST JIANGXI ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, asparagus is one of the important health-care vegetable crops rich in flavonoids. At present, there is no relevant literature report on asparagus CHI gene and its encoded protein, and the sequence of asparagus CHI gene and its encoded protein is still unclear.

Method used

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  • Asparagus Chalcone Isomerase Gene, Its Encoded Protein and Its Application
  • Asparagus Chalcone Isomerase Gene, Its Encoded Protein and Its Application
  • Asparagus Chalcone Isomerase Gene, Its Encoded Protein and Its Application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Cloning of AoCHI1 gene

[0028] Based on the analysis of the genome-wide high-throughput sequencing results of the new asparagus variety 'Jinggang 111' (JK111), the specific primers P1 forward primer: 5'-ATGGTGATGGTGGGTGATAT-3' and P2 reverse primer: 5'-CTAAGCAGAAGATAAAATGG- 3' (SEQ ID NO:3-4), using the commonly used CTAB method (refer to "Plant Genetic Engineering", edited by Wang Guanlin, Fang Hongyun) from the asparagus variety 'Jinggang 111' to extract tender stem total RNA, and synthesize cDNA by reverse transcription , using the above primers P1 and P2 to amplify the CDS sequence of the asparagus chalcone isomerase gene AoCHI1 shown in SEQ ID NO: 1 from the cDNA obtained by RNA reverse transcription, the full length of the CDS sequence of the gene AoCHI1 is 621bp.

[0029] Specific steps are as follows:

[0030] (1) Add CTAB (cetyltrimethylammonium bromide) extraction buffer [2% (W / V) CTAB, NaCl 1.4mol / L, EDTA (ethylenediaminetetraacetic acid) 20mmol / ...

Embodiment 2

[0038] Example 2 Construction and genetic transformation of AoCHI1 gene overexpression vector

[0039] In order to better analyze the biological function of the gene AoCHI1, the gene was further overexpressed in tobacco, and the biological function of the gene AoCHI1 was verified from the phenotypic characteristics of the total flavonoid content of the transgenic plants. Specific steps are as follows:

[0040] First, the pMD18-AoCHI plasmid obtained in Example 1 was double digested with BamH I and Knp I, and the target fragment was recovered; at the same time, the genetic transformation vector pCAMBIA2301 carrying the double tobacco mosaic virus promoter 35S was digested in the same way. After enzyme digestion, the enzyme-digested fragment containing the AoCHI1 gene and the enzyme-digested pCAMBIA2301 vector were used for ligation reaction to transform Escherichia coli DH5α (purchased from Treasure Bioengineering Dalian Co., Ltd.). Positive clones were screened by enzyme dige...

Embodiment 3

[0058] Example 3 RT-PCR detection of AoCHI1 gene transgenic T0 generation in the field

[0059] In order to verify whether the change of the total flavonoid content of transgenic tobacco is related to the transferred AoCHI1 gene, RT-PCR method was used to detect the expression of AoCHI1 gene in some transgenic tobacco plants, the results are shown in figure 1 . Specific steps are as follows:

[0060] Using TRIZOL reagent (purchased from Treasure Bioengineering Dalian Co., Ltd.) to extract the total RNA of the plant from transgenic tobacco No. 1-5 strains (extraction method refers to the operation of the TRIZOL reagent manual), using reverse transcriptase (purchased from Thermo Fisher Scientific company) It was reverse-transcribed to synthesize the first strand of cDNA, and the reaction conditions were 65°C for 5 minutes, 42°C for 50 minutes, and 70°C for 10 minutes. First use the internal reference gene Actin to detect and adjust the concentration of the cDNA obtained by rev...

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Abstract

The invention provides a chalcone isomerase gene of asparaguses, a protein encoded by the chalcone isomerase gene and application of the chalcone isomerase gene. The CDS sequence of the chalcone isomerase gene AoCHI1 is shown as SEQ ID NO: 1; the amino acid sequence of the protein encoded by the gene is represented by SEQ ID NO: 2. The chalcone isomerase gene AoCHI1 is cloned from the asparagus for the first time, and is one of key genes for synthesizing plant flavonoids; by the adoption of a method of gene engineering, the gene AoCHI1 is transferred into a target plant, so that the total flavonoids content of a transgenic plant can be increased, to improve the plant quality by using a gene engineering technology in the future; furthermore, an important theoretical basis is provided for obtaining medicines or food with high oxidization resistance, and the chalcone isomerase gene has a wide application prospect and high economic value.

Description

technical field [0001] The invention relates to the technical field of plant genetic engineering, in particular to asparagus chalcone isomerase gene and its coded protein and application. Background technique [0002] Plant active secondary metabolites are the products of a unique group of genes in their metabolic pathways. With the extensive and in-depth study of plant functional genomes, the study of functional genes related to the synthesis of plant secondary metabolism, which has unique characteristics and broad application prospects, has gradually become a research hotspot. Flavonoids are one of the important secondary metabolites in plants, which have important functions of anti-oxidation and free radical scavenging, and play an important role in improving human immunity. The study found that in the plant flavonoid biosynthesis pathway, the chalcone isomerase gene CHI is a very critical rate-limiting enzyme, which controls the synthesis and component differentiation o...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/61C12N9/90C12N15/82C12N1/21A01H5/00A01H6/82
CPCC12N9/90C12N15/8205C12N15/8243C12Y505/01006
Inventor 张岳平陈光宇瞿华香罗绍春赵萍周劲松汤泳萍尹玉玲
Owner VEGETABLE & FLOWER INST JIANGXI ACADEMY OF AGRI SCI
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