Asparagus chalcone synthase gene and its encoded protein and application

A chalcone synthase, gene technology, applied in the application, genetic engineering, plant genetic improvement and other directions, can solve the problem of unclear protein sequence, achieve great economic value, promote the content of total flavonoids, and promote the effect of increasing

Active Publication Date: 2019-06-07
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, there is no relevant literature report on asparagus CHS gene and its encoded protein, and the sequence of asparagus CHS gene and its encoded protein is still unclear.

Method used

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  • Asparagus chalcone synthase gene and its encoded protein and application
  • Asparagus chalcone synthase gene and its encoded protein and application
  • Asparagus chalcone synthase gene and its encoded protein and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Cloning of AoCHS1 gene

[0028] Based on the analysis of the genome-wide high-throughput sequencing results of the new asparagus variety 'Jinggang 111' (JK111), the design of specific primers P1 forward primer: 5'-ATGGCTGCAACATCAATG-3' and P2 reverse primer: 5'-CTATATAGGCACACTGTGAAG- 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 synthase gene AoCHS1 shown in SEQ ID NO: 1 from the cDNA obtained by RNA reverse transcription, the full length of the CDS sequence of the gene AoCHS1 is 1191bp.

[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)...

Embodiment 2

[0038] Example 2 Construction and genetic transformation of AoCHS1 gene overexpression vector

[0039] In order to better analyze the biological function of the gene AoCHS1, the gene was further overexpressed in tobacco, and the biological function of the gene AoCHS1 was verified from the phenotypic characteristics of the total flavonoid content of the transgenic plants. Specific steps are as follows:

[0040] First, the pMD18-AoCHS 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 with the same method. After enzyme digestion, the enzyme-digested fragment containing the AoCHS1 gene and the enzyme-digested pCAMBIA2301 vector were used for ligation reaction to transform Escherichia coli DH5α (the strain was purchased from Treasure Bioengineering Dalian Co., Ltd.). Positive clones were scr...

Embodiment 3

[0058] Example 3 RT-PCR detection of AoCHS1 gene transgenic T0 generation in the field

[0059] In order to verify whether the change of total flavonoid content in transgenic tobacco is related to the transferred AoCHS1 gene, RT-PCR method was used to detect the expression of AoCHS1 gene in some transgenic tobacco plants, the results are shown in figure 1 . Specific steps are as follows:

[0060] TRIZOL reagent (purchased from Treasure Bioengineering Dalian Co., Ltd.) was used to extract the total RNA of plants from transgenic tobacco No. 1-7 strains (the extraction method was operated according to the instructions of TRIZOL reagent), and reverse transcriptase (purchased from Thermo Fisher Scientific company) was used. 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 reported internal reference gene Actin to detect and adjust the concentration of...

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Abstract

The invention provides an asparagus chalcone synthetase gene, an encoded protein and applications thereof. The CDS sequence of asparagus chalcone synthetase gene (AoCHS1) is represented by the SEQ ID No.1. The amino acid sequence that encodes the protein is represented by the SEQ ID No.2. For the first time, asparagus chalcone synthetase gene (AoCHS1) is cloned from asparagus. The gene is one of the key genes in the synthesis route of plant flavonoid. The gene AoCHS1 can be transformed into target plants through a gene engineering method to increase the total flavonoid of transgenic plants. The gene is advantageously used for gene engineering to improve the plant quality so as to obtain drugs or foods with a high performance on resisting oxidation. The gene has a wide application prospect and can generate a great economic value.

Description

technical field [0001] The invention relates to the technical field of plant genetic engineering, in particular to asparagus chalcone synthase 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. Studies have found that in the biosynthetic pathway of plant flavonoids, chalcone synthase is the first key enzyme, also known as the rate-limiting enzyme, which catalyzes the 3 acetate groups of malo...

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汤泳萍赵萍周劲松谢启鑫
Owner VEGETABLE & FLOWER INST JIANGXI ACADEMY OF AGRI SCI
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