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Sweet potato chalcone isomerase IbCHIL1, as well as coding gene and application thereof

A technology of chalcone isomerase and gene, applied in the field of plant biology, to achieve the effects of enhancing memory, regulating body immunity, and great economic value

Inactive Publication Date: 2018-07-03
海南省农业科学院粮食作物研究所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are two CHI genes reported in sweet potato, both of which belong to type I, and other types of CHI genes have not been reported yet.

Method used

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  • Sweet potato chalcone isomerase IbCHIL1, as well as coding gene and application thereof
  • Sweet potato chalcone isomerase IbCHIL1, as well as coding gene and application thereof
  • Sweet potato chalcone isomerase IbCHIL1, as well as coding gene and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Cloning of sweet potato chalcone isomerase gene IbCHIL1:

[0032] Based on the analysis of the previous sweet potato transcriptome sequencing results, the specific primers P1 forward primer 5′-ATGGGTACTGAAATGGTGAT-3′ and P2 reverse primer 5′-TTAGACAGGCAGTGTGTTGGC-3′ were designed, using the first-strand cDNA of purple sweet potato root as a template The CDS sequence of IbCHIL1 gene was amplified by PCR. The amplification conditions were: pre-denaturation at 94°C for 5 minutes; denaturation at 94°C for 30 sec, annealing at 53°C for 30 sec, extension at 72°C for 1 min, and a total of 35 cycles; extension at 70°C for 8 min. The amplified PCR product was connected to the pMD18-T vector, transformed into Escherichia coli competent cells, positive clones were screened and sequenced to obtain the full-length coding sequence of the sweet potato chalcone isomerase gene IbCHIL1.

Embodiment 2

[0034] Sequence analysis of sweet potato chalcone isomerase gene IbCHIL1:

[0035] The full-length open reading frame of the sweet potato chalcone isomerase gene IbCHIL1 is 621bp, and the detailed sequence is shown in SEQ ID NO:1. The amino acid sequence of IbCHIL1 was deduced according to the open reading frame sequence, with a total of 213 amino acid residues (see SEQ ID NO: 2 for the detailed sequence), a molecular weight of 22.86, and a theoretical isoelectric point of 4.97.

[0036] 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 IbCHIL1. Use MEGA6 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 be divided into four branches, and IbCHIL1 is ...

Embodiment 3

[0038] Correlation analysis between IbCHIL1 expression and anthocyanin accumulation:

[0039] (1) Determination of anthocyanin content in different flesh-colored sweet potato varieties

[0040] Weigh different flesh-colored sweet potato tubers and air-dry them in an air-dried oven at 80°C for 8 hours; crush the air-dried samples, pass through an 80-mesh sieve, take 0.3g, add 20ml of 1% hydrochloric acid solution, shake well and let it stand for more than 2 hours, and take 1ml of supernatant 12000rpm centrifugal 5min; Get supernatant respectively and measure its OD value at 525nm on spectrophotometer; Calculation method and analysis calculation formula of sweet potato anthocyanin content:

[0041] C=1 / 958×V / 100×1 / m×ABS×1000000

[0042] In the formula, 958 is the empirical coefficient of converting ABS into mass-to-volume ratio; C is the anthocyanin content, and the unit is m g / kg fresh potato; V is the constant volume, which is 100m L; m is the sample mass (g); Absorbance of ...

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Abstract

The invention relates to the technical field of plant biology, and particularly relates to sweet potato chalcone isomerase IbCHIL1, as well as a coding gent and application thereof. The nucleotide sequence of the gene comprises a nucleotide sequence as shown in SEQ ID NO:1, or a nucleotide sequence which is the nucleotide sequence as shown in the SEQ ID NO:1 substituted, lost and / or added with oneor more proteins expressing the same function; or a nucleotide sequence which has more than 90 percent of homology with the nucleotide sequence of (a) or (b) and proteins expressing the same function; the amino acid sequence of the chalcone isomerase IbCHIL1 is as shown in SEQ ID NO:2, or is the amino acid sequence as shown in the SEQ ID NO:2, which is formed by substituting, losing or adding oneor more amino acids and has the same function. The chalcone isomerase gene is transferred into sweet potatoes by a biological technology, or expression of the gene is regulated, so that the content of anthocyanin of sweet potatoes can be increased, and the gene has a wide application prospect and a great economical value.

Description

technical field [0001] The invention relates to the field of plant biotechnology, in particular to a sweet potato chalcone isomerase IbCHIL1 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 is a key enzyme in the biosynthesis of flavonoids, which catalyzes the isomerization of chalcones into (2S)-flavanones, which are then converted into various types of flavonoids. The chalcone isomerase superfamily includes four types of CHI proteins: type I and type II CHI proteins are true CHIs with CHI pro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/61C12N9/90C12N15/10C12N15/82A01H5/00A01H6/00
CPCC12N9/90C12N15/1096C12N15/825C12Y505/01006C12Q2531/113
Inventor 徐靖朱红林韩义胜王效宁唐力琼张更武陈明阳
Owner 海南省农业科学院粮食作物研究所
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