CN110791510A - Screening identification and application of acetaldehyde dehydrogenase coding gene participating in crocetin synthesis - Google Patents

Abstract

The invention relates to acetaldehyde dehydrogenase GjALDH2C3 participating in crocetin synthesis in a crocin biosynthesis pathway of gardenia, and a coding gene and a function thereof. Belongs to the technical field of genetic engineering. The invention discloses an open reading frame sequence of a gardenia acetaldehyde dehydrogenase GjALDH2C3 gene and an amino acid sequence coded by the same. The invention obtains GjALDH2C3 gene based on gardenia genome and transcriptome data screening, and verifies the catalytic function of GjALDH2C3 protein by constructing pCold-GjALDH2C3 prokaryotic expression vector and inducing expression and taking crocetin dialdehyde as substrate. The result proves that GjALDH2C3 can efficiently catalyze the crocetin dialdehyde to generate the crocetin, and lays a foundation for the biosynthesis pathway and synthetic biology research of the medicinal active ingredients crocetin and crocin.

Description

Screening and identification of a gene encoding acetaldehyde dehydrogenase involved in the synthesis of crocus acid and application

technical field

The invention belongs to the technical field of plant genetic engineering, in particular to a method for screening, functional verification and application of crocus acid biosynthesis genes in Gardenia.

Background technique

Crocin is an apo-carotenoid compound, which was originally extracted from saffron known as "red gold", mainly accumulated in the stigma of saffron, and has high medicinal value. Crocus acid is the precursor material for the synthesis of crocin, which is formed by combining with different numbers of sugar groups. Crocus acid has great potential medicinal value, and modern pharmacological studies have proved that it plays a role in anti-tumor, anti-oxidation, anti-hypertension, anti-atherosclerosis, and anti-depression. At the same time, crocus acid is also used as a food coloring. However, the source of saffron acid mainly depends on the extraction, separation and purification from saffron. Due to the shortage of saffron resources and the low extraction efficiency, its large-scale application is greatly limited.

It is worth noting that the fruits of Gardenia, a Rubiaceae plant, are rich in crocin. Compared with saffron, gardenia is rich in resources and easy to obtain. It is considered to be an ideal alternative resource for saffron. The research on the biosynthetic pathway of crocin has attracted international attention. This research aims to find a breakthrough in the biosynthetic pathway of crocin, the precursor for crocin synthesis, and to reveal that Gardenia is involved in the biosynthesis of crocus acid. The function of the key enzyme gene ALDH in the pathway lays the foundation for the synthesis of crocus acid and crocin.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide an acetaldehyde dehydrogenase gene that catalyzes the synthesis of crocus acid from dialdehyde of crocetin and its encoded protein.

Another object of the present invention is to provide a screening method for the key enzyme gene of crocus acid synthesis in Gardenia.

The third object of the present invention is to provide a method for functional verification of the above screened key enzyme gene GjALDH2C3.

The nucleotide sequence of the GjALDH2C3 gene provided by the present invention is shown in SEQ ID No. 1, or its mutant sequence.

The amino acid sequence of the protein encoded by the GjALDH2C3 gene provided by the present invention is shown in SEQ ID No. 2, or a mutant sequence thereof.

The object of the present invention can be achieved through the following technical solutions, technical solution 1: based on transcriptome-based screening of the gene screening of key enzymes in the synthesis of gardenia crocus acid, the steps are as follows:

1) Based on the gardenia genome, identify the members of the gardenia ALDH gene family, construct an evolutionary tree to analyze the evolutionary relationship between gardenia and other species ALDH, and screen the crocus acid dialdehyde catalyzed in the biosynthetic pathway of gardenia crocin. The key enzyme protein sequence to crocus acid.

2) Based on the transcriptome data of different tissue parts of Gardenia, analyze the differential gene expression, and further screen the key enzyme genes that catalyze the conversion of crocus acid dialdehyde to crocus acid in the biosynthetic pathway of crocin in Gardenia japonica.

Technical scheme 2: functional verification of the key enzyme gene GjALDH2C3. The function of acetaldehyde dehydrogenase GjALDH2C3 was identified in vitro using a prokaryotic expression system with crocetin dialdehyde as a substrate.

The invention discloses a screening and functional identification system of the acetaldehyde dehydrogenase GjALDH2C3 gene for synthesizing crocus acid dialdehyde to crocus acid in Gardenia, and it is verified that GjALDH2C3 has the function of oxidizing crocetin dialdehyde to crocus acid , which provides a molecular basis for elucidating the biosynthetic pathway of crocus acid and crocin, and has great application value.

Description of drawings

Figure 1: Identification and evolutionary relationship of acetaldehyde dehydrogenase GjALDH family members based on the gardenia genome.

Figure 2: Differential expression of GjALDH-encoding gene in different tissue parts of Gardenia. Flower: flower; Leaf: leaf; Root: root; Stem: stem; Fruitlet: young fruit; GreenFruit: green fruit; RedFruit: red fruit.

Figure 3: In vitro functional validation of GjALDH2C3. A: GjALDH2C3 can catalyze the synthesis of crocus acid dialdehyde, and the intermediate product crocetin semialdehyde was detected in the reaction; B: The presumed synthesis pathway of crocus acid.

Figure 4: Identification of the products crocus acid and crocus acid semialdehyde by LC-MS.

specific implementation

The present invention will be described in detail below with reference to examples. The implementation is for a better understanding of the present invention, but is not limited to the present invention. The experimental methods in the following implementation methods are all conventional methods, and the experimental reagents involved are all conventional biochemical reagents.

Example 1 Screening and phylogenetic analysis of GjALDH gene based on gardenia genome and transcriptome data

1.1 Experimental method

Based on the gardenia genome data, the ALDH protein sequence of Arabidopsis thaliana was extracted from the TAIR database, and the gardenia protein sequence library was searched by BLASTP, and the identified homologous gene GjALDHs was further manually corrected. The GjALDH protein sequences of Gardenia and Arabidopsis were aligned by ClustalW2, and the NJ phylogenetic tree was constructed using Mega6 software, and boostrap selection was repeated 1000 times. The RNA-Seq transcriptome data of gardenia roots, stems, leaves, flowers, young fruits, green fruits, and red fruits were compared to the gardenia genome using HiSAT2, and differential expression analysis was calculated using Cuffiinks. Previous studies have shown that crocin is specifically distributed in the green and red fruits of Gardenia, and the candidate GjALDH gene that is consistent with the distribution of crocin was screened based on co-expression analysis.

1.2 Results and Analysis

Based on gardenia genome data, 19 GjALDHs were screened, mainly distributed in 10 subfamilies, including 7 ALDH2, 2 ALDH3, 1 ALDH5, 3 ALDH6, 1 ALDH10, 1 ALDH11, and 1 ALDH12 , 1 ALDH18, 1 ALDH22. Through transcriptome data analysis, it was found that GjALDH2C3 gene was specifically expressed in gardenia fruit and flower, and had the highest expression level. Therefore, it is speculated that it is the key enzyme gene that catalyzes the conversion of crocus acid dialdehyde to crocus acid in the biosynthetic pathway of gardenia crocin.

The functional verification of embodiment 2GjALDH2C3

2.1 Experimental method

The GjALDH2C3 encoding gene was cloned into pCold I vector to form pCold I-GjALDH2C3 recombinant expression vector, and the vector was transformed into BL21 E. coli expression strain. Take 2 mL of the overnight induced bacterial solution and add it to 50 mL of LB liquid culture concentrate (containing 50 μg/mL ampicillin-resistant medium), activate at 37 °C, 200 rpm for 2-3 h, and when its OD ₆₀₀ reaches 0.4-0.5, stimulate it at a low temperature of 15 °C , and add IPTG with a final concentration of 0.3mM for induction, 15°C, 130rpm. After 24 hours of induction, the bacterial pellets were collected by centrifugation for protein purification, and the concentration was determined using the BCA protein kit.

BEH C18column(1.7μm，100×2.1mm)，柱温：30℃。色谱条件：UV 440nm，流动相：(A)：乙腈(含0.1％甲酸)，(B)：水(含0.1％甲酸)，流速：0.3mL/min，洗脱程序：0-5min，10％A线性增长至50％A；5-8min，50％A线性增长至90％A；8-10min，90％A线性增长至100％A，100％A持续一分钟后回到起始状态。The purified protein was functionally verified in vitro according to the following reaction system (50 μL): 100 mM Tris-HCl (pH 8.5), 50 μg purified protein, 1 mM NADP ⁺ , and 40 μM crocetin dialdehyde. After terminating the reaction with methanol, it was mixed evenly through a 0.22 μm filter membrane, and its chemical composition was detected by UPLC. Instrument model: Thermo Ultimate 3000. Injection volume 10μL, chromatographic column: Waters Acquity

BEH C18column (1.7 μm, 100×2.1 mm), column temperature: 30°C. Chromatographic conditions: UV 440nm, mobile phase: (A): acetonitrile (containing 0.1% formic acid), (B): water (containing 0.1% formic acid), flow rate: 0.3 mL/min, elution program: 0-5min, 10% A linearly increases to 50%A; 5-8min, 50%A linearly increases to 90%A; 8-10min, 90%A linearly increases to 100%A, 100%A returns to the initial state after one minute.

2.2 Results and Analysis

The in vitro catalysis results show that GjALDH2C3 can convert crocus acid dialdehyde, and the reaction produces two new chromatographic peaks, one has the same chromatographic behavior as crocus acid (such as retention time, spectrum, etc.), and the other is between Between crocus acid and crocus acid dialdehyde, the qualitative analysis of the catalytic product requires liquid quality detection.

The qualitative analysis of embodiment 3 catalytic product

3.1 Experimental method

BEH C18column(1.7μm，100×2.1mm)，柱温：30℃。色谱条件：UV 440nm，流动相：(A)：乙腈(0.1％甲酸)，(B)：水(0.1％甲酸)，流速：0.3mL/min，洗脱程序：0-5min，10％A线性增长至50％A；5-8min，50％A线性增长至90％A；8-10min，90％A线性增长至100％A，100％A持续一分钟后回到起始状态。6545Q-TOF质谱参数为：干燥气体温度为325℃，流速6L/min；鞘气为350℃，流速12.0L/min；喷雾器为45psig；VCap为4000V。Use Agilent Technologies 1290Infinity II and 6545Q-TOF LC/MS instruments to detect the products extracted from the resulting reaction and conduct qualitative analysis. Injection volume 10μL, chromatographic column: Waters Acquity

BEH C18column (1.7 μm, 100×2.1 mm), column temperature: 30°C. Chromatographic conditions: UV 440nm, mobile phase: (A): acetonitrile (0.1% formic acid), (B): water (0.1% formic acid), flow rate: 0.3 mL/min, elution program: 0-5 min, 10% A linear Increase to 50%A; 5-8min, 50%A linearly increase to 90%A; 8-10min, 90%A linearly increase to 100%A, 100%A returns to the starting state after one minute. 6545Q-TOF mass spectrometry parameters are: drying gas temperature is 325°C, flow rate is 6L/min; sheath gas is 350°C, flow rate is 12.0L/min; nebulizer is 45psig; VCap is 4000V.

3.2 Results and Analysis

LC-MS test results The conversion of crocetin dialdehyde into crocetin ([M+H] ⁺ : m/z327.1601) and crocetin half under the catalysis of ALDH2C3 Aldehyde (Crocetinsemidialdehyde, [MH] ⁺ : m/z 311.1686).

The above are only the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, without departing from the technical principles of the present invention, several improvements and modifications can be made. These improvements and modifications It should also be regarded as the protection scope of the present invention.

Claims (6)

1. acetaldehyde dehydrogenase GjALDH2C3 gene in a kind of gardenia, is characterized in that it is following gene (a) or gene (b): Gene (a): a gene having the nucleotide sequence shown in SEQ ID No. 1. Gene (b): a protein having the function of oxidizing crocetin dialdehyde to crocus acid obtained by substitution, deletion or addition of one or more bases in the nucleotide sequence shown in SEQ ID No. 1. 2. a protein encoded by acetaldehyde dehydrogenase GjALDH2C3 gene in the gardenia, is characterized in that it is following protein (a) or protein (b): Protein (a): a protein having the amino acid sequence shown in SEQ ID No. 2. Protein (b): a protein having the function of oxidizing saffron dialdehyde to saffron acid obtained by replacing, deleting or adding one or more amino acids in the amino acid sequence shown in SEQ ID No. 2. 3. A method for catalyzing the GjALDH2C3 gene of crocinic acid dialdehyde to crocinic acid in the biosynthetic pathway of gardenia crocin in the screening using genome and transcriptome data, the concrete steps are as follows: 1) Based on the gardenia genome, identify the members of the gardenia ALDH gene family, construct an evolutionary tree to analyze the evolutionary relationship between gardenia and other species ALDH, and screen the crocus acid dialdehyde catalyzed in the biosynthetic pathway of gardenia crocin. The key enzyme protein sequence to crocus acid. 2) Based on the transcriptome data of different tissue parts of Gardenia, analyze the differential gene expression, and further screen the key enzyme genes that catalyze the conversion of crocus acid dialdehyde to crocus acid in the biosynthetic pathway of crocin in Gardenia japonica. 4. The functional verification method of Gardenia GjALDH2C3, the specific steps are as follows: 1) The pCold I-GjALDH2C3 expression vector was transformed into Escherichia coli BL21 strain, selected with ampicillin, induced by 0.2-1.0 mM IPTG, and induced at 15°C for 24 hours in the dark. 4°C, 7830 rpm, centrifugation for 15 min, after ultrasonication, the target protein with His tag was purified by nickel column, and the concentration was determined by BAC protein analysis kit after desalting and concentration. 2) The purified protein was subjected to functional verification in vitro according to the following reaction system (50 μL): 100 mM Tris-HCl (pH 8.5), 50 μg of purified protein, 1 mM NADP ⁺ , and 40 μM crocus acid dialdehyde. After terminating the reaction with methanol, it was mixed evenly through a 0.22 μm filter membrane, and its chemical composition was detected by UPLC. Instrument model, Thermo Ultimate 3000. Injection volume 10μL, chromatographic column: Waters Acquity BEH C18 column (1.7 μm, 100×2.1 mm), column temperature: 30°C. Chromatographic conditions: UV440nm, mobile phase: (A): acetonitrile (containing 0.1% formic acid), (B): water (containing 0.1% formic acid), flow rate: 0.3 mL/min, elution program: 0-5min, 10% A Linearly increase to 50%A; 5-8min, 50%A linearly increase to 90%A; 8-10min, 90%A linearly increase to 100%A, 100%A returns to the initial state after one minute. Use Agilent Technologies 1290Infinity II and 6545Q-TOF LC/MS instruments to detect the products extracted from the resulting reaction and conduct qualitative analysis. 5. The application of the acetaldehyde dehydrogenase gene or its mutant gene for synthesizing crocinic acid dialdehyde to crocinic acid in the biosynthetic pathway of gardenia crocin in claim 1 in plant genetic engineering. It is characterized by the application of the gardenia acetaldehyde dehydrogenase GjALDH2C3 gene in bacteria, fungi and higher plants to synthesize crocus acid by means of genetic engineering. 6. in the application described in claim 5, the introduction of exogenous gene into host cell needs a kind of plasmid that carries gardenia acetaldehyde dehydrogenase GjALDH2C3 gene, it is characterized in that: plasmid can be selected from pET series, pGEX series, pCold series etc. Prokaryotic expression vectors, yeast expression vectors such as pPIC9, pHIL-D2, pPIC3.5, and plant expression vectors such as PBI series and pCAMBIA series, and contain the nucleotide sequence or mutant sequence of claim 1.

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