CN112239764B - Screening identification and application of carotenoid-cleaved dioxygenase coding gene participating in honeysuckle flower color change - Google Patents

Abstract

The invention relates to carotenoid-cleaved dioxygenase LjCCD4 closely related to honeysuckle flower color and a coding gene and a function thereof. The present invention belongs to the field of gene engineering technology. The invention discloses an open reading frame sequence of a gene LjCCD4 of a honeysuckle carotenoid-lyase dioxygenase and an amino acid sequence coded by the same. The LjCCD4 gene is obtained by screening based on honeysuckle genome and transcriptome data, and a catalytic mechanism of LjCCD4 is identified in vitro by constructing pET32a-LjCCD4 prokaryotic expression vector and taking beta-carotene and lutein as substrates. LjCCD4 can catalyze beta-carotene and lutein to generate 10 ' -Apo-beta-carotenal, beta-ionone and 3-hydroxy-10 ' -Apo-beta-carotenal, 3-OH-10 ' -Apo-alpha-carotenal, 3-hydroxy-beta-ionone and 3-hydroxy-alpha-ionone respectively. The invention discloses an internal molecular mechanism of carotenoid pigment degradation and flower color change in honeysuckle, and lays a foundation for flowering phase regulation, molecular breeding and the like of the honeysuckle.

Description

Screening, identification and application of a carotenoid-cleaving dioxygenase-encoding gene involved in honeysuckle flower color change

technical field

The invention belongs to the technical field of plant genetic engineering, and in particular relates to a method for screening, functional verification and application of a carotenoid-cleaving dioxygenase gene involved in the change of honeysuckle flower color.

Background technique

Plant natural products are important sources of medicines, and different types of natural products also give plants various colors, such as anthocyanins, carotenoids, etc. The formation of plant flower color is closely related to its pollination, breeding and development of natural products, and its molecular mechanism is an international research hotspot. The synthesis and degradation of carotenoids are closely related to the yellow flowers displayed by plants. Studies have shown that the white and yellow color of chrysanthemum is affected by the carotenoid cleavage dioxygenase CmCCD4a in the degradation pathway; CitCCD4b1 in citrus acts on zeaxanthin and β-cryptoxanthin to produce β-orange pigments. The main C30 apocarotenoid, which gives citrus its bright orange-red color. Therefore, by analyzing the molecular change mechanism of plant flower color, it can lay a foundation for the use of molecular means to study its flowering period regulation, flower color and molecular breeding.

Honeysuckle is a dry bud or flower with early blooming of the honeysuckle plant. It can be used to treat fever, flu, etc. It is a traditional Chinese medicinal material. Honeysuckle can bloom many times within a year, and the flower color changes from green to white, and then from white to yellow, and the change is particularly obvious. During this period, the purpose of this study was to reveal the intrinsic molecular mechanism of honeysuckle flower color change, and to provide preconditions for the flowering regulation and molecular breeding of honeysuckle.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a carotenoid cleavage dioxygenase encoding gene and its encoded protein involved in the change of honeysuckle flower color.

Another object of the present invention is to provide a screening method for the key gene of carotenoid cleavage dioxygenase in honeysuckle.

The third object of the present invention is to provide a method for functional verification of the key enzyme gene LjCCD4 screened out above.

The nucleotide sequence of the LjCCD4 gene provided by the present invention is shown in SEQ ID No. 1, or a mutant sequence thereof.

The amino acid sequence of the protein encoded by the LjCCD4 gene provided by the present invention is shown in SEQ ID No.2.

The object of the present invention can be achieved through the following technical solutions. Technical solution 1: Screening of key genes of honeysuckle carotenoid cleavage dioxygenase based on transcriptome, the steps are as follows:

1) Based on the honeysuckle genome, identify the members of the honeysuckle CCD gene family, construct an evolutionary tree to analyze the evolutionary relationship between honeysuckle and other species of CCD, and screen the honeysuckle carotenoid cleavage dioxygenase amino acid sequence.

2) Based on the transcriptome data of the flower organs of honeysuckle in different developmental stages, the differential gene expression was analyzed, and the key genes of carotenoid cleavage dioxygenase of honeysuckle were further screened.

Technical scheme 2: functional verification of the key enzyme LjCCD4 involved in the change of honeysuckle flower color. Using a prokaryotic expression system, with β-carotene and lutein as substrates, the function of honeysuckle carotenoid-cleaving dioxygenase LjCCD4 was identified in vitro.

The invention discloses the screening and functional identification of the honeysuckle carotenoid-cleaving dioxygenase LjCCD4. LjCCD4 has the ability to catalyze β-carotene and lutein to generate 10'-Apo-β-carotene aldehyde, β-ionone and 3-carotene, respectively. Functions of hydroxy-10'-Apo-β-carotene aldehyde, 3-OH-10'-Apo-α-carotene aldehyde, 3-hydroxy-β-ionone, 3-hydroxy-α-ionone, revealed The intrinsic molecular mechanism of honeysuckle flower color change lays the foundation for honeysuckle flowering regulation and molecular breeding.

Description of drawings

Figure 1: Identification and evolutionary analysis of carotenoid-cleaving dioxygenase LjCCD4 family members based on the honeysuckle genome;

Figure 2: Differential expression of LjCCD4 encoding gene in flower organs of honeysuckle at different developmental stages;

Figure 3: The catalytic mechanism of LjCCD4 protein related to flower color change of honeysuckle. A: LjCCD4 can catalyze β-carotene to produce 10'-Apo-β-carotene aldehyde, LjCCD4 can catalyze lutein to produce 3-hydroxy-10'-Apo-β-carotene aldehyde and 3-OH-10'- Apo-α-carotene aldehyde; B: putative carotenoid cleavage pathway;

Figure 4: Identification of LjCCD4 catalytic substrates and products by LC-MS: β-carotene, lutein, 10'-Apo-β-carotene aldehyde, 3-hydroxy-10'-Apo-β-carotene aldehyde and 3-OH-10'-Apo-α-carotene aldehyde.

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 LjCCD4 gene based on honeysuckle genome and transcriptome data

1.1 Experimental method

The flower organs of six different developmental stages were dried at 45°C, and ground into fine powder with a Mixer Mill MM400. Weigh 0.1g of the powder and add 4mL of methanol, then add 200μL of 60% potassium hydroxide, and heat at 60°C. After cooling for 20 minutes, extract with ethyl acetate:petroleum ether=1:1. After the upper extract was concentrated to dryness, it was redissolved in methanol and detected by UV spectrophotometer and liquid phase. Each sample was repeated three times.

Based on the honeysuckle genome data, the CCD protein sequence of Arabidopsis thaliana was extracted from the TAIR database, and the honeysuckle LjCCDs were identified by BLASTP. The protein sequences were aligned by MUSCLE to construct the NJ phylogenetic tree, and the bootstrap selection was repeated 1000 times. Using HiSAT2, the RNA-Seq transcriptome data of six flower organs of different developmental stages of honeysuckle were compared to the honeysuckle genome, and Cufflinks was used to calculate the FPKM value of gene expression.

1.2 Results and Analysis

The analysis of the total carotenoid content showed that carotenoids accumulated a lot when the flower color changed from white to yellow, and the liquid phase detection results showed that β-carotene and lutein were the two main carotenoids.

Based on the honeysuckle genome data, 7 LjCCD4s were screened, mainly distributed in 4 subfamilies, including 3 LjCCD1s, 1 LjCCD4, 1 LjCCD7 and 2 LjCCD8s, as shown in Figure 1. Through transcriptome data analysis, it was found that the expression of LjCCD4 gene was negatively correlated with the accumulation of total carotenoid content in honeysuckle at different flowering stages. As the flower color of honeysuckle changed from white to yellow, LjCCD4 gene expression showed a significant downward trend, as shown in Figure 2. Therefore, it is speculated that it is a key enzyme gene involved in the change of honeysuckle flower color.

Example 2 Functional verification of the key enzyme LjCCD4 involved in the change of honeysuckle flower color

2.1 Experimental method

The LjCCD4 encoding gene was cloned into the pET32a vector to form the pET32a-LjCCD4 recombinant expression vector, and the vector was transformed into the BL21(DE3) E. coli expression strain. Take 2mL of the overnight cultured bacterial solution and add it to 50mL LB liquid culture concentrate (containing 50μg/mL ampicillin-resistant medium), 37°C, 200rpm, activate for 2-3h, and when its OD600 reaches 0.4-0.5, add the final concentration of 0.3 Induction with mM IPTG, 16°C, 130rpm. After 24 hours of induction, the cell pellet was collected by centrifugation, and the supernatant obtained after ultrasonication was the crude enzyme solution, which could be used for in vitro catalytic reaction.

BEH C18 column(1.7μm，100×2.1mm)，柱温：30℃。色谱条件：UV440nm，流动相：检测叶黄素及其裂解产物条件：A：0.1％甲酸乙腈，B：0.1％甲酸水，流速：0.3mL/min，洗脱程序：0-5min，10％A增长至50％A；5-8min，50％A增长至90％A；8-10min，90％A增长至100％A，100％A持续一分钟后回到起始状态；检测β-胡萝卜素及其裂解产物条件：A：0.1％甲酸乙腈，B：0.1％甲酸水，流速：0.3mL/min，洗脱程序：0-5min，10％A增长至50％A；5-8min，50％A增长至90％A；8-10min，90％A增长至100％A，维持20min后回到起始状态。The crude enzyme solution was tested in vitro according to the following reaction system (50 μL): 100 mM Hepes (pH 8.0), 100 μL crude enzyme solution, 1 mM Fe ²⁺ , 100 μM β-carotene or lutein. 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: detection of lutein and its cleavage products Conditions: A: 0.1% formic acid acetonitrile, B: 0.1% formic acid water, flow rate: 0.3 mL/min, elution program: 0-5min, 10% A Increase to 50%A; 5-8min, 50%A increases to 90%A; 8-10min, 90%A increases to 100%A, 100%A lasts for one minute and then returns to the initial state; Beta-carotene is detected and its cleavage product conditions: A: 0.1% formic acid acetonitrile, B: 0.1% formic acid water, flow rate: 0.3mL/min, elution program: 0-5min, 10%A increases to 50%A; 5-8min, 50% A increases to 90%A; 8-10min, 90%A increases to 100%A, and returns to the initial state after maintaining for 20min.

2.2 Results and Analysis

The in vitro catalysis results show that LjCCD4 can catalyze β-carotene to generate a chromatographic peak with the same chromatographic behavior (such as retention time, spectrum, etc.) as 10'-Apo-β-carotene aldehyde, as shown in Figure 3; When flavin is the substrate, two new compounds can be produced, and the qualitative analysis of the catalytic product requires liquid mass detection.

Example 3 Identification of LjCCD4 catalytic substrates and products by liquid phase-mass spectrometry

3.1 Experimental method

BEH C18 column(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：检测叶黄素及其裂解产物条件：A：0.1％甲酸乙腈，B：0.1％甲酸水，流速：0.3mL/min，洗脱程序：0-5min，10％A增长至50％A；5-8min，50％A增长至90％A；8-10min，90％A增长至100％A，100％A持续一分钟后回到起始状态；检测β-胡萝卜素及其裂解产物条件：A：0.1％甲酸乙腈，B：0.1％甲酸水，流速：0.3mL/min，洗脱程序：0-5min，10％A增长至50％A；5-8min，50％A增长至90％A；8-10min，90％A增长至100％A，维持20min后回到起始状态。6545 Q-TOF质谱参数为：干燥气体温度为350℃，流速5.0L/min；喷雾器为40psig；VCap为4000V。The products extracted from the resulting reaction were detected by Agilent Technologies 1290 Infinity II and 6545 Q-TOF LC/MS for qualitative analysis. Injection volume 10μL, chromatographic column: Waters Acquity

BEH C18 column (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 procedure: detection of lutein and its cleavage products Conditions: A: 0.1% formic acid in acetonitrile, B: 0.1% formic acid in water, flow rate: 0.3mL/min, elution program: 0-5min, 10%A increases to 50%A; 5-8min, 50%A increases to 90 %A; 8-10min, 90%A increased to 100%A, 100%A continued for one minute and then returned to the initial state; β-carotene and its cleavage products were detected Condition: A: 0.1% formic acid acetonitrile, B: 0.1 % formic acid water, flow rate: 0.3mL/min, elution program: 0-5min, 10%A increases to 50%A; 5-8min, 50%A increases to 90%A; 8-10min, 90%A increases to 100% A, return to the initial state after maintaining for 20 minutes. 6545 Q-TOF mass spectrometry parameters are: drying gas temperature is 350°C, flow rate is 5.0L/min; nebulizer is 40psig; VCap is 4000V.

3.2 Results and Analysis

The results of liquid and mass spectrometry showed that: LjCCD4 could catalyze β-carotene to generate 10'-Apo-β-carotene aldehyde and β-ionone; catalyze lutein to generate 3-hydroxy-10'-Apo-β-carotene aldehyde, The functions of 3-OH-10'-Apo-α-carotene aldehyde, 3-hydroxy-β-ionone, and 3-hydroxy-α-ionone are shown in Figure 3 and Figure 4.

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 (3)

1. A carotenoid-cleaved dioxygenase LjCCD4 encoding gene participating in honeysuckle flower color formation has a nucleotide sequence shown in SEQ ID No. 1.

2. The carotenoid cleavage dioxygenase LjCCD4 as claimed in claim 1, wherein the amino acid residue sequence of LjCCD4 is shown in SEQ ID No. 2.

3. Use of the carotenoid-cleaving dioxygenase LjCCD4 as claimed in claim 1 or 2 in plant genetic engineering, characterized in that the carotenoid-cleaving dioxygenase LjCCD4 is capable of catalyzing the production of 10' -Apo- β -carotenal, β -ionone from β -carotene; catalyzing the lutein to generate 3-hydroxy-10 '-Apo-beta-carotenal, 3-OH-10' -Apo-alpha-carotenal, 3-hydroxy-beta-ionone and 3-hydroxy-alpha-ionone.

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