CN113088498A - Screening identification and application of carotenoid-cleaved dioxygenase coding gene participating in formation of volatile components - Google Patents

Screening identification and application of carotenoid-cleaved dioxygenase coding gene participating in formation of volatile components Download PDF

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CN113088498A
CN113088498A CN202010017042.1A CN202010017042A CN113088498A CN 113088498 A CN113088498 A CN 113088498A CN 202010017042 A CN202010017042 A CN 202010017042A CN 113088498 A CN113088498 A CN 113088498A
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ljccd1b
beta
ionone
honeysuckle
carotenoid
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CN113088498B (en
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宋经元
浦香东
徐志超
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Institute of Medicinal Plant Development of CAMS and PUMC
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Abstract

The invention belongs to the technical field of genetic engineering, and relates to carotenoid-cleaved dioxygenase LjCCD1b closely related to formation of volatile components, and a coding gene and a function thereof. The invention discloses an open reading frame sequence of a carotenoid cleavage dioxygenase LjCCD1b gene and an amino acid sequence coded by the same. Based on honeysuckle genome and transcriptome data, the LjCCD1b gene is obtained by screening, and the catalytic function of LjCCD1b is identified in vitro by constructing pET32a-LjCCD1b prokaryotic expression vector and taking beta-carotene, 10' -Apo-beta-carotenal and lutein as substrates. The results show that LjCCD1b is capable of cleaving β -carotene and 10' -Apo- β -carotene aldehyde to form β -ionone; catalyzing the lutein to generate 3-hydroxy-beta-ionone and 3-hydroxy-alpha-ionone. The invention discloses an internal molecular mechanism for forming volatile components in honeysuckle, and lays a foundation for fragrance component regulation and molecular breeding research of honeysuckle.

Description

Screening identification and application of carotenoid-cleaved dioxygenase coding gene participating in formation of volatile components
Technical Field
The invention belongs to the technical field of plant genetic engineering, and particularly relates to a screening and function verification method and application of a carotenoid cracking dioxygenase gene participating in formation of volatile components.
Background
Volatile organic compounds released by plants mainly include terpenes, benzene/phenylpropanoids and fatty acid derivatives, these low molecular weight organic substances play an extremely important role in plant-plant, plant-insect and even interaction with the surrounding environment. The cracking of carotenoid compounds and the formation of volatile components of plant terpenes are closely related. Research shows that plant CCD1 family is involved in the cracking of carotenoid compounds, and the produced volatile C13 carbon-lowering sesquiterpene and its derivative (norrisoprenoids) play an important role in the formation of plant aroma and flavor substances. For example: the VvCCD1 of grapes is proved to participate in the formation of volatile substances in their fruit; zmcc 1 is involved in the formation of volatile substances in corn fruits; SlCCD1a and SlCCD1b are closely related to the aroma components of tomatoes.
Flos Lonicerae is dried flower bud or flower with initial blossom of Caprifoliaceae plant, is a traditional Chinese medicinal material, and can be used for treating fever, influenza, etc. Honeysuckle flowers bloom many times within one year, have strong fragrance, and are also cultivated as horticultural plants, and the volatile components of the honeysuckle flowers are mainly alcohols, aldehydes, terpenes, ketones, esters, such as beta-ionone, (E, E) -2, 4-nonadienal, (E, E) -2, 4-decadienal, (E) -2-nonenal, 1-octen-3-ol, geraniol, citronellol, linalool, (E) -2-hexenal, geranylacetone, n-hexanal, phenylacetaldehyde, n-octanol and the like. Therefore, the method lays a foundation for flower fragrance regulation and molecular breeding of the honeysuckle by disclosing an internal molecular mechanism formed by volatile components such as beta-ionone in the honeysuckle.
Disclosure of Invention
The invention aims to provide a carotenoid-cleaved dioxygenase encoding gene involved in formation of volatile components and a protein encoded by the gene.
The second purpose of the invention is to provide a screening method of key genes of carotenoid-cleaved dioxygenase in honeysuckle.
The third object of the present invention is to provide a method for verifying the function of the above-selected key enzyme gene LjCCD1 b.
The nucleotide sequence of the LjCCD1b gene provided by the invention is shown in SEQ ID No.1 or a mutant sequence thereof.
The amino acid sequence of the protein coded by the LjCCD1b gene provided by the invention is shown in SEQ ID No. 2.
The purpose of the invention can be realized by the following technical scheme: based on honeysuckle genome and transcriptome, screening key genes of carotenoid-cleaved dioxygenase, comprising the following steps:
1) and (3) identifying all CCD genes in the honeysuckle based on honeysuckle genome analysis, and constructing an evolutionary tree to analyze the evolutionary relationship between the honeysuckle CCD and other CCD species.
2) Analyzing the differential expression of the CCD gene based on the transcriptome data of the floral organs of the honeysuckle in different development periods, and further screening the carotenoid cracking dioxygenase gene participating in the formation of the volatile components of the honeysuckle.
The second technical scheme is as follows: the functional verification of a key enzyme LjCCD1b participating in the formation of honeysuckle volatile components. The function of the honeysuckle carotenoid-splitting dioxygenase LjCCD1b is identified in vitro by adopting an escherichia coli prokaryotic expression system and taking beta-carotene, 10' -Apo-beta-carotenal and lutein as substrates.
The invention discloses a screening and function identification method of a honeysuckle carotenoid-splitting dioxygenase LjCCD1b, wherein LjCCD1b is used for catalyzing beta-carotene and 10' -Apo-beta-carotene aldehyde to generate beta-ionone; the function of catalyzing xanthophyll to generate 3-hydroxy-beta-ionone and 3-hydroxy-alpha-ionone. Discloses an internal molecular mechanism for forming volatile components in the honeysuckle, and lays a foundation for the fragrance component regulation and control of the honeysuckle, molecular breeding and the like.
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FIG. 1: the LjCCD1b coding gene is differentially expressed in the floral organs of the honeysuckle at different development stages, and JB, WB, SF, GF and GWF respectively represent the bud stage, bud green stage, white stage, silver flower stage, golden flower stage and withering stage of the honeysuckle.
FIG. 2: gas-phase mass spectrometry is used for identifying the LjCCD1b catalytic product: a, catalyzing beta-carotene to generate beta-ionone; b, catalyzing 10' -Apo-beta-carotene aldehyde to generate beta-ionone; and C and D, catalyzing the lutein to generate 3-hydroxy-alpha-ionone and 3-hydroxy-beta-ionone respectively.
FIG. 3: honeysuckle LjCCD1b protein catalytic mechanism. LjCCD1b catalyzes beta-carotene and 10' -Apo-beta-carotene aldehyde to generate beta-ionone; catalyzing the lutein to generate 3-hydroxy-beta-ionone and 3-hydroxy-alpha-ionone.
Detailed description of the preferred embodiments
The present invention is described in detail below with reference to examples. The practice is for a better understanding of the invention but is not limiting. The experimental methods in the following implementation methods are all conventional methods, and the involved experimental reagents are all conventional biochemical reagents.
Example 1 screening and phylogenetic analysis based on Lonicera japonica genome and transcriptome data LjCCD1b Gene
1.1 Experimental methods
Based on honeysuckle genome data, an arabidopsis CCD protein sequence is extracted from a TAIR database, and LjCCDs of honeysuckle are identified by a BLASTP method. Protein sequences are compared by MUSCLE to construct an ML phylogenetic tree, and bootstrap selection is repeated 1000 times. RNA-Seq transcription group data of flower organs of honeysuckle at 6 different development stages are compared to honeysuckle genome by HiSAT2, and gene expression FPKM value is calculated by Cufflinks.
1.2 results and analysis
7 CCD genes are screened from honeysuckle genome and are mainly distributed in 4 subfamilies, wherein the 7 CCD genes comprise 3 LjCCD1s, 1 LjCCD4, 1 LjCCD7 and 2 LjCCD8 s. Further transcriptome data analysis result shows that the LjCCD1b gene is stably expressed in bud stage, white stage, silver flower stage, golden flower stage and withering stage of honeysuckle, as shown in FIG. 1. Therefore, it is presumed to be a key enzyme gene involved in the formation of honeysuckle aroma.
Example 2 honeysuckle LjCCD1b function verification method
2.1 Experimental methods
The LjCCD1b gene is cloned into pET32a expression vector through KpnI and EcoRI restriction enzyme cutting sites to construct pET32a-LjCCD1b prokaryotic expression vector, and the vector is further transformed into BL21(DE3) strain. Culturing the bacterial liquid at 37 ℃ and 200rpm overnight; transferring into LB liquid culture medium (containing 50 ug/mL ampicillin) 2-50 mL of overnight-cultured bacterial liquid, culturing at 37 deg.C and 200rpm for 2-3h to obtain bacterial liquid OD 600-0.4-0.5; adding IPTG with final concentration of 0.3mM, and inducing at 16 deg.C and 130rpm for 24 h; centrifuging to obtain thallus precipitate, adding 10mM phosphate buffer solution, and ultrasonically crushing to obtain crude enzyme solution.
The beta-carotene, 10' -Apo-beta-carotene aldehyde and lutein are respectively used as catalytic substrates, and the crude enzyme liquid is subjected to function verification (200 mu L) in vitro according to the following reaction system: 100mM Hepes (pH 8.0), 100. mu.L of crude enzyme solution, 1mM Fe 2+100. mu.M beta-carotene (10' -Apo-beta-carotenal or lutein). After the reaction solution reacted at 30 ℃ for 3 hours, the volatile components generated by the reaction were extracted by solid phase microextraction using SPME (65 μm PDMS/DVB, Supelco, USA) cartridge.
Example 3 gas phase-mass spectrometry coupled identification of LjCCD1b catalytic product
3.1 Experimental methods
The catalytic products were detected by an Agilent Technologies 7890 GC and 5977A MS gas chromatography-mass spectrometer for qualitative analysis. When detecting beta-ionone: the flow rate of the carrier gas was 1.0mL min-1Keeping the column temperature at 50 ℃ for 5 min; at 25 ℃ for min-1The speed of (2) is increased to 340 ℃; when 3-hydroxy-beta-ionone and 3-hydroxy-alpha-ionone are detected: the flow rate of the carrier gas is 1.5mLmin-1Maintaining the column temperature at 40 deg.C for 3 min; at 5 ℃ for min-1The rate of (2) increased to 250 ℃. The electron impact energy was 70 eV. Data analysis used MassHunter (vcrison B.07.00).
3.2 results and analysis
LjCCD1b can catalyze beta-carotene and 10' -Apo-beta-carotene aldehyde to generate beta-ionone; the function of catalyzing xanthophyll to generate 3-hydroxy-beta-ionone and 3-hydroxy-alpha-ionone is shown in figure 2 and figure 3.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the technical principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Figure ISA0000199694690000011
Figure ISA0000199694690000021
Figure ISA0000199694690000031
Figure ISA0000199694690000041
Figure ISA0000199694690000051
Figure ISA0000199694690000061
Figure ISA0000199694690000071
Figure ISA0000199694690000081

Claims (4)

1. A carotenoid-cleaved dioxygenase LjCCD1b encoding gene participating in volatile component formation, the nucleotide sequence of which is shown in SEQ ID No. 1.
2. The carotenoid cleavage dioxygenase LjCCD1b according to claim 1, wherein the amino acid residue sequence of the LjCCD1b protein is shown as SEQ ID No. 2.
3. Has protein with similar functions obtained by replacing, deleting or adding one or more amino acids in the amino acid sequence shown in SEQ ID No. 2.
4. Use of the coding genes and proteins described in claims 1, 2 and 3 in genetic engineering. Characterized by the ability to catalytically cleave, but not limited to, carotenoids such as beta-carotene and 10' -Apo-beta-carotenal to beta-ionone; catalyzing the lutein to generate 3-hydroxy-beta-ionone and 3-hydroxy-alpha-ionone.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116042663A (en) * 2022-07-06 2023-05-02 中国科学院昆明植物研究所 Dendrobium candidum beta-ionone synthesis key enzyme gene DoCCD1 and cloning method and application thereof

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CN105368850A (en) * 2015-11-25 2016-03-02 天津大学 Lycium carotenoid cleavage dioxygenase enzyme gene with function of generating beta-ionone aroma substances and application of lycium carotenoid cleavage dioxygenase enzyme gene with function of generating beta-ionone aroma substances
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CN105368850A (en) * 2015-11-25 2016-03-02 天津大学 Lycium carotenoid cleavage dioxygenase enzyme gene with function of generating beta-ionone aroma substances and application of lycium carotenoid cleavage dioxygenase enzyme gene with function of generating beta-ionone aroma substances
CN112239764A (en) * 2019-07-16 2021-01-19 中国医学科学院药用植物研究所 Screening identification and application of carotenoid-cleaved dioxygenase coding gene participating in honeysuckle flower color change

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116042663A (en) * 2022-07-06 2023-05-02 中国科学院昆明植物研究所 Dendrobium candidum beta-ionone synthesis key enzyme gene DoCCD1 and cloning method and application thereof

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