CN113151368A

CN113151368A - Coenzyme Q synthesis pathway benzene ring 6-position hydroxylase and application thereof

Info

Publication number: CN113151368A
Application number: CN202110291291.4A
Authority: CN
Inventors: 许晶晶; 杨蕾; 范航; 陈晓亚; 李辰意
Original assignee: SHANGHAI CHENSHAN BOTANICAL GARDEN
Current assignee: SHANGHAI CHENSHAN BOTANICAL GARDEN
Priority date: 2021-03-18
Filing date: 2021-03-18
Publication date: 2021-07-23
Anticipated expiration: 2041-03-18
Also published as: CN113151368B

Abstract

The invention obtains the benzene ring 6-position hydroxylase CoqF of arabidopsis thaliana through research. The activity is further verified by complementing 6-site hydroxylase gene mutant strains of saccharomyces cerevisiae, and the synthesis of coenzyme Q in plants is proved by analyzing arabidopsis RNAi transgenic plants. Therefore, the benzene ring 6-position hydroxylase CoqF provided by the invention can be used for improving the content of coenzyme Q, and has a great application value.

Description

Coenzyme Q synthesis pathway benzene ring 6-position hydroxylase and application thereof

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a benzene ring 6-position hydroxylase in a coenzyme Q synthesis way.

Background

Coenzyme Q (CoQ), also known as ubiquinone (ubiquinone), is a class of terpene benzoquinones that are present in all eukaryotes and in a subset of bacteria (α, β, γ proteobacterium). CoQ consists of a quinone ring parent nucleus and a polyisoprene side chain (FIG. 1), with different numbers of side chain isoprene units in different organisms, humanClass is 10 isoprene units, Synthesis of CoQ₁₀Coli synthesis of CoQ₈Synthesis of CoQ by Saccharomyces cerevisiae₆Synthesis of CoQ from Arabidopsis thaliana₉。

CoQ has an important physiological function, and is located centrally in the electron transport chain (respiratory chain), and transfers electrons from NADH-Q reductase (complex I) and succinate-Q reductase (complex II) to cytochrome reductase (complex III), thereby promoting oxidative phosphorylation and the transfer of electrons, thereby generating ATP. CoQ has other various functions in cells, for example, CoQ is found in the membrane structure of almost all eukaryotic cells, and can protect the stability of the biological membrane structure as a fat-soluble antioxidant.

The lack of CoQ in humans can lead to very serious diseases. The mutation of CoQ synthetic pathway gene can cause primary CoQ deficiency, affect energy-consuming organs such as brain, heart, kidney, skeletal muscle and the like, and cause a series of diseases such as encephalopathy, epilepsy, cerebellar ataxia, hypertrophic cardiomyopathy, nephrotic syndrome, myopathy and the like. Factors such as statins, age increase, other diseases, etc., contribute to secondary deficiencies in CoQ. Exogenous supplementation of CoQ may improve the symptoms of CoQ deficiency to some extent.

However, the biosynthesis of CoQ is a complex process and the synthetic pathway has not been completely resolved to date. It is known that enzymes of the CoQ synthetic pathway are mainly identified in Escherichia coli (Escherichia coli) or Saccharomyces cerevisiae (Saccharomyces cerevisiae). Coli are named Ubi (UbiA-J and UbiX) and yeast are named Coq (Coq 1-11). The biosynthetic pathway of CoQ includes four links: the specific routes of the synthesis of the benzene ring, the synthesis of the side chain, the connection of the side chain and the benzene ring and the modification of the benzene ring are not completely the same in different organisms.

The precursor of the benzene ring is usually p-hydroxybenzoic acid. In E.coli, chorismic acid is converted to p-hydroxybenzoic acid by the action of chorismic acid lyase Ubic. Human, yeast and plant p-hydroxybenzoic acid is from the tyrosine pathway, which currently resolves only the first and last steps. In addition, the plant para-hydroxybenzoic acid is also derived in large part from the phenylpropanoid pathway.

Precursors of the polyisoprene side chain are isopentenyl diphosphate (IPP) and dimethylallyl Diphosphate (DMAPP), which are supplied by the mevalonate pathway (MVA pathway) in eukaryotes and 2-C-methyl-D-erythritol 4-phosphate pathway (2-C-methyl-D-erythritol 4-phosphate pathway, MEP pathway) in prokaryotes. Polyprenyl diphosphate synthase (PPS) produces polyisoprene pyrophosphate by condensation reaction of IPP with a propenyl acceptor at the 1' -4 position. The synthesis of eukaryotic CoQ proceeds from this reaction all on the matrix side of the inner mitochondrial membrane.

The side chain of polyisoprene pyrophosphate is condensed with p-hydroxybenzoic acid under the action of 4-hydroxyphenyl pyrophosphate transferase (PPT) to form 3-polyprenyl-4-hydroxybenzoic acid (3-polyprenyl-4-hydroxybenzoate), and the enzymes in E.coli and yeast are UbiA and Coq2, respectively.

In the final process of CoQ synthesis, a benzene ring needs to be subjected to a series of modifications, including one-step decarboxylation, three-time hydroxylation modification and three-time methylation modification. In eukaryotes, 3-polyprenyl-4-hydroxybenzoic acid first introduces a hydroxyl group at the 5-position of the benzene ring, then converts to a methoxy group, and then decarboxylates by unknown enzymes. The subsequent reaction steps are consistent between eukaryotes and prokaryotes, and include introduction of a hydroxyl group at the 1-position of the benzene ring, followed by introduction of a methyl group at the 2-position, and finally introduction of a hydroxyl group at the 6-position, and conversion of the hydroxyl group at the 6-position to a methoxy group, thereby completing the synthesis of CoQ (fig. 2).

The enzymes of the CoQ synthesis process are generally conserved across species, with the exception of the benzene ring 6 hydroxylase. Three enzymes are known to have hydroxylation activity at the 6-position of the benzene ring, namely UbiF, UbiM and Coq 7. UbiF belongs to the class a Flavinoid Monooxygenase (FMO), was first found in e.coli, and is also present in some other gamma proteobacteria. Ubim is currently found only in a subset of proteobacteria, not a specific 6-hydroxylase, but also has 1-and 5-position hydroxylating activity, and is also a retinoid monooxygenase. Coq7 is a diiron hydroxylase (diiron hydroxylase) containing conserved sequences that bind iron ligands and is known to occur in yeast, humans, nematodes and some proteus species.

There are no homologous proteins of the above three enzymes in plants, and it has not been known which enzyme catalyzes the 6-position hydroxylation reaction of the benzene ring, and a specific 6-position hydroxylase may exist in plants.

Disclosure of Invention

The invention discovers a novel coenzyme Q synthesis pathway benzene ring 6-position hydroxylase CoqF in plant Arabidopsis thaliana, participates in the synthesis of coenzyme Q in plants, and no comment is made in a gene database to realize the function. The invention discovers that the new coenzyme Q synthesis pathway benzene ring 6-position hydroxylase CoqF is widely present in plants, green algae, unequal flagellates, vesicular worms, foraminous worms, dinoflagellates, crypthecodinium and other eukaryotes.

The invention provides application of a benzene ring 6-position hydroxylase gene in generation of coenzyme Q in plants, bacteria, fungi and algae, and is characterized in that the benzene ring 6-position hydroxylase gene is derived from plants, green algae, SAR super group, algae of vervain and cryptophyte.

Preferably, the hydroxylase gene at the 6 th position of the benzene ring is from arabidopsis thaliana, rice, tomato, liverwort, freshwater microalgae (Chlorella sorokiniana), marine microalgae (Ostreococcus tauri), Saprolegnia parasitica (Saprolegnia parasitica), duckweed thorny beetle (styrylonychia lemnae); more preferably, its GenBank accession numbers are NP _173844.2, XP _015620261.1, XP _004239375.1, PTQ40024.1, PRW56607.1, XP _003079408.2, XP _012203255.1, CDW 87725.1.

Further, the hydroxylase gene at the 6-position of the benzene ring is:

(1) a nucleotide sequence of SEQ ID No.1 or degenerate sequence thereof;

(2) the sequence obtained by adding, deleting and replacing one or more nucleotides in SEQ ID No.1, and the sequence codes for the protein with the same function as SEQ ID No. 2.

In one embodiment, the application is to produce coenzyme Q by fermentation using a microorganism into which the gene for the hydroxylase at the 6-position of the benzene ring is introduced and which is capable of expressing, preferably the microorganism is Escherichia coli, yeast.

In another embodiment, the use is to increase the level of coenzyme Q in transgenic plants into which the benzene ring 6 hydroxylase gene has been introduced and expressed by transgenic means. Preferably, the plant is arabidopsis thaliana.

The invention also provides application of the benzene ring 6-position hydroxylase in generating coenzyme Q, wherein the benzene ring 6-position hydroxylase is coded by a benzene ring 6-position hydroxylase gene derived from plants, green algae, SAR super groups, vervain and cryptophyte. Preferably from Arabidopsis thaliana, rice, tomato, liverwort, freshwater microalgae (Chlorella sorokiniana), marine microalgae (Ostreococcus tauri), Saprolegnia parasitica (Saprolegnia parasitica), Spirodela spinosa (Stylonychia lemnae), more preferably with GenBank accession numbers NP-173844.2, XP-015620261.1, XP-004239375.1, PTQ40024.1, PRW56607.1, XP-003079408.2, XP-012203255.1, CDW 87725.1.

Further preferably, the amino acid sequence of the benzene ring 6-position hydroxylase is shown as SEQ ID No. 2.

The research of the invention finds that the gene with the activity of the benzene ring 6-position hydroxylase CoqF from plants can be used for improving the content of coenzyme Q, for example, the coenzyme Q is produced by microbial fermentation, and provides a foundation for improving the content of the coenzyme Q in organisms. The invention enriches the diversity of the synthetic routes of the eukaryotic coenzyme Q and lays a foundation for designing medicines, insecticides, pesticides and the like of the targeted coenzyme Q route. Therefore, the invention has great application value.

Drawings

FIG. 1 Structure of coenzyme Q.

FIG. 2 eukaryotic CoQ synthesis pathway.

FIG. 3 knock-out of E.coli UbiF gene. Wherein, (A) a design drawing for knocking out the UbiF gene; (B) the wild type and mutant strains were PCR-tested, primers F/R are shown in A, and the band of the mutant strain Δ ubiF was smaller than that of the wild type.

FIG. 4AtCoqF complemented mutant E.coli.DELTA.ubiF. Wherein (A) from top to bottom, pTrc99a empty vector is used for transforming wild strain, pTrc99a empty vector and pTrc99a containing AtCoqF and AtCoq6 are used for transforming delta ubiF mutant strain respectively.Culturing positive transformant to bacterial liquid OD₆₀₀The cells were diluted in a 10-fold gradient at 0.5, inoculated in M9 medium containing 0.4% glucose or 0.4% succinate as a carbon source, and cultured overnight at 37 ℃. (B) CoQ₈LC-MS (MRM mode) detection profile of (1). (C) Quantitative determination of CoQ₈Data are mean ± standard deviation of three biological replicates.

FIG. 5AtCoqF complemented the Saccharomyces cerevisiae Δ coq7 mutant strain. Wherein (A) the wild type strains are transferred by pRS426 and pRS423 empty vectors, the pRS426 and pRS423 empty vectors, pRS426-AtCoqF and pRS423-ScCoq8, and the mutant strain delta coq7 is transferred by pRS 426-ScCoq 8 from top to bottom. Culturing positive transformant to bacterial liquid OD₆₀₀The cells were diluted in a gradient of 10 times to 1, and inoculated on SD-Ura-His medium containing glucose or glycerol and ethanol as carbon sources, and cultured at 30 ℃. (B) CoQ₆LC-MS (MRM mode) detection profile of (1). (C) Quantitative determination of CoQ₆Data are mean ± standard deviation of three biological replicates.

FIG. 6 detection of CoQ content in RNAi transgenic plants of AtCoqF. Wherein (A) qRT-PCR detects the relative expression of AtCoqF genes in three transgenic strains. (B) The plant aerial parts were assayed for coenzyme Q9 content for 4 weeks and the data were mean. + -. standard deviation of four biological replicates.

FIG. 7 CoqF Gene complementation of E.coli.DELTA.ubiF mutants by different species CoqF to generate CoQ₈And (4) data.

Detailed Description

The invention is further described below by means of specific embodiments in order to facilitate a better understanding of the invention, without however constituting a limitation thereto. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1: cloning of Arabidopsis thaliana CoqF Gene and complementation of E.coli Delta ubiF mutant Strain

The gene of the plant coenzyme Q synthetic pathway has the characteristic of co-expression, and a plurality of candidate hydroxylase genes are screened through the co-expression analysis with the known gene of the Arabidopsis coenzyme Q pathway. The activity of these candidate genes was examined by complementing the Escherichia coli 6-hydroxylase gene UbiF mutant. It was found that the transfer of one of the candidate genes (At1g24340, NP-173844.2, GeneID:839050, nucleotide sequence as SEQ ID NO.1, amino acid sequence as SEQ ID NO.2) could complement the growth of the Ubif mutant strain, and thus it was named CoqF. CoqF is a gene of Arabidopsis with unknown function. The specific experimental procedure is as follows.

To obtain the E.coli Δ ubiF mutant, the UbiF gene of E.coli MG1655 strain was knocked out by CRISPR/Cas9 gene editing technique. Designing a knockout target spot in the UbiF gene, designing homologous repair templates at positions which are about 150bp away from the two sides of the target spot, deleting about 300bp of the repaired coding region of the UbiF gene, and carrying out PCR identification (AGTATGAGCGTTATCAGC, AGCGGATGGATGGTATGC) to obtain a positive strain. As shown in FIG. 3, A is a design drawing of the knock-out UbiF gene, in which primer F/R is as shown in the drawing, and the band of the.DELTA.ubiF mutant strain is smaller than that of the wild type. B is an electrophoresis picture of PCR detection of wild type and mutant strains. The specific experimental procedure is as follows.

Plasmid pCas was transformed into MG 1655. Positive clones were picked, electroporation competent cells were prepared, and electroporation into plasmid pTargetT containing the homologous fragment. Construction of pTargetT: a fragment containing N20 and sgRNA (CGGACTAGTGCAGACTGGAAACGTGGGAGGTTTTAGAGCTAGAAATAGC, CTCAAAAAAAGCACCGACTCGG), a homologous fragment 1(GGATTACCCTGTTATCCCTATTGAGCAGGTGTTCAGC, CGAATCACTGGTCAATCATCCTGTTAG) and a homologous fragment 2(TGATTGACCAGTGATTCGCGCGAAGCT, CGCGTCGACACGGGAAATGCTTCGCGA) of UbiF were amplified respectively, and the three-segment PCR product was subjected to overlap amplification, digested with SpeI/SalI, and cloned into a plasmid pCB003, thereby obtaining pTargetT.

The UbiF mutant E.coli is reported to grow on a medium containing succinic acid as a carbon source and on a medium containing glucose as a carbon source, but at a lower growth rate than the wild type. The Δ ubiF mutant strain transferred into empty vector was observed to grow slowly on glucose medium and not on succinic acid medium, consistent with literature reports.

The Arabidopsis thaliana CoqF gene (i.e., AtCoqF) was cloned into the E.coli expression vector pTrc99a using primer CGGGGTACCATGGCGATTCTAGGGCTT, GCTCTAGATCATTGTTTCCCTAGTATGAT, KpnI/XbaI and transformed into the above-described mutant strain (. DELTA.ubiF). The resulting positive transformants were inoculated on M9 medium (A in FIG. 4) with 0.4% glucose or 0.4% succinate as a carbon source, respectively.

In addition, the known Arabidopsis thaliana ATCoq6, which is a benzene ring 5-hydroxylase, was used as a control (primer CGGAATTCATGTCTAAGGACAGGGTG, CGGGATCCTCAAGAAAATAGCGGCAAAG, EcoRI/BamHI cut), cloned into the vector pTrc99a, and transformed into a.DELTA.ubiF mutant strain, which failed to recover growth, indicating that AtCoq6 had no benzene ring 6-hydroxylase activity.

Several of the above transformants were cultured overnight in liquid LB medium (containing 0.4% glucose), and CoQ was extracted and detected by LC-MS. The results show that the wild type strains synthesize CoQ₈The mutant strain of Δ ubiF hardly contains CoQ₈The mutant strain transferred into AtCoqF can synthesize CoQ₈And there was no change after the transfer to AtCoq6 (B in FIG. 4). The results of the quantitative determination revealed that the mutant strain into which AtCoqF was transferred had a CoQ content similar to that of the wild type (C in FIG. 4). The above results indicate that AtCoqF has a benzene ring 6-hydroxylase activity.

In this example, the CoQ of Escherichia coli was measured₈The detection method of (3) is as follows. Culturing overnight in 10mL LB liquid culture medium (containing 0.4% glucose), centrifuging to collect thallus, weighing wet weight, adding isopropanol proportionally, ultrasonic extracting at low temperature in dark place for 1h, and then injecting the filtered sample into liquid phase-mass spectrometry for analysis. The liquid phase conditions were as follows: HPLC-DAD-MS (1260Infinity II-6460, Agilent) is matched with a ZORBAX Eclipse XDB C18 column (3.5 mu m, 2.1 multiplied by 50mm), isopropanol is taken as a mobile phase (A), and (B) 87.5% acetonitrile water solution (containing 10mmol/L ammonium acetate) is taken as a mobile phase, and the conditions of the mobile phase are 0-6min and 65-15% B; the flow rate was 0.4 mL/min. The mass spectrometry conditions were as follows: ESI ion source is adopted, the capillary voltage is set to be 4.0kV, the atomizer pressure is set to be 35psi, high-purity nitrogen is used as carrier gas, and the flow rate is 11L/min; selecting positive ion mode pair CoQ₈(parent ion m/z 727.6, daughter ion m/z 197.1) under the conditions of a fragmentation voltage of 182V and a collision energy of 32V.

Example 2: arabidopsis thaliana CoqF gene complementation Saccharomyces cerevisiae delta coq7 mutant strain

The arabidopsis CoqF gene (amplification primer ACGAGATGTAAGAGCACAATGGCGATTCTAGGGCTT, ATCGAATTCCTGCAGCCCTCATTGTTTCCCTAGTAT) was fused to the N-terminus of the mitochondrial signal peptide (amplification primer TTCGATATGGGATCCCCCATGTTGTTAAGATCTAGA, TGTGCTCTTACATCTCGT) of yeast Coq3 and cloned into the yeast expression vector pRS426, expression of which was regulated by the promoter (TTGCGGCCGCGATCCGGGTGTTCGG, CGCGGATCCCATATCGAACGATATCTT) and terminator (GACGTCGACAATACTTCCCCGCTATTTG, CGCTCGAGGCTATTGGCAGAAGGATT) of yeast Coq 8.

The Coq8 gene (CCGCGGCCGCATGGTTACAAATATGGTGAAAT, GGCTCGAGTTAAACTTTATAGGCAAAAATC) of yeast was cloned into expression vector pRS 423.

The two vectors were co-transferred into a Saccharomyces cerevisiae Δ coq7 mutant strain (Y12381, purchased from EUROSCARF), and the resulting positive transformants were inoculated on SD medium (A in FIG. 5) containing 2% glucose or 3% glycerol and 2.5% ethanol as carbon sources, respectively, and the results showed that they could grow on medium containing glycerol and ethanol as carbon sources. Whereas the mutant strain Δ coq7 transferred to an empty vector or only CoqF could not grow on a medium with glycerol and ethanol as carbon sources.

The wild strain BY4742 was transferred to two empty vectors pRS426 and pRS423 as controls. CoQ of the above several transformants was extracted and detected by LC-MS. The results show that the wild type strain can synthesize CoQ₆Whereas the mutant strain Δ CoQ7 hardly contained CoQ₆The mutant strain transferred to both AtCoqF and ScCoq8 can synthesize CoQ₆(B and C in FIG. 5). The above results indicate that AtCoqF has a benzene ring 6-hydroxylase activity.

In this example, the CoQ of Saccharomyces cerevisiae was measured₆The method of (3) is as follows. Culturing with 10mL SD-Ura-His liquid culture medium (containing 2% glucose) overnight, centrifuging to collect thallus, weighing wet weight, cracking thallus with methanol, adding isopropanol at a certain ratio, and extracting and detecting. CoQ₆(parent ion m/z 591.4, daughter ion m/z 197.1), fragmentation voltage 182V, collision energy 28V.

Example 3: RNAi transgenic plant of Arabidopsis CoqF with reduced CoQ content

Constructing an RNAi vector: about 300bp fragment of coding region of AtCoqF (GGGGACAAGTTTGTACAAAAAAGCAGGCT) was amplified by PCRTCAACAGTTGGTTCGATCCT, GGGGACCACTTTGTACAAGAAAGCTGGGTTTCTTGAACCTGGTTCAGC) was cloned into the vector pDOnr207 by BP reaction and then both fragments were cloned back into the vector pK7GWIWG2R (II) by LR reaction. The wild type Arabidopsis thaliana Col-0 was transformed by the floral dip method. Obtaining 3 strains with obvious down-regulation, and detecting CoQ₉The content, significantly lower than that of the wild type, indicates that AtCoqF is involved in CoQ synthesis in plants.

As shown in FIG. 6, qRT-PCR is shown in A to detect the relative expression of AtCoqF genes in three transgenic lines. The coenzyme Q9 content of the aerial parts of the plants measured for 4 weeks is shown in B, and the data are the mean values. + -. standard deviation of four biological replicates. The coenzyme Q content of the three lines is obviously reduced and is about 68-73% of that of wild plants.

In this example, Arabidopsis CoQ was measured₉The method of (3) is as follows. And (3) carrying out freeze drying on the plants at a low temperature, then carrying out vibration grinding by using a ball mill, weighing 10mg of powder, adding 1mL of isopropanol, and carrying out ultrasonic extraction for 1h at a low temperature in a dark place. The detection method is the same as above. CoQ₉(parent ion m/z 795.6, daughter ion m/z 197.1), fragmentation voltage 216V, collision energy 36V.

Example 4: CoqF is ubiquitous in many types of eukaryotes

Protein sequence analysis indicated that CoqF is ubiquitous in plants and green algae, and is also present in other eukaryotes, such as SAR super groups (Stramenopila, Alveolata, Rhizaria), Dinophyta (Haptista), Crypthecodinium (Cryptista). Representative species such as three plants of rice (Oryza sativa, XP _015620261.1), tomato (Solanum lycopersicum, XP _004239375.1) and liverwort (Marchantia polymorpha, PTQ40024.1), two green algae of fresh water microalgae (PRW 56607.1) and marine microalgae (Ostreococcus tauri, XP _003079408.2), two green algae of parasitic Saprolegnia parasitica (XP _012203255.1) and duckweed (Stylonychia lemnae, CDW87725.1) are selected as species of SAR super-cluster, and CoqF gene is synthesized and transferred to Escherichia coli expression vector pTrc99a, delta. ubiF mutant.

The results of the quantitative assay showed that after transfer of the CoqF genes from these species, the Δ ubiF mutants were able to produce CoQ, indicating that they all have benzene ring 6 hydroxylase activity (fig. 7, where the data are the mean ± standard deviation of three biological replicates). The results showed that the CoqF of three plants, rice (Oryza sativa), tomato (Solanum lycopersicum) and liverwort (Marchantia polymorpha), was transformed into E.coli.DELTA.ubiF mutant strains, and the ability to synthesize coenzyme Q was restored to 33% to 94% of that of wild type. CoqF of two green algae, namely freshwater microalgae (Chlorella sorokiniana) and marine microalgae (Ostreococcus tauri), is transferred into the Escherichia coli delta ubiF mutant strain, and the capability of synthesizing coenzyme Q is recovered and is 80-137% of that of a wild type. In the mutant strain of the escherichia coli delta ubiF, two kinds of CoqF belonging to species of a super group SAR (Saprolegnia parasitica) or Lemna minor (Stylonychia lemnae) are transferred, and the capability of synthesizing coenzyme Q is recovered and is 94% -109% of that of the wild type.

Sequence listing

<110> Shanghai mountain plant garden

<120> coenzyme Q synthesis pathway benzene ring 6-position hydroxylase and application thereof

<160> 26

<170> PatentIn version 3.5

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<213> Arabidopsis thaliana

<400> 5

cggactagtg cagactggaa acgtgggagg ttttagagct agaaatagc 49

<210> 6

<211> 22

<212> DNA

<213> Artificial sequence

<400> 6

ctcaaaaaaa gcaccgactc gg 22

<210> 7

<211> 37

<212> DNA

<213> Arabidopsis thaliana

<400> 7

ggattaccct gttatcccta ttgagcaggt gttcagc 37

<210> 8

<211> 27

<212> DNA

<213> Artificial sequence

<400> 8

cgaatcactg gtcaatcatc ctgttag 27

<210> 9

<211> 27

<212> DNA

<213> Artificial sequence

<400> 9

tgattgacca gtgattcgcg cgaagct 27

<210> 10

<211> 27

<212> DNA

<213> Arabidopsis thaliana

<400> 10

cgcgtcgaca cgggaaatgc ttcgcga 27

<210> 11

<211> 27

<212> DNA

<213> Artificial sequence

<400> 11

cggggtacca tggcgattct agggctt 27

<210> 12

<211> 29

<212> DNA

<213> Artificial sequence

<400> 12

gctctagatc attgtttccc tagtatgat 29

<210> 13

<211> 26

<212> DNA

<213> Artificial sequence

<400> 13

cggaattcat gtctaaggac agggtg 26

<210> 14

<211> 28

<212> DNA

<213> Artificial sequence

<400> 14

cgggatcctc aagaaaatag cggcaaag 28

<210> 15

<211> 36

<212> DNA

<213> Artificial sequence

<400> 15

acgagatgta agagcacaat ggcgattcta gggctt 36

<210> 16

<211> 36

<212> DNA

<213> Arabidopsis thaliana

<400> 16

atcgaattcc tgcagccctc attgtttccc tagtat 36

<210> 17

<211> 36

<212> DNA

<213> Artificial sequence

<400> 17

ttcgatatgg gatcccccat gttgttaaga tctaga 36

<210> 18

<211> 18

<212> DNA

<213> Artificial sequence

<400> 18

tgtgctctta catctcgt 18

<210> 19

<211> 25

<212> DNA

<213> Artificial sequence

<400> 19

ttgcggccgc gatccgggtg ttcgg 25

<210> 20

<211> 27

<212> DNA

<213> Artificial sequence

<400> 20

cgcggatccc atatcgaacg atatctt 27

<210> 21

<211> 28

<212> DNA

<213> Artificial sequence

<400> 21

gacgtcgaca atacttcccc gctatttg 28

<210> 22

<211> 26

<212> DNA

<213> Artificial sequence

<400> 22

cgctcgaggc tattggcaga aggatt 26

<210> 23

<211> 32

<212> DNA

<213> Artificial sequence

<400> 23

ccgcggccgc atggttacaa atatggtgaa at 32

<210> 24

<211> 30

<212> DNA

<213> Artificial sequence

<400> 24

ggctcgagtt aaactttata ggcaaaaatc 30

<210> 25

<211> 49

<212> DNA

<213> Artificial sequence

<400> 25

ggggacaagt ttgtacaaaa aagcaggctt caacagttgg ttcgatcct 49

<210> 26

<211> 48

<212> DNA

<213> Artificial sequence

<400> 26

ggggaccact ttgtacaaga aagctgggtt tcttgaacct ggttcagc 48

Claims

1. The application of the benzene ring 6-position hydroxylase gene in generating coenzyme Q in plants, bacteria, fungi and algae is characterized in that the benzene ring 6-position hydroxylase gene is derived from plants, green algae, SAR super groups, vervain and cryptophyte.

2. The use according to claim 1, wherein the hydroxylase gene at the 6-position of the benzene ring is derived from arabidopsis thaliana, rice, tomato, liverwort, fresh water microalgae (Chlorella sorokiniana), marine microalgae (ostrococcus tauri), Saprolegnia parasitica (Saprolegnia parasitica), duckweed thorny beetle (styronychia lemnae); preferably, their GenBank accession numbers are NP _173844.2, XP _015620261.1, XP _004239375.1, PTQ40024.1, PRW56607.1, XP _003079408.2, XP _012203255.1, CDW 87725.1.

3. The use according to claim 1 or 2, wherein the hydroxylase gene in the 6-position of the benzene ring is:

(1) a nucleotide sequence of SEQ ID No.1 or degenerate sequence thereof;

4. Use according to any one of claims 1 to 3, wherein coenzyme Q is produced by fermentation using a microorganism into which said benzene ring 6 hydroxylase gene is introduced and which is capable of expressing, preferably E.coli, yeast.

5. The use according to any one of claims 1 to 3, wherein the level of coenzyme Q is increased by transgenic means in a transgenic plant into which the benzene ring 6 hydroxylase gene is introduced and expressed.

6. The use of claim 4, wherein the plant is Arabidopsis thaliana.

7. The application of the benzene ring 6-position hydroxylase in the production of coenzyme Q is characterized in that the benzene ring 6-position hydroxylase is coded by a benzene ring 6-position hydroxylase gene derived from plants, green algae, SAR super groups, vervain and cryptophyte.

8. The use according to claim 6, wherein the hydroxylase at the 6-position of the benzene ring is derived from Arabidopsis thaliana, rice, tomato, liverwort, freshwater microalgae (Chlorella sorokiniana), marine microalgae (Ostreococcus tauri), Saprolegnia parasitica (Saprolegnia parasitica), Lemna minor (Stylonychia lemnae), more preferably, the GenBank accession numbers thereof are NP-173844.2, XP-015620261.1, XP-004239375.1, PTQ40024.1, PRW56607.1, XP-003079408.2, XP-012203255.1, CDW 87725.1.

9. The use of claim 8, wherein: the amino acid sequence of the benzene ring 6-position hydroxylase is shown in SEQ ID No. 2.