WO2021190632A1 - 一种羟化酶基因及其应用 - Google Patents

一种羟化酶基因及其应用 Download PDF

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WO2021190632A1
WO2021190632A1 PCT/CN2021/083269 CN2021083269W WO2021190632A1 WO 2021190632 A1 WO2021190632 A1 WO 2021190632A1 CN 2021083269 W CN2021083269 W CN 2021083269W WO 2021190632 A1 WO2021190632 A1 WO 2021190632A1
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seq
nucleotide sequence
host bacteria
mrf3
expression vector
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李鲜
邢梦云
曹运琳
徐昌杰
孙崇德
陈昆松
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浙江大学
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C12N9/0083—Miscellaneous (1.14.99)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/02—Oxygen as only ring hetero atoms
    • C12P17/06—Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein

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  • the invention belongs to the field of molecular biology, relates to recombinant protein and genetic engineering, and specifically relates to a hydroxylase gene and its coded protein and application.
  • Flavonoid 3'5'-hydroxylase (F3'5'H) belongs to the CYP45075A subfamily and catalyzes the hydroxylation of the flavonoid B ring.
  • the research on the function of F3'5'H gene mainly focuses on the synthesis of plant purple anthocyanin, and the research on other functions of F3'5'H gene is relatively limited.
  • Chinese bayberry (Morella rubra) is a characteristic fruit in my country, which has good medicinal activity, which is inseparable from its high content of flavonoids.
  • Myricetin is the main flavonoid compound in bayberry. It was isolated from the bark of bayberry for the first time. Under this name, it usually exists in vacuoles in the form of glycoside derivatives.
  • myricetin's anti-oxidant, anti-tumor, prevention of cardiovascular disease, anti-inflammatory and other pharmaceutical activities Plants rich in myricetin can be used to produce health foods and medicines, and have broad development and application prospects.
  • the identification of the gene F3'5'H involved in myricetin biosynthesis is of great significance for elucidating the myricetin biosynthesis mechanism, laying a foundation for the development of engineered microorganisms, and can be used for the improvement of myricetin components in other plants based on genetic engineering technology. It has important application value for increasing the content of myricetin in food and increasing the health function of food.
  • the present invention isolates a new hydroxylase gene MrF3'5'H (flavonoid 3'5'-hydroxylase of Morella rubra) from bayberry for the first time, which is a key gene for myricetin synthesis in bayberry.
  • MrF3'5'H flavonoid 3'5'-hydroxylase of Morella rubra
  • F3'5'H hydroxylase that has identified functions in other plants
  • MrF3'5'H shows strong myricetin synthesis ability.
  • MrF3'5'H's preference for kaempferol and high catalytic activity are important factors for myricetin enrichment.
  • the present invention provides a completely new isolated nucleotide, which encodes the F3'5'H hydroxylase gene, and comprises a nucleotide sequence having at least one of the following characteristics:
  • the nucleotide sequence shown in SEQ ID NO.1 is extracted and purified from the bayberry fruit, even if one or more nucleotide sequences shown in SEQ ID NO.1 are substituted, deleted, inserted and/or added
  • the nucleotide sequence of the base, or the nucleotide sequence of SEQ ID NO.1 has 80% or more homology, 85% or more homology, 90% or more homology, or 95% or more homology, as long as It is a nucleotide sequence encoding a protein with F3'5'H hydroxylation function, which is included in the hydroxylase gene involved in the present invention.
  • MrF3'5'H The hydroxylase gene is MrF3'5'H, which can also be called CYP75A.
  • MrF3'5'H flavonoid 3'5'-hydroxylase of Morella rubra
  • MrF3'5'H gene The applicant successfully cloned the MrF3'5'H gene, the cDNA sequence of MrF3'5'H (see the nucleotide sequence of SEQ ID NO.1 in the sequence list), and then used the specificity for the MrF3'5'H cDNA sequence PCR amplification was carried out with sex primers, and the sequence was constructed into a eukaryotic expression vector (see Example 3), and MrF3'5'H polypeptide or protein was expressed in eukaryotic cells (see SEQ ID NO. 2 sequence in the sequence table of the specification). And Example 4).
  • MrF3'5'H flavonoid 3'5'-hydroxylase of Morella rubra protein expressed in yeast host bacteria can catalyze the 3'-site hydroxylation reaction of naringenin, which is a monohydroxyl group in the B ring.
  • MrF3'5'H protein can catalyze the hydroxylation of the B-ring monohydroxy kaempferol at the 3'position to produce B-ring dihydroxy quercetin (EIC 301 ), and hydroxylation at the 3'and 5'positions to generate B-ring trihydroxy myricetin (EIC 317); MrF3'5'H protein can promote the B-ring dihydroxy eriorrhizin to 5'site The hydroxylation reaction produces the B-ring trihydroxy pentahydroxy dihydroflavonoid (EIC 303); MrF3'5'H protein can promote the B-ring dihydroxy dihydroquercetin to undergo the 5'position hydroxylation reaction to generate the B-ring trihydroxy Dihydromyricetin (EIC 319); MrF3'5'H protein can catalyze the hydroxylation reaction of the B-ring dihydroxy quercetin at the 5'position to produce the B-ring trihydroxymy
  • the present invention also provides a polypeptide or protein encoded by the F3'5'H hydroxylase gene, comprising at least one of the following characteristics:
  • the polypeptide or protein of the present invention is expressed from the MrF3'5'H gene through intracellular transcription and translation processes.
  • the transcription refers to the process in which the cell uses the nucleotides of the MrF3'5'H gene as a template, according to the principle of nucleotide complementary pairing, and uses ribonucleotides as raw materials to synthesize the corresponding MrF3'5'H mRNA.
  • the translation refers to a process in which the cell further uses the MrF3'5'H mRNA as a template and amino acids as raw materials to synthesize the corresponding MrF3'5'H polypeptide or protein.
  • MrF3'5'H is the closest to VvF3'5'H in the phylogenetic tree, the nucleotide sequence homology is 78.21%, the amino acid sequence homology is 82.87%, and the distance to GtF3'5'H is the farthest The nucleotide homology is 66.41%, and the amino acid homology is 71.71%, both of which belong to the CYP75A family (see Figure 3 and Figure 6).
  • MrF3'5'H and VvF3'5'H proteins have similar functions, and both can catalyze the synthesis of B-ring monohydroxy flavonoids and/or B-ring dihydroxy flavonoids B-ring trihydroxyflavonoids (see Figure 7).
  • the present invention also provides a hydroxylase, which is an enzyme that can be extracted from plants capable of producing myricetin, and is characterized in that it contains an amino acid sequence having at least one of the following 1) to 4):
  • the hydroxylase has the function of catalyzing the hydroxylation reaction of flavonoids at 3'and/or 5'positions.
  • the hydroxylase is MrF3'5'H, which can catalyze the hydroxylation reaction of the flavonoids of the B-ring monohydroxy group and/or the B-ring dihydroxy group to the 3'position and/or the 5'position.
  • the present invention also provides a gene expression vector which contains the nucleotide sequence of the F3'5'H gene as described above or the nucleoside containing the MrF3'5'H gene as described above Acid sequence.
  • the genes of the present invention can be inserted into existing eukaryotic or prokaryotic expression vectors, and suitable vectors include bacterial plasmids, lentiviruses, adenoviruses, adeno-associated viruses, retroviruses, and the like.
  • the vector is a circular DNA molecule, capable of autonomous replication and transcription expression in cells, and is the most commonly used tool in genetic engineering.
  • the present invention also provides a transgenic cell line or host bacteria, said transgenic cell line or host bacteria containing the aforementioned gene expression vector with F3'5'H nucleotide sequence.
  • the vector containing the F3'5'H gene of the present invention can be used to transform appropriate cell lines or host bacteria, the cell lines are derived from animal or plant cells, such as insect cells, mammalian cells, and the host bacteria can be It is genetically engineered bacteria, such as yeast, E. coli, etc.
  • the present invention also provides a method for preparing B-ring trihydroxyflavonoids.
  • the method is to introduce the aforementioned gene expression vector containing MrF3'5'H nucleotide sequence into the host bacteria, and give the host
  • the bacteria provide raw materials B-ring monohydroxy flavonoids and/or B-ring dihydroxy flavonoids to synthesize B-ring trihydroxy flavonoids.
  • the B-ring trihydroxy flavonoids can be extracted from the host bacteria; the host bacteria An expression vector containing the nucleotide sequence of MrF3'5'H as described above.
  • the raw materials naringenin and/or eriochiol are provided to the yeast host bacteria expressing MrF3'5'H protein, and pentahydroxyflavone is produced. ; Provide the raw material dihydrokaempferol and/or dihydroquercetin to the yeast host bacteria expressing MrF3'5'H protein, and then generate dihydromyricetin; to the yeast host bacteria expressing MrF3'5'H protein Provided with raw materials kaempferol and/or quercetin, myricetin is produced.
  • the present invention provides a preparation method of myricetin, the preparation method is selected from any one of the following methods: 1) Introducing the F3'5'H core containing F3'5'H into the plant body or plant tissue.
  • the gene expression vector containing the F3'5'H nucleotide sequence as described above is introduced into the bacteria, and the raw materials kaempferol and/or quercetin are provided to the host bacteria to synthesize myricetin; preferably, it can be obtained from the Myricetin is extracted from cell lines or host bacteria.
  • the myricetin content in the flowers and leaves of tobacco overexpressing the MrF3'5'H gene is significantly increased.
  • the myricetin is produced by providing raw materials kaempferol and/or quercetin to the yeast host bacteria expressing MrF3'5'H protein.
  • the present invention provides a method for preparing pentahydroxyflavone, the method is to introduce the gene expression vector containing the MrF3'5'H nucleotide sequence into the host bacteria, and to the host bacteria
  • the raw materials naringenin and/or holy grass are provided to synthesize pentahydroxyflavone.
  • the pentahydroxyflavone can be extracted from the host bacteria.
  • Figure 5 and Example 4 taking the yeast host bacteria as an example, the raw materials naringenin and/or eriochiol are provided to the yeast host bacteria expressing MrF3'5'H protein, and pentahydroxyflavone is produced. .
  • the present invention provides a method for preparing dihydromyricetin, which is characterized in that: the method is to introduce the gene expression vector containing the MrF3'5'H nucleotide sequence into the host bacteria, and
  • the host bacteria are provided with raw materials dihydrokaempferol and/or dihydroquercetin to synthesize dihydromyricetin.
  • dihydromyricetin can be extracted from the host bacteria.
  • Example 4 taking the yeast host bacteria as an example, if the raw materials dihydrokaempferol and/or dihydroquercetin are provided to the yeast host bacteria expressing MrF3'5'H protein, dihydromyricetin is produced .
  • the present invention provides the nucleotide sequence encoding MrF3'5'H gene as described above, the expression vector of MrF3'5'H gene as described above, and the nucleotide sequence containing MrF3'5'H as described above
  • the application of sequenced cell lines or host bacteria in genetic engineering refers to the genetic technology of constructing an expression vector containing MrF3'5'H nucleotide sequence in vitro, and then introducing it into living cells to change the original genetic characteristics of organisms, obtain new varieties, and produce new products.
  • the applications of the genetic engineering include the production of genetic engineering drugs, the cultivation of new plant varieties, and the development of the food industry.
  • the present invention provides a method for cultivating a transgenic plant.
  • the cultivating method is to introduce a specific nucleotide into a recipient plant and allow the recipient plant to express the nucleotide to obtain a transgenic plant.
  • the specific nucleotide is selected from the aforementioned expression vector containing MrF3'5'H gene.
  • the plant is tobacco. As described in Example 7, when the MrF3'5'H gene was introduced into tobacco, compared with wild-type tobacco, the myricetin content in the flowers and leaves of tobacco overexpressing the MrF3'5'H gene was significantly increased.
  • the present invention also provides a transgenic tobacco, which is characterized in that specific nucleotides are introduced and expressed in the transgenic tobacco, and the specific nucleotides are selected from the gene expression vector of claim 6.
  • the present invention provides a hydroxylase gene F3'5'H involved in myricetin biosynthesis and its encoding protein and application.
  • the hydroxylase gene is MrF3'5'H, which is derived from the CYP450 family of bayberry.
  • MrF3'5'H is derived from the CYP450 family of bayberry.
  • a key gene involved in myricetin synthesis lays the foundation for the engineering of myricetin synthesis. Its nucleotide sequence is shown in SEQ ID NO. 1, and its amino acid sequence is shown in SEQ ID NO. 2.
  • MrF3'5'H has the function of P450 hydroxylase, which can catalyze the hydroxylation reaction of kaempferol at the 3'site to produce quercetin, and the hydroxylation reaction at the 3'and 5'sites occurs.
  • Myricetin is produced, and it can also catalyze the hydroxylation reaction of quercetin at the 5'site to produce myricetin, and its catalytic activity on kaempferol is higher than its homologous protein VvF3'5'H (see Figure 4 and Figure 7) .
  • Another object of the present invention is to provide a gene MrF3'5'H involved in the synthesis of B-ring trihydroxyflavonoids and its coded protein and applications.
  • MrF3'5'H can respectively catalyze the synthesis of B-ring monohydroxy naringenin, dihydrokaempferol, and kaempferol to synthesize B-ring dihydroxy erichol, dihydroquercetin, Quercetin and B-ring trihydroxy pentahydroxy dihydroflavonoids, dihydroquercetin, dihydromyricetin can also catalyze the synthesis of B-ring dihydroxy eriochohol, dihydroquercetin, and quercetin
  • the trihydroxy pentahydroxy dihydroflavonoids, dihydroquercetin and dihydromyricetin provide intermediate products for the synthesis of other B-ring trihydroxyflavonoids.
  • the codable cDNA sequence of MrF3'5'H gene is shown in SEQ ID NO. 1.
  • the coding sequence has a total length of 1530 nucleotides and can encode a protein of 509 amino acids. Its amino acid sequence is shown in SEQ ID NO. 2, which contains conserved proline-rich, SRS, CR, E ⁇ R and heme binding domains, and belongs to the CYP450 family.
  • MrF3'5'H has the function of P450 hydroxylase, which can catalyze kaempferol and quercetin to myricetin, respectively, and has catalytic activity on kaempferol Higher than its homologous protein VvF3'5'H.
  • the invention has guiding significance for studying the myricetin shunt mechanism, and lays a foundation for the development of engineered microorganisms or the improvement of plant myricetin components based on genetic engineering technology.
  • Figure 1 is a diagram of the basic structure of flavonoids.
  • the basic structure of flavonoids consists of ring A, ring B and ring C.
  • Figure 2 is an analysis diagram of the content of myricetin and quercetin in different tissues of'Biqi' and'Dongkui' bayberry.
  • Figure 3 is the result of MrF3'5'H amino acid sequence alignment; SlF3'5'H (ACF32346), PhF3'5'H-Hf1 (CAA80266), PhF3'5'H-Hf2 (CAA80265), F3'5' H protein contains conserved proline-rich, SRS, CR, E ⁇ R and heme binding domains.
  • Figure 4 is the LC-MS spectrum analysis diagram of the in vitro activity of the recombinant protein MrF3'5'H.
  • Figure 4 shows that MrF3'5'H has the function of catalyzing the hydroxylation reaction at the 3'position and/or the 5'position.
  • MrF3'5'H can catalyze the B-ring monohydroxy flavonoid naringenin, dihydrokaempferol, and kaempferol to produce the B-ring dihydroxy flavonoids at the 3'position respectively to generate the B-ring dihydroxyflavonoid ericola (EIC 287), dihydroquercetin Cortin (EIC 303) and Quercetin (EIC 301), which undergo hydroxylation reactions at the 3'and 5'positions respectively to generate the B-ring trihydroxyflavonoid pentahydroxy dihydroflavonoid (EIC 303) and dihydromyricetin (EIC 319) and Myricetin (EIC 317); MrF3'5'H can catalyze the B-ring dihydroxyflavonoid saccharol, dihydroquercetin and quercetin to undergo 5'hydroxylation reaction to form the B ring, respectively Trihydroxyflavonoids pentahydroxyflavone (EIC303), di
  • Nar naringenin; Eri: saccharol (EIC 287); PHF: pentahydroxydihydroflavone (EIC 303); DHK: dihydrokaempferol; DHQ: dihydroquercetin (EIC 303); DHM: two Hydromyricetin (EIC 319); K: Kaempferol; Q: Quercetin (EIC 301); M: Myricetin (EIC 317).
  • Figure 5 is the structural formula of the catalytic substrate and product of the recombinant protein MrF3'5'H.
  • Naringenin, dihydrokaempferol, and kaempferol belong to the B-ring monohydroxy flavonoids
  • holy moor a moor
  • dihydroquercetin and quercetin belong to the B-ring dihydroxy flavonoids
  • pentahydroxy dihydroflavonoids dihydro Myricetin and myricetin are B-ring trihydroxy flavonoids.
  • Figure 6 is the phylogenetic tree analysis diagram of MrF3'5'H; CrF3'5'H (Q9ZRY0), GhF3'5'H (AAP31058), GmF3'5'H (AAM51564), GtF3'5'H (Q96581), NtF3 '5'H(XP_016452599), PhF3'5'H(Hf1)(CAA80266), PhF3'5'H(Hf2)(CAA80265), SlF3'5'H(ACF32346), StF3'5'H(NP_001274807), VmF3'5'H (BAC97831), VvF3'5'H (CAI54277).
  • Figure 7 is an analysis diagram of the catalytic properties of MrF3'5'H and VvF3'5'H substrates.
  • Figure 8 is an analysis diagram of the phenotype and flavonoid content of tobacco overexpressing MrF3'5'H. Among them, (a) represents the change in flower color of overexpression of MrF3'5'H tobacco; (b) represents that overexpression of MrF3'5'H significantly increases the content of myricetin, quercetin and kaempferol in tobacco leaves, and significantly increases the content of flowers. Myricetin and delphinium content.
  • Water chestnut and Dongkui bayberry tissues (fruits, flowers, leaves) were harvested on the same day, frozen in liquid nitrogen and stored in a -80°C refrigerator. Each tissue sample was set up with 3 biological replicates, each with 7-8 fruits, each The mass of each repeated flower is more than 500g, and each repeat has 10-15 whole leaves.
  • the mobile phases used are all chromatographic grade.
  • the myricetin content was as high as 10 mg g -1 FW, and the myricetin content showed a downward trend during fruit development (Figure 2).
  • the bayberry tissue sample is ground into powder in a liquid nitrogen environment, and the total RNA is extracted by the ordinary CTAB method. After the electrophoresis test is qualified, refer to the TURBO DNAase Kit (Ambion) manual to remove the DNA. According to the requirements of iScript cDNA Synthesis Kit (Bio-Rad), Take 1.0 ⁇ g RNA and reverse transcribed into cDNA.
  • RNA-Seq database of Chinese water chestnut bayberry use Flavonoid 3',5'-hydroxylase as a keyword to search for genes related to myricetin synthesis, and use the amino acid sequence of SlF3'5'H with clear functions in tomato as a reference, through CLUSTALX
  • the software homology comparison screened out a gene Unigene5190 (MrF3'5'H) that may be involved in the synthesis of myricetin from myrica fruit.
  • the application sequence is: SEQ ID NO.1 and passed BLAST (https://blast.ncbi.nlm) .nih.gov/Blast.cgi) online analysis to confirm that it is a full-length sequence.
  • Design full-length cloning primers SEQ ID NO.3 and SEQ ID NO.4, PCR reaction system is 50 ⁇ L, the components are: 0.5 ⁇ L Roche high-fidelity enzyme, 5 ⁇ L buffer (10 ⁇ ), 4 ⁇ L dNTP (2.5mM), Upstream and downstream primers (10 ⁇ M, Hua Gene) each 2 ⁇ L, 4 ⁇ L cDNA, 32.5 ⁇ L H 2 O.
  • the reaction procedure is: pre-denaturation at 95°C, 2min; denaturation at 95°C, 30s; annealing at 58°C, 30s; extension at 72°C, 90min, 35 cycles; extension at 72°C for 10min, storage at 4°C.
  • the PCR amplified product was ligated to the T-easy vector, transformed into E. coli DH5 ⁇ , and verified by colony PCR to obtain positive colonies for sequencing.
  • the cloning results were verified by sequencing, and the full-length sequence of MrF3'5'H matching the transcriptome database was obtained as shown in SEQ ID NO.1 and contained 1530 nucleotides.
  • Online translation into amino acid sequence http://web.expasy.org/translate/), namely: SEQ ID NO.2.
  • the amino acid sequence of MrF3'5'H was compared with the published P45075A family hydroxylase with 3'5' site hydroxylase, and the results are shown in Figure 3.
  • MrF3'5'H gene that is, the cDNA sequence of MrF3'5'H (see SEQ ID NO. 1 in the Sequence Listing).
  • the MrF3'5'H gene undergoes intracellular transcription and translation processes to synthesize the corresponding polypeptide or protein, that is, the amino acid sequence of MrF3'5'H (see SEQ ID NO. 2 in the sequence list).
  • Amino acid sequence analysis shows that SEQ ID NO. 2 has typical proline-rich, SRS, CR, E ⁇ R and heme binding conserved domains.
  • the primer sequence containing the BamHI and EcoRI restriction sites was designed: SEQ ID NO.5 And SEQ ID NO. 6, the primer design contains a start codon and a stop codon, and amplifies the MrF3'5'H sequence containing BamHI and EcoRI restriction sites.
  • the PCR reaction system is 50 ⁇ L, and the components are: 1 ⁇ L Phanta high-fidelity enzyme (Vazyme), 25 ⁇ L buffer (2 ⁇ ), 1 ⁇ L dNTP (10mM), 2 ⁇ L each of the upstream and downstream primers (10 ⁇ M, Hua Gene), 1 ⁇ L cDNA, 18 ⁇ L H 2 O.
  • the reaction procedure is: pre-denaturation at 95°C, 2min; denaturation at 95°C, 15s; annealing at 58°C, 15s; extension at 72°C, 1min, 35 cycles; complete extension at 72°C for 5min, and storage at 4°C.
  • ligase connects the target gene fragment to the pYES2 vector.
  • the ligation reaction system is 10 ⁇ L, and the components are: 1 ⁇ L II Ligase (Vazyme), 2 ⁇ L buffer (5 ⁇ ), 1 ⁇ L PCR product, 3 ⁇ L vector, 3 ⁇ L H 2 O. After mixing, connect for 0.5h at 37°C and place on ice for 5min. Transform the ligation product into DH5 Harbor competent (Takara), spread it on a culture plate containing Amp and cultivate overnight at 37°C, pick out positive clones, and send them to Shanghai Hua Gene for sequencing. The sequencing results are analyzed, and those containing the correct target gene sequence
  • the vector is the successfully constructed pYES2-MrF3'5'H recombinant plasmid.
  • the successfully constructed pYES2-MrF3'5'H recombinant plasmid or pYES2 empty vector was transformed into the Saccharomyces cerevisiae strain INVScI (Invitrogen) by the yeast transformation kit (Clontech) using the LiAC method. Then spread it on the SD/-Ura culture plate at 30°C for 3 days, pick a single colony, and detect the recombinant plasmid or empty by PCR.
  • the single colony with the correct PCR band is selected as the Saccharomyces cerevisiae INVScI containing the pYES2-MrF3'5'H recombinant plasmid, which is stored in the refrigerator at -80°C with 25% glycerol for later use.
  • Liquid chromatography detection of mobile phase A: water (0.1% formic acid) B: acetonitrile (0.1% formic acid); injection volume: 10 ⁇ L; flow rate: 0.3 mL/min; column temperature: 25° C.; detection wavelength is 370 nm; elution Gradient: 0-7min, 90%-50%A, 7-10min, 50%A, 10-15min, 50%-0%A, 15-15.1min, 0-90%A, 15.1-21min, 90%A.
  • MrF3'5'H has the activity of the P45075A family of hydroxylases, which can catalyze the hydroxylation reaction of the B-ring monohydroxy naringenin at the 3'site to generate the B-ring dihydroxy ericolaol (EIC 287).
  • the 3'position and the 5'position hydroxylation reaction will generate the B-ring trihydroxy pentahydroxy dihydroflavonoid (EIC 303); MrF3'5'H protein can promote the B-ring monohydroxy dihydrokaempferol to produce 3
  • The'site hydroxylation reaction generates the B-ring dihydroxy dihydroquercetin (EIC 303), and the 3'site and 5'site hydroxylation reactions generate the B-ring trihydroxy dihydromyricetin (EIC 319) ;
  • MrF3'5'H protein can catalyze the hydroxylation of the B-ring monohydroxy kaempferol at the 3'position to generate the B-ring dihydroxy quercetin (EIC 301), and generate 3'and 5'positions
  • the hydroxylation reaction produces B-ring trihydroxy myricetin (EIC 317); MrF3'5'H protein can catalyze the B-ring dihydroxy saccharol to undergo 5'hydroxylation reaction to generate B-ring trihydroxy pen
  • the phylogenetic tree construction is completed by MEGA7.0 (Mega Software, USA) software.
  • MEGA7.0 Mega Software, USA
  • MrF3'5'H does belong to the P45075A family of proteins. It is the closest VvF3'5'H to grapes on the evolutionary tree.
  • the nucleotide homology is 78.21%, and the amino acid homology is 82.87%, which is similar to that of GtF3'5.
  • 'H is the farthest distance, the nucleotide homology is 66.41%, and the amino acid homology is 71.71% ( Figure 6).
  • Grape VvF3'5'H was separated from the skin of'Xiahei', and the specific operation was the same as in Example 3.
  • the operation of expressing VvF3'5'H in Saccharomyces cerevisiae is the same as in Example 4.
  • MrF3'5'H and VvF3'5'H have different catalytic properties on naringenin, eriochiol, dihydrokaempferol, dihydroquercetin, kaempferol and quercetin.
  • MrF3'5 The catalytic activity of'H on kaempferol is significantly stronger than that of VvF3'5'H ( Figure 7).
  • the primer combination SEQ ID NO.7 and SEQ ID NO.8 was used to amplify the full-length sequence of F3'5'H (SEQ ID NO.1), and load it onto the pGreenII002962-SK expression vector to construct the recombinant expression vector SK-F3 '5'H.
  • This PCR reaction system is the same as the PCR reaction system in Example 3.
  • the finally constructed correct expression vector was transferred into Agrobacterium strain GV3101::pSoup by electroporation, and 3 positive clones were selected and stored at -80°C with sterilized glycerol at a final concentration of 25%.
  • the CsF3'5'H derived from tea was heterologously expressed in tobacco, and the delphinium content in the flowers of the transgenic lines increased significantly, and the detected myricetin content was too low to be quantified (Wang et al., 2014 Functional analysis of flavonoid 3',5'-hydroxylase from tea plant (Camellia sinensis): critical role in the accumulation of catechins. BMC Plant Biol. 14:347.), indicating that MrF3'5'H has a strong ability to synthesize myricetin.
  • MrF3'5'H is clustered in the CYP75A family on the evolutionary tree, and is the closest to VvF3'5'H.
  • the nucleotide sequence homology of MrF3'5'H and VvF3'5'H is 78.21%, MrF3'5
  • the amino acid sequence homology of'H and VvF3'5'H is 82.87% ( Figure 3 and Figure 6).
  • MrF3'5'H and VvF3'5'H proteins Comparing the functions of MrF3'5'H and VvF3'5'H proteins, it can be seen that MrF3'5'H and VvF3'5'H proteins have similar functions, and both can catalyze the synthesis of B-ring monohydroxy flavonoids and/or B-ring dihydroxy flavonoids
  • the B-ring trihydroxyflavonoids ( Figure 7) indicate that the hydroxylase proteins clustered in the same family have similar functions.
  • the substrate catalytic properties of MrF3'5'H and VvF3'5'H are different, and the catalytic activity of MrF3'5'H on kaempferol is significantly higher than that of VvF3'5'H ( Figure 7).

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Abstract

本发明提供了一种羟化酶基因F3'5'H及其编码蛋白、含有该F3'5'H核苷酸序列的表达载体以及含有该表达载体的转基因细胞系和宿主菌。本发明还提供了上述物质在杨梅素及其他B环三羟基类黄酮合成和基因工程中的用途。

Description

一种羟化酶基因及其应用 技术领域
本发明属于分子生物学领域，涉及重组蛋白和基因工程，具体涉及一种羟化酶基因及其编码蛋白和应用。
背景技术
细胞色素P450，简称CYP450，在许多物质的生物合成中起着重要作用，比如参与次生产物(如类黄酮)和激素的生物合成。类黄酮3'5'-羟化酶(F3'5'H)属于CYP45075A亚家族，催化类黄酮B环发生羟化反应。目前，有关F3'5'H基因功能的研究主要集中在植物紫色花色苷合成，有关F3'5'H基因的其他功能研究比较有限。
杨梅(Morella rubra)属于我国特色水果，具有很好的医药学活性，这与其较高的类黄酮化合物含量密不可分，而杨梅素是杨梅中主要的类黄酮化合物，首次从杨梅树皮中分离并以此命名，通常以糖苷衍生物形式存在于液泡中。大量研究报道了杨梅素抗氧化、抗肿瘤、预防心血管疾病、消炎等医药学活性。富含杨梅素的植物可用于生产保健食品、药品，具有广阔的开发应用前景。
鉴别出参与杨梅素生物合成的基因F3'5'H，对于阐明杨梅素生物合成机制具有重要意义，为开发工程微生物菌奠定基础，且可用于其他植物基于基因工程技术的杨梅素组分改良，对提高食物中的杨梅素含量，增加食物的保健功能，有重要的应用价值。
发明内容
本发明首次从杨梅中分离出一种全新的羟化酶基因MrF3'5'H(flavonoid 3'5'-hydroxylase ofMorella rubra)，是杨梅中杨梅素合成的关键基因。与其他植物中已鉴定功能的F3'5'H羟化酶相比(Wang et al.,2014Functional analysis of flavonoid 3′,5′-hydroxylase from tea plant(Camellia sinensis):critical role in the accumulation of catechins.BMC Plant Biol.14:347.)，MrF3'5'H表现出较强的杨梅素合成能力。此外，MrF3'5'H对山奈酚的偏好性及较高的催化活性是杨梅富集杨梅素的重要因素。
本发明提供了一种全新的分离的核苷酸，所述分离的核苷酸编码F3'5'H羟化酶基因，包含至少具有下列特征之一的核苷酸序列：
1)序列表中的SEQ ID NO.1的核苷酸序列；
2)编码序列表中SEQ ID NO.2蛋白质序列的多核苷酸；
3)与序列表中SEQ ID NO.1的核苷酸序列具有80％以上同源性或85％以上同源性或90％以上同源性或95％以上同源性，且编码相同功能蛋白质的核苷酸序列；
4)与序列表中的SEQ ID NO.1限定的DNA序列杂交的核苷酸序列
5)与SEQ ID NO.1编码相同功能蛋白质的核苷酸序列。
序列SEQ ID NO.1所示的核苷酸序列是从杨梅果实中提取纯化的，即使对SEQ ID NO.1所示的核苷酸序列置换、缺失、插入和/或添加了一个或数个碱基的核苷酸序列，或与SEQ ID NO.1的核苷酸序列具有80％以上同源性或85％以上同源性或90％以上同源性或95％以上同源性，只要是编码具有F3'5'H羟化功能蛋白质的核苷酸序列，就包含在本发明涉及的羟化酶基因中。
所述的羟化酶基因为MrF3'5'H，也可称为CYP75A。申请人通过系统研究MrF3'5'H(flavonoid 3'5'-hydroxylase of Morella rubra)基因的生物学功能，首次验证了MrF3'5'H在B环三羟基类黄酮合成中的独特作用。申请人成功克隆MrF3'5'H基因，即MrF3'5'H的cDNA 序列(参见序列表中的SEQ ID NO.1的核苷酸序列)，然后使用针对MrF3'5'H cDNA序列的特异性引物进行PCR扩增，将该序列构建至真核表达载体(参见实施例3)，在真核细胞中表达MrF3'5'H多肽或蛋白质(参见说明书序列表中的SEQ ID NO.2序列和实施例4)。进一步地，申请人发现了酵母宿主菌中表达的MrF3'5'H(flavonoid 3'5'-hydroxylase of Morella rubra)蛋白可催化B环单羟基的柚皮素发生3’位点羟化反应生成B环双羟基的圣草酚(EIC 287)，并发生3’位点和5’位点羟化反应生成B环三羟基的五羟基双氢黄酮(EIC 303)；MrF3'5'H蛋白可催B环单羟基的二氢山柰酚发生3’位点羟化反应生成B环双羟基的二氢槲皮素(EIC 303)，并发生3’位点和5’位点羟化反应生成B环三羟基的二氢杨梅素(EIC 319)；MrF3'5'H蛋白可催B环单羟基的山柰酚发生3’位点羟化反应生成B环双羟基的槲皮素(EIC 301)，并发生3’位点和5’位点羟化反应生成B环三羟基的杨梅素(EIC 317)；MrF3'5'H蛋白可催B环双羟基的圣草酚发生5’位点羟化反应生成B环三羟基的五羟基双氢黄酮(EIC 303)；MrF3'5'H蛋白可催B环双羟基的二氢槲皮素发生5’位点羟化反应生成B环三羟基的二氢杨梅素(EIC 319)；MrF3'5'H蛋白可催化B环双羟基的槲皮素发生5’位点羟化反应生成B环三羟基的杨梅素(EIC 317)(参见附图4和附图5)。进一步地，申请人还发现异源过表达MrF3'5'H基因的烟草叶中杨梅素和槲皮素含量显著增加，异源过表达MrF3'5'H基因的烟草花中杨梅素和飞燕草素含量显著增加(参见附图8)。
本发明还提供了一种F3'5'H羟化酶基因编码的多肽或蛋白质，包含至少具有下列特征之一：
1)序列表中的SEQ ID NO.2的氨基酸序列；
2)与序列表中SEQ ID NO.2的氨基酸残基序列具有80％以上同源性或85％以上同源性或90％以上同源性或95％以上同源性，且显示羟化功能的多肽或蛋白质。
3)将序列表中的SEQ ID NO.2的氨基酸残基序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且显示羟化功能的蛋白质；
4)具有proline-rich、SRS、CR、E××R和heme binding保守结构域。
本发明所述的多肽或蛋白质是由所述的MrF3'5'H基因经细胞内的转录和翻译过程表达而来。所述的转录是指细胞以所述MrF3'5'H基因的核苷酸为模板，根据核苷酸互补配对原则，以核糖核苷酸为原料，合成对应的MrF3'5'H mRNA的过程。所述的翻译是指，细胞进一步以所述的MrF3'5'H mRNA为模板，以氨基酸为原料，合成出对应MrF3'5'H多肽或蛋白质的过程。所述的MrF3'5'H在进化树上与VvF3'5'H距离最近，核苷酸序列同源性为78.21％，氨基酸序列同源性为82.87％，与GtF3'5'H距离最远，核苷酸同源性为66.41％，氨基酸同源性为71.71％，均属于CYP75A家族(参见附图3和附图6)。对比MrF3'5'H和VvF3'5'H蛋白功能可知，MrF3'5'H和VvF3'5'H蛋白功能相似，均可催化B环单羟基类黄酮和/或B环双羟基类黄酮合成B环三羟基类黄酮(参见附图7)。
本发明还提供了一种羟化酶，是可以从能生成杨梅素的植物体内提取的酶，其特征在于：包含至少具有下列1)～4)特征之一的氨基酸序列：
1)SEQ ID NO.2表示的氨基酸序列；
2)与SEQ ID NO.2具有80％以上同源性，且显示羟化功能的多肽或蛋白质；
3)将序列表中的SEQ ID NO.2的氨基酸残基序列经过一个或2个以上氨基酸残基的取代和/或缺失和/或添加且显示羟化功能的蛋白质；
4)具有proline-rich、SRS、CR、E××R和heme binding保守结构域的氨基酸序列。
所述的羟化酶具有催化类黄酮发生3’位点和/或5’位点羟化反应的功能。优选的，所述的羟化酶为MrF3'5'H，可催化B环单羟基和/或B环双羟基的类黄酮发生3’位点和/或5’位点羟化反应。
本发明还提供了一种基因表达载体，所述的基因表达载体含有如前所述的F3'5'H基因的核苷酸序列或含有如前所述的MrF3'5'H基因的核苷酸序列。本发明所述的基因可插入到现有的真核或原核表达载体中，适宜的载体包括细菌质粒、慢病毒、腺病毒、腺相关病毒、逆转录病毒等。所述的载体是一种环状DNA分子，能够在细胞内自主复制和转录表达，是基因工程中最常用的工具。
本发明还提供了一种转基因细胞系或宿主菌，所述的转基因细胞系或宿主菌含有如前所述的具有F3'5'H核苷酸序列的基因表达载体。本发明所述的含有F3'5'H基因的载体可以用来转化适当细胞系或宿主菌，所述的细胞系来自动物或植物细胞，例如昆虫细胞、哺乳动物细胞，所述的宿主菌可以是基因工程菌，例如酵母、大肠杆菌等。
本发明还提供了一种B环三羟基类黄酮制备的方法，所述的方法是向宿主菌中导入如前所述的含有MrF3'5'H核苷酸序列的基因表达载体，并给宿主菌提供原料B环单羟基类黄酮和/或B环双羟基类黄酮，从而合成B环三羟基类黄酮，优选的，可以从所述宿主菌提取B环三羟基类黄酮；所述的宿主菌含有如前所述的MrF3'5'H核苷酸序列的表达载体。如图5和实施例4所示，以酵母宿主菌为例，向表达MrF3'5'H蛋白的酵母宿主菌中提供原料柚皮素和/或圣草酚，则生成了五羟基双氢黄酮；向表达MrF3'5'H蛋白的酵母宿主菌中提供原料二氢山柰酚和/或二氢槲皮素，则生成了二氢杨梅素；向表达MrF3'5'H蛋白的酵母宿主菌中提供原料山柰酚和/或槲皮素，则生成了杨梅素。
进一步的，本发明提供了一种杨梅素的制备方法，所述的制备方法选自以下方法的任何一种：1)向植物体或植物组织导入如前所述的含有F3'5'H核苷酸序列的基因表达载体，从而实现植物系统杨梅素的合成；优选的，可以从所述植物体或植物组织提取杨梅素；优选的，所述的植物是烟草；2)向细胞系或宿主菌中导入如前所述的含有F3'5'H核苷酸序列的基因表达载体，并向宿主菌提供原料山奈酚和/或槲皮素，从而合成杨梅素；优选的，可以从所述细胞系或宿主菌提取杨梅素。如实施例7所述，向烟草中导入MrF3'5'H基因，与野生型烟草相比，过表达MrF3'5'H基因的烟草花和叶中杨梅素含量显著增加。如图5和实施例4所示，以酵母宿主菌为例，向表达MrF3'5'H蛋白的酵母宿主菌中提供原料山柰酚和/或槲皮素，则生成了杨梅素。
进一步的，本发明提供了一种五羟基双氢黄酮的制备方法，所述的方法是向宿主菌中导入所述的含有MrF3'5'H核苷酸序列的基因表达载体，并向宿主菌提供原料柚皮素和/或圣草酚，从而合成五羟基双氢黄酮，优选的，可以从所述宿主菌提取五羟基双氢黄酮。如图5和实施例4所示，以酵母宿主菌为例，向表达MrF3'5'H蛋白的酵母宿主菌中提供原料柚皮素和/或圣草酚，则生成了五羟基双氢黄酮。
进一步的，本发明提供了一种二氢杨梅素的制备方法，其特征在于：所述的方法是向宿主菌中导入所述的含有MrF3'5'H核苷酸序列的基因表达载体，并向宿主菌提供原料二氢山奈酚和/或二氢槲皮素，从而合成二氢杨梅素，优选的，可以从所述宿主菌提取二氢杨梅素。如实施例4所示，以酵母宿主菌为例，向表达MrF3'5'H蛋白的酵母宿主菌中提供原料二氢山柰酚和/或二氢槲皮素，则生成了二氢杨梅素。
本发明提供了如前所述的编码MrF3'5'H基因的核苷酸序列、如前所述的MrF3'5'H基因表达载体、如前所述的含有MrF3'5'H核苷酸序列的细胞系或宿主菌在基因工程中的应用。所述的基因工程是指在体外构建含有MrF3'5'H核苷酸序列的表达载体，然后导入活细胞，以改变生物原有的遗传特性、获得新品种、生产新产品的遗传技术。所述的基因工程的应用包括生产基因工程药品、培育植物新品种、开发食品工业等。
进一步的，本发明提供了一种转基因植物的培育方法，所述的培育方法是向受体植物中导入特定的核苷酸，并使受体植物表达该核苷酸以获得转基因植物。所述的特定的核苷酸选自如前所述的含有MrF3'5'H基因的表达载体。优选的，所述的植物是烟草。如实施例7所 述，向烟草中导入MrF3'5'H基因，与野生型烟草相比，过表达MrF3'5'H基因的烟草花和叶中杨梅素含量显著增加。
本发明还提供了一种转基因烟草，其特征在于：所述的转基因烟草中导入并表达了特定的核苷酸，所述的特定的核苷酸选自权利要求6所述的基因表达载体。
本发明提供了一种参与杨梅素生物合成的羟化酶基因F3'5'H及其编码蛋白和应用，所述的羟化酶基因为MrF3'5'H,来源于杨梅的CYP450家族，是一种参与杨梅素合成的关键基因，为杨梅素合成的工程化奠定基础，其核苷酸序列如SEQ ID NO.1所示，其氨基酸序列如SEQ ID NO.2所示。酵母体内进行的重组蛋白实验表明，MrF3'5'H具有P450羟化酶功能，可催化山奈酚发生3’位点羟化反应生成槲皮素，发生3’点和5’位点羟化反应生成杨梅素，也可催化槲皮素发生5’位点羟化反应生成杨梅素，且对山奈酚的催化活性高于其同源蛋白VvF3'5'H(参见附图4和附图7)。利用基因工程技术获得过表达MrF3'5'H的烟草转基因植株，相较于野生型，转基因株系叶片中杨梅素、槲皮素、和山奈酚含量显著增加，转基因株系花中杨梅素和飞燕草素含量显著增加，槲皮素和山奈酚含量无显著变化。
本发明的另一个目的是提供一种参与B环三羟基类黄酮合成基因MrF3'5'H及其编码蛋白和应用。在酵母中进行的体外功能验证表明，MrF3'5'H可分别催化B环单羟基的柚皮素、二氢山奈酚、山奈酚合成B环双羟基的圣草酚、二氢槲皮素、槲皮素和B环三羟基的五羟基双氢黄酮、二氢槲皮素、二氢杨梅素，也可催化B环双羟基的圣草酚、二氢槲皮素、槲皮素合成B环三羟基的五羟基双氢黄酮、二氢槲皮素、二氢杨梅素，为其他B环三羟基类黄酮的合成提供中间产物。
本发明提供的基因特征如下：
(1)基因序列特征：MrF3'5'H基因的可编码cDNA序列如SEQ ID NO.1所示，编码序列全长为1530个核苷酸，可编码一个含509个氨基酸的蛋白。其氨基酸序列如SEQ ID NO.2所示，含有保守的proline-rich、SRS、CR、E××R和heme binding结构域，属于CYP450家族。
(2)基因功能特征：在酵母中进行的体外功能验证表明，MrF3'5'H具有P450羟化酶功能，可以将山奈酚和槲皮素分别催化为杨梅素，且对山奈酚的催化活性高于其同源蛋白VvF3'5'H。利用基因工程技术获得高表达F3'5'H的烟草转基因植株，相较于野生型，转基因株系叶片中杨梅素、槲皮素、和山奈酚含量显著增加，转基因株系花中杨梅素和飞燕草素含量显著增加，槲皮素和山奈酚含量无显著变化。
本发明对研究杨梅素分流机制具有指导意义，为开发工程微生物菌或基于基因工程技术的植物杨梅素组分改良奠定基础。
附图说明
图1是类黄酮基本结构图，类黄酮基本结构由A环、B环和C环组成。
图2是‘荸荠(Biqi)’和‘东魁(Dongkui)’杨梅不同组织中杨梅素和槲皮素含量分析图。
图3是MrF3'5'H氨基酸序列比对结果图；SlF3'5'H(ACF32346)，PhF3'5'H-Hf1(CAA80266)，PhF3'5'H-Hf2(CAA80265)，F3'5'H蛋白含有保守的proline-rich、SRS、CR、E××R和heme binding结构域。
图4是重组蛋白MrF3'5'H体外活性LC-MS图谱分析图。图4显示了MrF3'5'H具有催化3’位点和/或5’位点发生羟化反应的功能。MrF3'5'H可催化B环单羟基类黄酮柚皮素、二氢山柰酚和山柰酚分别发生3’位点生成B环双羟基类黄酮圣草酚(EIC 287)、二氢槲皮素(EIC 303)和槲皮素(EIC 301)，分别发生3’位点和5’位点羟化反应生成B环三羟基类黄酮五羟基双氢黄酮(EIC 303)、二氢杨梅素(EIC 319)和杨梅素(EIC 317)；MrF3'5'H可催化B环双羟基类黄酮圣草酚、二氢槲皮素和槲皮素分别发生5’位点羟化反应生成B环三羟基类黄酮五羟基双氢黄酮(EIC 303)、二氢杨梅素(EIC 319)和杨梅素(EIC 317)。Nar：柚皮 素；Eri：圣草酚(EIC 287)；PHF：五羟基双氢黄酮(EIC 303)；DHK：二氢山奈酚；DHQ：二氢槲皮素(EIC 303)；DHM：二氢杨梅素(EIC 319)；K：山奈酚；Q：槲皮素(EIC 301)；M：杨梅素(EIC 317)。
图5是重组蛋白MrF3'5'H催化底物和产物的结构式。柚皮素、二氢山奈酚、山奈酚属于B环单羟基的类黄酮，圣草酚、二氢槲皮素、槲皮素属于B环双羟基的类黄酮，五羟基双氢黄酮、二氢杨梅素、杨梅素属于B环三羟基的类黄酮。
图6是MrF3'5'H进化树分析图；CrF3'5'H(Q9ZRY0)，GhF3'5'H(AAP31058)，GmF3'5'H(AAM51564)，GtF3'5'H(Q96581)，NtF3'5'H(XP_016452599)，PhF3'5'H(Hf1)(CAA80266)，PhF3'5'H(Hf2)(CAA80265)，SlF3'5'H(ACF32346)，StF3'5'H(NP_001274807)，VmF3'5'H(BAC97831)，VvF3'5'H(CAI54277)。
图7是MrF3'5'H和VvF3'5'H底物催化特性分析图。
图8是过量表达MrF3'5'H的烟草表型和类黄酮含量分析图。其中，(a)图代表过量表达MrF3'5'H烟草花色改变；(b)图代表过量表达MrF3'5'H显著提高烟草叶片中杨梅素、槲皮素和山奈酚含量，显著提高花中杨梅素和飞燕草素含量。
具体实施方式
本发明结合附图和实施例作进一步的说明。
说明：本发明中涉及的目的基因引物设计、全长克隆、表达载体构建、RNA提取、cDNA合成、测序分析和鉴定以及PCR产物的分离纯化等基本操作，可按照本领域已知的技术进行，若未特别说明，实施例中的技术手段为本领域技术人员熟知的常规手段。
实施例1：杨梅不同组织黄酮醇含量检测
1.杨梅组织材料
荸荠和东魁杨梅组织(果实、花、叶片)当天采收，经液氮冷冻后存放于-80℃冰箱，每个组织样品设置3个生物学重复，每个重复7-8个果实，每个重复花的质量在500g以上，每个重复10-15片整叶。
2.黄酮醇含量检测
分别称量0.3g左右鲜样粉末，按1:10(g：mL)溶于80％甲醇(1％HCL)中，浸泡2h，超声30min，重复3次，合并上清液，得粗提液6mL。取1mL提取液30℃旋转真空浓缩到只剩水相，10000rpm离心5min，定容到200μL色谱甲醇(1％HCL)中，过0.22μM水系滤膜(LABMAX)滤膜，备用。检测色谱柱为固定相(SunfireC18 5μm(4.6*250mm))，流动相为A：(0.1％甲酸)B：乙腈：水(0.1％甲酸)＝1:1，进样体积10μL，流速1mL/min，柱温25℃，体系：0-20min 28％B，20-25min 28-36％B，25-40min 36％B，40-45min 36-50％B，45-60min 50-100％B，60-65min 100％B，65-70min 100-28％B，70-75min 28％B。所用流动相均为色谱级。
在荸荠和东魁杨梅叶片和花中，杨梅素含量高达10mg g -1FW，果实发育过程中，杨梅素含量呈下降趋势(附图2)。
实施例2：MrF3'5'H基因全长获得及鉴定
1.RNA提取和cDNA合成
杨梅组织样品在液氮环境中研磨成粉末，利用普通CTAB法提取总RNA，经电泳检测合格后，参照TURBO DNAase Kit(Ambion)说明书，去除DNA，根据iScript cDNA Synthesis Kit(Bio-Rad)要求，取1.0μg RNA，反转录成cDNA。
3.MrF3'5'H基因全长获得
在荸荠杨梅的RNA-Seq数据库中，以Flavonoid 3',5'-hydroxylase为关键词搜索与杨梅素合成相关的基因，并以番茄中功能明确的SlF3'5'H氨基酸序列为参考，通过CLUSTALX 软件同源比对，筛选出一个可能参与杨梅果实杨梅素合成的基因Unigene5190(MrF3'5'H)，应用序列为：SEQ ID NO.1，并通过BLAST(https://blast.ncbi.nlm.nih.gov/Blast.cgi)在线分析，确认其为全长序列。设计全长克隆引物：SEQ ID NO.3和SEQ ID NO.4，PCR反应体系为50μL，成分分别为：0.5μL Roche高保真酶，5μL缓冲液(10×)，4μL dNTP(2.5mM)，上下游引物(10μM，Hua Gene)各2μL，4μL cDNA，32.5μL H 2O。反应程序为：预变性95℃，2min；变性95℃，30s；退火58℃，30s；延伸72℃，90min，35个循环；72℃延伸10min，4℃保存。
4.MrF3'5'H基因全长鉴定及序列分析
将PCR扩增产物连接到T-easy载体，转化大肠杆菌DH5α，进行菌落PCR验证，获得阳性菌落进行测序。克隆结果经测序验证，获得与转录组数据库相匹配的MrF3'5'H全长序列如SEQ ID NO.1所示含有1530个核苷酸。在线翻译成氨基酸序列(http://web.expasy.org/translate/)，即：SEQ ID NO.2。利用MrF3'5'H氨基酸序列与已发表具有3’5’位点羟化的P45075A家族羟化酶比对，结果如图3所示。
申请人成功克隆了MrF3'5'H基因，即MrF3'5'H的cDNA序列(参见序列表SEQ ID NO.1)。MrF3'5'H基因经细胞内的转录和翻译过程合成对应的多肽或蛋白质，即MrF3'5'H的氨基酸序列(参见序列表SEQ ID NO.2)。氨基酸序列分析表明，SEQ ID NO.2具有典型的proline-rich、SRS、CR、E××R和heme binding保守结构域。
实施例3：pYES2-MrF3'5'H表达载体的构建
根据pYES2 NT/C(Invitrogen)载体多克隆位点序列及MrF3'5'H(SEQ ID NO.1)全长基因序列，设计包含BamHI和EcoRI酶切位点的引物序列：SEQ ID NO.5和SEQ ID NO.6，该引物设计包含起始密码子和终止密码子，扩增得到含有BamHI和EcoRI酶切位点的MrF3'5'H序列。PCR反应体系为50μL，成分分别为：1μL Phanta高保真酶(Vazyme)，25μL缓冲液(2×)，1μL dNTP(10mM)，上下游引物(10μM，Hua Gene)各2μL，1μL cDNA，18μL H 2O。反应程序为：预变性95℃，2min；变性95℃，15s；退火58℃，15s；延伸72℃，1min，35个循环；72℃彻底延伸5min，4℃保存。分别用BamHI(NEB)和EcoRI(NEB)双酶切pYES2载体，使用
Figure PCTCN2021083269-appb-000001
II连接酶(Vazyme)将目的基因片段连接到pYES2载体上。连接反应体系为10μL，成分分别为：1μL
Figure PCTCN2021083269-appb-000002
II连接酶(Vazyme)，2μL缓冲液(5×)，1μL PCR回收产物，3μL载体，3μL H 2O。混匀后，37℃连接0.5h后冰上放置5min。将连接产物转化到DH5а感受态(Takara)中,涂布于含有Amp的培养板上37℃过夜培养，挑取阳性克隆菌株，送往上海Hua Gene测序，分析测序结果，含有正确目的基因序列的载体，则为构建成功的pYES2-MrF3'5'H重组质粒。
实施例4：酿酒酵母异源表达MrF3'5'H
1.重组载体酵母转化
将构建成功的pYES2-MrF3'5'H重组质粒或pYES2空载体，通过酵母转化试剂盒(Clontech)用LiAC法转化至酿酒酵母株系INVScI(Invitrogen)。然后涂布于SD/-Ura的培养板上30℃培养3天，挑取单菌落，PCR检测重组质粒或空载。选取PCR条带正确的单菌落，则为含有pYES2-MrF3'5'H重组质粒的酿酒酵母INVScI，用25％甘油保存于-80℃冰箱备用。
2.MrF3'5'H诱导表达
挑取单菌落于5mL SD/-Ura+20g/L glucose培养液，在30℃，250rpm摇床上培养12h。室温条件下700g离心5min收集酵母菌体，然后加入SD/-Ura+20g/L galactose培养液重悬浮菌体至OD 600为0.4。取2mL重悬浮菌体后的培养液至两个新的离心管，各自加入1mM NADPH(Sigma)和100μM反应底物，16℃，250rpm摇床上培养12h。以上操作均以空载酵母作为对照。
3.类黄酮检测
诱导完成后，加入1∶1体积的乙酸乙酯溶液终止反应，涡旋混匀，1000g离心5min，取上清到新的10mL离心管，重复操作一次，合并上清，真空旋转蒸干有机相，加入150μL的色谱甲醇溶解备用。液相色谱检测流动相：A：水(0.1％甲酸)B：乙腈(0.1％甲酸)；进样体积：10μL；流速：0.3mL/min；柱温：25℃；检测波长为370nm；洗脱梯度：0-7min 90％-50％A，7-10min 50％A，10-15min 50％-0％A，15-15.1min 0-90％A，15.1-21min 90％A。
检测结果表明，MrF3'5'H具有P45075A家族羟化酶的活性，可催化B环单羟基的柚皮素发生3’位点羟化反应生成B环双羟基的圣草酚(EIC 287)，并发生3’位点和5’位点羟化反应生成B环三羟基的五羟基双氢黄酮(EIC 303)；MrF3'5'H蛋白可催B环单羟基的二氢山柰酚发生3’位点羟化反应生成B环双羟基的二氢槲皮素(EIC 303)，并发生3’位点和5’位点羟化反应生成B环三羟基的二氢杨梅素(EIC 319)；MrF3'5'H蛋白可催B环单羟基的山柰酚发生3’位点羟化反应生成B环双羟基的槲皮素(EIC 301)，并发生3’位点和5’位点羟化反应生成B环三羟基的杨梅素(EIC 317)；MrF3'5'H蛋白可催B环双羟基的圣草酚发生5’位点羟化反应生成B环三羟基的五羟基双氢黄酮(EIC 303)；MrF3'5'H蛋白可催B环双羟基的二氢槲皮素发生5’位点羟化反应生成B环三羟基的二氢杨梅素(EIC 319)；MrF3'5'H蛋白可催化B环双羟基的槲皮素发生5’位点羟化反应生成B环三羟基的杨梅素(EIC 317)(附图4和附图5)。
实施例5：MrF3'5'H进化树构建
进化树构建由MEGA7.0(Mega Software,美国)软件完成。首先使用MEGA 7.0内置的内置的ClustalW工具比对F3'5'H的氨基酸序列，之后用邻接法(Neighbor–Joining method)构建系统进化树树，并用步长检验法(Bootstrap method)评估进化树的质量，检验次数设为1000。
结果表明，MrF3'5'H确实属于P45075A家族蛋白，在进化树上与葡萄距离最近VvF3'5'H，核苷酸同源性为78.21％，氨基酸同源性为82.87％，与GtF3'5'H距离最远，核苷酸同源性为66.41％，氨基酸同源性为71.71％(附图6)。
实施例6：酿酒酵母异源表达VvF3'5'H
葡萄VvF3'5'H分离于‘夏黑’果皮，具体操作同实施例3。酿酒酵母表达VvF3'5'H操作同实施例4。
结果表明，MrF3'5'H和VvF3'5'H对柚皮素、圣草酚、二氢山奈酚、二氢槲皮素、山奈酚和槲皮素的催化特性有所不同，MrF3'5'H对山奈酚的催化活性显著强于VvF3'5'H(附图7)。
实施例7：转基因烟草过量表达MrF3'5'H促进杨酶素积累
1.转基因载体构建
应用引物组合SEQ ID NO.7和SEQ ID NO.8，扩增F3'5'H(SEQ ID NO.1)的全长序列，搭载到pGreenⅡ002962-SK表达载体上，构建重组表达载体SK-F3'5'H。此PCR反应体系同实施例3中的PCR反应体系。将最终构建正确的表达载体电击法转入农杆菌株系GV3101∷pSoup，挑选3个阳性克隆菌株，用终浓度25％灭菌甘油保存于-80℃。
2.转基因植株鉴定
经基因工程技术转化烟草普通植株获得转基因植株后，需进一步通过PCR手段验证。利用CTAB法，提取烟草叶片总DNA，结合引物对SEQ ID NO.9和SEQ ID NO.10，进行PCR扩增鉴定转基因烟草植株，所得阳性植株进行后续培养，经过两代筛选，获得T1代转基因烟草植株。
3.黄酮醇含量检测
充分研磨烟草花，准确称取0.1g粉末加入1mL 50％甲醇水溶液中，超声30min，然后11000rpm离心15min，吸取700μL上清液至新管中，并加入300μL的3N HCL溶液，70℃水浴1h。然后13000rpm离心15min，吸取100μL上清液用于HPLC分析。检测过程同实施例5中黄酮醇检测。
结果表明，与野生型烟草相比，转基因植株叶片中杨梅素、槲皮素和山奈酚含量显著提高，转基因植株花色变深，杨梅素和飞燕草素被显著诱导，证明MrF3'5'H编码的蛋白在植物体内具有生物学功能，参与了杨酶素和飞燕草素生物合成(附图8)。来源于茶的CsF3'5'H异源表达到烟草中，转基因株系花中飞燕草素含量显著增加，检测到的杨梅素因含量太低而无法定量(Wang et al.,2014 Functional analysis of flavonoid 3′,5′-hydroxylase from tea plant(Camellia sinensis):critical role in the accumulation of catechins.BMC Plant Biol.14:347.)，表明MrF3'5'H合成杨梅素的能力较强。
申请人首次验证了MrF3'5'H在杨梅素合成中的独特作用。MrF3'5'H在进化树上聚类在CYP75A家族，与VvF3'5'H距离最近，MrF3'5'H和VvF3'5'H的核苷酸序列同源性为78.21％，MrF3'5'H和VvF3'5'H的氨基酸序列同源性为82.87％(附图3和附图6)。对比MrF3'5'H和VvF3'5'H蛋白功能可知，MrF3'5'H和VvF3'5'H蛋白功能相似，均可催化B环单羟基类黄酮和/或B环双羟基类黄酮合成B环三羟基类黄酮(附图7)，说明聚类在同一家族上的羟化酶蛋白功能相似。但是，MrF3'5'H和VvF3'5'H的底物催化特性有所差异，MrF3'5'H对山奈酚的催化活性显著高于VvF3'5'H(附图7)。利用基因工程技术获得过表达MrF3'5'H的烟草转基因植株，相较于野生型，转基因株系叶片中杨梅素、槲皮素、和山奈酚含量显著增加，转基因株系花中杨梅素和飞燕草素含量显著增加，槲皮素和山奈酚含量无显著变化。本工作对研究杨梅素分流机制具有指导意义，为开发工程微生物菌或基于基因工程技术的植物杨梅素组分改良奠定基础。
以上对本发明的具体实施例进行了描述，需要理解的是，对于本领域普通技术人员来说，在权利要求范围内，可以根据上述说明加以改进或变换，这并不影响本发明的实质内容。

Claims (16)

  1. 一种分离的核苷酸序列，其特征在于，
    所述分离的核苷酸序列编码F3'5'H羟化酶基因，包含至少具有下列1)～5)特征之一的核苷酸序列：
    1)SEQ ID NO.1表示的核苷酸序列；
    2)编码SEQ ID NO.2氨基酸序列的核苷酸序列；
    3)与SEQ ID NO.1的核苷酸序列具有80％以上同源性，且编码相同功能蛋白质的核苷酸序列；
    4)与SEQ ID NO.1表示的DNA序列杂交的核苷酸序列；
    5)与SEQ ID NO.1编码相同功能蛋白质的核苷酸序列。
  2. 根据权利要求1所述的核苷酸序列，其特征在于，所述羟化酶基因是MrF3'5'H，所述羟化酶基因的核苷酸序列是SEQ ID NO.1。
  3. 一种多肽或蛋白质，其特征在于，包含至少具有下列1)～4)特征之一的氨基酸序列：
    1)SEQ ID NO.2表示的氨基酸序列；
    2)与SEQ ID NO.2具有80％以上同源性，且显示羟化功能的多肽或蛋白质；
    3)将序列表中的SEQ ID NO.2的氨基酸残基序列经过一个或2个以上氨基酸残基的取代和/或缺失和/或添加且显示羟化功能的蛋白质；
    4)具有proline-rich、SRS、CR、E××R和heme binding保守结构域的氨基酸序列。
  4. 一种从植物组织中提取的羟化酶，所述植物能生成杨梅素，其特征在于：
    包含至少具有下列1)～4)特征之一的氨基酸序列：
    1)SEQ ID NO.2表示的氨基酸序列；
    2)与SEQ ID NO.2具有80％以上同源性，且显示羟化功能的多肽或蛋白质；
    3)将序列表中的SEQ ID NO.2的氨基酸残基序列经过一个或2个以上氨基酸残基的取代和/或缺失和/或添加且显示羟化功能的蛋白质；
    4)具有proline-rich、SRS、CR、E××R和heme binding保守结构域的氨基酸序列。
  5. 根据权利要求4所述的羟化酶，在催化类黄酮发生3’位点和/或5’位点羟化反应中的应用。
  6. 一种基因表达载体，其特征在于：所述的载体含有权利要求1所述核苷酸具有的核苷酸序列。
  7. 一种基因表达载体，其特征在于：所述的载体含有权利要求2所述的羟化酶基因具有的核苷酸序列。
  8. 一种转基因细胞系或宿主菌，其特征在于：所述的转基因细胞系或宿主菌含有权利要求6所述的基因表达载体。
  9. 一种转基因细胞系或宿主菌，其特征在于：所述的转基因细胞系或宿主菌含有权利要求7所述的基因表达载体。
  10. 一种B环三羟基类黄酮的制备方法，其特征在于，包括以下步骤：
    向宿主菌中转导权利要求7所述的基因表达载体；
    向宿主菌提供原料B环单羟基类黄酮和/或B环双羟基类黄酮。
  11. 一种杨梅素的制备方法，其特征在于：所述的制备方法选自以下1)～2)的任 何一种：
    1)向植物体或植物组织转入权利要求6所述的基因表达载体；
    从所述植物体或植物组织或细胞系或宿主菌提取杨梅素；
    2)向细胞系或宿主菌转入权利要求6所述的基因表达载体；
    向细胞系或宿主菌提供原料山奈酚和/或槲皮素。
  12. 一种五羟基双氢黄酮的制备方法，其特征在于，包括以下步骤：
    向宿主菌中转导权利要求7所述的基因表达载体；
    向宿主菌提供原料柚皮素和/或圣草酚。
  13. 一种二氢杨梅素的制备方法，其特征在于，包括以下步骤：
    向宿主菌中转导权利要求7所述的基因表达载体；
    向宿主菌提供原料二氢山奈酚和/或二氢槲皮素。
  14. 权利要求2所述的核苷酸序列、权利要求7所述的表达载体、权利要求9所述的细胞系或宿主菌在基因工程中的应用。
  15. 一种转基因植物的培育方法，其特征在于，包括以下步骤：
    向植物中导入权利要求7所述基因表达载体，获得转基因植物。
  16. 一种转基因烟草，其特征在于：包括以下步骤：
    向烟草中导入权利要求6所述基因表达载体，获得转基因烟草。
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