CN105039274A - Gene cluster participating in synthesis of cucurbitacin E of watermelon and application of gene cluster - Google Patents
Gene cluster participating in synthesis of cucurbitacin E of watermelon and application of gene cluster Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
- C12N9/0036—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6)
- C12N9/0038—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6) with a heme protein as acceptor (1.6.2)
- C12N9/0042—NADPH-cytochrome P450 reductase (1.6.2.4)
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1025—Acyltransferases (2.3)
- C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/90—Isomerases (5.)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y106/00—Oxidoreductases acting on NADH or NADPH (1.6)
- C12Y106/02—Oxidoreductases acting on NADH or NADPH (1.6) with a heme protein as acceptor (1.6.2)
- C12Y106/02004—NADPH-hemoprotein reductase (1.6.2.4), i.e. NADP-cytochrome P450-reductase
Abstract
The invention relates to a gene cluster participating in synthesis of cucurbitacin E of watermelon and an application of the gene cluster. According to the invention, the gene cluster participating in synthesis of cucurbitacin E is found in the watermelon genome for the first time, the gene cluster comprises eight genes, wherein six of the eight genes are positioned within the range of the No.6 chromosome 57kb. A yeast system is adopted for analyzing the reaction from the step 1 to the step 4 of the synthesis of cucurbitacin E. The invention further discloses a molecular mechanism for forming the bitter taste of the watermelon, so that a theoretical basis and a molecule assistant breeding objective are provided for the breeding of the watermelon without the bitter taste, and valuable experiences are provided for heterologous biosynthesis and development and utilization of cucurbitacine. The technology provided by the invention can replace the traditional method for extracting bitter principle from a plant material, so that synthesis of the bitter principle in vitro is realized by utilizing synthetic biology.
Description
Technical field
The present invention relates to genetically engineered and biology field, specifically, relate to the gene cluster and application thereof that participate in the synthesis of watermelon Cucurbitacin E.
Background technology
The bitter taste of watermelon is called that the triterpenoid of Cucurbitacin E causes by a class.Mouthfeel and quality that Cucurbitacin E can have a strong impact on watermelon is accumulated in Watermelon Fruit.Early-stage Study utilizes the means such as genomics, molecular biology, biological chemistry to disclose the route of synthesis of the cucumber bitter substance cucurbitacin C participated in by 9 genes; comprising 1 squalene oxide cyclase Bi; 7 cytochrome P450 genes and 1 acetyl transferase gene; and utilize biochemical method to illustrate four-step reaction completely; its research for the cucurbitacin Biosynthetic pathway of other types in cucurbitaceous plant has important directive significance, also for the path parsing of other triterpenoids in plant provides new approaches.Cucurbitacin has pest-resistant effect, and medical research proves that cucurbitacin can suppress multiple growth of cancer cells, and current cucurbitacin mainly extracts and obtains from plant, does not synthesize by heterologous organisms.And participate in the biosynthetic gene of Cucurbitacin E in watermelon and be not also cloned, the parsing of Cucurbitacin E Biosynthetic pathway will be exploitation
novelcancer therapy drug provides basis.
Summary of the invention
The object of this invention is to provide the gene cluster and application thereof that participate in the synthesis of watermelon Cucurbitacin E.
In order to realize the object of the invention, the invention provides the albumen participating in the synthesis of watermelon Cucurbitacin E, comprise Cla007077, Cla007078, Cla007079, Cla007080, Cla007081, Cla007082, Cla008354 and Cla008355, their aminoacid sequence is respectively as shown in SEQIDNo.1-8.
The present invention also provides the gene cluster participating in the synthesis of watermelon Cucurbitacin E, described gene cluster is by following 8 genomic constitutions, Cla007077, Cla007078, Cla007079, Cla007080, Cla007081, Cla007082, Cla008354 and Cla008355, their nucleotide sequence is respectively as shown in SEQIDNo.10-17.
The present invention also provides the carrier of the gene cluster containing the synthesis of described participation watermelon Cucurbitacin E, engineering bacteria and host cell.
The application of the gene cluster that the present invention also provides described participation watermelon Cucurbitacin E to synthesize in the synthesis of regulation and control watermelon bitter taste.
The application of the gene cluster that the present invention also provides described participation watermelon Cucurbitacin E to synthesize in the external synthesis of regulation and control cucurbitacin.
The gene cluster that the present invention also provides described participation watermelon Cucurbitacin E to synthesize is without the application in bitter taste watermelon molecular breeding.
The means such as genomics, molecular biology, biological chemistry are utilized to disclose the route of synthesis of the cucumber bitter substance cucurbitacin C participated in by 9 genes in early-stage Study, based on this, by the cyclase gene in watermelon and the squalene oxide cyclase gene Bi in cucumber are carried out phylogenetic analysis, have found a gene C la007080 (Bi) the highest with cucumber Bi genetic homology, utilizing yeast expression system to demonstrate this genes encoding cucurbit dienol synthetic enzyme, is the key enzyme of cucurbitacin synthesis.Cucurbit dienol also needs oxidized and acetylize further to generate cucurbitacin.In order to resolve other steps of Cucurbitacin E synthesis, utilize comparative genomics method in watermelon genome, have found a gene cluster being similar to cucumber cucurbitacine C and synthesizing: to be made up of 6 P450 genes (Cla007077, Cla007078, Cla007079, Cla007082, Cla008354 and Cla008355), 1 acetyl transferase gene (Cla007081) and 1 Bi gene (Cla007080).The expression status of these 8 genes in each tissue of watermelon is closely similar, only great expression in root.In addition, except gene C la008354 and Cla008355, other genes and Bi gene are all positioned at the scope of No. 6 karyomit(e) 57kb, form the structure of a similar gene cluster.At present, participate in the synthesis of important secondary metabolite with the form of gene cluster and extensively found in higher plant, therefore infer that these 8 candidate genes probably take part in the synthesis of Cucurbitacin E.
Utilize yeast expression system further, in yeast, express these candidate genes, by the accurate molecular weight of UPLC-Q-TOF instrument assay products, infer contingent oxidation reaction product, resolve Cucurbitacin E synthetic mesophase step.First, two specific product have been found in the yeast of expressing Bi, reductase enzyme CPR and Cla008354, a molecular weight is 441.3727 [M+H], another is 457.3676 [M+H], infer that Cla008354 is multi-functional P450 through MS, MS/MS interpretation of result, first carbonylation in the synthesis of cucurbit dienol, rehydroxylation.Out, utilize nucleus magnetic resonance (NMR) to resolve its structure this product purifies and separates from yeast further, find that carbonylation (C first occurs No. 11 carbon atoms of cucurbit dienol
30h
48o
2), then on No. 20 carbon atoms, there is hydroxylation (C
30h
48o
3), and then card Cla008354 can continuous oxidation cucurbit dienol, the second step that catalysis Cucurbitacin E synthesizes and the 3rd step.Due to gene C la008354 and Cla008355 very high homology, through identical method validation gene C la008355, also there is identical function, namely also No. 11 carbon atoms of catalysis cucurbit dienol carbonylation (C can first occur
30h
48o
2), then on No. 20 carbon atoms, there is hydroxylation (C
30h
48o
3).Continue to utilize yeast expression system, express more candidate gene, utilize similar method, only in the yeast extract of expressing Bi, CPR, Cla008354 (or Cla008355) and Cla007079, find that molecular weight is the specific product of 473.3625 [M+H], infer that previous step catalysate is by Cla007079 hydroxylation, by this product, from yeast, purifies and separates is out further, utilize nucleus magnetic resonance (NMR) to resolve its structure, find to there occurs hydroxylation (C on No. 2 carbon atoms
30h
48o
4), and then demonstrate the 4th step of Cla007079 catalysis Cucurbitacin E synthesis.
The present invention finds the gene cluster participating in Cucurbitacin E synthesis first in watermelon genome, and altogether by 8 genomic constitutions, wherein 6 genes are all positioned at the scope of No. 6 karyomit(e) 57kb.Yeast system is utilized successfully to resolve the 1st to the 4th step reaction of Cucurbitacin E synthesis.Invention further discloses the molecular mechanism that watermelon bitter taste is formed, for providing theoretical foundation and marker assisted selection target without bitter taste watermelon breeding, can be used for Large-scale Screening breeding material, greatly accelerate the breeding process of high-quality watermelon; Simultaneously also for the external synthetic of cucurbitacin provides invaluable experience, the alternative traditional method extracting bitter principle (cucurbitacin) from vegetable material of the present invention, utilize synthetic biology thus realize external synthesis bitter principle.
Accompanying drawing explanation
fig. 1for the present invention is by comparative genomics and RNA-Seq data analysis, find to synthesize relevant gene cluster to bitter principle (Cucurbitacin E) from watermelon genome.
fig. 2for the present invention utilizes UPLC-Q-TOF to detect the result of participation bitter principle synthesis second step and three-step reaction; Wherein, A is the specific product of Cla008354 and Cla008355 that UPLC-Q-TOF detects, B is the MS/MS detected result of this specific product, and C is nuclear magnetic resonance nmr structure elucidation result.
fig. 3for the present invention utilizes UPLC-Q-TOF detection participation bitter principle to synthesize the result of four-step reaction; Wherein, A is the specific product of the Cla007079 that UPLC-Q-TOF detects, B is the MS/MS detected result of this specific product, and C is nuclear magnetic resonance nmr structure elucidation result.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.If do not specialize, embodiment is experiment condition all conveniently, as Sambrook equimolecular Cloning: A Laboratory Manual (SambrookJ & RussellDW, Molecularcloning:alaboratorymanual, 2001) condition of, or according to manufacturer's specification sheets advising.
Embodiment 1 participates in excavation and the acquisition of the gene cluster of watermelon Cucurbitacin E synthesis
1, the excavation of gene cluster
Based on bunch research of cucumber bitter substance cucurbitacin C synthetic gene, utilize comparative genomics method, the gene cluster that one is similar to cucumber cucurbitacine C synthesis is have found in watermelon genome, find 8 genes altogether, comprise 1 cucurbit dienol synthase gene Bi (Cla007080), 6 P450 gene (Cla007077, Cla007078, Cla007079, Cla007082, Cla008354 and Cla008355) and 1 acetyl transferase gene (Cla007081), except Cla008354 and Cla008355 gene, other genes are all positioned at the scope of No. 6 karyomit(e) 57kb, form the structure of a similar gene cluster, and these 8 genes special coexpression in watermelon root, be proportionate with Cucurbitacin E content in individual tissue (
fig. 1), think that this gene cluster participates in the synthesis of Cucurbitacin E thus.
2, the acquisition of watermelon Cla007077, Cla007078, Cla007079, Cla007080, Cla007081, Cla007082, Cla008354 and Cla008355 gene
First extract total mRNA in watermelon root, and reverse transcription becomes cDNA, (primer sequence is shown in then to utilize forward primer and reverse primer to carry out pcr amplification
table 1).
table 1primer sequence (5 '-3 ')
| Cla007077-TVector-F | GCTGCAGAAGAATGCTTCACCAAAA |
| Cla007077-TVector-R | GCAGCTTGGTTGCAATTTCTCTTGC |
| Cla007078-TVector-F | GGATGGTTGGCTAACAGCGCTCC |
| Cla007078-TVector-R | ACAAGCCTTGGAGACAAGTAAGCA |
| Cla007079-TVector-F | TGGAGACCACCACCTATTTTCCATTT |
| Cla007079-TVector-R | TGGGACGAGCTCTACACAATGCC |
| Cla007080-TVector-F | AGTTGGGGCCGAGAGCGTTG |
| Cla007080-TVector-R | ACCCGATGGAAATACTCTCCAAGAGCC |
| Cla007081-TVector-F | TCCATTCCAACGCCTCAAACCCA |
| Cla007081-TVector-R | TGTTGGAGCTGAAGAACATTGGGA |
| Cla007082-TVector-F | CGCCGGGATTAGCCATCGCC |
| Cla007082-TVector-R | AGCTTTTGCTGAGAAGCTGACATGA |
| Cla008354-TVector-F | GGACAATCTTGCTCGGTTTGGCG |
| Cla008354-TVector-R | CACATGTAGCCCATCTTCAAACCTCA |
| Cla008355-TVector-F | CTTTCTCGGTTTCGTGACGCTGACA |
| Cla008355-TVector-R | TGAAGTTCACATGTAGCCCATCAACA |
PCR reaction system is counted with 20 μ l: 10-20ng/ μ l template 1 μ l, and the forward and reverse primer of 10pmol/ μ l each 1 μ l, 10mmol/LdNTPmix0.4 μ l, 0.5U/ μ L high-fidelity Taq DNA polymerase 1 μ l, 10 × PCR reaction buffer 2 μ l, surplus is water.
PCR reaction conditions is: 94 DEG C 5 minutes; 94 DEG C 20 seconds, 55 DEG C 20 seconds, 72 DEG C 2 points 30 seconds, 35 circulations; 72 DEG C 10 minutes.
By increasing, the fragment obtained is connected with T-carrier (TAKARA), and order-checking confirms not sudden change.The nucleotide sequence of watermelon Cla007077, Cla007078, Cla007079, Cla007080, Cla007081, Cla007082, Cla008354 and Cla008355 gene is respectively as shown in SEQIDNo.10-17.
Embodiment 2 utilizes yeast expression system to verify the function of Cucurbitacin E synthesis candidate gene
Cla007077, Cla007078, Cla007079, Cla007080, Cla007081, Cla007082, Cla008354 and Cla008355 gene and reductase enzyme CPR gene are building up on expression vector pYES2 (Invitrogen) respectively, and transformed yeast bacterium.Induction yeast expression albumen, collects yeast.Sherwood oil extract compounds is used after cracking.Detect with UPLC-Q-TOF after sample preparation is good.Result respectively
as Fig. 2,
fig. 3shown in.
fig. 2the result participating in bitter principle synthesis second step and three-step reaction is detected for utilizing UPLC-Q-TOF; Wherein, A is the specific product of Cla008354 and Cla008355 that UPLC-Q-TOF detects, B is the MS/MS detected result of this specific product, and C is nuclear magnetic resonance nmr structure elucidation result.
fig. 3the result participating in bitter principle and synthesize four-step reaction is detected for utilizing UPLC-Q-TOF; Wherein, A is the specific product of the Cla007079 that UPLC-Q-TOF detects, B is the MS/MS detected result of this specific product, and C is nuclear magnetic resonance nmr structure elucidation result.
Cla018830 genes encoding reductase enzyme CPR (SEQIDNo.9) is the reductase gene of P450 gene when oxidation substrates, adds CPR and is conducive to improving efficiency.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Sequence illustrates:
SEQIDNo.1-9 is respectively the aminoacid sequence of Cla007077, Cla007078, Cla007079, Cla007080, Cla007081, Cla007082, Cla008354, Cla008355 and CPR albumen.
SEQIDNo.10-18 is respectively the nucleotide sequence of gene C la007077, Cla007078, Cla007079, Cla007080, Cla007081, Cla007082, Cla008354, Cla008355 and CPR.
Reference:
1.SekiH,etal.(2008)Licoriceβ-amyrin11-oxidase,acytochromeP450withakeyroleinthebiosynthesisofthetriterpenesweetenerglycyrrhizin.ProceedingsoftheNationalAcademyofSciences105(37):14204.
2.vanHerpenTWJM,etal.(2010)NicotianabenthamianaasaProductionPlatformforArtemisininPrecursors.PLoSOne5(12):e14222.
3.YiShang,etal.(2014)Biosynthesis,regulation,anddomesticationofbitternessincucumber,Science,346(6213):1084-1088。
Claims (7)
1. participate in the albumen of watermelon Cucurbitacin E synthesis, it is characterized in that, comprise Cla007077, Cla007078, Cla007079, Cla007080, Cla007081, Cla007082, Cla008354 and Cla008355, their aminoacid sequence is respectively as shown in SEQIDNo.1-8.
2. participate in the gene cluster of watermelon Cucurbitacin E synthesis, it is characterized in that, described gene cluster is by following 8 genomic constitutions, Cla007077, Cla007078, Cla007079, Cla007080, Cla007081, Cla007082, Cla008354 and Cla008355, their nucleotide sequence is respectively as shown in SEQIDNo.10-17.
3. the carrier containing gene cluster described in claim 2.
4. the engineering bacteria containing gene cluster described in claim 2.
5. the application of gene cluster described in claim 2 in the synthesis of regulation and control watermelon bitter taste.
6. the application of gene cluster described in claim 2 in the external synthesis of regulation and control Cucurbitacin E.
7. gene cluster described in claim 2 is without the application in bitter taste watermelon molecular breeding.
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Cited By (8)
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| CN110305855A (en) * | 2019-06-26 | 2019-10-08 | 昆明理工大学 | Rhizoma Gastrodiae GeCPR gene and its application |
| WO2020096907A1 (en) * | 2018-11-07 | 2020-05-14 | Firmenich Incorporated | Methods for making high intensity sweeteners |
| CN111411099A (en) * | 2020-05-14 | 2020-07-14 | 云南农业大学 | Hemsleya amabilis acetyl transferase, coding gene thereof and application of hemsleya amabilis acetyl transferase in preparation of cucurbitacin |
| CN112063647A (en) * | 2020-09-17 | 2020-12-11 | 云南农业大学 | Construction method of saccharomyces cerevisiae recombinant strain Cuol01, saccharomyces cerevisiae recombinant strain Cuol02 and application |
| US11060124B2 (en) | 2017-05-03 | 2021-07-13 | Firmenich Incorporated | Methods for making high intensity sweeteners |
| CN113215121A (en) * | 2021-06-07 | 2021-08-06 | 云南师范大学 | Method for efficiently and heterogeneously synthesizing mogroside V precursor |
| CN114573672A (en) * | 2022-03-21 | 2022-06-03 | 中国农业科学院农业基因组研究所 | Transport protein of watermelon bitter substance cucurbitacin E and application thereof |
| US11357246B2 (en) | 2015-10-29 | 2022-06-14 | Firmenich Incorporated | High intensity sweeteners |
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| US11758933B2 (en) | 2015-10-29 | 2023-09-19 | Firmenich Incorporated | High intensity sweeteners |
| US11060124B2 (en) | 2017-05-03 | 2021-07-13 | Firmenich Incorporated | Methods for making high intensity sweeteners |
| WO2020096907A1 (en) * | 2018-11-07 | 2020-05-14 | Firmenich Incorporated | Methods for making high intensity sweeteners |
| CN110305855A (en) * | 2019-06-26 | 2019-10-08 | 昆明理工大学 | Rhizoma Gastrodiae GeCPR gene and its application |
| CN110305855B (en) * | 2019-06-26 | 2022-03-22 | 昆明理工大学 | Gastrodia elata GeCPR gene and application thereof |
| CN111411099A (en) * | 2020-05-14 | 2020-07-14 | 云南农业大学 | Hemsleya amabilis acetyl transferase, coding gene thereof and application of hemsleya amabilis acetyl transferase in preparation of cucurbitacin |
| CN111411099B (en) * | 2020-05-14 | 2022-09-27 | 云南农业大学 | Hemsleya amabilis acetyl transferase, coding gene thereof and application of hemsleya amabilis acetyl transferase in preparation of cucurbitacin |
| CN112063647A (en) * | 2020-09-17 | 2020-12-11 | 云南农业大学 | Construction method of saccharomyces cerevisiae recombinant strain Cuol01, saccharomyces cerevisiae recombinant strain Cuol02 and application |
| CN112063647B (en) * | 2020-09-17 | 2023-05-02 | 云南农业大学 | Construction method of saccharomyces cerevisiae recombinant Cuol01, saccharomyces cerevisiae recombinant Cuol02 and application |
| CN113215121B (en) * | 2021-06-07 | 2021-12-17 | 云南师范大学 | Method for efficiently and heterogeneously synthesizing mogroside V precursor |
| CN113215121A (en) * | 2021-06-07 | 2021-08-06 | 云南师范大学 | Method for efficiently and heterogeneously synthesizing mogroside V precursor |
| CN114573672A (en) * | 2022-03-21 | 2022-06-03 | 中国农业科学院农业基因组研究所 | Transport protein of watermelon bitter substance cucurbitacin E and application thereof |
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