CN114438109A - Osmanthus gene OfTPS13.2 and application thereof - Google Patents
Osmanthus gene OfTPS13.2 and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of molecular biology and discloses an osmanthus fragrans geneOfTPS13.2And the application thereof, wherein the beta-bisabolene synthetase gene highly expressed in the petals of the osmanthus fragrans is screened through genome data bioinformatics analysisOfTPS13.2，OfTPS13.2The full-length sequence of gene CDS is shown as SEQ ID NO.1 and contains geneOfTPS13.2The expression vector of the method is used for transforming saccharomyces cerevisiae and fermenting and culturing, the yield of the beta-bisabolene reaches 35.21mg/L, and the method has industrial production value.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to an osmanthus fragrans gene OfTPS13.2 and application thereof in production of beta-bisabolene
Background
beta-Bisabolene (beta-Bisabolene) is a sesquiterpene isolated from Paeonia lactiflora (Commiphora guidotti) and is a colorless oily liquid, which is insoluble in water, dissolved in organic solvents such as ethanol, and has warm, woody, citrus, floral, fruity, green, and sweet balsamic aroma. The beta-bisabolene can be used for preparing edible essences such as oranges, tropical fruits, bananas, pomelos, apples, raw pears and the like. Meanwhile, the beta-Bisabylene is an anti-tumor agent (anti-cancer agent) and can be used for researching breast cancer.
The sweet osmanthus is a famous sweet osmanthus plant and contains abundant terpenoids, and the sweet osmanthus can not only bring pleasant mental feeling to people, but also have health care values of sterilization, inflammation diminishing, oxidation resistance, aging resistance and the like. The genome of osmanthus fragrans contains a large number of aroma genes, but no related report of beta-bisabolene synthetase genes exists in osmanthus fragrans. The invention screens the beta-bisabolene synthetase gene in the osmanthus by utilizing the bioinformatics technology and performs functional verification in saccharomyces cerevisiae.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention screens a beta-bisabolene synthetase gene OfTPS13.2 highly expressed in the petals of the osmanthus fragrans from the genome of the osmanthus fragrans, and performs functional verification in saccharomyces cerevisiae.
In order to achieve the purpose, the invention adopts the following technical scheme:
sweet osmanthus beta-bisabolene synthetase gene OfTPS13.2: after the genomic data bioinformatics analysis, a terpenoid gene OfTPS13.2 highly expressed in the petals of the osmanthus fragrans is screened, and the CDS sequence of the gene is shown as SEQ ID NO. 1.
An expression vector contains a beta-bisabolene synthetase gene OfTPS13.2.
The recombinant saccharomyces cerevisiae expressing the OfTPS13.2 gene and the application thereof are as follows: transforming saccharomyces cerevisiae with the yeast expression vector containing the gene OfTPS13.2, and obtaining recombinant saccharomyces cerevisiae expressing the gene OfTPS13.2 through a resistance plate and sequencing screening; and (3) inoculating the recombinant saccharomyces cerevisiae into a YPD culture medium containing galactose, adding isopropyl myristate for covering, and extracting the beta-bisabolene after the fermentation is finished.
Compared with the prior art, the invention has the following advantages and beneficial effects:
the research of the beta-bisabolene synthetase gene in plants is very little, the beta-bisabolene synthetase gene is excavated in the osmanthus fragrans for the first time and is subjected to functional verification in saccharomyces cerevisiae, the yield of the beta-bisabolene reaches 35.21mg/L, and the method has industrial production value.
Drawings
FIG. 1 is a clone gel map of OfTPS13.2 from example 1
FIG. 2 is a map of the expression vector plasmid in example 2.
FIG. 3 shows the GC-MS detection of β -bisabolene.
Fig. 4 is a fragment diagram of β -bisabolene ions.
Detailed Description
The technical solutions of the present invention are described below with reference to the drawings and the specific embodiments, and the examples are only for explaining the present invention and are not used to limit the scope of the present invention.
Example 1 Gene screening and cloning
Gene screening and cloning: through the analysis of genome data bioinformatics, a beta-bisabolene synthetase gene (OfTPS13.2) highly expressed in the petals of the osmanthus fragrans is screened, specific primers OfTPS13.2-CDS-F and OfTPS13.2-CDS-R (shown in Table 1) for amplifying the full length of the OfTPS13.2 gene CDS are designed by Primer5.0 software, and amplification primers are synthesized by Beijing optisco Biotech limited. Taking the osmanthus fragrans cDNA as a template, amplifying a target gene CDS by referring to Phanta high-fidelity enzyme instruction, wherein PCR reaction systems and programs are shown in tables 2 and 3.
TABLE 1 primer List
TABLE 2 PCR reaction System
TABLE 3 PCR reaction procedure
Gel electrophoresis detection and gene sequencing: 30ml TAE added 0.45g agarose powder, microwave oven boiling after adding 3 u l 10000X nucleic acid dye, pouring into the rubber plate, after solidification sample. After electrophoresis at 120V for 30min at 150mA, the bands were observed on a Gel-Logie200 Gel scanning imager (as shown in FIG. 1). And recovering the target band, connecting the recovered target band with a T-vector, transforming escherichia coli for sequencing, wherein the sequence of the OfTPS13.2 gene CDS is shown as SEQ ID No.1, and extracting a Plasmid from a bacterial liquid with correct sequencing, wherein the Plasmid is named as OfTPS13.2-CDS-Plasmid and is used as a template for constructing a vector later.
Example 2 vector construction
Construction of a yeast vector: OfTPS13.2-CDS-Plasmid is used as a template, an upstream primer and a downstream primer of OfTPS13.2-BsaI-F/R (table 1) carrying BsaI enzyme cutting sites are designed to clone an OfTPS13.2 gene fragment, and the obtained PCR product and an expression vector Plasmid (a Plasmid map is shown in figure 2 and is presented to the Master group of Liu Tiangang) required to be constructed are subjected to Goldengate ligation. The PCR reaction system and the procedure are shown in tables 2 and 3, the product is directly transformed into escherichia coli, monoclonal positive detection is carried out, the positive single colony is shaken to extract plasmids, then enzyme digestion verification is carried out, the plasmids with correct enzyme digestion verification are sequenced, and the plasmids with correct sequencing are named as: OfTPS13.2-Pkz762-Plasmid for the subsequent transformation of s.cerevisiae.
Example 3 Yeast transformation and plate screening
YPD solid medium preparation: 2% of peptone, 1% of yeast extract and 10g/L of agar, adding distilled water, heating and dissolving, and fixing the volume to 0.9 time of the final volume; sterilizing at 115 deg.C for 30min, and adding 0.1 volume of 20% glucose solution to obtain YPD medium. SC-URA screening medium: 0.67% yeast nitrogen source basic culture medium YNB, weighing the amino acid mixture lacking uracil according to the following table 4, adjusting the pH to 6.5 by using 2M NaOH solution, adding distilled water, heating for dissolving, fixing the volume to 0.9 time, and sterilizing at 115 ℃ for 30 min; after sterilization, a 0.1-fold volume of 20% glucose solution was added.
TABLE 4 amino acid mixture formula
10 × TE configuration: 100mM Tris and 10mM EDTA, adjusted to pH 7.5 with hydrochloric acid, and finally autoclaved at 115 ℃ for 30min, and stored at room temperature.
10 × LiAc (lithium acetate) configuration: 1M LiAc, adjusted to pH 7.5 with glacial acetic acid (acetic acid), then sterilized by filtration through a 0.22. mu.M sterile filter and kept in a freezer at 4 ℃ until use.
50% PEG 4000: weighing 40g of PEG4000, adding a proper amount of water, heating until the PEG4000 is completely dissolved, and adding water to a constant volume of 80 mL; sterilizing at 115 deg.C for 30min, packaging with 600 μ l/tube, and storing in refrigerator at 4 deg.C.
DMSO, DMSO: filtering with 0.22 μ M sterile filter membrane, sterilizing, and storing in refrigerator at 4 deg.C.
Single-stranded fish sperm dna (ssdna): purchased from kulai bokoku technologies, beijing.
The saccharomyces cerevisiae strain YZL141 transformation and plate screening method comprises the following specific steps:
1. selecting about 5 single clones of Saccharomyces cerevisiae YZL141, placing in a PA bottle containing 5ml YPD liquid culture medium, and shaking for overnight culture at 30 ℃; 50 μ l of the bacterial solution was diluted 20 times with sterile water, and OD was measured600Value (20 times dilution, not able to make OD value greater than 1, otherwise would be inaccurate); by OD600The volume x is 10 (e.g. 5ml if OD 2, typically between 500 and 1000. mu.l) is transferred to 50ml fresh YPD medium and incubated at 30 ℃ and 220rpm for 3.5 h; when the OD600 is 0.5-0.8, centrifuging at 3000rpm for 5min to collect thallus; adding 20ml of 1 × TE solution to resuspend the cells, centrifuging at 3000rpm for 5min, and discarding the supernatant; adding 2 to the precipitated cellsml 1×LiAc/0.5×TE(1700μl H2O + 200. mu.l of 10 XLiAc + 100. mu.l of 10 XTE), left at room temperature for 10min (preferably 25 ℃);
2. boiling the single-chain milt DNA in water for 10min, quickly putting back to ice for cooling to ensure that the milt DNA is in a single-chain state, taking 100 mu l of yeast strain YZL141 suspension, adding 10 mu l of denatured single-chain milt DNA, adding 300ng of OfTPS13.2-Pkz762-Plasmid which is diluted for later use in advance, slightly flicking and uniformly mixing, and finally adding 700 mu l of 1 XLiAc/40% PEG4000/1 XTE solution (560 mu l of PEG4000+70 mu l of 10 XLiAc +70 mu l of 10 XTE) and uniformly mixing; culturing at 30 deg.C for 30 min; adding 88 μ l DMSO, mixing, and performing heat shock at 42 deg.C for 9 min; centrifuging at 10000rpm for 30s, and discarding the supernatant; adding 1ml YPD culture medium to resuspend the thallus, centrifuging at 8000rpm for 30s, and discarding the supernatant; adding 1ml YPD culture medium to suspend thallus, and incubating for 30min at 30 deg.C by shaking bed; centrifuging at 10000rpm for 30s, and discarding the supernatant; adding 1ml of 1 × TE solution to resuspend the thalli, centrifuging at 10000rpm for 30s, and discarding the supernatant;
3. and coating the transformed YZL141 on an SC-URA screening plate, drying the liquid, and culturing in an incubator at 30 ℃ until obvious single colonies are visible for about 2-3 days.
EXAMPLE 4 Positive detection of Yeast transformed colonies
Single colonies of the transformed yeast were picked into 10. mu.l of sterile water, 2. mu.l was taken out, 10. mu.l of 20mM NaOH solution was added, treated with PCR instrument at 99 ℃ for 20min, placed on ice for template application, and the template was vortexed for 2s to prevent sedimentation before use.
PCR System (10. mu.l): 2 × Taq Master Mix: mu.l of OfTPS13.2-Yeast transformation Positive detection-F/R0.5. mu.l each, template 1. mu.l, water 3. mu.l, PCR conditions were: pre-denaturation at 94 ℃ for 2min, denaturation at 94 ℃ for 20S, annealing at 55 ℃ for 20S, extension at 72 ℃ for 1min/kb, setting 30 cycles, and final extension at 72 ℃ for 5 min. And (3) observing a band after the PCR product is electrophoresed, and performing yeast fermentation and beta-bisabolene GC-MS detection if a band (a target band is 1626bp) is obviously positive.
Example 5 Yeast fermentation and beta-bisabolene GC-MS detection
1. Shake flask fermentation culture
YPD liquid medium preparation: the final concentration of peptone (Angel brand) is 20g/L, the final concentration of yeast powder (Angel brand) is 10g/L, the final concentration of glucose is 10g/L, the final concentration of galactose is 10g/L (glucose and galactose are separately prepared and added into a culture medium after sterilization), and isopropyl myristate (IPM) is filtered by a sterile filter membrane before use.
Inoculation: from the transformed plate, 5 single colonies positive for detection were picked up in 5ml of YPD liquid medium at 220rpm and 30 ℃ overnight. The next day, overnight culture broth is extracted and added into 50ml liquid culture medium according to a certain proportion to make initial OD600The value was about 0.1, and 10% isopropyl myristate was added for covering, and the cells were incubated at 30 ℃ for 72 hours with shaking at 220 rpm.
GC-MS quantitative detection of beta-bisabolene
After the fermentation is finished, centrifuging at 4000rpm of 4 ℃ for 10 minutes, taking 200 mu L of isopropyl myristate, putting the isopropyl myristate into a sample bottle, detecting the product by GC-MS, and simultaneously adding 1 mu L of methyl nonanoate with the concentration of 4.75mg/L as an internal standard for quantitative calculation, wherein the unconverted saccharomyces cerevisiae strain YZL141 is used as a negative control, and the quantitative calculation formula is as follows: C1/V1 ═ C2/V2(C1 is the peak area of methyl nonanoate, V1 is the concentration of methyl nonanoate, C2 is the peak area of β -bisabolene, and V2 is the concentration of β -bisabolene).
And (3) GC-MS detection:
GC setting: the injection port temperature is 250 ℃, the injection mode is no shunt, the initial temperature of the temperature rise program is 40 ℃, the temperature is kept for 3.5min, the temperature rise rate of 10 ℃/min is increased to 100 ℃, the temperature is kept for 3min, and then the temperature is increased to 280 ℃ at the rate of 5 ℃/min and is kept for 5 min. The total program time was 46.5 min.
Setting of the MS: the interface temperature is 280 ℃, the loading gas is helium, the flow rate is 1.0ml/min, the ion source temperature is 220 ℃, the EI ionization mode is adopted, the electron energy is 70ev, and the scanning range is 50-500 amu.
The sample injection mode is a non-flow splitting mode, the temperature of the sample injection port is maintained at 230 ℃, and the temperature of the transmission line is 240 ℃. Electron energy 70eV, scan range 40-450 amu, ion source temperature 150 ℃, high purity helium (99.999%) as carrier gas, flow rate 0.8 mL/min. The initial column temperature was 40 deg.C, held for 3min, then raised to 80 deg.C at a rate of 1 deg.C/min, held for 3min, then raised to 220 deg.C at a rate of 10 deg.C/min, and held for 15 min.
GC-MS detection shows that the peak of the target product is beta-bisabolene, the target peak-off time is 10.98min, and the final concentration of the beta-bisabolene is 35.21 mg/L.
The above-mentioned embodiments are merely illustrative of the technical ideas and features of the present invention, and the purpose thereof is to enable those skilled in the art to understand the contents of the present invention and implement the present invention, and not to limit the protection scope of the present invention. Any modification, equivalent replacement, simple improvement and the like of the above embodiments according to the technical spirit of the present invention shall be covered within the scope of the present invention.
Sequence listing
<110> university of agriculture in Huazhong
<120> osmanthus fragrans gene OfTPS13.2 and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1626
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
atggaggcaa ggagatctgg aaactatgaa ccaagtgttt ggaatgacga ttatgtgcag 60
tcaatattta ctccgtatgc gggaaaagag tacacgcaac tcgttgagaa tctgaaagaa 120
aaaataagaa ctatcatcaa cgaaacggaa gatgtgcttc atcaacttga gcttattgat 180
aatttgcaaa ggcttgatgt ttgtaaccac tttaaggatg aaataaagaa aatattggag 240
cttatatatc taactaataa agattccaat aatcaaaatg aaaaggattt gtatccaaca 300
gctctaaaat ttagactcct tcgacaacat ggataccatg tccctcaaga agttttctgc 360
agtttcatgg aagaggaagg aaatttcaat gcagaacttt ctggggatat tgtaggaatt 420
ctatctctgt acgaagcttc atttctatcg ttggaaaatg aaggcatcct ggatgaggct 480
agaaatttca caactcatca tctcaaggaa agactccagc atattacaga ccaaggtctt 540
gctatgcaag tcagccatgc attagagctt ccgctgcact ggagagtgca gaaacttgaa 600
gcaaaatggt tcatatatgt atacgagaat agaaatgatg cagactataa tttacttgaa 660
ttcgctaagt tggatttcaa catcgtacag gctatatatc aagatgaaat aaagcaattg 720
tcaaggtggt ataaagaaac ccgtcttaca gagaagttga gcttcgctcg gcacagattg 780
gtggagagct tcctgtgggc attgggattc actccagagc cacaatttcg atacagtagg 840
aggatttcaa ccaatattat tgccctaata acaattattg atgacttata tgacgtatat 900
gggagcttag acgaacttga gctatttact gatatagtgg agaggtggga catcaacgca 960
ttggaccagc ttccagaata cttgaggatt tgtttccttg ctctcttcaa cttcattaat 1020
gaaatggctt acgatgttct taaagaccat aatttcaaca taatcccaaa cgccaagaaa 1080
ttgtgggcgg atctttgtag agcctacttg acagaagcta gatggtataa tagtgggtat 1140
tttcccagcc tgggtgagta cctcaacaca gcctggatat ccgtagcagg acctttagtt 1200
cttttccatg gatatttttg cacaataaac ccgttaacaa agaaggatct gggatgtttg 1260
gagcaatacc ctggtgtcat tcactggcca tcactggttc ttcgtctagc agatgacttg 1320
ggaacttcat ctgatgaact caagagaggt gatgttccaa aatcaatcca gtgttacatg 1380
aatgacacag gttgttccga agaggatgct agagactaca ttaagtatct aatagatgtg 1440
acattgaaga aaatgaataa ggatatacta atggactgcc ccttcaagga tttcgttggc 1500
catgcaatga atgtagcacg gatttctcaa tgcatgtacc agtatggaga tggatttgga 1560
gttcctcatc tcgagactaa aaagaactta atttcgttaa ttgttgaacc aattccactg 1620
caataa 1626
Claims (5)
1. The osmanthus beta-bisabolene synthetase gene OfTPS13.2 is characterized in that the nucleotide sequence of the gene OfTPS13.2 is shown as SEQ ID NO. 1.
2. An expression vector comprising the β -bisabolene synthase gene oftps13.2 according to claim 1.
3. A recombinant saccharomyces cerevisiae is characterized in that a yeast expression vector containing a gene OfTPS13.2 is transformed into saccharomyces cerevisiae, and the recombinant saccharomyces cerevisiae expressing the gene OfTPS13.2 is obtained through a resistant plate and sequencing screening.
4. Use of the gene oftps13.2 according to claim 1 or the expression vector according to claim 2 or the recombinant saccharomyces cerevisiae according to claim 3 for the synthesis of β -bisabolene.
5. A method for synthesizing beta-bisabolene by yeast is characterized by comprising the following steps: the recombinant Saccharomyces cerevisiae of claim 3 was inoculated into YPD medium containing galactose, and isopropyl myristate was added to cover the medium, and after the fermentation was completed, β -bisabolene was extracted.
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| CN111088175A (en) * | 2019-11-26 | 2020-05-01 | 天津科技大学 | Yarrowia lipolytica for producing bisabolene and construction method and application thereof |
| CN112877228A (en) * | 2021-01-22 | 2021-06-01 | 浙江工业大学 | Saccharomyces cerevisiae engineering bacterium for high yield of bisabolene and application thereof |
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| CN111088175A (en) * | 2019-11-26 | 2020-05-01 | 天津科技大学 | Yarrowia lipolytica for producing bisabolene and construction method and application thereof |
| CN112877228A (en) * | 2021-01-22 | 2021-06-01 | 浙江工业大学 | Saccharomyces cerevisiae engineering bacterium for high yield of bisabolene and application thereof |
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