CN113699139B - Lagerstroemia terpene synthase gene and application thereof - Google Patents

Lagerstroemia terpene synthase gene and application thereof Download PDF

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CN113699139B
CN113699139B CN202110944260.4A CN202110944260A CN113699139B CN 113699139 B CN113699139 B CN 113699139B CN 202110944260 A CN202110944260 A CN 202110944260A CN 113699139 B CN113699139 B CN 113699139B
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蔡明�
章寒
张启翔
潘会堂
朱梓宁
续言
吴宜静
张一鸣
王佳
程堂仁
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Beijing Forestry University
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Abstract

The invention discloses a crape terpene synthase gene and application thereof. The terpene synthase gene is obtained by cloning and separating the gene in the crape plant for the first time, the catalytic function of the terpene synthase gene is explored through in vitro expression, the molecular mechanism of the biosynthesis of the flower fragrance substance of the crape plant is primarily known, and a foundation is provided for further research on the biosynthesis and regulation of the flower fragrance of the crape plant.

Description

Lagerstroemia terpene synthase gene and application thereof
Technical Field
The invention relates to the field of plant genetic engineering, in particular to a crape terpene synthase gene and application thereof.
Background
Lagerstroemia species Lagerstroemia cauda (Lagerstroemia caudata) and Lagerstroemia speciosa ' Bai Yunying nepheline (L.indica ' Baiyunningxia ') are white flowers, and monoterpenes are the main different volatile components of flowers in the full bloom stage of the Lagerstroemia speciosa and Lagerstroemia speciosa. The monoterpene substances in the full-open period of the lagerstroemia indica are aroma characteristic components and have high content, the monoterpene substances in the lagerstroemia indica 'Bai Yunying nepheline' are few in types and low in concentration, and the terpene synthase is a key enzyme of a terpene substance synthesis pathway in plants. At present, no research report on terpene synthase genes is found in Lagerstroemia plants.
Disclosure of Invention
The invention aims to provide a crape terpene synthase gene and application thereof.
To achieve the object of the present invention, in a first aspect, the present invention provides a terpene synthase gene LcTPS14 (from lagerstroemia cauda), which is a gene encoding the following protein (a) or (B):
(A) A protein consisting of the amino acid sequence shown in SEQ ID NO. 1; or (b)
(B) The protein which is derived from (A) and has the same function and is obtained by substituting, deleting or adding one or more amino acids in the sequence shown in SEQ ID NO. 1.
In a second aspect, the invention provides a biological material comprising said gene LcTPS14, said biological material including but not limited to recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors, engineering bacteria or non-regenerable plant parts.
In a third aspect, the invention provides any one of the following applications of the gene LcTPS14 or a biomaterial containing the gene:
(1) For the preparation of transgenic plants;
(2) Is used for plant breeding.
Wherein the breeding purpose is to improve the floral fragrance of plants.
In the present invention, the plant includes a lagerstroemia plant.
In a fourth aspect, the invention provides the use of said gene LcTPS14 in plant floral material biosynthesis; wherein the floral material includes, but is not limited to, monoterpenes, sesquiterpenes.
Monoterpenes include, but are not limited to, linalool, sesquiterpenes include, but are not limited to, E-nerolidol.
In a fifth aspect, the invention provides any one of the following uses of a banaba terpene synthase encoded by the gene LcTPS 14:
(1) In vitro catalysis of geranyl pyrophosphate (GPP) to linalool;
(2) In vitro catalysis of farnesyl pyrophosphate (FPP) to E-nerolidol.
In a sixth aspect, the invention provides a lagerstroemia terpene synthase gene, liTPS14 (from lagerstroemia indica 'Bai Yunying nepheline'), which is a gene encoding either protein (a) or (b):
(a) A protein consisting of the amino acid sequence shown in SEQ ID NO. 2; or (b)
(b) And (b) a protein which is derived from (a) and has equivalent functions and is obtained by substituting, deleting or adding one or more amino acids in the sequence shown in SEQ ID NO. 2.
In a seventh aspect, the present invention provides a biological material comprising said gene LiTPS14, said biological material comprising but not limited to recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors, engineering bacteria or non-regenerable plant parts.
In an eighth aspect, the present invention provides any one of the following applications of the gene LiTPS14 or a biomaterial comprising the gene:
(1) For the preparation of transgenic plants;
(2) Is used for plant breeding.
Wherein the breeding purpose is to improve the floral fragrance of plants.
In the present invention, the plant includes a lagerstroemia plant.
In a ninth aspect, the present invention provides an application of the gene LiTPS14 in biosynthesis of floral substances of plants; wherein the floral material includes, but is not limited to, monoterpenes, sesquiterpenes.
Monoterpenes include, but are not limited to, linalool, sesquiterpenes include, but are not limited to, E-nerolidol.
In a tenth aspect, the invention provides any one of the following uses of a lagerstroemia terpene synthase encoded by the gene LiTPS 14:
(1) In vitro catalysis of geranyl pyrophosphate (GPP) to linalool;
(2) In vitro catalysis of farnesyl pyrophosphate (FPP) to E-nerolidol.
The terpene synthase gene is obtained by cloning and separating the gene in the crape plant for the first time, the catalytic function of the terpene synthase gene is explored through in vitro expression, the molecular mechanism of the biosynthesis of the flower fragrance substance of the crape plant is primarily known, and a foundation is provided for further research on the biosynthesis and regulation of the flower fragrance of the crape plant.
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FIG. 1 shows the results of full-length amplification of terpene synthase genes in a preferred embodiment of the invention.
FIG. 2 is a phylogenetic tree analysis of terpene synthase genes according to a preferred embodiment of the invention. Wherein Aa, artemisia annua; ag, fir; am, goldfish grass; ar, wrinkled giant hyssop; cb, clarkia brew; cc, clarkia confcinna; ci, chicory; cl, lemon; cm, winter squash; ct, cinnamomum tenuifolium; eo, american oil palm; ga, tree cotton; gb, ginkgo; l, lavender latifolia; ls, lettuce; md, apple; ml, peppermint; ob, basil; oe, oletum Canarium; pf, tempering; ps, north american spruce; pt, loblolly pine; sf, salvia fructiose; so, salvinia; st, yang Yu; vv, grape; zm, maize; zz, red ball ginger.
FIG. 3 shows the expression pattern of TPS gene in different flowering stages according to the preferred embodiment of the present invention.
FIG. 4 shows the expression patterns of TPS gene at different parts of flowers according to the preferred embodiment of the present invention.
FIG. 5 shows the result of detection of TPS gene recombinant vector in the preferred embodiment of the present invention.
FIG. 6 is a subcellular localization view of TP14 in accordance with the preferred embodiment of the present invention. Wherein Green column indicates GFP fluorescence in Green channel, red column indicates chlorophyll autofluorescence in Red channel, mered column is superposition of bright field image, green channel and Red channel, BF column is bright field, CK indicates no-load control; the scale is 50. Mu.m.
FIG. 7 shows in vitro enzymatic products of recombinant proteins LcTPS14 and LiTPS14 according to a preferred embodiment of the invention. Wherein, 1,4,5 is a non-terpenoid; 2. trans-nerolidol; 3. nerolidol acetate; 6. linalool; 7. linalyl formate.
FIG. 8 is an ion peak diagram of the in vitro enzymatic reaction product in a preferred embodiment of the invention.
Detailed Description
The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise indicated, the examples are in accordance with conventional experimental conditions, such as the molecular cloning laboratory Manual of Sambrook et al (Sambrook J & Russell DW, molecular Cloning: a Laboratory Manual, 2001), or in accordance with the manufacturer's instructions.
EXAMPLE 1 cloning of the terpene synthase Gene LcTPS14, liTPS14 and bioinformatics analysis of Lagerstroemia
The invention provides a method for sequencing Lagerstroemia cauda L.sub.f.sub.f.crape (Lagerstroemia caudata) and Lagerstroemia cauda L.sub.f. Bai Yunying nepheline '(L.indica' Baiyunningxia ') which are white flowers, wherein monoterpenes are main different volatile components of flowers in the full bloom period, but genes and functions related to biosynthesis of the substances are not clear in Lagerstroemia, and the method is used for analyzing molecular mechanisms of the biosynthesis of the monoterpenes in Lagerstroemia, carrying out full-length transcriptome sequencing on Lagerstroemia cauda L.sub.f.sub.f.to obtain differentially expressed terpene synthase genes by screening, taking Lagerstroemia cauda L.sub.f.and Lagerstroemia cauda L.sub.f. Bai Yunying nepheline' as materials, separating the terpene synthase genes, analyzing the expression rules of the genes, and carrying out preliminary verification on the functions of the terpene synthase genes.
1. Materials and methods
1.1 plant material: reference Xu Wan (2019) is made to research on different parts of flowers and full-open periods of Lagerstroemia speciosa in each stage, and the different parts of flowers and full-open periods of Lagerstroemia speciosa and Lagerstroemia speciosa 'Bai Yunying' are collected, packed by a centrifuge tube, quick-frozen by liquid nitrogen and stored in a laboratory ultralow-temperature refrigerator.
1.2 reagents and consumables
Total plant RNA extraction kit (DP 441), DNA recovery kit (DP 209), E.coli (Escherichia coli) competent DH 5. Alpha., ampicillin (RT 501) were purchased from Tiangen Biochemical technology (Beijing);max DNA Polymerase (R045A), antigenome-deleted reverse transcription reagent (RR 047), TB ∈>Premix Ex Taq TM II (RR 820A), DNA Marker purchased from Baoli doctor's technique (Beijing)) A limited company; TOPO Blunt (Blunt ended) cloning vector (cv 17) was purchased from Beijing Edley Biotech Co., ltd; exogenous rnase scavengers were purchased from the sciences company of the biowarfarin (beijing). Primer synthesis and sequencing are all undertaken by Beijing qingke new industry biotechnology Co., ltd; other consumables are available from Beijing bayer di Biotechnology Co.
1.3 test methods
(1) Total RNA extraction from plants
Before extracting RNA, exogenous RNase scavenger is used to treat consumable, table top and instrument, high temperature and high pressure sterilizing equipment, etc. to reduce interference of RNA decomposing enzyme. Samples were ground thoroughly with liquid nitrogen to a powder, total RNA was extracted by the method of reference Chen Zhilin (2017), and lysates were prepared using DTT (working concentration 1 mmol/L).
(2) Detection of RNA integrity and concentration
The film was photographed and analyzed under UV conditions in a gel imaging system using 1.2% TBE common agarose gel electrophoresis. RNA extract was detected by an ultra-micro ultraviolet spectrophotometer, and OD was recorded 260 /OD 280 The readings and concentration, RNA is temporarily stored on ice for subsequent reversal experiments, and stored for a long time in a refrigerator at-80 ℃.
(3) Reverse synthesis of cDNA and amplification of full-length target gene
cDNA was obtained by reverse transcription in an amount of 0.5. Mu.g total RNA per tube according to the protocol of the reverse transcription kit. Amplification primers (upstream Primer 5'-ATGGATTGTGTGGAAAGCTT-3' and downstream Primer 5'-TTAGGCTAAACCATTAATCAGC-3') were designed using Primer premier 5 based on full length transcriptome sequence data. cDNA of flowers in full bloom stage of Lagerstroemia speciosa and Lagerstroemia speciosa 'Bai Yunying' are used as templates to amplify target genes. The PCR reaction system is as follows:
the PCR reaction procedure was as follows: 98 ℃ for 1min;98℃for 10s,55℃for 15s,72℃for 30s,35 cycles; 72 ℃ for 2min; preserving at 4 ℃.
The products were detected by agarose gel electrophoresis, and gel recovery was performed with reference to the DNA recovery kit instructions. Agarose gel electrophoresis and detection of the recovered product using an ultra-micro ultraviolet spectrophotometer.
(4) TOPO cloning vector for target gene connection
Adopts the Edley end connection kit, the connection system is 10 XEnhance 0.5 mu L, TOPO carrier 0.5 mu L, target gene 20-40ng, ddH 2 O was made up to 5. Mu.L. The connection is carried out at 25℃for 5min.
Converting Escherichia coli DH5 alpha, mixing 5 μl of the ligation solution with 50 μl of fungus solution, flicking, ice-bathing for 25min, heat-shocking at 42deg.C for 45sec-1min, standing on ice for 2min, adding 500 μl of LB liquid without antibiotics, and renaturating at 37deg.C with shaking table 200rpm for 30min. The cells were collected by centrifugation at 5000rpm, the supernatant was removed in an ultra clean bench environment, 50. Mu.L of liquid was retained, the resuspended cells were aspirated at the tip, and the cells were spread on solid LB medium containing Amp (working concentration 50. Mu.g/mL) and cultured upside down at 37℃overnight.
Single colony with proper and round size is selected and cultured in 1mL of liquid LB culture medium containing 50 mug/mL Amp at 37 ℃ and 200rpm for 4 hours, bacterial liquid PCR detection is carried out, and positive bacterial liquid is sent to a company for sequencing.
(5) Bioinformatics analysis of sequences
Bioinformatic analysis is performed on the obtained sequence information. Open reading frame searches were performed using an ORF Finder, and Blastp analyzed for multiple sequence homology and protein domains. Multiple sequence alignment was performed using DNAMAN and ClustalW software, MEGA7 constructed a phylogenetic tree.
The nucleotide sequence of the gene LcTPS14 is shown as SEQ ID NO. 3, the nucleotide sequence after codon optimization is shown as SEQ ID NO. 4, and the amino acid sequence of the protein encoded by the gene LcTPS14 is shown as SEQ ID NO. 1.
The nucleotide sequence of the gene LiTPS14 is shown as SEQ ID NO. 5, the nucleotide sequence after codon optimization is shown as SEQ ID NO. 6, and the amino acid sequence of the gene LcTPS14 encoded protein is shown as SEQ ID NO. 2.
(6) qRT-PCR verification of Gene expression
Designing quantitative Primer in Primer premier 5 software according to Primer design principle, diluting cDNA 5 times by adopting EF-1 alpha (Chen Zhilin, 2017) as reference gene,using a 10. Mu.L system (dye 5. Mu.L+cDNA 1. Mu.L+upstream and downstream primers 0.4. Mu.L+ddH each) 2 O) carrying out fluorescent quantitative reaction, preparing dye liquor and cDNA according to the reaction number +3 in advance, fully and uniformly mixing and centrifuging. Primer Advance and ddH 2 O is uniformly mixed according to the reaction number of +3. The temperature program is as follows: pre-denaturation at 95 ℃ for 30s;95 ℃ for 5s and 60 ℃ for 15s, and 40 cycles are total; heating from 60 ℃ to 95 ℃ at 5 ℃/s, Δt=1 ℃, collecting 6s. 3 technical and biological replicates were performed with 2 -△△Ct Analytical data were calculated by the method.
2. Results and analysis
2.1 extraction results of Total RNA from plants
OD measured from total RNA extracted 260 /OD 280 In the range of 1.8-2.1, the concentration is in the range of 100-800 ng/. Mu.L, and the agarose gel electrophoresis result of RNA shows that the total RNA extracted has clear bands and no obvious tailing phenomenon, and can be used for downstream experiments.
2.2 terpene synthase sequence analysis
Candidate terpene synthase sequences were obtained by early screening, gene CDS was searched for using NCBI ORF Finder and full length primers were designed, lcTPS14 was successfully isolated in lagerstroemia cauda, and LiTPS14 was isolated in lagerstroemia indica 'Bai Yunying nepheline' by homologous cloning (fig. 1).
(1) LcTPS14 and LiTPS14 sequence analysis
LcTPS14 and LiTPS14 are 1575bp in length, encode 525 amino acids (SEQ ID NO:1 and 2), have a nucleotide sequence similarity of 96.83% and an amino acid sequence similarity of 95.24%, and contain DDXXD and DTE/NSE domains.
(2) TPS gene coding protein homology comparison and phylogenetic tree construction
The two gene sequence information are respectively subjected to blastp comparison analysis at NCBI, and multiple sequence comparison shows that LcTPS14 and LiTPS14 amino acid sequences contain DDXXD domains and lack RRX 8 W domain. NCBI protein conserved domain analysis both Terene_cycle_plant_C1 (LcTPS 14 in the 4-522 site interval, liTPS14 in the 1-522 site interval) and Terene_synth_C (208-470 site) domains, two aspartic acid rich regions and metal Mg 2+ Binding sites.
Construction of phylogenetic tree (FIG. 2) with terpene synthase genes that have been validated for function (Neighbor-Joining, boottrap 1000, partial separation 50), lcTPS14 and LiTPS14 are in the TPS-g subfamily, whereas TPS-g subfamily is usually deleted for RRX 8 W domain.
2.3 analysis of Gene expression
Candidate gene expression rules are explored for different flowering stages and different parts of flowers in full bloom periods of lagerstroemia indica and lagerstroemia indica 'Bai Yunying' by utilizing a fluorescence quantification technology. Semi-quantitative PCR initially screened for fluorescent quantitative primers, the primers screened should make the band single clear, without primer dimer (Table 1).
TABLE 1 fluorescent quantitative primers
(1) Transcriptome data validation
And comparing the qRT-PCR data of the lagerstroemia indica with FPKM data of the same flowering stage in the second generation transcriptome, and ensuring that the differential expression gene data of the second generation transcriptome of the lagerstroemia indica is more reliable.
(2) Expression analysis at different flowering stages
The expression change rule of the genes in different periods is detected by selecting eight periods of leaf buds, inflorescence buds, immature flower buds, mature flower buds, bud stage, full-bloom end stage and decay stage (figure 3).
The expression rule of LcTPS14 in different flowering stages of Lagerstroemia speciosa is as follows: the leaf bud-mature bud-full bloom stage-full bloom end stage-decay stage show a tendency of no difference from descending to ascending to peak value, then descending to lower expression quantity. The expression quantity is higher in the stage of leaf buds, inflorescence buds and full-bloom stage.
The expression rule of the LiTPS14 in different flowering stages of the crape myrtle 'Bai Yunying nepheline' is leaf bud-immature flower bud-mature flower bud-full-open period-full-open end-decay period, the expression rule of ascending and then descending to peak value and then descending to no difference is shown, the expression quantity is relatively highest in the full-open period, the expression quantity is extremely low in the immature flower bud period and the leaf bud period.
(3) Expression analysis of different parts of flowers
The expression analysis was performed on different parts of flowers in full bloom (fig. 4), and it was found that LcTPS14 was expressed very high in the pistil of crape myrtle, and hardly expressed in other parts. While LiTPS14 is highly expressed in the petals and pistils of Lagerstroemia indica 'Bai Yunying nepheline', and expressed in stamens, calyx and pedicel, but the expression level is not high.
In the embodiment, key terpene synthase genes of the biological synthesis paths of Lagerstroemia cauda and Lagerstroemia indica 'Bai Yunying nepheline' terpenoid substances are obtained by adopting a molecular biological means, a phylogenetic tree is constructed, lcTPS14 and LiTPS14 are found to be divided into TPS-g subfamilies, the expression of the LcTPS14 and LiTPS14 in cytoplasm is predicted, and the LcTPS14 is possibly sesquiterpene synthase or multifunctional enzyme with the function of catalyzing and generating different terpenoids, and the specific functions of the LcTPS14 and LiTPS need to be further verified.
qRT-PCR detection and expression analysis shows that the expression quantity of LcTPS14 and LiTPS14 is highest in the full open period, the expression quantity of LcTPS14 in pistil is extremely high, the expression quantity of LiTPS14 in petals and pistil is high, and the expression quantity of LiTPS14 in other parts is relatively low.
EXAMPLE 2 subcellular localization of terpene synthase genes
Terpene synthases are key enzymes that catalyze the last step in the terpenoid reaction pathway, and their gene functions are associated with subcellular localization. GFP is used for constructing LcTPS14 and LiTPS14 plant expression vectors, agrobacterium tumefaciens is used for mediating and infecting Nicotiana benthamiana leaves, and subcellular localization of genes is explored to further understand functions of the two TPS genes.
1. Materials and methods
1.1 plant Material
Benshi tobacco (Nicotiana benthamiana) at 6 weeks of age.
1.2 reagents and consumables
Agarose gel DNA recovery kit (DP 209) was purchased from Tiangen Biochemical technology (Beijing) Co. Agrobacterium GV3101 and E.coli DH 5. Alpha. Were purchased from Shanghai Weidi Biotechnology Co. Restriction endonucleases Kpn I (1068S), xba I (1093S), DNA Ligation Kit (602)3Q) was purchased from baori doctor materials technology (beijing) limited. DMSO (CAS: 67-68-5), kana (INALCO, CAS: 133-92-6), rif (INALCO, CAS: 13292-46-1), AS (Sigma, CAS: 2478-38-8), MES.H 2 Reagents such as O (Amresco, CAS: 145224-94-8) were purchased from Beijing Bayer Di Biotechnology Co. The seamless cloning kit (CV 1901) was purchased from Beijing Edley Biotechnology Co. Primer synthesis and sequencing are all undertaken by Beijing qingke new industry biotechnology Co.
The recombinant plasmid pCAMBIA Super1300:: GFP (hereinafter referred to as pSuper 1300) was stored by the laboratory.
1.3 test methods
(1) Solution preparation
100mg/mL Kana mother liquor: 3g Kana+30mL ddH 2 O, filter sterilized (0.22 μm), -storing at 20 ℃.
10mg/mL Rif mother liquor: 100mg of the powder was dissolved in 10mL of DMSO, sterilized by filtration (0.22 μm), and stored at-20 ℃.
1mol/L MES solution: 10.66g MES H 2 O powder is dissolved in 50mL distilled water, filtered and sterilized (0.22 μm) in an ultra-clean bench, and sub-packaged and stored at low temperature.
50mmol/L AS solution: 0.196g of As powder was dissolved in 20mL of DMSO, sterilized by filtration (0.22 μm), and stored at-20 ℃.
1mol/L MgCl 2 Solution: dissolving in water, sterilizing at high temperature under high pressure, and preserving at 4deg.C.
(2) Amplification of the Gene fragment of interest
Primers were designed according to the instructions of the seamless cloning kit, and primers (upstream primer 5'-ATACTAGTGGATCCGGTACCATGGATTGTGTGGAAAGCTTGC-3' and downstream primer 5'-CCTTGCTCACCATGGTACCGGCTAAACCATTAATCAGCAATGAC-3') were amplified using the TOPO recombinant plasmid containing the target gene as a template. The PCR reaction system is as follows:
the PCR reaction procedure was as follows: 98 ℃ for 1min;98℃for 10s,55℃for 15s,72℃for 30s,35 cycles; 72 ℃ for 2min; preserving at 4 ℃.
(3) Enzyme cutting of carrier
Restriction enzyme Kpn I is used for cutting plasmid pSuper1300, GFP is cut for 4 hours at 37 ℃, and the cutting system is as follows:
(4) Construction of recombinant vector by seamless cloning connection
Mixing the target genes LcTPS14 and LiTPS14 recovered by enzyme digestion with a single enzyme digestion recovered vector according to a molar ratio of 3:1, and connecting for 30min at 50 ℃, wherein a connecting system is as follows:
e.coli DH 5. Alpha. Was transformed and renatured for 1h at 37℃with shaking table 200 rpm. The cells were collected by centrifugation at 5000rpm for 1min, the supernatant was removed in an ultra clean bench, 50. Mu.L of liquid was retained, the resuspended cells were sucked at the tip of the gun, and the cells were spread on a solid LB medium containing Kana (working concentration 50. Mu.g/mL) and cultured upside down at 37℃overnight.
Selecting single colony with proper and round size, culturing in 1mL liquid LB medium containing 50 μg/mL Kana at 37deg.C and 200rpm for 4h, detecting bacterial liquid by PCR, selecting positive bacterial liquid, and delivering to Beijing qingke new industry biotechnology Co.
(5) Agrobacterium transformation and leaf infection of Nicotiana benthamiana
The 4 recombinant vectors which are unloaded and successfully constructed are respectively transformed into agrobacterium GV3101,1 mu L of recombinant plasmid is mixed with 50 mu L of bacterial liquid, the mixture is uniformly mixed at the bottom of a light bullet pipe, the agrobacterium transformation is carried out according to instructions, the bacterial liquid is coated on solid LB (50 mu g/mL Kana+30 mu g/mL Rif), and the solid LB is subjected to inversion culture at 28 ℃ for 60 hours.
Single colonies with proper and round sizes are picked up and cultured in 1mL of liquid-containing LB medium (50 mug/mL Kana+30 mug/mL Rif) at 28 ℃ for 24 hours at 200rpm, and bacterial liquid PCR detection is carried out.
Mixing positive bacterial liquid 1mL with 15mL LB medium containing 50 mug/mL Kana+30 mug/mL Rif+10mmol/L MES+20 mug/L AS, and 20 ℃ at 28 DEG CInduced at 0rpm overnight. The cells were collected by centrifugation at 5000rpm and fresh prepared invading solution (sterile water +10mmol/L MES +200. Mu. Mol/L AS +10mmol/L MgCl) 2 ) Resuspension of the cells and modulation of OD 600 And standing in dark for 2-3 h until the temperature reaches 0.5-0.8. The disposable injector sucks the dyeing liquid, injects the dyeing liquid on the back of the leaf of the Nicotiana benthamiana, and places the tobacco leaf into a climatic chamber after dark treatment for 24 hours.
(6) Fluorescence observation by laser confocal microscope
After 72 hours of infection, 1cm×1cm leaves were prepared with clear water, the back of the leaves was facing upwards, no load was used as a control, and the green fluorescent protein expression was observed on the machine.
2. Results and analysis
2.1 plant expression vector containing the Gene of interest and transformed Agrobacterium
(1) Obtaining LcTPS14 and LiTPS14 recombinant vector by seamless cloning method
The target genes LcTPS14 and LiTPS14 are removed with stop codons, amplification primers are designed by a seamless cloning method, the amplification primers are connected with an expression vector subjected to Kpn I single digestion, and a correct recombinant vector is obtained through company sequencing.
(2) Obtaining Agrobacterium containing recombinant vector
pSuper1300 is empty and used as a control, agrobacterium is transformed by the recombinant plasmid, after the complete colony is selected for culturing for 24 hours, bacterial liquid PCR detection (figure 5) is carried out, bacterial liquid containing the band with the target size is screened for subsequent expansion culture and infection of the Nicotiana benthamiana leaf.
2.2 observing the Gene expression site with a laser confocal microscope
The leaf blade of Nicotiana benthamiana is infected by agrobacterium, fluorescence is detected by a machine after injection for 72 hours, and the lower epidermis of the leaf blade of Nicotiana benthamiana is observed under a Leica SP8 laser confocal microscope. The results showed that the leaves (CK) injected with empty vector found GFP expression in both cytoplasm and nucleus, and no green fluorescent signal was detected in plastids, and that GFP fluorescence was not coincident with plastid autofluorescence. The expression of two TPS14 gene fusion proteins was observed, and GFP signal expression was detected in the cytoplasm similar to the no-load expression results, indicating that both the fusion proteins of LcTPS14 and LiTPS14 were expressed in the cytoplasm of the lamina of nicotiana benthamiana (fig. 6), and that both proteins were functional in the cytoplasm, consistent with the pre-prediction results.
In this example, subcellular localization of LcTPS14 and LiTPS14 was obtained by tobacco leaf transient expression techniques, with both LcTPS14 and LiTPS14 being expressed in the cytoplasm.
EXAMPLE 3 prokaryotic expression of terpene synthase Gene and in vitro functional Studies
In this example, prokaryotic expression vectors of the target genes LcTPS14 and LiTPS14 are constructed, the target genes are expressed in escherichia coli to obtain recombinant proteins, in-vitro enzymatic reaction is carried out by taking FPP/GPP as a substrate, and the generated substances are analyzed by GC-MS technology so as to obtain the functions of the recombinant proteins.
1. Materials and methods
1.1 vectors and strains
Arctic-Express BL21 (DE 3) and BL21 (DE 3) PLySs expression bacteria and expression vectors pCZN1, pET30a, pGEX-4T-1 are purchased from Nanjing's tripod biotechnology Co. Coli competent TOP10 was purchased from Shanghai Biotechnology Inc.
1.2 reagents and consumables
Protein Marker (Thermo), GPP ammonium salt (Sigma, CAS:763-10-0, cat# G6772), FPP ammonium salt (Sigma, CAS:13058-04-3, cat# F6892), IPTG (Sigma, CAS: 367-93-1), arc (Sigma, CAS: 79-06-1), bis (Sigma, CAS: 110-26-9), tris (Sigma, CAS: 77-86-1), SDS (Amresco, CAS: 151-21-3), TEMED (BIO-RAD, CAS: 110-18-9), peptone (OXOID), LB (OXOID), 0.22 μmNylon syringe filter (Membrane Solutions), 0.22 μm sterile filter->And dialysis bags (Millipore), ni-IDA affinity chromatography gel (Novagen), n-hexane (chromatography purity, CAS: 110-54-3), and other reagents were purchased from Beijing blue chemical products, inc.
1.3 test methods
(1) Expression vector construction
The gene LcTPS14 and LiTPS14 are respectively subjected to sequence optimization according to the preference of the escherichia coli codons. Sequence synthesis was completed by Nanjing tripod biotechnology Co., ltd, and LcTPS14 and LiTPS14 were constructed between Nde I-Xba I sites of vector pCZN1, between Nde I-Xho I sites of pET30a, and between EcoR I-Xho I sites of pGEX-4T-1, respectively, using PAS-based method. The recombinant plasmid is identified by enzyme digestion, and the enzyme digestion system is as follows:
the plasmid containing the target gene is transformed into an escherichia coli TOP10 strain, and positive colony sequencing is selected.
(2) Transformation of recombinant vector into E.coli
The recombinant plasmid was transformed into E.coli Arctic Express BL (DE 3) or BL21 (DE 3) PLySs, the plasmid and competent bacteria were mixed at a volume ratio of 1:100, transformation was described as being performed at 37℃for 1h by renaturation at 220rpm, recombinant vectors of pCZN1 and pGEX-4T-1 were coated on LB solid medium with Amp resistance (working concentration 50. Mu.g/mL), recombinant vector of pET30a was coated on LB solid medium with Kana (working concentration 50. Mu.g/mL), and inverted overnight at 37 ℃.
(3) IPTG-induced expression of recombinant proteins
The single clone on the transformation plate was picked up and inoculated in 3mL of LB liquid medium containing the corresponding antibiotic (50. Mu.g/mL), and shaken at 37℃and 220rpm overnight. Referring to Green et al (2012), 0.5mM IPTG was used to induce expression, 1mL OD at 37℃respectively 600 Reaching 0.6-0.8 bacterial liquid, inducing the bacterial liquid overnight, performing ultrasonic disruption on bacterial cells, performing 12% SDS-PAGE on supernatant and precipitation to detect protein expression condition, and developing coomassie brilliant blue.
(4) Ni column affinity purification of recombinant proteins
And (3) selecting the recombinant strain with the best expression effect for protein purification, and purifying and recovering the protein by using an Ni affinity chromatography column method by taking no-load as a control, and analyzing by 12% SDS-PAGE. The purified protein solution is preserved in an ultralow temperature refrigerator at the temperature of-80 ℃.
(7) In vitro catalytic reaction of recombinant proteins
100. Mu.L of the total reaction system, buffer containing 13-35. Mu.g of recombinant protein (PBS+20 mM MgCl) 2 +5mM DTT), mixing with substrate GPP or FPP (working concentration 0.5 mM), covering with 100 μl of n-hexane, catalytic reacting at 30deg.C for 1 hr, stopping the reaction by vortexing for 2min, centrifuging to promote liquid delamination, absorbing upper organic phase, and anhydrous MgSO 4 Fully dried, filtered through a 0.22 μm nylon membrane, and subjected to GC-MS analysis on a 1. Mu.L liquid machine, and repeated 3 times. The GC-MS detection conditions are shown in Table 2, and the products were compared with NIST147 for qualitative analysis.
TABLE 2 GC-MS detection conditions
2. Results and analysis
2.1 construction of prokaryotic expression vectors
LcTPS14 and LiTPS14 sequences were optimized according to E.coli codon preference, and N-his tag fusion expression protein (6Xhis+NdeI+target protein+taa+Xba I), N-GST and C-his tag fusion expression protein (EcoRI+target protein+6Xhis+Xho I) and C-his tag fusion expression protein (NdeI+target protein+6Xhis+Xho I) were constructed with vector pCZN1, pGEX-4T-1 vector, respectively, and the recombinant protein molecular weights were shown in Table 3.
TABLE 3 molecular weight of recombinant proteins
Sequencing and double enzyme digestion verification are carried out on the constructed recombinant vector, and recombinant plasmids pCZN1-LcTPS14, pGEX-4T-1-LcTPS14, pET30a-LcTPS14, pCZN1-LiTPS14, pGEX-4T-1-LiTPS14 and pET30a-LiTPS14 containing target genes are obtained.
2.2 functional analysis of recombinant proteins
The recombinant protein is respectively mixed with different substrates GPP or FPP for catalytic reaction, and GC-MS is carried out on-machine analysis. A large amount of linalool was detected in the reaction of the recombinant proteins LcTPS14 and LiTPS14 with the substrate GPP, and trans-nerolidol was detected in the reaction of the substrate FPP (FIG. 7). The ion peak diagram is shown in fig. 8. Indicating that LcTPS14 and LiTPS14 are bifunctional enzymes, and have the functions of catalyzing FPP to generate trans-nerolidol and catalyzing GPP to generate linalool.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Reference is made to:
[1] chen Zhilin cloning and expression pattern research [ D ] of MADS-box family B and C genes of Lagerstroemia speciosa, university of Beijing forestry, university of Shuoshi treatise.2017.
[2] Xu Wan the characteristic aroma component release rule of Lagerstroemia speciosa and the study of MEP channel key gene [ D ] doctor's thesis of Beijing university of forestry, 2019.
Sequence listing
<110> Beijing university of forestry
<120> Lagerstroemia terpene synthase gene and use thereof
<130> KHP211118368.1
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 525
<212> PRT
<213> Lagerstroemia speciosa (Lagerstroemia caudata)
<400> 1
Met Asp Cys Val Glu Ser Leu His Glu Arg Arg Leu Lys Glu Ala Arg
1 5 10 15
His Leu Val Arg Arg Val Glu Lys Gly Ser Leu Glu Ser Leu Val Met
20 25 30
Val Asp Ala Leu Gln Arg Leu Gly Ile Ala Tyr His Phe Glu Glu Glu
35 40 45
Thr Arg Ser Leu Leu Gln Glu His Leu Leu Ser Ala Tyr Asn Gly His
50 55 60
Ser Ser Ser Asp Ser Leu Asn Lys Val Ala Leu Arg Phe Arg Leu Leu
65 70 75 80
Arg Gln Glu Gly Tyr Asn Val Pro Ala Gly Ile Phe Glu Gly Phe Lys
85 90 95
Ser Lys Asp Asp Asn Asn Gly Arg Phe Thr Phe Asp Arg Lys Leu Tyr
100 105 110
Lys Asp Ile Val Gly Leu Met Ser Leu Tyr Glu Ala Ser His Leu Gly
115 120 125
Thr Gln Gly Glu Asp Ile Leu Asp Glu Ala Ala Ser Phe Ser Lys Lys
130 135 140
Ala Leu Thr Gly Thr Leu Asp Arg Leu Leu Cys Ala Gly Asn Asp Thr
145 150 155 160
Asn Gly Ile Ser Ser Pro Ile Leu Arg Glu Leu Val Lys Asn Thr Leu
165 170 175
Ser Asn Pro Phe His Lys Ser Leu Pro Arg Phe Thr Ser Glu Thr Phe
180 185 190
Gln Val Tyr Phe Gly Gly Pro Phe Glu Trp Ile Gly Val Phe Arg Glu
195 200 205
Leu Ala Val Leu Asp Ser Glu Leu Val Ala Ser Ile Asn Arg Asn Glu
210 215 220
Ile Leu Gln Val Ser Lys Trp Trp Lys Asp Leu Gly Leu Ala Lys Glu
225 230 235 240
Leu Lys Phe Ala Arg Asp Gln Pro Met Lys Trp Tyr Leu Trp Pro Met
245 250 255
Ala Val Leu Pro Asp Pro Lys Leu Ser Gln Glu Arg Val Asp Ile Thr
260 265 270
Lys Pro Ile Ala Met Val Tyr Ile Ile Asp Asp Ile Phe Asp Val Tyr
275 280 285
Gly Ser Leu Asp Glu Leu Thr Leu Phe Thr Glu Ala Val Lys Arg Trp
290 295 300
Glu Cys Ile Glu Glu Leu Pro Asp Tyr Met Lys Arg Cys Phe Arg Ala
305 310 315 320
Leu Asp Asp Ile Thr Ser Glu Ile Ser Phe Asn Val Tyr Lys Lys His
325 330 335
Gly Trp Ser Pro Met Asp Leu Leu Lys Glu Ser Trp Lys Ser Leu Phe
340 345 350
Asp Ala Phe Leu Leu Glu Thr Arg Trp Phe Arg Cys His Asn Ser Pro
355 360 365
Ser Ala Asp Glu Tyr Leu Asn Asn Ala Ile Val Thr Ser Gly Val Pro
370 375 380
Leu Val Ile Val His Ile Phe Ala His Leu Cys Glu Gly Leu Asn Lys
385 390 395 400
Gln Cys Leu Asp Lys Leu Ser Gly Phe Ser Glu Ile Ser Ser Ser Thr
405 410 415
Ala Lys Ile Leu Arg Leu Trp Asp Asp Leu Gly Ser Ala Lys Asp Glu
420 425 430
Asn Gln Glu Gly His Asp Gly Ser Tyr Leu Asp Tyr Tyr Met Asn Glu
435 440 445
Asn Pro Ser Cys Ser Leu Glu Gln Ala Thr Asp Arg Val Lys Glu Met
450 455 460
Ile Leu Asp Ala Trp Lys Ser Leu Asn Lys Glu Cys Leu Phe Ser His
465 470 475 480
Thr Phe Ser Thr Ser Val Ala Gln Ala Ser Leu Asn Thr Ala Arg Met
485 490 495
Val Pro Leu Met Tyr Asp Tyr Asp Glu Asn His Cys Leu Pro Arg Ile
500 505 510
Glu Asp Tyr Met Lys Ser Leu Leu Ile Asn Gly Leu Ala
515 520 525
<210> 2
<211> 525
<212> PRT
<213> Bai Yunying Cynanchum (L. Indica 'Baiyunyingxia')
<400> 2
Met Asp Cys Val Glu Ser Leu His Glu Thr Arg Leu Lys Glu Ala Arg
1 5 10 15
His Leu Val Arg Gly Val Glu Lys Gly Ser Leu Glu Ser Leu Val Met
20 25 30
Val Asp Ala Leu Gln Arg Leu Gly Ile Ala Tyr His Phe Glu Glu Glu
35 40 45
Thr Arg Ser Leu Leu Gln Glu His Leu Leu Ser Ala Tyr Asn Gly His
50 55 60
Ser Ser Ser Asp Ser Leu Asn Glu Val Ala Leu Arg Phe Arg Leu Leu
65 70 75 80
Arg Gln Glu Gly Tyr Asn Val Pro Ala Gly Ile Phe Glu Gly Phe Lys
85 90 95
Ser Lys Asp Asp Asn Ser Gly Arg Phe Thr Phe Asp Arg Lys Leu Tyr
100 105 110
Lys Asp Ile Val Gly Leu Met Ser Leu Tyr Glu Ala Ser His Leu Gly
115 120 125
Thr Gln Gly Glu Asp Ile Leu Asp Asp Ala Ser Ser Phe Ser Lys Lys
130 135 140
Ala Leu Ser Gly Thr Gln Asp Arg Leu Leu Asp Ala Gly Asn Asp Ile
145 150 155 160
Asp Gly Ile Ser Ser Pro Ile Leu Ile Glu Phe Val Arg Asn Thr Leu
165 170 175
Ser Asn Pro Phe His Lys Ser Leu Pro Arg Phe Thr Ser Glu Thr Phe
180 185 190
Gln Val Tyr Phe Gly Gly Pro Tyr Glu Trp Ile Gly Val Phe Arg Glu
195 200 205
Leu Ala Val Leu Asp Ser Glu Leu Ile Ala Ser Ile Asn Arg Asn Glu
210 215 220
Ile Leu Gln Val Ser Lys Trp Trp Lys Asp Leu Gly Leu Ala Lys Glu
225 230 235 240
Leu Lys Phe Ala Arg Asp Gln Pro Met Lys Trp Tyr Leu Trp Pro Met
245 250 255
Ala Val Leu Pro Asp Pro Lys Leu Ser Gln Glu Arg Val Asp Ile Thr
260 265 270
Lys Pro Ile Ala Met Val Tyr Ile Ile Asp Asp Ile Phe Asp Val Tyr
275 280 285
Gly Ser Leu Asp Glu Leu Thr Leu Phe Thr Glu Ala Ile Lys Arg Trp
290 295 300
Glu Cys Ile Glu Glu Leu Pro Asp Tyr Met Lys Arg Cys Phe Arg Ala
305 310 315 320
Leu Asp Asp Ile Thr Ser Glu Ile Ser Phe Asn Val Tyr Lys Lys His
325 330 335
Gly Trp Ser Pro Met Asp Ser Leu Lys Glu Ser Trp Lys Ser Leu Phe
340 345 350
Asp Ala Phe Leu Val Glu Thr Arg Trp Phe Arg Cys Arg Asn Ser Pro
355 360 365
Ser Ala Asp Glu Tyr Leu Asn Asn Ala Ile Val Thr Ser Gly Val Pro
370 375 380
Leu Val Ile Val His Ile Phe Ala His Leu Cys Glu Gly Leu Asn Lys
385 390 395 400
Gln Cys Leu Asp Lys Leu Ser Ser Phe Ser Glu Ile Ser Ser Ser Thr
405 410 415
Ala Lys Ile Leu Arg Leu Trp Asp Asp Leu Gly Ser Ala Lys Asp Glu
420 425 430
Asn Gln Glu Gly His Asp Gly Ser Tyr Leu Asp Tyr Tyr Met Asn Glu
435 440 445
Asn Pro Ser Cys Ser Leu Glu Gln Ala Arg Asp Arg Val Lys Glu Met
450 455 460
Ile Ser Asp Ala Trp Lys Asn Leu Asn Lys Glu Cys Leu Phe Ser His
465 470 475 480
Thr Phe Ser Thr Ser Val Ala Gln Ala Ser Leu Asn Thr Ala Arg Met
485 490 495
Val Pro Leu Met Tyr Asp Tyr Asp Glu Asn His Cys Leu Pro Lys Ile
500 505 510
Glu Asp Tyr Met Lys Ser Leu Leu Ile Asn Gly Leu Ala
515 520 525
<210> 3
<211> 1578
<212> DNA
<213> Lagerstroemia speciosa (Lagerstroemia caudata)
<400> 3
atggattgtg tggaaagctt gcacgagagg aggttgaagg aagcgaggca tttggttcgg 60
agagtggaaa agggatcctt agaaagtctg gtaatggtgg acgctctcca acgccttggc 120
attgcctacc actttgagga agagactcgg agccttctgc aggaacattt gctctccgcc 180
tacaatggcc actccagcag tgacagcctt aacaaggtcg cgcttcgttt tcgacttctc 240
cgacaggaag gctacaatgt tcccgcaggt atttttgagg gcttcaagag caaagacgac 300
aacaatggca gatttacgtt tgaccgaaag ctgtacaagg acattgtcgg attaatgagc 360
ttgtatgaag cttcccatct gggcacacaa ggggaagata tactcgacga ggctgcaagt 420
tttagcaaaa aggccctaac tggtacacta gatagattgt tatgtgctgg taatgatact 480
aacggcataa gttctccgat actaagagag cttgtgaaga acacattgtc aaatcccttc 540
cacaagagct tgccaaggtt cacttctgag actttccaag tttatttcgg tgggcccttc 600
gagtggatcg gagtattcag ggagctggcc gttttggact cggaattggt tgcttccatt 660
aatcggaacg aaatcttaca agtctccaag tggtggaaag atctaggatt agcaaaggag 720
ttgaagttcg caagagatca gcccatgaaa tggtacctgt ggccaatggc agttctaccg 780
gacccgaaat tatcacagga gagagtggat ataacaaagc cgatagccat ggtctacatc 840
atcgacgaca tcttcgatgt ctatgggtcc cttgatgaac tcactctctt cactgaagcc 900
gtcaaaagat gggagtgtat cgaagaactg ccggattaca tgaagagatg cttcagggct 960
ttagatgaca ttacgagtga aatcagcttc aacgtctata aaaagcatgg ctggagtcca 1020
atggatttac tcaaagaatc atggaagagc ttgtttgatg cattcctgct ggaaacgaga 1080
tggtttcgtt gccacaactc tccatctgca gatgagtact tgaacaatgc aatagtcacc 1140
tccggggtgc ccctagtgat tgttcacata tttgcgcacc tgtgtgaagg tctaaataag 1200
cagtgtcttg acaagctgag tggtttctcg gaaatttctt cctcgacagc aaagattcta 1260
cgtctatggg atgacctcgg aagtgccaag gatgagaatc aagaaggtca tgatggctcg 1320
tacttggact actacatgaa cgaaaaccct agttgctcac tcgagcaggc aacagaccgt 1380
gtgaaggaga tgatcttaga tgcatggaag agcctaaaca aagaatgtct cttctcccac 1440
acattctcta cttccgtggc tcaagcttct cttaacactg ccagaatggt ccctctcatg 1500
tacgattacg atgagaacca ttgcctccca agaattgagg attatatgaa gtcattgctg 1560
attaatggtt tagcctaa 1578
<210> 4
<211> 1578
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
atggattgcg tggaaagtct gcatgaacgt cgtctgaaag aagcacgcca tctggtgcgt 60
cgtgttgaaa aaggtagcct ggaaagcctg gttatggttg atgcactgca gcgtctgggt 120
attgcatatc attttgaaga agaaacccgt agcctgctgc aggaacatct gctgagcgcc 180
tataatggtc atagcagtag cgatagcctg aataaggtgg ccctgcgctt tcgtctgctg 240
cgccaggaag gctataatgt tccggccggc atttttgaag gctttaaaag taaagatgac 300
aacaatggtc gctttacctt tgatcgcaaa ctgtataaag atattgtggg cctgatgagc 360
ctgtatgaag ccagtcatct gggcacccag ggtgaagata ttctggatga agccgcaagc 420
tttagtaaaa aagccctgac cggcaccctg gatcgtctgc tgtgtgcagg taatgatacc 480
aatggtatta gcagtccgat tctgcgtgaa ctggtgaaaa ataccctgag caatccgttt 540
cataaaagcc tgccgcgctt taccagtgaa acctttcagg tgtattttgg tggtccgttt 600
gaatggattg gcgtttttcg cgaactggca gttctggata gcgaactggt tgccagtatt 660
aatcgcaatg aaattctgca ggttagtaaa tggtggaaag atctgggcct ggcaaaagaa 720
ctgaaatttg cacgtgatca gccgatgaaa tggtatctgt ggccgatggc cgttctgccg 780
gatccgaaac tgagccagga acgcgtggat attaccaaac cgattgcaat ggtgtatatt 840
attgatgata tcttcgacgt ttacggcagt ctggatgaac tgaccctgtt taccgaagcc 900
gtgaaacgct gggaatgcat tgaagaactg ccggattata tgaaacgctg ctttcgtgcc 960
ctggatgata ttaccagtga aattagcttt aacgtgtata aaaagcacgg ttggagtccg 1020
atggatctgc tgaaagaaag ctggaaaagc ctgtttgatg cctttctgct ggaaacccgt 1080
tggtttcgtt gtcataatag cccgagcgca gatgaatatc tgaataatgc aattgtgacc 1140
agtggtgtgc cgctggttat tgttcatatt tttgcacatc tgtgcgaagg tctgaataag 1200
cagtgtctgg ataaactgag cggctttagt gaaatttcaa gtagcaccgc caaaattctg 1260
cgcctgtggg atgatctggg cagtgcaaaa gatgaaaatc aggaaggtca tgatggtagt 1320
tatctggatt attatatgaa cgaaaacccg agctgcagtc tggaacaggc aaccgatcgc 1380
gttaaagaaa tgattctgga tgcatggaaa agtctgaata aggaatgcct gtttagtcat 1440
acctttagca ccagtgttgc acaggcaagt ctgaataccg cacgtatggt tccgctgatg 1500
tatgattatg atgaaaatca ttgcctgccg cgtattgaag attatatgaa gagcctgctg 1560
attaatggtc tggcataa 1578
<210> 5
<211> 1578
<212> DNA
<213> Bai Yunying Cynanchum (L. Indica 'Baiyunyingxia')
<400> 5
atggattgtg tggaaagctt gcacgagacg aggttgaagg aagcgaggca tttggttcgg 60
ggagtggaaa agggatcctt agaaagtctg gtaatggtgg acgctctcca acgccttggc 120
attgcctacc actttgagga agagactcgg agccttctgc aggaacattt gctctccgcc 180
tacaatggcc actccagtag tgacagcctt aacgaggttg cacttcgttt tcgacttctc 240
cgacaggaag gctacaatgt tcccgcaggt atttttgagg gcttcaagag caaagacgac 300
aacagtggca gatttacgtt tgaccgaaag ctgtacaagg atattgtcgg attaatgagc 360
ttgtatgaag cttcccatct gggcacgcaa ggggaagata tactcgacga tgcttcaagt 420
tttagcaaaa aggccctcag tggtacacaa gatagattgt tagatgctgg taatgatatc 480
gacggcataa gttctccgat actaatagag tttgtgagga acacattgtc aaatcccttc 540
cacaagagct taccaaggtt cacttctgag accttccaag tttatttcgg agggccctac 600
gagtggatcg gagtattcag ggagctggcc gttttggact cggaattgat tgcttccatt 660
aatcgaaatg aaatcttaca agtctccaag tggtggaaag atctagggtt agcaaaggag 720
ttgaagttcg caagagatca gcccatgaaa tggtacctgt ggccaatggc agttctacca 780
gacccgaaat tatcgcaaga gagagtggat ataacaaagc caatagccat ggtctacatc 840
atcgacgaca tcttcgatgt ctatgggtcc cttgatgaac tcactctctt cactgaagcc 900
atcaaaagat gggagtgtat cgaagaactg ccggattaca tgaagagatg tttcagggct 960
ttagatgaca ttacgagtga aatcagcttc aacgtctata aaaagcatgg ctggagtcca 1020
atggattcac tcaaagaatc ctggaagagc ttgtttgatg cattcctggt ggaaacgaga 1080
tggtttcgtt gccgcaactc tccatctgca gatgagtact tgaacaatgc aatagtcacc 1140
tccggggtgc ccctggtgat cgttcacata tttgcgcacc tgtgtgaagg tctgaataag 1200
cagtgtcttg acaagctgag tagtttctcg gaaatttctt cctcgacagc aaagattcta 1260
cgtctatggg atgacctcgg aagtgccaag gatgagaatc aagaaggtca tgatggctcg 1320
tacttggact actacatgaa cgaaaaccct agttgctcac tcgagcaggc aagggaccgt 1380
gtgaaggaga tgatctcaga tgcatggaag aacctaaaca aagaatgtct cttctcccac 1440
acattctcta cttccgtggc tcaagcttct cttaacactg ccagaatggt ccctctcatg 1500
tacgattacg atgagaacca ttgcctccca aaaatcgagg attatatgaa gtcattgctg 1560
attaatggtt tagcctaa 1578
<210> 6
<211> 1596
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 6
atggattgcg tggaaagtct gcatgaaacc cgtctgaaag aagcacgcca tctggtgcgt 60
ggcgtggaaa aaggtagcct ggaaagtctg gttatggtgg atgcactgca gcgtctgggc 120
attgcatatc attttgaaga agaaacccgt agcctgctgc aggaacatct gctgagcgca 180
tataatggtc atagtagcag cgatagcctg aatgaagtgg ccctgcgttt tcgtctgctg 240
cgccaggaag gttataatgt gccggcaggt atttttgaag gctttaaaag taaagacgac 300
aatagtggtc gttttacctt tgatcgtaaa ctgtataaag acattgtggg cctgatgagt 360
ctgtatgaag ccagccatct gggcacccag ggtgaagata ttctggatga tgcaagtagc 420
tttagtaaaa aagccctgag tggtacccag gatcgtctgc tggatgcagg taatgatatt 480
gatggcatta gtagcccgat tctgattgaa tttgtgcgca ataccctgag caatccgttt 540
cataaaagtc tgccgcgttt taccagcgaa acctttcagg tgtattttgg tggtccgtat 600
gaatggattg gtgtgtttcg tgaactggcc gttctggata gcgaactgat tgccagcatt 660
aatcgcaatg aaattctgca ggtgagcaaa tggtggaaag atctgggtct ggcaaaagaa 720
ctgaaatttg cacgcgatca gccgatgaaa tggtatctgt ggccgatggc agtgctgccg 780
gatccgaaac tgagccagga acgtgtggat attaccaaac cgattgccat ggtttatatt 840
attgatgata tcttcgacgt gtacggtagc ctggatgaac tgaccctgtt taccgaagca 900
attaagcgct gggaatgtat tgaagaactg ccggattata tgaaacgttg ctttcgtgcc 960
ctggatgata ttaccagcga aattagtttt aacgtttaca aaaagcacgg ttggagtccg 1020
atggatagcc tgaaagaaag ctggaaaagt ctgtttgatg cctttctggt tgaaacccgt 1080
tggtttcgtt gccgcaatag cccgagcgca gatgaatatc tgaataatgc aattgttacc 1140
agcggcgtgc cgctggttat tgtgcatatt tttgcccatc tgtgtgaagg tctgaataag 1200
cagtgcctgg ataaactgag tagctttagc gaaatttcaa gcagcaccgc caaaattctg 1260
cgcctgtggg atgatctggg tagtgccaaa gatgaaaatc aggaaggtca tgatggtagt 1320
tatctggatt attatatgaa cgaaaacccg agttgcagtc tggaacaggc acgtgatcgc 1380
gttaaagaaa tgattagtga tgcatggaaa aacctgaata aggaatgcct gtttagtcat 1440
acctttagta ccagtgttgc ccaggccagc ctgaataccg cccgtatggt tccgctgatg 1500
tatgattatg atgaaaatca ttgcctgccg aaaattgaag attatatgaa gagcctgctg 1560
attaatggtc tggcacatca tcatcatcac cattaa 1596

Claims (10)

1. Lagerstroemia terpene synthase geneLcTPS14The method is characterized in that the amino acid sequence of the encoded protein is shown as SEQ ID NO. 1.
2. Comprising the gene according to claim 1LcTPS14The biological material is recombinant DNA, an expression cassette, a transposon, a plasmid vector, a viral vector or engineering bacteria.
3. The gene of claim 1LcTPS14Or any one of the following applications of the biomaterial of claim 2:
(1) For the preparation of transgenic plants;
wherein the purpose for preparing transgenic plants is to improve the floral fragrance of the plants; the material for controlling the fragrance is trans-nerolidol and linalool;
(2) Used for plant breeding;
the breeding purpose is to improve the flower fragrance of plants; the material for controlling the fragrance is trans-nerolidol and linalool;
the plant is Lagerstroemia plant.
4. The gene of claim 1LcTPS14Application in biosynthesis of plant floral substances; wherein the floral material is linalool, trans-nerolidol;
the plant is Lagerstroemia plant.
5. A gene as set forth in claim 1LcTPS14Any of the following uses of the encoded crape terpene synthases:
(1) In vitro catalyzing geranyl pyrophosphate to generate linalool;
(2) In vitro catalysis of farnesyl pyrophosphate to trans-nerolidol.
6. Lagerstroemia terpene synthase geneLiTPS14The method is characterized in that the amino acid sequence of the encoded protein is shown as SEQ ID NO. 2.
7. Comprising the gene according to claim 6LiTPS14The biological material is recombinant DNA, an expression cassette, a transposon, a plasmid vector, a viral vector or engineering bacteria.
8. The gene according to claim 6LiTPS14Or any one of the following applications of the biomaterial of claim 7:
(1) For the preparation of transgenic plants;
wherein the purpose for preparing transgenic plants is to improve the floral fragrance of the plants; the material for controlling the fragrance is trans-nerolidol and linalool;
(2) Used for plant breeding;
the breeding purpose is to improve the flower fragrance of plants; the material for controlling the fragrance is trans-nerolidol and linalool;
the plant is Lagerstroemia plant.
9. The gene according to claim 6LiTPS14Application in biosynthesis of plant floral substances; wherein the floral material is linalool, trans-nerolidol;
the plant is Lagerstroemia plant.
10. A gene as set forth in claim 6LiTPS14Any of the following uses of the encoded crape terpene synthases:
(1) In vitro catalyzing geranyl pyrophosphate to generate linalool;
(2) In vitro catalysis of farnesyl pyrophosphate to trans-nerolidol.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108660128A (en) * 2018-05-18 2018-10-16 江苏省农业科学院 A kind of alfalfa sesquiterpene synthases, its encoding gene, carrier, polyclonal antibody and its application
CN108893482A (en) * 2018-06-22 2018-11-27 中国医学科学院药用植物研究所 Radix Salviae Miltiorrhizae Terpene synthase gene SmTPS8, its cloning primer, expression vector, catalysate and application

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108660128A (en) * 2018-05-18 2018-10-16 江苏省农业科学院 A kind of alfalfa sesquiterpene synthases, its encoding gene, carrier, polyclonal antibody and its application
CN108893482A (en) * 2018-06-22 2018-11-27 中国医学科学院药用植物研究所 Radix Salviae Miltiorrhizae Terpene synthase gene SmTPS8, its cloning primer, expression vector, catalysate and application

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