Lagerstroemia terpene synthase gene and application thereof
阅读说明：本技术 紫薇属萜烯合酶基因及其应用 (Lagerstroemia terpene synthase gene and application thereof ) 是由 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 于 2021-08-17 设计创作，主要内容包括：本发明公开了紫薇属萜烯合酶基因及其应用。本发明首次在紫薇属植物中克隆分离获得了萜烯合酶基因并通过体外表达探究其催化功能,初步了解紫薇属植物花香物质生物合成的分子机制,为进一步研究紫薇属植物花香生物合成及调控提供基础。(The invention discloses a lagerstroemia terpene synthase gene and application thereof. The terpene synthase gene is obtained by cloning and separating in the lagerstroemia plant for the first time, the catalytic function of the terpene synthase gene is explored through in vitro expression, the molecular mechanism of the lagerstroemia plant flower fragrance substance biosynthesis is preliminarily known, and a foundation is provided for further researching the lagerstroemia plant flower fragrance biosynthesis and regulation.)
1. Lagerstroemia terpene synthase gene LcTPS14, characterized in that it is a gene encoding the following protein (a) or (B):
(A) consisting of SEQ ID NO:1, and the protein consists of an amino acid sequence shown in the specification; or
(B) SEQ ID NO:1 by substituting, deleting or adding one or more amino acids, and the protein derived from (A) has equivalent functions.
2. Biomaterial containing the gene LcTPS14 of claim 1, said biomaterial being a recombinant DNA, an expression cassette, a transposon, a plasmid vector, a viral vector or an engineered bacterium.
3. Use of the gene LcTPS14 of claim 1 or the biomaterial of claim 2 for any of the following applications:
(1) for the preparation of transgenic plants;
(2) for plant breeding;
wherein, the breeding aim is to improve the flower fragrance of the plants;
the plant comprises a lagerstroemia plant.
4. Use of the gene LcTPS14 of claim 1 in the biosynthesis of floral substances; wherein the floral material comprises monoterpenes and sesquiterpenes; the monoterpene compound comprises linalool, and the sesquiterpene compound comprises E-nerolidol;
the plant comprises a lagerstroemia plant.
5. Use of the lagerstroemia terpene synthase encoded by the gene LcTPS14 of claim 1, in any of the following applications:
(1) catalyzing geranyl pyrophosphate in vitro to generate linalool;
(2) catalyzing farnesyl pyrophosphate in vitro to generate E-nerolidol.
6. Lagerstroemia terpene synthase gene LiTPS14, characterized in that it is a gene encoding the following protein (a) or (b):
(a) consisting of SEQ ID NO:2, and 2, or a pharmaceutically acceptable salt thereof; or
(b) SEQ ID NO:2 by substituting, deleting or adding one or more amino acids, and has equivalent functions.
7. Biological material containing the gene LiTPS14 according to claim 1, which is a recombinant DNA, an expression cassette, a transposon, a plasmid vector, a viral vector or an engineered bacterium.
8. Use of the gene LiTPS14 according to claim 1 or the biomaterial according to claim 2 for any of the following applications:
(1) for the preparation of transgenic plants;
(2) for plant breeding;
wherein, the breeding aim is to improve the flower fragrance of the plants;
the plant comprises a lagerstroemia plant.
9. Use of the gene LiTPS14 according to claim 1 for the biosynthesis of floral substances; wherein the floral material comprises monoterpenes and sesquiterpenes; the monoterpene compound comprises linalool, and the sesquiterpene compound comprises E-nerolidol;
the plant comprises a lagerstroemia plant.
10. Use of the lagerstroemia terpene synthase encoded by the gene LiTPS14 according to claim 1, in any one of the following applications:
(1) catalyzing geranyl pyrophosphate in vitro to generate linalool;
(2) catalyzing farnesyl pyrophosphate in vitro to generate E-nerolidol.
Technical Field
The invention relates to the field of plant genetic engineering, in particular to a lagerstroemia terpene synthase gene and application thereof.
Background
Lagerstroemia caudate (Lagerstroemia caudate) and Lagerstroemia caudate 'white cloud sunset' (L.indica 'Baiyunyingxia') are both white flowers, and monoterpene is the main difference volatile component of flowers in full bloom stage of the Lagerstroemia caudate and the Lagerstroemia caudate. The monoterpene substances in the full-bloom stage of lagerstroemia caudate are characteristic aroma components and have high content, the lagerstroemia caudate 'Caesalpinia indica' has few types and low concentration of the monoterpene substances, and the terpene synthase is a key enzyme of a terpene substance synthesis pathway in a plant body. At present, no research report about terpene synthase gene is found in lagerstroemia plant.
Disclosure of Invention
The invention aims to provide a lagerstroemia terpene synthase gene and application thereof.
To achieve the object of the present invention, in a first aspect, the present invention provides a lagerstroemia terpene synthase gene LcTPS14 (from lagerstroemia caudate), which is a gene encoding the following protein (a) or (B):
(A) 1, a protein consisting of an amino acid sequence shown in SEQ ID NO; or
(B) 1, protein which is derived from (A) and has the same function 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 containing said gene LcTPS14, including but not limited to recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors, engineered 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 biological material containing the gene:
(1) for the preparation of transgenic plants;
(2) is used for plant breeding.
Among them, the breeding aim is to improve plant floral aroma.
In the present invention, the plant includes a lagerstroemia plant.
In a fourth aspect, the invention provides an application of the gene LcTPS14 in plant floral substance biosynthesis; wherein, the flower fragrance substance includes but is not limited to monoterpene and sesquiterpene compound.
Monoterpenes include, but are not limited to, linalool, and sesquiterpenes include, but are not limited to, E-nerolidol.
In a fifth aspect, the invention provides any one of the following uses of the lagerstroemia terpene synthase encoded by the gene LcTPS 14:
(1) catalyzing geranyl pyrophosphate (GPP) in vitro to generate linalool;
(2) catalyzing farnesyl pyrophosphate (FPP) in vitro to generate E-nerolidol.
In a sixth aspect, the present invention provides a lagerstroemia terpene synthase gene LiTPS14 (from lagerstroemia indica 'cloud enanthes') which is a gene encoding a protein (a) or (b) as follows:
(a) a protein consisting of an amino acid sequence shown as SEQ ID NO. 2; or
(b) 2, protein which is derived from (a) and has the same function 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 including but not limited to recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors, engineered bacteria or non-regenerable plant parts.
In an eighth aspect, the invention provides any one of the following applications of the gene LiTPS14 or the biological material containing the gene:
(1) for the preparation of transgenic plants;
(2) is used for plant breeding.
Among them, the breeding aim is to improve plant floral aroma.
In the present invention, the plant includes a lagerstroemia plant.
In a ninth aspect, the invention provides an application of the gene LiTPS14 in plant floral substance biosynthesis; wherein, the flower fragrance substance includes but is not limited to monoterpene and sesquiterpene compound.
Monoterpenes include, but are not limited to, linalool, and sesquiterpenes include, but are not limited to, E-nerolidol.
In a tenth aspect, the present invention provides any one of the following uses of the lagerstroemia terpene synthase encoded by the gene LiTPS 14:
(1) catalyzing geranyl pyrophosphate (GPP) in vitro to generate linalool;
(2) catalyzing farnesyl pyrophosphate (FPP) in vitro to generate E-nerolidol.
The terpene synthase gene is obtained by cloning and separating in the lagerstroemia plant for the first time, the catalytic function of the terpene synthase gene is explored through in vitro expression, the molecular mechanism of the lagerstroemia plant flower fragrance substance biosynthesis is preliminarily known, and a foundation is provided for further researching the lagerstroemia plant flower fragrance biosynthesis and regulation.
Drawings
FIG. 1 shows the result of full-length amplification of terpene synthase gene in a preferred embodiment of the present invention.
FIG. 2 is a phylogenetic tree analysis of terpene synthase genes according to a preferred embodiment of the present invention. Wherein, Aa, artemisia annua; ag, fir; am, snapdragon; ar, Agastache rugosa; cb, Clarkia breweri; cc, Clarkia concinna; ci, chicory; cl, lemon; cm, winter squash; ct, cinnamomum tenuipilum; eo, american oil palm; ga, tree cotton; gb, ginkgo biloba; ll, Lavandula latifolia; ls, lettuce; md, apple; ml, peppermint; ob, basil; oe, trifolium olea; pf, tempering; ps, spruce north america; pt, loblolly pine; sf, Salvia fructicose; so, saervia; st, Yangliu; vv, grape; zm, maize; zz, Zingiber zerumbet.
FIG. 3 shows the expression rules of TPS gene in different flowering stages in the preferred embodiment of the present invention.
FIG. 4 shows the expression pattern of TPS gene in different parts of flower in the preferred embodiment of the present invention.
FIG. 5 shows the result of detecting TPS gene recombinant vector in the preferred embodiment of the present invention.
FIG. 6 is a view showing the subcellular localization of TP14 in a preferred embodiment of the invention. Wherein, Green column represents GFP fluorescence under a Green channel, Red column represents chlorophyll autofluorescence under a Red channel, Merged column represents superposition of a bright field image, the Green channel and the Red channel, BF column represents a bright field, and CK represents no-load contrast; the scale bar is 50 μm.
FIG. 7 shows the in vitro enzymatic products of the recombinant proteins LcTPS14 and LiTPS14 in a preferred embodiment of the invention. Wherein, 1,4 and 5 are non-terpenoid compounds; 2. trans-nerolidol; 3. nerolidol acetate; 6. linalool; 7. linalyl formate.
FIG. 8 is a graph showing ion peaks of in vitro enzymatic reaction products in a preferred embodiment of the present invention.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular Cloning handbook, Sambrook et al (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual,2001), or the conditions as recommended by the manufacturer's instructions.
Example 1 cloning and bioinformatic analysis of Lagerstroemia terpene synthase genes LcTPS14 and LiTPS14
The Lagerstroemia indica species Lagerstroemia caudate (Lagerstroemia caudate) and the odor-free Lagerstroemia indica 'white cloud astraga' (L.indica 'Baiyunyingxia') are both white flowers, monoterpene substances are main different volatile components of flowers in full bloom stages of the Lagerstroemia caudate and the odor-free Lagerstroemia caudate, but related genes and functions of biosynthesis of the substances in the Lagerstroemia caudate are not clear, and in order to analyze a molecular mechanism of biosynthesis of the Lagerstroemia single-terpene substances, the invention carries out full-length transcription group sequencing on the Lagerstroemia caudate, screens to obtain the terpene synthase genes with different expressions, uses the Lagerstroemia caudate and the Lagerstroemia caudate 'white cloud astraga' as materials, separates the terpene synthase genes, analyzes the expression rules of the genes, and carries out preliminary verification on the functions of the terpene synthase genes.
1. Materials and methods
1.1 plant material: referring to the study of xuwang (2019) on flowers of lagerstroemia caudate at each stage and different parts of flowers in full bloom stage, lagerstroemia caudate and lagerstroemia caudate 'Baiyunnangxia' at different stages and different parts of flowers in full bloom stage are collected, packaged by a centrifugal tube, quick-frozen by liquid nitrogen, and stored in an ultra-low temperature refrigerator in a laboratory.
1.2 reagents and consumables
Total plant RNA extraction kit (DP441), DNA recovery kit (DP209), Escherichia coli (Escherichia coli) competent DH5 alpha, ampicillin (RT501) were purchased from Tiangen Biochemical technology (Beijing) Ltd;max DNA Polymerase (R045A), antigenomic reverse transcription reagent (RR047), TBPremix Ex TaqTMII (RR820A), DNA Marker available from Baozi physician technology (Beijing) Ltd; TOPO Blunt (Blunt-ended) cloning vector (cv17) was purchased from Erdela biotech, Inc. of Beijing; exogenous RNAse scavengers were purchased from Wash-ocean organisms (Beijing) technologies, Inc. The primer synthesis and sequencing are all undertaken by Beijing Optimalaceae New industry biotechnology Limited; other consumables are available from Beerdy Biotechnology Inc., Beijing.
1.3 test methods
(1) Plant total RNA extraction
Before RNA extraction, exogenous RNA enzyme scavenger is used to treat consumables, table tops, instruments, high-temperature and high-pressure sterilizing equipment and the like, so that interference of RNA decomposition enzyme is reduced. The sample was ground well into powder with liquid nitrogen, total RNA was extracted by the method of Chenzhilin (2017), and a lysate was prepared using DTT (working concentration 1 mmol/L).
(2) Detection of RNA integrity and concentration
Detection was performed by 1.2% TBE normal agarose gel electrophoresis, and the film was photographed and analyzed under UV conditions on a gel imaging system. Detecting RNA extractive solution with ultramicro ultraviolet spectrophotometer, and recording OD260/OD280Reading and concentration, RNA temporary storage on ice and subsequent reversal experiment, long-term storage in-80 ℃ refrigerator.
(3) Reverse synthesis of cDNA and amplification of the full Length of the Gene of interest
cDNA was obtained by reverse transcription at 0.5. mu.g total RNA per tube following the reverse transcription kit protocol. Amplification primers (upstream 5'-ATGGATTGTGTGGAAAGCTT-3' and downstream 5'-TTAGGCTAAACCATTAATCAGC-3') were designed using Primer premier 5 based on full length transcriptome sequence data. And respectively using cDNA of flowers of lagerstroemia caudate and lagerstroemia indica 'white cloud enanthes' full bloom stage as templates to amplify target genes. The PCR reaction system is as follows:
the PCR reaction procedure was as follows: 1min at 98 ℃; 10s at 98 ℃, 15s at 55 ℃, 30s at 72 ℃ and 35 cycles; 2min at 72 ℃; storing at 4 ℃.
The products were detected by agarose gel electrophoresis and gel recovery was performed according to the DNA recovery kit instructions. Agarose gel electrophoresis and detection of the recovered product using an ultra-micro UV spectrometer.
(4) Target gene connected TOPO cloning vector
The end of the Edelapin is connected with a kit, the connector system is 10 XEnhancer 0.5 muL, TOPO carrier 0.5 muL, target gene 20-40ng, ddH2The content of O is filled to 5 mu L. Ligation was performed at 25 ℃ for 5 min.
Transforming Escherichia coli DH5 alpha, mixing 5 μ L of the connecting solution with 50 μ L of the bacterial solution, flicking, ice-cooling for 25min, heat-shocking for 45sec-1min at 42 deg.C, standing for 2min on ice, adding 500 μ L of LB liquid without antibiotic, and renaturing at 200rpm in a shaker at 37 deg.C for 30 min. And (3) centrifugally collecting thalli at 5000rpm, removing supernatant in an ultra-clean workbench environment, remaining 50 mu L of liquid, sucking and beating the resuspended thalli by a gun head, coating the bacterial liquid on a solid LB culture medium containing Amp (working concentration of 50 mu g/mL), and carrying out inverted culture at 37 ℃ for overnight.
Selecting a round single colony with proper size in 1mL of liquid LB culture medium containing 50 mu g/mL Amp, culturing at 37 ℃ and 200rpm for 4h, carrying out PCR detection on bacterial liquid, and sending positive bacterial liquid to a company for sequencing.
(5) Bioinformatic analysis of sequences
Bioinformatics analysis was performed on the obtained sequence information. ORF Finder was used for open reading frame search, and Blastp analyzed multiple sequence homology and protein domains. The phylogenetic tree was constructed using DNAMAN and ClustalW software for multiple sequence alignments, MEGA 7.
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 coded 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 coding protein is shown as SEQ ID NO. 2.
(6) Verification of Gene expression by qRT-PCR
Designing quantitative primers in Primer premier 5 software according to the Primer design principle, diluting reference genes by EF-1 alpha (Chenzhilin, 2017) and cDNA by 5 times, and using a 10 mu L system (dye 5 mu L + cDNA1 mu L + upstream and downstream primers 0.4 mu L + ddH respectively2O) carrying out fluorescent quantitative reaction, preparing the dye solution and the cDNA in advance according to the reaction number +3, fully mixing and centrifuging. Primer Advance and ddH2And mixing the O uniformly according to the reaction number + 3. The temperature program is: pre-denaturation at 95 ℃ for 30 s; 5s at 95 ℃ and 15s at 60 ℃ for 40 cycles; the temperature was raised from 60 ℃ to 95 ℃ at 5 ℃/s, Δ T ═ 1 ℃, and 6s were collected. Performing 3 technical and biological replicates, using 2-△△CtAnd (4) calculating and analyzing data.
2. Results and analysis
2.1 extraction results of Total RNA from plants
OD measured from extracted Total RNA260/OD280In 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 the RNA shows that the extracted total RNA band is clear and has no obvious tailing phenomenon, and can be used for downstream experiments.
2.2 terpene synthase sequence analysis
Candidate terpene synthase sequences are obtained by early screening, NCBI ORF Finder is used for searching gene CDS and designing full-length primers, LcTPS14 is successfully separated and obtained from lagerstroemia caudate, and LiTPS14 is separated and obtained from lagerstroemia indica 'Caesalpinia enanthes' by a homologous cloning method (figure 1).
(1) LcTPS14 and LiTPS14 sequence analysis
The length of the LcTPS14 gene and the LiTPS14 gene are both 1575bp, 525 amino acids (SEQ ID NO:1 and 2) are coded, the similarity of the nucleic acid sequences of the LcTPS14 gene and the LiTPS14 gene is 96.83%, the similarity of the amino acid sequences of the LcTPS14 gene and the LiTPS14 gene is 95.24%, and the LcTPS14 gene and the LiTPS14 gene contain DDXXD and DTE/NSE structural domains.
(2) TPS gene coding protein homology comparison and phylogenetic tree construction
Performing blastp comparison analysis on the information of the two gene sequences in NCBI respectively, and finding that amino acid sequences of LctPS14 and LiTPS14 contain DDXXD structural domains and lack RRX through multiple sequence comparison8A W domain. The NCBI protein conserved domain analysis has a Terphene _ cycle _ plant _ C1(LcTPS14 is in the 4-522 locus interval, LiTPS14 is in the 1-522 locus interval) and a Terphene _ synth _ C (208-470 locus) domain, an aspartic acid-rich region and a metal Mg2+A binding site.
Construction of phylogenetic trees (Neighbor-Joining, bootstrapping 1000, partial deletion 50) (FIG. 2), LctPS14 and LiTPS14 in the TPS-g subfamily, which is usually deficient in RRX, along with a validated functional terpene synthase gene(s)8A W domain.
2.3 Gene expression analysis
The method utilizes a fluorescence quantitative technology to explore the expression rule of candidate genes of different flowering stages and different parts of flowers of lagerstroemia caudate and lagerstroemia indica 'white cloud Xiagxia'. The fluorescent quantitative primers were initially screened by semi-quantitative PCR, and the screened primers should make the band single and clear without primer dimer (Table 1).
TABLE 1 fluorescent quantitation primers
(1) Transcriptome data validation
The qRT-PCR data of the lagerstroemia caudate is compared with the FPKM data in the same flowering stage in the second-generation transcriptome, and the rule is consistent, so that the differentially expressed gene data of the second-generation lagerstroemia caudate transcriptome is reliable.
(2) Expression analysis at different flowering stages
Selecting eight periods of leaf bud, inflorescence bud, immature bud, mature bud, bud stage, full bloom terminal stage and decay stage, and detecting expression change rule of gene at different periods (figure 3).
The expression rule of LcTPS14 in different flowering stages of lagerstroemia caudate is as follows: leaf bud-mature bud-full bloom stage-full bloom end stage-decay stage shows the trend of descending first, then ascending to peak value, then descending to lower expression level and no difference. The expression level is higher in leaf bud, inflorescence bud and full bloom stage.
The expression rule of the LiTPS14 in different flowering stages of the lagerstroemia indica 'Baiyunshuxia' is that the leaf bud-immature flower bud-mature flower bud-full bloom stage-full bloom final stage-decay stage shows that the expression rule firstly rises and then falls to a peak value and then falls to no difference, the expression quantity is relatively highest in the full bloom stage, and the expression quantity is extremely low in the leaf bud stage in the immature flower bud stage.
(3) Expression analysis of different parts of flowers
Expression analysis was performed on different parts of the blooming flowers (FIG. 4), and LcTPS14 was found to be highly expressed in the pistil of Lagerstroemia indica Lagerstroemia caudate and hardly expressed in other parts. The LiTPS14 is highly expressed in crape myrtle 'white cloud enantiomorpha' petals and pistils, and is expressed in stamens, calyces and pedicels, but the expression amount is not high.
In the embodiment, a molecular biology method is adopted to obtain key terpene synthase genes of a lagerstroemia caudate and lagerstroemia indica' white cloud enanthrine biosynthetic pathway, a phylogenetic tree is constructed, LcTPS14 and LiTPS14 are found to be divided into a TPS-g subfamily and are predicted to be expressed in cytoplasm, the genes may be sesquiterpene synthases or multifunctional enzymes which catalyze and generate different terpenes, and specific functions of the enzymes need to be further verified.
qRT-PCR detection expression analysis shows that the LcTPS14 and the LiTPS14 have the highest expression level in the full bloom stage, the LcTPS14 has the highest expression level in pistil, the LiTPS14 has the high expression level in petals and pistil, and the expression level in other parts is lower.
Example 2 subcellular localization of terpene synthase genes
Terpene synthases are key enzymes catalyzing the last step of the terpenoid reaction pathway, and the functions of the genes are related to subcellular localization. In the embodiment, plant expression vectors pCAMBIA Super1300, namely GFP, are used for constructing LcTPS14 and LiTPS14 plant expression vectors, agrobacterium-mediated infection is carried out on leaves of Nicotiana benthamiana, and subcellular localization of genes is researched, so that functions of the two TPS genes can be further known.
1. Materials and methods
1.1 plant Material
6 weeks old Nicotiana benthamiana (Nicotiana benthamiana).
1.2 reagents and consumables
Agarose gel DNA recovery kit (DP209) was purchased from Tiangen Biochemical technology, Inc. (Beijing). Agrobacterium GV3101 and Escherichia coli DH5 α were purchased from Shanghai Diego Biotech Ltd. Restriction endonucleases Kpn I (1068S), Xba I (1093S), DNA Ligation Kit (6023Q) were purchased from Baozi Biotech (Beijing) Ltd. DMSO (CAS: 67-68-5), Kana (INALCO, CAS: 133-92-6), Rif (INALCO, CAS: 13292-46-1), AS (Sigma, CAS: 2478-38-8), MES. H2Reagents such as O (Amresco, CAS: 145224-94-8) were purchased from Bearddy Biotechnology Inc., Beijing. The seamless cloning kit (CV1901) was purchased from Erdela biotech, Beijing. The primer synthesis and sequencing are all undertaken by Beijing Optimalaceae New Biotechnology Co.
The recombinant plasmid pCAMBIA Super1300: GFP (hereinafter referred to as pSuper1300) was stored in this laboratory.
1.3 test methods
(1) Solution preparation
100mg/mL Kana stock solution: 3g Kana +30mL ddH2O, filtering and sterilizing (0.22 μm), and 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. H2Dissolving O powder in 50mL of distilled water, and performing ultra-clean workFiltering and sterilizing (0.22 μm), and packaging at low temperature.
50mmol/L AS solution: 0.196g As powder was dissolved in 20mL DMSO, sterile filtered (0.22 μm), and stored at-20 ℃.
1mol/L MgCl2Solution: dissolving in water, sterilizing at high temperature and high pressure, and storing at 4 deg.C.
(2) Amplification of target Gene fragments
Primers are designed according to the requirements of the seamless cloning kit specification, and primers (an upstream primer 5'-ATACTAGTGGATCCGGTACCATGGATTGTGTGGAAAGCTTGC-3' and a downstream primer 5'-CCTTGCTCACCATGGTACCGGCTAAACCATTAATCAGCAATGAC-3') are amplified by taking TOPO recombinant plasmid containing a target gene as a template. The PCR reaction system is as follows:
the PCR reaction procedure was as follows: 1min at 98 ℃; 10s at 98 ℃, 15s at 55 ℃, 30s at 72 ℃ and 35 cycles; 2min at 72 ℃; storing at 4 ℃.
(3) Vector cleavage
The plasmid pSuper1300 is digested by the restriction enzyme Kpn I, GFP and the digestion is carried out for 4 hours at 37 ℃, and the digestion system is as follows:
(4) seamless cloning connection construction of recombinant vector
Mixing the target genes LcTPS14 and LiTPS14 recovered by enzyme digestion and the carrier recovered by single enzyme digestion according to the molar ratio of 3:1, connecting for 30min at 50 ℃, wherein the connecting system is as follows:
the transformed E.coli DH 5. alpha. was renatured at 37 ℃ for 1h with a shaker at 200 rpm. The thalli is collected by centrifugation at 5000rpm for 1min, supernatant is removed in a clean bench, 50 mu L of liquid is remained, the resuspended thalli is sucked and beaten by a gun head, the bacterial liquid is coated on a solid LB culture medium containing Kana (working concentration 50 mu g/mL), and the solid LB culture medium is inversely cultured at 37 ℃ overnight.
Selecting a round single colony with proper size to be cultured in 1mL of liquid LB culture medium containing 50 mu g/mL Kana for 4h at 37 ℃ under 200rpm, carrying out PCR detection on bacterial liquid, and selecting positive bacterial liquid to be sent to Beijing Optimalaceae New Biotechnology Limited for sequencing.
(5) Agrobacterium transformation and Nicotiana benthamiana leaf infection
Respectively transforming 4 recombinant vectors with no load and successful construction into agrobacterium GV3101, mixing 1 muL recombinant plasmid with 50 muL bacterial liquid, flicking the tube bottom uniformly, transforming the agrobacterium according to the instruction, coating the bacterial liquid on solid LB (50 mug/mL Kana +30 mug/mL Rif), and performing inverted culture at 28 ℃ for 60 h.
Single round colonies with appropriate size were picked and cultured in 1mL of liquid-containing LB medium (50. mu.g/mL Kana + 30. mu.g/mL Rif) at 28 ℃ and 200rpm for 24h, and the PCR detection was performed on the bacterial solution.
1mL of positive bacterial liquid is mixed with 15mL of LB culture medium containing 50. mu.g/mL Kana + 30. mu.g/mL Rif +10mmol/L MES + 20. mu. mol/L AS, and the mixture is induced overnight at 28 ℃ and 200 rpm. The cells were collected by centrifugation at 5000rpm, and freshly prepared invader solution (sterile water +10mmol/L MES + 200. mu. mol/L AS +10mmol/L MgCl) was used2) Resuspending the cells and adjusting OD600Standing for 2-3 h in the dark until the temperature is 0.5-0.8 ℃. The disposable injector sucks the staining solution to inject on the back of the Nicotiana benthamiana leaf, and after dark treatment for 24h, the tobacco leaves are placed into a climatic chamber.
(6) Fluorescence observation by laser confocal microscope
After the infection for 72 hours, 1cm multiplied by 1cm leaves are taken and sliced by clear water, the back surfaces of the leaves face upwards, and the green fluorescent protein expression condition is observed on a machine by taking no-load as a contrast.
2. Results and analysis
2.1 plant expression vectors containing target genes and transformed Agrobacterium
(1) The LcTPS14 and LiTPS14 recombinant vectors are obtained by a seamless cloning method
The termination codon of the target genes LcTPS14 and LiTPS14 is removed, an amplification primer is designed by a seamless cloning method, the amplification primer is connected with an expression vector subjected to Kpn I single enzyme digestion, and a correct recombinant vector is obtained by company sequencing.
(2) Obtaining Agrobacterium containing recombinant vector
No-load of pSuper1300 is taken as a control, agrobacterium is transformed by recombinant plasmids, after a complete colony is picked and cultured for 24 hours, the PCR detection of bacterial liquid (figure 5) is carried out, and bacterial liquid containing a band with a target size is screened for subsequent amplification culture and is infected on the leaf of Nicotiana benthamiana.
2.2 confocal laser scanning microscopy of Gene expression sites
The agrobacterium infects the lamina bengalensis, fluorescence is detected on a machine after injection for 72 hours, and the epidermis under the lamina bengalensis is observed under a Leica SP8 laser confocal microscope. The results showed that the leaf (CK) injected with the empty vector found GFP expression both in the cytoplasm and in the nucleus, and no green fluorescence signal was detected in the plastids, indicating that GFP fluorescence does not coincide with plastid autofluorescence. Observing the expression of the two TPS14 gene fusion proteins, similar to the no-load expression result, GFP signal expression is detected in cytoplasm, and the results show that the fusion proteins of LcTPS14 and LiTPS14 are both expressed in the cytoplasm of Nicotiana benthamiana leaves (figure 6), and the two proteins play a role in the cytoplasm and are consistent with the previous prediction result.
In the embodiment, the subcellular localization of LcTPS14 and LiTPS14 is obtained by a tobacco leaf transient expression technology, and both LcTPS14 and LiTPS14 are expressed in cytoplasm.
EXAMPLE 3 prokaryotic expression and in vitro functional Studies of terpene synthase Gene
In this example, prokaryotic expression vectors of target genes LcTPS14 and LiTPS14 were constructed, the target genes were expressed in E.coli to obtain recombinant proteins, FPP/GPP was used as a substrate for in vitro enzymatic reaction, and GC-MS technology was used to analyze the resultant, in order to obtain the function of recombinant proteins.
1. Materials and methods
1.1 vectors and strains
Arctic-Express BL21(DE3) and BL21(DE3) PLySs expression bacteria and expression vectors pCZN1, pET30a and pGEX-4T-1 are all purchased from Nanjing Dingding biotechnology limited company. Coli competent TOP10 was purchased from Shanghai Toshidi Biotech Ltd.
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 filters (Membrane Solutions), 0.22 μm sterile filtersAnd dialysis bags (Millipore), Ni-IDA affinity chromatography gel (Novagen), n-hexane (chromatogrAN _ SNhy, CAS: 110-54-3) and other reagents were purchased from Beijing Lange chemical products, Inc.
1.3 test methods
(1) Expression vector construction
The genes LcTPS14 and LiTPS14 are subjected to sequence optimization according to the codon preference of escherichia coli. Sequence synthesis is completed by Nanjing Belding biotechnology limited, LcTPS14 and LiTPS14 are respectively 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 by a PAS-based method. And (3) enzyme digestion and identification of the recombinant plasmid, wherein an enzyme digestion system is as follows:
the plasmid containing the target gene is transformed into an escherichia coli TOP10 strain, and positive colonies are picked for sequencing.
(2) Transformation of the recombinant vector into E.coli
The recombinant plasmid is transformed into Escherichia coli Arctic Express BL21(DE3) or BL21(DE3) PLySs, the plasmid and competent bacteria are mixed uniformly according to the volume ratio of 1:100, the mixture is transformed as described, the recombinant vectors of pCZN1 and pGEX-4T-1 are renatured for 1h at the temperature of 37 ℃ and are coated on an Amp resistant LB solid culture medium (working concentration is 50 mu g/mL), the recombinant vector of pET30a is coated on an LB solid culture medium containing Kana (working concentration is 50 mu g/mL), and the mixture is inverted overnight at the temperature of 37 ℃.
(3) Expression of IPTG-inducible recombinant protein
Single colonies on the transformation plates were picked and inoculated into 3mL of LB liquid medium containing the corresponding antibiotic (50. mu.g/mL) and shaken overnight at 220rpm at 37 ℃. Referring to Green et al (2012), 0.5mM IPTG induced expression was performed by taking 1mL of 37 ℃ OD600The bacterial liquid reaches 0.6 to 0.8, after overnight induction, the supernatant and the precipitate after the thalli are subjected to ultrasonic disruption are subjected to 12 percent SDS-PAGE to detect the protein expression condition, and coomassie brilliant blue is developed.
(4) Ni column affinity purification of recombinant proteins
And (3) selecting a recombinant strain with the best expression effect to purify the protein, taking no-load as a control, purifying and recovering the protein by a Ni affinity chromatography column method, and analyzing by 12% SDS-PAGE. The purified protein solution is stored in a refrigerator at the ultralow temperature of-80 ℃.
(7) In vitro catalytic reaction of recombinant proteins
100 μ L Total reaction containing 13-35 μ g recombinant protein in buffer (PBS +20mM MgCl. sub.G)2+5mM DTT), mixing with substrate GPP or FPP (working concentration 0.5mM), covering with 100 μ L of chromatographically pure n-hexane, catalyzing reaction at 30 deg.C for 1h, vortexing and shaking for 2min to stop reaction, centrifuging to promote liquid separation, sucking upper organic phase, and collecting anhydrous MgSO4Drying thoroughly, filtering with 0.22 μm nylon membrane, collecting 1 μ L liquid, loading on a machine, performing GC-MS analysis, and repeating for 3 times. The GC-MS detection conditions are shown in Table 2, and the products are compared with NIST147 for qualitative analysis.
TABLE 2 GC-MS detection conditions
2. Results and analysis
2.1 prokaryotic expression vector construction
LcTPS14 and LiTPS14 sequences are optimized according to codon preference of escherichia coli, and respectively construct N-his tag fusion expression protein (6 x his + Nde I + target protein + taa + Xba I) with a vector pCZN1, construct N-GST and C-his tag fusion expression protein (EcoR I + target protein +6 x his + taa + Xho I) with a pGEX-4T-1 vector, construct C-his tag fusion expression protein (Nde I + target protein +6 x his + taa + Xho I) with a pET30a vector, and obtain the molecular weight of the recombinant protein shown in Table 3.
TABLE 3 recombinant protein molecular weight
Sequencing and double enzyme digestion verification are carried out on the constructed recombinant vector to obtain recombinant plasmids pCZN1-LcTPS14, pGEX-4T-1-LcTPS14, pET30a-LcTPS14, pCZN1-LiTPS14, pGEX-4T-1-LiTPS14 and pET30a-LiTPS14 containing target genes.
2.2 functional analysis of recombinant proteins
The recombinant protein is respectively mixed with different substrates GPP or FPP for catalytic reaction, and is analyzed on a GC-MS machine. A large amount of linalool was detected in the reaction solution of the recombinant proteins LcTPS14 and LiTPS14 with the substrate GPP, and trans-nerolidol was detected in the reaction solution of the substrate FPP (FIG. 7). The ion peak pattern is shown in FIG. 8. The LcTPS14 and the LiTPS14 are shown to be bifunctional enzymes and have the functions of catalyzing FPP to generate trans-nerolidol and catalyzing GPP to generate linalool.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Reference documents:
[1] chenzhilin, Lagerstroemia speciosa MADS-box family B-class and C-class gene cloning and expression pattern research [ D ]. Beijing university of forestry Master academic paper 2017.
[2] Xuwang, research on characteristic aroma component release rule of lagerstroemia caudate and MEP pathway key gene [ D ] thesis of doctor academic university of Beijing 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 caudate (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> cloud enantiomorphous bana (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 caudate (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> cloud enantiomorphous bana (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