CN108486078B - Pyrophosphate synthase gene derived from anoectochilus formosanus - Google Patents

Abstract

The invention provides a pyrophosphatase gene from anoectochilus formosanus, and relates to the technical field of genetic engineering. A pyrophosphatase synthase gene derived from Anoectochilus formosanus has a nucleotide sequence shown in SEQ ID NO.1 and an amino acid sequence shown in SEQ ID NO. 2. The sequence is a new pyrophosphate synthase gene sequence, and is a new orchid amplified pyrophosphate synthase gene except dendrobium officinale of orchid plants. The method has important significance for researching the enrichment basis of specific target secondary metabolites in anoectochilus formosanus.

Description

Pyrophosphate synthase gene from anoectochilus formosanus

Technical Field

The invention relates to the field of genetic engineering, and in particular relates to a pyrophosphatase synthase gene derived from anoectochilus formosanus.

Background

Anoectochilus formosanus (Aoectochilus formosanus) is a perennial herb, grows in Taiwan area of China, and belongs to Anoectochilus formosanus of Labiatae. The important pharmacological active substances in anoectochilus formosanus are mainly as follows: flavonoids, steroids, triterpenes, saccharides, alkaloids, cardiac glycosides, esters, taurine, various amino acids, trace elements, inorganic elements and the like. Folk agents are called "Yaowang", "gold grass", "Shencao", "bird ginseng" and the like. Among them, polysaccharides, flavonoids, steroids, etc. in taiwan anoectochilus formosanus are considered as important active substances, and the synthesis of these substances directly affects the medicinal value of taiwan anoectochilus formosanus. The analysis of the synthetic pathway of the secondary metabolite is the premise and the basis for developing related synthetic biology research.

Farnesyl-diphosphate synthase (Farnesyl-diphosphate synthase) is one of the key enzymes in the anabolic pathway of active substances. FPS is a main chain-lengthening enzyme, and the FPS catalyzes hydrocarbon parts of DMPP, C5 and C10 in turn to generate isopentenyl pyrophosphate (IPP) and C5 to synthesize E-farnesyl pyrophosphate (FPP). FPS is the flow of branch point key enzymes that direct carbon and precursors. FPS in both the MVA and MEP pathways catalyze the synthesis of FPP, providing synthetic precursors for subsequent products. The research on the cloning of steroid metabolic synthesis pyrophosphate synthase (FPS) genes in anoectochilus formosanus is the basis for researching the enrichment of specific target secondary metabolites in the anoectochilus formosanus and has important significance.

Disclosure of Invention

The invention aims to provide a pyrophosphoric acid synthase gene derived from anoectochilus formosanus, a protein coded by the gene and a recombinant plasmid containing the gene, which provide a foundation for steroid metabolic synthesis in the anoectochilus formosanus.

The technical problem to be solved by the invention is realized by adopting the following technical scheme.

A pyrophosphate synthase gene derived from anoectochilus formosanus, wherein the nucleotide sequence of the pyrophosphate synthase gene is shown as SEQ ID NO: 1 is shown.

The amino acid sequence of the protein coded by the pyrophosphate synthase gene is shown as SEQ ID NO: 2, respectively.

A recombinant plasmid containing the pyrophosphate synthase gene.

Optionally, the plasmid is pMD18-T, pBI121 or pMD 19-T.

Compared with the prior art, the invention has the beneficial effects that:

the pyrophosphoric acid synthase gene provided by the invention is obtained by cloning from anoectochilus formosanus for the first time. The gene can be used for carrying out genetic engineering transformation on the anoectochilus formosanus plants, improving components such as steroids in the plants through transgenosis, or carrying out plant variety identification, screening and the like by using the gene to obtain plant varieties with high steroid content and the like, and provides a basis for enriching specific target secondary metabolites in the anoectochilus formosanus. Meanwhile, the FPS gene is cloned in the anoectochilus formosanus, so that the method is a basis for researching steroid anabolic pathway in the anoectochilus formosanus, can provide important basis for variety breeding of the anoectochilus formosanus and also provides a basis for research on steroid-enriched transgenic strains.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 is a diagram showing the result of electrophoresis detection of FPS gene CDS sequence of Anoectochilus formosanus provided in embodiment 4 of the present invention;

FIG. 2 is a diagram of the FPS-predicted protein structure of Anoectochilus formosanus in Taiwan according to embodiment 5 of the present invention;

FIG. 3 is a FPS protein occurrence tree analysis of Anoectochilus formosanus in Taiwan according to embodiment 5 of the present invention;

FIG. 4 is a diagram showing the expression patterns of different tissues of the FPS gene of Anoectochilus formosanus in example 6 of the present invention;

FIG. 5 is a diagram showing an expression pattern of the FPS gene of Anoectochilus formosanus in embodiment 6 in 24 hours under 100nM NaCl;

FIG. 6 is a diagram showing an expression pattern of the FPS gene of Anoectochilus formosanus in example 6 of the present invention within 24 hours under ultraviolet stress at 253.7 nm;

FIG. 7 is a diagram showing an expression pattern of the FPS gene of Anoectochilus formosanus in example 6 of the present invention within 12 hours under 650nm infrared stress.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The following specifically describes embodiments of the present invention.

In each of the following examples, the pyrophosphate synthase gene of anoectochilus formosanus was abbreviated as AoFPS, and the pyrophosphate synthase sequence derived from anoectochilus formosanus (shown as SEQ ID No. 1) and the corresponding amino acid sequence (shown as SEQ ID No. 2) were finally obtained by experimental examples.

Example 1

The embodiment provides total RNA extraction of anoectochilus formosanus, and the Trizol extraction kit of Dalianbao biological wired company is adopted to extract total RNA of anoectochilus formosanus leaves, and the method comprises the following steps:

(1) approximately 100mg of fresh leaves were ground to a powder in liquid nitrogen and transferred to a 1.5mL centrifuge tube, followed immediately by the addition of 1mL of RNAasso Plus, and the mixture was inverted and mixed to give a homogenate.

(2) The well-mixed homogenate was allowed to stand at room temperature for 5min and then centrifuged at 12000g at 4 ℃ for 5 min.

(3) Sucking 800ml of supernatant, transferring into a new centrifuge tube, adding 200ml of chloroform into the centrifuge tube, violently shaking and mixing until the homogenate is emulsified into milk white, and standing at room temperature for 5min to obtain a mixed solution.

(4) The mixture was centrifuged at 12000g at 4 ℃ for 15min, at which time the centrifuged homogenate was divided into three layers. From top to bottom are respectively: supernatant (containing RNA), middle white layer (mostly DNA) and lower colored organic phase.

(5) Sucking 400ml of the supernatant in the step (4) and transferring the supernatant into a new centrifuge tube without touching the middle layer. Then 400ml of isopropanol was added and mixed by inversion, and then left to stand at room temperature for 10min.

(6) The solution left standing in step (5) was centrifuged at 12000g at 4 ℃ for 10min, whereupon white flocculent RNA was observed. The supernatant was carefully discarded and 1mL of 75% ethanol was added, and the supernatant was discarded after washing the RNA upside down.

(7) After the RNA was dried at room temperature for a few minutes by opening the centrifuge cap, 30. mu.l of RNase-free water was added to dissolve the RNA.

(8) Total RNA concentration was calculated by measuring the value of A260 using a ultramicrospectrophotometer (Bio-Rad, USA), and the value of OD260/OD280 was read to estimate total RNA purity and integrity. The RNA quality was rapidly checked by electrophoresis on a 1.2% agarose gel at 135V.

Example 2

This example provides transcriptome sequencing of anoectochilus formosanus, comprising the following steps:

according to the method provided in example 1, total RNA of leaves of Anoectochilus formosanus was extracted. Detecting the RNA extraction quality, meeting the requirement of library construction (RNA concentration)>250 ng/. mu.L, total amount>20μg，OD ₂₆₀ /OD ₂₈₀ Between 1.8 and 2.2, good integrity and RIN>6.5). Then, poly (A) mRNA is enriched by magnetic beads and is broken into segment fragments, the segment fragments are used as templates, the 1 st cDNA chain and the 2 nd cDNA chain are sequentially synthesized, and a sequencing joint is connected after purification, elution, end repair and poly (A) addition. Selecting fragments with the size of 200 bp-700 bp for PCR amplification, establishing a cDNA sequencing library, and sequencing by using IIIuma HiSeq 2000. The part of the experiment is finished by the Mei-Nei science and technology service cable company.

Using short reads assembly software Trinity (v2.4.0) to carry out De novo assembly, after obtaining a Contig assembly fragment without N, using tgicl (v2.1) to carry out redundancy removal, removing sequences with low quality and uncertainty in the sequences, and reserving sequences larger than 200bp for subsequent analysis. The gene structure of the splicing result is predicted by using a transdecoder (v2.0.1), and the CDS sequence of the FPS gene is obtained.

Example 3

This embodiment provides a first strand of anoectochilus formosanus cDNA, comprising the following steps:

using the total RNA of Anoectochilus roxburghii leaves provided in example 1 as a template, oligo (dT)18 as a Reverse transcription primer, PrimeScript Reverse Transcriptase Transcriptase (Takara China) according to SMART ^TM PCR cDNA Synthesis Kit (Clontech USA) protocol describes the first strand cDNA Synthesis. The total volume of the reaction system was 20. mu.L.

(1) Preparing a reverse transcription mixed solution 1 in a 0.2mL PE tube according to the reagents in the table 1;

(2) preparing a reverse transcription mixed solution 2 in another 0.2mL PE tube according to the reagents in the table 2;

(3) preserving the heat of the reverse transcription mixed solution 1 in the step (1) at 65 ℃ for 5min, rapidly cooling on ice for 2min, and centrifuging for several seconds to enable the mixed solution of template RNA, primers and the like to gather at the bottom of a PE tube;

(4) adding the reverse transcription mixed solution 2 prepared in the step (2) into the reverse transcription mixed solution 1 reacted in the step (3), gently mixing by using a pipette gun, and reacting for 90min at 42 ℃;

(5) and (4) preserving the temperature of the reaction solution obtained in the step (4) for 5min at 80 ℃, and cooling on ice to obtain a cDNA solution.

TABLE 1 composition of reverse transcription mixture 1

TABLE 2 reverse transcription Mixed solution 2 composition

Example 4

This example provides a method for cloning AoFPS gene, comprising the steps of:

(1) primer design

On the basis of obtaining the CDS sequence of the FPS gene by transcriptome sequencing and splicing in example 2, primers were designed by using Primer Premier 5.0 software, and the designed primers were analyzed for the specificity of the Primer in Oligo6.0.

5'-ATGGAGGAAGGGGACAGGA-3' as upstream primer FPSF;

the downstream primer FPSR: 5'-CTACTTTTGCCTTTTATAAATCTTATGA-3'.

(2) PCR reaction

The reaction procedure is as follows: pre-denaturation at 95 ℃ for 3 min; then, the mixture is denatured at 95 ℃ for 30s, annealed at 52 ℃ for 30s, and extended at 72 ℃ for 1min for amplification for 38 cycles. A50. mu.L system was used for PCR reactions, as shown in Table 3.

TABLE 3 PCR reaction System

*: first strand cDNA as provided in example 3 was diluted 5-10 fold.

50ul of the resulting amplified product was subjected to 180V electrophoresis on 1% non-denaturing agarose gel for 30min, stained with GoLdenView, and then examined with ultraviolet rays for the amplified fragment, the results are shown in FIG. 1.

(3) Recovery of the fragment of interest

The specific band amplified in step (2) was quickly and accurately removed from the agarose using a clean blade under an ultraviolet lamp, and placed in a 1.5ml centrifuge tube, and the DNA fragment in the Gel was recovered using a Gel recovery Kit (E.Z.N.A.TM. Gel Extraction Kit from OMEGA).

(4) Ligation reaction

The recovered DNA fragment was cloned into the Pmd19-T vector of TaKaRa. Ligation was carried out overnight at 16 ℃ in a total volume of 10ul using a ligation kit (TaKaRa).

(5) Preparation of competent cells:

and (3) selecting a newly activated E.CoLiDH5a single colony from an LB plate, inoculating the colony in 3-5ml of an LB liquid culture medium, and carrying out shake culture at 37 ℃ and 225r/min for about 12h until the late logarithmic growth stage. The suspension was mixed with a suspension of 1: the culture medium is inoculated into 100ml LB liquid culture medium in the proportion of 10-1:50, and is cultured for 2-3h under shaking at 37 ℃ until OD600 is about 0.35-0.5.

Transferring the culture solution into a centrifuge tube, standing on ice for 10min, and centrifuging at 4 deg.C at 3000r/min for 10min.

The supernatant was discarded, and the cells were gently suspended in 10ml of a pre-cooled 0.05mol/L CaCl2 solution, and after standing on ice for 15-30min, centrifuged at 3000r/min at 4 ℃ for 10min.

The supernatant was discarded, 4ml of a pre-cooled 0.05mol/L CaCl2 solution containing 15% glycerol was added, the cells were gently suspended, and the suspension was allowed to stand on ice for several minutes to obtain a competent cell suspension.

The competent cells were divided into 100ul aliquots and stored at-70 ℃ for half a year.

(6) Transformation of plasmid DNA

Taking a tube of the E.coli competent cells DH5a in step (5), and thawing the tube on ice. And then adding 10ml of the ligation reaction solution obtained in the step (4) under an aseptic condition, shaking gently and mixing uniformly, and then placing on ice for 30 min. Heating in 42 deg.C water bath for 90s without shaking, and rapidly cooling in ice bath for 2-3 min. Adding 890ul of LB liquid culture medium without Amp, mixing uniformly, shaking and culturing at 37 ℃ and 150r/min, and incubating for 1h to obtain the transformed bacterial liquid.

Spreading 100ul of transformed bacterial liquid on a culture plate of LB Amp, standing for 30min with the front side facing upwards, sealing with a sealing film after the bacterial liquid is completely absorbed by the culture medium, then inverting the culture dish, and culturing at 37 ℃ in the dark for 12-16 h; subsequently, positive colonies were screened.

(7) Identification and preservation of recombinant colonies

The generally white and round colonies were positive in appearance and were further characterized by PCR using bacterial suspension, as follows:

several white colonies were picked up on overnight-cultured plates with a sterilized small gun head, and seeded in 0.1g/L Amp LB liquid medium 1.5ml centrifuge tubes, respectively, and cultured at 37 ℃ for 4-6h with shaking at 150 r/min.

Taking 1ul of bacterial suspension as a template, carrying out PCR amplification by using a primer FPSF/FPSR, prolonging the cracking time in the PCR reaction condition to 5min, and detecting the amplification product by using 1% non-denaturing agarose gel electrophoresis. If the product is the target band, the colony is a positive clone, otherwise, the colony is a negative clone. Meanwhile, a negative control without adding bacterial suspension is set.

750ul of colony suspension of the identified positive clone is taken, 250ul of sterilized glycerol is added, the mixture is uniformly mixed, and then the mixture is quickly frozen by liquid nitrogen and stored in a refrigerator at the temperature of 70 ℃ below zero for later use.

(8) Sequencing

And (4) sending the bacterial colony of the positive clone identified and stored in the step (7) to a Invitrogen biotechnology company for sequencing, and comparing and analyzing the obtained sequence in BLAstn of NCBI to verify that the cloned fragment is correct.

The product obtained by amplification is an AoFPS gene CDS sequence, and the obtained nucleotide sequence is shown in SEQ ID NO.1 and the amino sequence is shown in SEQ ID NO. 2.

Example 5

This example provides a protein bioinformatic analysis of AoFPS genes, including the steps of:

the AoFPS gene was analyzed for physical and chemical properties using ProtParam (http:// web. expasy. org/ProtParam /). The total length of the FPS gene of anoectochilus formosanus is 1047bp, 349 amino acids are coded, the physicochemical property of the FPS gene coding protein is predicted by using an online tool ProtParam provided by ExPASY Proteomics Server, and the protein is presumed to have the relative molecular mass of 40.4kDa and the isoelectric point pI of 5.23.

Secondary structure of FPS gene was predicted using GOR IV (http:// npsa-pbil. ibcp. fr/cgi-bin/npsa _ Automat. plpage ═ npsa _ gor4. html). The results of the on-line prediction of secondary structure showed that the Alpha-helix (Alpha helix) accounted for 35.92% of the total amino acids, the Extended strand (Extended strand) accounted for 20.4% of all amino acids, and the Random coil (Random coil) accounted for 43.68% of the total amino acids.

The FPS gene of Anoectochilus formosanus was predicted for protein tertiary structure by SWISS-MODEL (https:// swissminor. expay. org /), mapped by PyMOL Viewer, and the three-dimensional MODEL is shown in FIG. 2.

Finally, finding out the FPS amino acid sequence of the plant with higher homology, comparing the amino acid sequences by using a ClustalW method, and constructing a Neighbor-join phylogenetic tree through MEGA7.0 as shown in figure 3.

Example 6

This example provides an endogenous expression assay for an AoFPS gene comprising the steps of:

(1) material treatment

Ultraviolet irradiation treatment:

transplanting the tissue culture seedling of anoectochilus formosanus cultured for 4 months into a flowerpot filled with nutrient soil (nutrient soil: vermiculite: 3:1), and keeping the seedling relatively wet at the temperature of 25 ℃ (day)/20 ℃ (night)60-70% of the degree and 200 mu mol/m of illumination ² S (14 h)/dark (10 h). Adapting to 3-5 days, taking root, stem and leaf samples from untreated seedlings with uniform growth vigor, and quickly freezing the samples at-70 ℃ by liquid nitrogen for storage. And (5) carrying out ultraviolet irradiation treatment on the rest seedlings with uniform growth.

The treatment conditions were: placing potted anoectochilus formosanus in an ultraviolet incubator, and respectively treating for 0h, 0.5h, 1h, 2h, 4h, 8h, 12h and 24h by using ultraviolet radiation with the wavelength of 253.7 nm.

Each treatment was repeated 3 times, and samples were immediately taken at each treatment time point and snap frozen in liquid nitrogen at-70 ℃ for storage.

Red light irradiation treatment:

transplanting the tissue culture seedlings of anoectochilus formosanus cultured for 4 months into a flowerpot filled with nutrient soil (nutrient soil: vermiculite: 3:1), and culturing at the temperature of 25 ℃ (day)/20 ℃ (night) with the relative humidity of 60-70% and the illumination of 200 mu mol/m ² S (14 h)/dark (10 h). Adapting to 3-5 days, taking root, stem and leaf samples from untreated seedlings with uniform growth vigor, and quickly freezing the samples at-70 ℃ by liquid nitrogen for storage. And (5) carrying out ultraviolet irradiation treatment on the rest seedlings with uniform growth.

The treatment conditions were: placing potted anoectochilus formosanus in a red light incubator, and treating for 0h, 0.5h, 1h, 2h, 4h, 8h and 12h respectively at the red light wavelength of 650 nm.

Each treatment was repeated 3 times, and samples were immediately taken at each treatment time point and snap frozen in liquid nitrogen at-70 ℃ for storage.

Salt stress treatment:

transplanting the tissue culture seedlings of the anoectochilus formosanus cultured for 4 months to a plastic foam plate with holes, culturing for 3-5 days by using Hoagland nutrient solution, and selecting the seedlings with uniform growth vigor for salt stress treatment. The salt stress treatment is to add NaCl into the Hoagland nutrient solution to a final concentration of 100mmol/L, and treat for 0h, 0.5h, 1h, 2h, 4h, 8h, 12h and 24h respectively. Each treatment was repeated 3 times, and samples were immediately taken at each treatment time point and snap frozen in liquid nitrogen at-70 ℃ for storage.

(2) RNA extraction

Leaves of the 3 types of stress-treated and control seedlings were respectively subjected to liquid nitrogen quick-freezing and grinding, and then total RNA was extracted using Trizol (TaKaRa, Dalian) total RNA extraction kit. The procedure is as in example 1.

(3) First Strand cDNA Synthesis

Using the extracted RNA as a template, adding sample according to a system in a table 4, reacting for 2min at 42 ℃, removing possible genome DNA (gDNA), adding sample according to a system in a table 5 by using a PrimeScript RT reagent Kit with gDNA Eraser Kit (TaKaRa, Dalian), bathing for 30min at 37 ℃, keeping 5s at 85 ℃ to terminate the reaction so as to synthesize cDNA by reverse transcription, and storing at-20 ℃ for later use.

TABLE 4 gDNA removal reaction System

TABLE 5 cDNA Synthesis System

(4) Specific detection of qRT-PCR primers

cDNA synthesized by a sample (a control sample) subjected to stress treatment for 0h is diluted by 3 times and used as a reaction template of qRT-PCR, and qRT-PCR reaction is carried out by adopting an annealing temperature gradient of 50-65 ℃ to determine the optimal annealing temperature, and a 20 mu L reaction system shown in Table 6 is adopted. The qRT-PCR primer sequences are shown in Table 7.

TABLE 6 qRT-PCR reaction System

TABLE 7 qRT-PCR primer sequences

The reaction tube used 0.2mL PCR plate after siliconization. qRT-PCR at iQ ^TM 5thermal cycler (Bio-Rad USA). The reaction procedure is as follows: pre-denaturation at 95 ℃ for 10 s; then performing denaturation for 10s at 95 ℃, and annealing for 20 at 50-65 DEG Cs, extension at 72 ℃ for 20s, and collection of fluorescence at this temperature, Plate reading (Plate read) procedure was performed for 46 cycles. Then, starting at 50 ℃, the temperature is increased to 95 ℃ with a temperature gradient of 0.5 ℃ per step, and the temperature is maintained for 5s per step.

(5) qRT-PCR reaction

The reverse transcribed cDNA samples of the different samples in step (3) of this example were diluted 3 times and used as a template for qRT-PCR amplification, siliconized 0.2mL PCR plates were used as reaction tubes, a 20. mu.L reaction system was used (Table 6), and a negative control using ddH2O as a template was set up. A single reverse transcribed cDNA sample was made in triplicate.

The qRT-PCR reaction program was: pre-denaturation at 95 ℃ for 10s, followed by annealing at 95 ℃ for 10s, annealing at the optimal annealing temperature for 20s, extension at 72 ℃ for 20s, and collection of fluorescence at this temperature, Plate reading (Plate read) was performed for 46 cycles. Then finally, starting at 50 ℃ and increasing the temperature to 95 ℃ at a rate of 0.5 ℃ per step, the melting curve is plotted for 5s per temperature.

(6) qRT-PCR data analysis

The experiment adopts a dual-standard curve method to relatively quantify the expression level of a key enzyme gene (AoFPS gene) in the pathway of the metabolism and synthesis of flavone. The relative Expression level of the Gene was calculated by DD CT (Normalized Gene Expression) method.

The expression difference between the 0h control and treatment and between treatments was analyzed using SPSS (version10.0inc. chicago, IL) statistical analysis software. Two levels of differential significance were established, P0.05 and P0.01. FIG. 4 shows the expression patterns of FPS gene of Anoectochilus formosanus in Taiwan.

FIG. 5 is a diagram showing an expression pattern of the FPS gene of Anoectochilus formosanus in 24 hours under 100nM NaCl. Under the treatment of 100nM NaCl, FPS gene expression changes irregularly, and the lowest expression value and the highest expression value appear in 2h and 24h respectively, and the difference is very obvious compared with 0 h.

FIG. 6 is a diagram showing the expression pattern of the FPS gene of Anoectochilus formosanus under 253.7nm UV stress for 24 h. It is seen that the FPS gene shows negative expression under UV stimulation, and the difference between the expression level of the 1hFPS gene and 0h is significant, and the difference between the expression levels of 4h, 8h, 12h and 24h is very significant compared with 0 h.

FIG. 7 is a diagram showing the expression pattern of the FPS gene of Anoectochilus formosanus in 12h under 650nm red light stress. As can be seen, FPS gene shows a change of increased and decreased expression under red light stress. The peak appears at 1h, and the wave peak value is about 2.5 times of the unstressed wave peak value (namely 0 h).

In conclusion, the expression of the FPS gene of anoectochilus formosanus can be improved through short-time red light irradiation, so that the steroid yield of the anoectochilus formosanus is improved.

The embodiments described above are some, not all embodiments of the invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

SEQUENCE LISTING

<110> Sanming academy of academic

<120> a pyrophosphate synthase gene derived from Anoectochilus formosanus

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<170> PatentIn version 3.5

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atggaggaag gggacaggaa ggcggtggat ctgaaagatg gcttcctcca gatctactac 60

cggctcaaat ccgagctcct cgaagatccg gccttcagct ttacagaaga ttcacggcaa 120

tggatcgata ggatgctgga gtataatgta ccaggtggga agctcaatcg tggcatctcc 180

gtgattgata gctataaaat tctaaaagga ggtcaactca acgatgatga gttttttctt 240

gcaagtgtac ttggttggtg tattgaatgg cttcaagctt attttctcgt tcttgatgat 300

atcatggaca actcccacac taggcgtggc catccttgtt ggtttagatt acctaaggtt 360

ggtattattg ccgcaaatga tgggatttta cttcgtaacc acatccctag gatgctcaat 420

cgtcatttca aggagaaaac ttactatgtt catctactgg acctatttaa tgaagtagaa 480

tttcagacag cttcaggaca gatgcttgat ctgatcacta ctcatgaagg agagaaggat 540

ctatccaaat ataaaatgcc agtgtacaat cgtattgttc agtacaaaac tgcatattat 600

tcattttatc ttcctgttgc atgtgccttg ttgatggctg gtgaaaattt agacaactat 660

gatgatgtaa aaacagttct tgtggagatg ggaacatact ttcaagtaca ggatgactac 720

ctagactgct ttggtgatcc tgaagtcatt ggtaagattg gaactgacat tgaagattat 780

aagtgttctt ggcttgttgt acaagcactt gaattagcaa atgacaacca actgaaaatt 840

ttgtttgaga actatgggaa gtcagatcca gcttgtgtca cgaaagttaa atccctttac 900

aaagatctca accttcaggt ggtattttcc aagtacgaga gtgctagcta cgagcagcta 960

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Met Glu Glu Gly Asp Arg Lys Ala Val Asp Leu Lys Asp Gly Phe Leu

1 5 10 15

Gln Ile Tyr Tyr Arg Leu Lys Ser Glu Leu Leu Glu Asp Pro Ala Phe

20 25 30

Ser Phe Thr Glu Asp Ser Arg Gln Trp Ile Asp Arg Met Leu Glu Tyr

35 40 45

Asn Val Pro Gly Gly Lys Leu Asn Arg Gly Ile Ser Val Ile Asp Ser

50 55 60

Tyr Lys Ile Leu Lys Gly Gly Gln Leu Asn Asp Asp Glu Phe Phe Leu

65 70 75 80

Ala Ser Val Leu Gly Trp Cys Ile Glu Trp Leu Gln Ala Tyr Phe Leu

85 90 95

Val Leu Asp Asp Ile Met Asp Asn Ser His Thr Arg Arg Gly His Pro

100 105 110

Cys Trp Phe Arg Leu Pro Lys Val Gly Ile Ile Ala Ala Asn Asp Gly

115 120 125

Ile Leu Leu Arg Asn His Ile Pro Arg Met Leu Asn Arg His Phe Lys

130 135 140

Glu Lys Thr Tyr Tyr Val His Leu Leu Asp Leu Phe Asn Glu Val Glu

145 150 155 160

Phe Gln Thr Ala Ser Gly Gln Met Leu Asp Leu Ile Thr Thr His Glu

165 170 175

Gly Glu Lys Asp Leu Ser Lys Tyr Lys Met Pro Val Tyr Asn Arg Ile

180 185 190

Val Gln Tyr Lys Thr Ala Tyr Tyr Ser Phe Tyr Leu Pro Val Ala Cys

195 200 205

Ala Leu Leu Met Ala Gly Glu Asn Leu Asp Asn Tyr Asp Asp Val Lys

210 215 220

Thr Val Leu Val Glu Met Gly Thr Tyr Phe Gln Val Gln Asp Asp Tyr

225 230 235 240

Leu Asp Cys Phe Gly Asp Pro Glu Val Ile Gly Lys Ile Gly Thr Asp

245 250 255

Ile Glu Asp Tyr Lys Cys Ser Trp Leu Val Val Gln Ala Leu Glu Leu

260 265 270

Ala Asn Asp Asn Gln Leu Lys Ile Leu Phe Glu Asn Tyr Gly Lys Ser

275 280 285

Asp Pro Ala Cys Val Thr Lys Val Lys Ser Leu Tyr Lys Asp Leu Asn

290 295 300

Leu Gln Val Val Phe Ser Lys Tyr Glu Ser Ala Ser Tyr Glu Gln Leu

305 310 315 320

Ile Ser Ala Ile Glu Ala Gln Pro Ser Asn Lys Val Gln Glu Val Leu

325 330 335

Lys Ser Phe Leu His Lys Ile Tyr Lys Arg Gln Lys

340 345

Claims (4)

1. A pyrophosphate synthase gene derived from Anoectochilus formosanus, characterized in that the nucleotide sequence of the pyrophosphate synthase gene is shown in SEQ ID NO: 1 is shown in the specification; the CDS sequence of the pyrophosphate synthase gene is obtained by transcriptome sequencing and splicing, wherein the expression of the pyrophosphate synthase gene can be improved by red light stress.

2. The protein encoded by the pyrophosphate synthase gene of claim 1, wherein said protein has the amino acid sequence of SEQ ID NO: 2, respectively.

3. A recombinant plasmid comprising the pyrophosphate synthase gene according to claim 1.

4. The recombinant plasmid of claim 3, wherein the plasmid is pMD18-T, pBI121 or pMD 19-T.

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