CN111909942B - Clone of cyclocarya paliurus gene CpSE1-like coding sequence and application thereof - Google Patents
Clone of cyclocarya paliurus gene CpSE1-like coding sequence and application thereof Download PDFInfo
- Publication number
- CN111909942B CN111909942B CN202010631113.7A CN202010631113A CN111909942B CN 111909942 B CN111909942 B CN 111909942B CN 202010631113 A CN202010631113 A CN 202010631113A CN 111909942 B CN111909942 B CN 111909942B
- Authority
- CN
- China
- Prior art keywords
- gene
- cyclocarya paliurus
- cpse1
- content
- cpse
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/124—Animal traits, i.e. production traits, including athletic performance or the like
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Analytical Chemistry (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Immunology (AREA)
- Mycology (AREA)
- Botany (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a cyclocarya paliurus geneCpSEThe cloning and application of coding sequence belongs to the field of plant molecular biology and gene cloning technology. Firstly, cyclocarya paliurus gene is obtainedCpSEObtaining cyclocarya paliurus geneCpSEAfter the coding region sequence is complete, a fluorescent quantitative primer of the gene is designed and the primer specificity is verified. Through the correlation analysis of the total triterpene content in the cyclocarya paliurus leaves and the gene expression level, the result shows that the total triterpene content is related to the gene expression levelCpSEThe expression level of (A) shows a significant positive correlation (P)<0.05). The gene expression of 16 primary selected excellent individual cyclocarya paliurus (divided into high-content group H (5 plants), medium-content group M (6 plants) and low-content group L (5 plants) according to the triterpene content) leaves is measured, and the result shows that the gene expression passes through the leaves in SeptemberCpSEThe relative expression quantity can obviously distinguish the cyclocarya paliurus high-triterpene content group from other groups, and the result can be applied to the breeding, popularization and application of cyclocarya paliurus high-triterpene content excellent plants.
Description
Technical Field
The invention relates to clone of a cyclocarya paliurus gene CpSE1-like coding sequence and application thereof, belonging to the technical field of plant molecular biology and gene cloning.
Background
Cyclocarya paliurus is a special multifunctional tree species integrating medicinal, material, ornamental and health-care values in China, and is popular to the public due to multiple pharmacological activities. The triterpene compound is used as an important chemical component with biological activity in the cyclocarya paliurus, and the content of the triterpene compound is an important index for evaluating the quality of the cyclocarya paliurus product. The method for obtaining triterpenoids from breeding excellent resources is a main target of directionally cultivating cyclocarya paliurus leaves by artificial forests, and the time-space expression characteristic of key genes in the metabolic pathway of triterpenoids is an important factor influencing the product accumulation. Research on the triterpene metabolic pathway shows that the SE gene is a key enzyme gene which is in a plurality of branches of the downstream of the terpene synthetic pathway and is led to a triterpene synthetic pathway and is closest to a target product, and the expression of the gene has the most direct influence on the total triterpene content of a plant. Heretofore, the result of measuring the content of the triterpene is only a method for screening a single plant with high triterpene content and good quality, and the method has the defects of large material sampling, complex operation, long experimental period and the like.
Disclosure of Invention
The technical problem to be solved by the invention is to overcome the defects of the prior art and provide clone of a cyclocarya paliurus gene CpSE1-like coding sequence, a quantitative primer probe is designed according to the coding sequence, the expression of key enzyme genes is monitored, only a few leaves are needed as detection objects, the detection result can be obtained on the same day of an experiment, and the method can more efficiently realize the breeding of good single plants of cyclocarya paliurus.
In order to solve the technical problem, the invention provides a cyclocarya paliurus gene CpSE1-like, wherein the nucleotide sequence of the CpSE1-like is shown as SEQ ID NO: 1 is shown.
The invention also provides a cloning primer of the cyclocarya paliurus gene CpSE1-like coding sequence, wherein the nucleotide sequence of the primer is shown as SEQ ID NO: 2 and SEQ ID NO: 3, respectively.
The invention also provides a cloning method of the cyclocarya paliurus gene CpSE1-like coding sequence, which comprises the following steps:
extracting total RNA of cyclocarya paliurus, and performing reverse transcription to obtain cDNA;
using cDNA as a template, and utilizing the primer to obtain the complete coding region sequence of the gene through PCR amplification;
and recovering and purifying the target fragment in the amplification product, and sequencing after connection transformation to obtain the coding sequence of the cyclocarya paliurus gene CpSE 1-like.
The invention also provides a fluorescent quantitative primer designed according to the cyclocarya paliurus gene CpSE1-like coding sequence and applied to screening of cyclocarya paliurus high-triterpene-content excellent individual plants. The nucleotide sequence of the primer is shown as SEQ ID NO: 4 and SEQ ID NO: 5, respectively.
The qRT-PCR reaction system is as follows: SYBR Green premix 10. mu.L, 10. mu. mol/L upstream and downstream primers 0.8. mu.L each, cDNA 2. mu.L, deionized water 6.4. mu.L, total volume 20. mu.L.
The qRT-PCR reaction program is as follows: at 95 ℃ for 40 s; denaturation at 95 ℃ for 15 s; annealing at 55 ℃ for 30 s; and (3) extending for 35s at 72 ℃, wherein 40 cycles of denaturation, annealing and extension are set.
The invention also provides application of the cyclocarya paliurus gene CpSE1-like probe primer to seasonal dynamic changes of CpSE1-like gene expression in a cyclocarya paliurus single plant.
The invention achieves the following beneficial effects:
(1) according to the invention, after a complete coding region sequence of cyclocarya paliurus gene CpSE1-like is obtained, a fluorescent quantitative primer of the gene is designed and the specificity of the primer is verified. Through the correlation analysis between the total triterpene content in the cyclocarya paliurus leaves and the gene expression level, the result shows that the expression of the CpSE1-like gene is obviously and positively correlated with the accumulation of the triterpene (P < 0.05).
(2) The invention takes the gene fluorescence quantitative primer as a detection probe to respectively carry out fluorescence quantitative detection on the primary selected excellent individual plants of cyclocarya paliurus (classified into a high-content group H, a medium-content group M and a low-content group L according to the content of triterpene). As a result, at 9 months, the quantitative result of the CpSE1-like in the leaf can successfully separate a high-content group from other groups, and the probe can be used for screening excellent individuals with high triterpene content of cyclocarya paliurus.
(3) The invention obtains the clone of the cyclocarya paliurus gene CpSE1-like coding sequence, designs a quantitative primer probe according to the coding sequence, monitors the expression of key enzyme genes, only needs a plurality of leaves as detection objects, and can obtain a detection result on the same day of an experiment.
Drawings
FIG. 1 shows the cloning of the coding region sequence of the cyclocarya paliurus gene CpSE1-like of interest, wherein lane M is DL2000 Marker; lane 1-4 shows the coding region sequence clone of cyclocarya paliurus gene CpSE 1-like;
FIG. 2 shows the fluorescent quantitative PCR sequence of the cyclocarya paliurus gene CpSE1-like, wherein lane M is DL2000 Marker; lanes 1-4 are cyclocarya paliurus gene CpSE1-like fluorescent quantitative PCR sequences;
FIG. 3 shows the result of PCR primer specificity test of cyclocarya paliurus gene CpSE1-like fluorescence quantification, wherein the only peak shows that the primer specificity is good;
FIG. 4 shows the seasonal dynamic change of the total triterpene content in the primary selected superior leaves of cyclocarya paliurus of 16 plants among different groups, wherein H, M, L represents the high, middle and low groups of the total triterpene content respectively. Different capital letters indicate significant difference between different groups of the same month (P <0.05), different lower case letters indicate significant difference between different months of the same group (P <0.05), and the following.
FIG. 5 shows seasonal dynamic changes in CpSE1-like gene expression levels in leaves of 16 primarily selected cyclocarya paliurus superior strains.
FIG. 6 is a correlation analysis result of the total triterpene content in the leaves of 16 primarily selected cyclocarya paliurus superior plants and the relative expression level of the CpSE1-like gene, and the result shows that the total triterpene content and the CpSE1-like gene present a positive correlation (R) 2 ＝0.0326)
Detailed Description
The invention is further described below with reference to the accompanying drawings. The following examples are only for illustrating the technical solutions of the present invention more clearly, and the protection scope of the present invention is not limited thereby.
The invention provides cloning and application of a cyclocarya paliurus CpSE1-like coding sequence, and comprises the following embodiments.
The materials and reagents used in the following examples are as follows:
materials: primary selection of 16 excellent individuals of cyclocarya paliurus, cloning Vector pMD18-T Vector (Takara) of PCR product, and Escherichia coli DH5 alpha (all-plant gold).
Reagent: omega plant RNA extraction kit; DNA recovery kit (Axygen), 2 × Taq PCR pre-mix (tiangen); takara reverse transcription kit, DTT (0.1mol/L), MgCl 2 (25mmol/L), dNTP (10mmol/L), Taq DNA polymerase, dNTP (10mM), Marker DL2000 and RNase-free deionized water; the primers used in the experiments were synthesized by Nanjing Optimus department Biometrics.
Example 1
Cloning of a cyclocarya paliurus gene CpSE1-like coding sequence, wherein the cloning of the cyclocarya paliurus CpSE1-like coding sequence is obtained according to the following method:
1. extraction of total RNA of cyclocarya paliurus
The method refers to the instruction of the plant RNA extraction kit provided by Omega company, and comprises the following specific steps:
(1) taking 500 mu L of RCL buffer solution to a 2mL centrifuge tube (RNase-free), and adding 10 mu L of beta-mercaptoethanol;
(2) grinding a fresh tissue sample (or a sample stored in a refrigerator at-80 ℃) by using about 100mg of liquid nitrogen, adding the ground powder into the centrifugal tube in the step (1), and uniformly mixing the powder by shaking;
(3) after water bath at 55 ℃ for 2min, cooling to room temperature, and rotating the centrifuge at the maximum speed for 5 min;
(4) transferring the supernatant into a gDNA filter column containing a 2mL collecting pipe at room temperature of 14000rpm for 2 min;
(5) adding RCB buffer solution (about 450 μ L) with the same volume into the collection tube in the previous step, reversing the upper part and the lower part for 5-10 times, and mixing with the filtrate obtained in the previous step;
(6) adding the mixed solution into a HiBindTM RNA column containing a collecting pipe with the volume of 2mL, and performing reaction at room temperature of 10000rpm for 1min, wherein the reaction can be performed twice if the mixed solution is not left for one time;
(7) the filtrate was discarded and 400. mu.l of RWC eluent was added at 1000rpm for 1min at room temperature. Removing the mobile phase and the collection tube, and placing the column on a new collection tube;
(8) adding 500 μ l RNA eluent II at room temperature 10000rpm for 1min, and discarding mobile phase;
(9) repeating the step (8), discarding the mobile phase, and centrifuging for 2min at the maximum rotating speed of the empty tube at room temperature;
(10) placing the column in a new 1.5mL centrifuge tube (RNase-free), adding 30-50 μ L of 65 deg.C preheated RNase-free deionized water into the center of the column, standing for 2min at room temperature of 10000rpm for 1min to obtain a liquid, namely RNA.
2. Reverse transcription
An ice box is prepared before the experiment, and the reverse transcription process of RNA needs to be operated in a low-temperature environment.
(1) For DNA removal, 0.2mL of EP tube (RNase-free) was first used as follows:
incubating at 42 deg.C for 2 min.
(2) Reverse transcription is carried out by the following system:
mixing the mixed solution with the reaction solution after removing DNA in the previous step, counting 20 mu L of the system, incubating for 15min at 37 ℃, and then treating for 5s at 85 ℃ to inactivate the enzyme.
The system size can be increased by a factor according to the dosage. After the reaction is finished, 2 mu L of cDNA stock solution is taken to carry out PCR (polymerase chain reaction) verification on whether the reverse transcription is successful through 18S primer sequences, and the verified available cDNA is diluted by 5-10 times, subpackaged and placed at-20 ℃ for later use.
3. Amplification of the CpSE1-like Gene with high Fidelity enzymes
The PCR system was as follows:
PCR reaction procedure:
98 ℃ for 10s, 68 ℃ for 1min/kb, for a total of 30 cycles.
Wherein, CpSE1-like forward primer (F): 5'-ATGGTAGATCTGTACGCGCTCG-3' (SEQ ID NO: 2);
CpSE1-like reverse primer (R): 5'-CTACAATCCCTTTATGGCATCT-3' (SEQ ID NO: 3).
4. Agarose gel electrophoresis
After completion of the PCR reaction, the reaction mixture was electrophoresed in 1% agarose gel to examine the amplification effect. The result of agarose gel electrophoresis detection of the PCR product is shown in FIG. 1, and a clear target band can be observed at 1620 bp. The band after being recovered and purified by cutting gel is subjected to connecting transformation (the vector used for connecting is a PCR product cloning vector pMD18-T, and the competent cell used for transforming is escherichia coli DH5 alpha), and recombinant sequencing is screened to determine the sequence.
Example 2
The invention provides an application of the clone of the cyclocarya paliurus gene CpSE1-like coding sequence in screening of cyclocarya paliurus high-triterpene content excellent strains, wherein the obtained cyclocarya paliurus gene CpSE1-like gene sequence is used for designing a fluorescent quantitative primer to detect specificity, and the specific steps are as follows:
1. the specificity of the primers was checked by ordinary PCR amplification. The result is shown in FIG. 2, the size of the agarose gel electrophoresis band of the product meets the expected size, the band is bright and single, and the designed primer has good specificity and meets the requirement.
2. The specificity of the melting curve is confirmed by applying the peak appearance condition of the melting curve in real-time fluorescent quantitative PCR, the result is shown in figure 3, the peak is single, the specificity is good, and the experimental requirements are met.
The expression pattern analysis of the CpSE1-like gene in the primarily selected superior individual leaves of cyclocarya paliurus is specifically carried out as follows:
diluting cDNA products obtained by reverse transcription by 5-10 times, taking the diluted reverse transcription cDNA as a template, and carrying out qRT-PCR experiments by adopting an SYBR Green I chimeric fluorescence method, wherein the reaction kit adopts an SYBR Green real-time fluorescence quantitative pre-mixed sample (TOYBO Toyobo Co., Ltd.), and the reaction system (20 mu L): SYBR Green premix (2X) 10. mu.L, 0.8. mu.L each of upstream and downstream primers (10. mu. mol/L), 2. mu.L of diluted cDNA, 6.4. mu.L of deionized water.
The PCR amplification procedure was as follows: at 95 ℃ for 40 s; denaturation at 95 ℃ for 15 s; annealing at 55 ℃ for 30 s; extension 72 ℃ for 35 s. The steps of denaturation, annealing and extension are set for 40 cycles.
Wherein, the upstream and downstream primer sequences are respectively:
CpSE1-like quantitative forward primer (F): 5'-GGAGAGTTGCTACAACCTGGAGGCT-3' (SEQ ID NO: 4);
CpSE1-like quantitative reverse primer (R): 5'-TCCCGCATCCTCTGTATGAACC-3' (SEQ ID NO: 5).
The invention takes the gene fluorescent quantitative primer as a detection probe to respectively carry out fluorescent quantitative detection on the initially selected 16 cyclocarya paliurus excellent individual plants (which are divided into a high-content group H (5 plants), a medium-content group M (6 plants) and a low-content group L (5 plants) according to the content of triterpenoids). As shown in fig. 5, it was found that the expression of each gene as a whole showed a tendency of increasing after decreasing. After 7 months, the expression level difference of different groups becomes larger gradually. At 9 months, the expression level of CpSE1-like in three groups of test strains showed the most significant difference, and the gene expression level of the high-content group was significantly higher than that of the other two groups (P < 0.05).
Example 3
The determination of the total triterpene content of the cyclocarya paliurus leaves is carried out by an ultraviolet spectrophotometry, and the specific operation steps are as follows:
1. preparation of samples
Weighing about 2g of dried cyclocarya paliurus powder, wrapping with filter paper, and performing Soxhlet extraction for defatting (extracting with petroleum ether at 70 deg.C under reflux for 4 hr). Recovering the extract, naturally drying the powder bag at room temperature, and taking the volatilized powder as a sample of the total triterpene extraction. 0.8g of the powder is weighed, mixed evenly with 10mL of 70% ethanol aqueous solution by vortexing, sonicated at 70 ℃ for 45min, the procedure is repeated once, and the two extracts are combined. The combined extract is rotated and evaporated to be nearly dry, then the residue is repeatedly cleaned by absolute ethyl alcohol and the volume is determined to be 10mL, the mixture is centrifuged for 10min at 10000rpm and 4 ℃ by a centrifuge, the supernatant is taken and filtered by a microporous filter membrane with the pore diameter of 0.22 mu m, and the filtrate is placed in a refrigerator at 4 ℃ for standby.
2. Determination of Total triterpene content
Accurately sucking 0.1mL of the alcohol extract to-be-detected solution in the step 1, sequentially adding 0.5mL of 5% vanillin-glacial acetic acid mixed solution and 1.4mL of perchloric acid, uniformly mixing, heating in a water bath at 60 ℃ for 15min, cooling to room temperature, diluting to 10mL with ethyl acetate, after the solution is developed stably, measuring absorbance at the wavelength of 550nm, and substituting into a standard curve equation to obtain the total triterpene content.
The result of seasonal dynamic change of the total triterpene content of the cyclocarya paliurus plant to be detected is shown in fig. 4, along with the change of seasons, the whole cyclocarya paliurus plant shows a change trend that the content gradually rises, and the content is the highest in September (harvesting season). And the initially selected individual plants are still clustered with a high content group (H), a medium content group (M) and a low content group (L) respectively after 7 months, and the difference between the groups is obvious (P)<0.05). By making the leaf of cyclocarya paliurusCorrelation analysis between total triterpene content and Gene expression levels showed that the total triterpene content was positively correlated with CpSE1-like Gene expression (R) 2 ＝0.0326)。
The total triterpene content and the expression of the CpSE1-like gene are in a positive correlation relationship, and in 9 months (harvesting season), the relative expression quantity of the CpSE1-like gene can obviously distinguish the individual plants of the high triterpene content group from the individual plants of other groups, so that the CpSE1-like gene probe designed in the invention can be used for more efficient breeding work of the individual plants of cyclocarya paliurus with high triterpene content.
The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, it is possible to make various improvements and modifications without departing from the technical principle of the present invention, and those improvements and modifications should be considered as the protection scope of the present invention.
Sequence listing
<110> Nanjing university of forestry
<120> clone of cyclocarya paliurus gene CpSE coding sequence and application thereof
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1620
<212> DNA
<213> Cyclocarya paliurus (Cyclocaraya palaurus)
<400> 1
atggtagatc tgtacgcgct cgggttgatc ttggcctcgg tgttggggtt cgtggtgttg 60
tacaatctga tggccaagaa gaaccgcagc agagtttctt cggaggcgag aagcgagggc 120
gtgcagaccg tcaccacgac aaccaacgga gaatgcagat ccattgaaag cgacgtcgac 180
gtcgacgtta tcattgtcgg agccggtgtc gccggctctg ccctcgctca cactctcggc 240
aaggatggac gtaaagttca tgtgattgaa agagatttat cggagcctga tagaattgtt 300
ggagagttgc tacaacctgg aggctacctc aagttgattg agttgggtct tcaagactgt 360
gtggaggaca ttgatgctca gcaagtgttt ggttatgctc tttttaagga tgggaaaaac 420
acccgactct cttatccgtt ggagaaattt cactcagatg tttctggaag gagctttcac 480
aatggccggt tcatacagag gatgcgggag aaggctgcat cccttcccaa tatacgattg 540
gagcaaggaa ctgttacttc tctgcttgga gaaaatggaa caattaaagg cgtgcagtac 600
aagactaaag atggaaaaga actggcagca catgcacctc taaccattgt ctgtgatggc 660
tgtttctcaa acttgcgtcg ctcactttgc aatcctcagg ttgatatgcc ttcttccttt 720
gttggtttgg ttttggagaa ttgtgaacta ccttatgcaa atcatgggca tgtcgtacta 780
gcagaccctt cccccatttt gttttatcct attagtagca cagaggttcg ttgcctggtt 840
gatgtacctg gacagaaagt cccatctatt tcaaatggcg aaatggcaaa gtatttgaaa 900
accatggtgg cccctcaggt tccccctgaa atatatgatt cttttgtagc tgccgttgat 960
agaggtaaca ttaggacaat gccaaacaga agcatgccag cggctccaca gcctactcct 1020
ggagccttgc taatggggga tgcattcaac atgcgccacc ccttaaccgg gggaggaatg 1080
actgtggcat tgtcagatat tgttgtgctt cgggatctcc tcaggccttt gcgagaccta 1140
aatgatgcac ccacactctg caagtatctt gaatccttct acaccttgcg taagccggta 1200
gcatctacaa ttaatactct ggcgggggcc ttgtacaagg tcttttgtgc ttcacccgat 1260
cgagcaagga acgaaatgcg ccaggcttgc tttgattatc taagtcttgg aggtgtattc 1320
tcaacgggac cagtttctct tctctcgggg ttgaaccctc gcccattgag tttggttctc 1380
cactttttcg ccgttgcagt ttatggtgtt ggccgtttat tggtgccctt cccttcaccc 1440
tcacgcatgt ggattggagc cagattaatt tcgggtgcat cagcaatcat cttccccatt 1500
atcaaggcag aaggagtgag acagatgttc ttccctgcga ccgttccagc ttattacaga 1560
gctcctcccg tcaactgtca agcgagatca ttaaaaccag atgccataaa gggattgtag 1620
<210> 2
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
atggtagatc tgtacgcgct cg 22
<210> 3
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
ctacaatccc tttatggcat ct 22
<210> 4
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
ggagagttgc tacaacctgg aggct 25
<210> 5
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
tcccgcatcc tctgtatgaa cc 22
Claims (4)
1. Cyclocarya paliurus geneCpSE1-likeThe application of the fluorescent quantitative primer in screening single cyclocarya paliurus strains with high triterpene content is characterized in that the cyclocarya paliurus geneCpSE1-likeThe nucleotide sequence of (a) is shown as SEQ ID NO: 1 is shown.
2. The use of claim 1, wherein the cyclocarya paliurus gene is used as a template for reverse transcription of cDNACpSE1-likeAnd (3) carrying out qRT-PCR by using fluorescent quantitative primers, wherein the nucleotide sequences of the primers are shown as SEQ ID NO: 4 and SEQ ID NO: 5, respectively.
3. The use according to claim 2, wherein the qRT-PCR reaction system is: SYBR Green Mix 10. mu.L, 10. mu. mol/L upstream and downstream primers 0.8. mu.L each, cDNA 2. mu.L, deionized water 6.4. mu.L, total volume 20. mu.L.
4. The use according to claim 2, wherein the qRT-PCR reaction procedure is: at 95 ℃ for 40 s; denaturation at 95 ℃ for 15 s; annealing at 55 ℃ for 30 s; and (3) extending for 35s at 72 ℃, wherein 40 cycles of denaturation, annealing and extension are set.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010631113.7A CN111909942B (en) | 2020-07-03 | 2020-07-03 | Clone of cyclocarya paliurus gene CpSE1-like coding sequence and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010631113.7A CN111909942B (en) | 2020-07-03 | 2020-07-03 | Clone of cyclocarya paliurus gene CpSE1-like coding sequence and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111909942A CN111909942A (en) | 2020-11-10 |
| CN111909942B true CN111909942B (en) | 2022-08-26 |
Family
ID=73227212
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010631113.7A Active CN111909942B (en) | 2020-07-03 | 2020-07-03 | Clone of cyclocarya paliurus gene CpSE1-like coding sequence and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111909942B (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109486781A (en) * | 2018-11-21 | 2019-03-19 | 中国农业科学院北京畜牧兽医研究所 | A kind of alfalfa is overexpressed the method and application of squalene synthase gene |
-
2020
- 2020-07-03 CN CN202010631113.7A patent/CN111909942B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109486781A (en) * | 2018-11-21 | 2019-03-19 | 中国农业科学院北京畜牧兽医研究所 | A kind of alfalfa is overexpressed the method and application of squalene synthase gene |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111909942A (en) | 2020-11-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107586867B (en) | Characteristic sequence, labeled primer and identification method of apocarya variety Pawnee | |
| CN107557434B (en) | Characteristic sequence, labeled primer and identification method of Carya illinoensis variety Van Deman | |
| CN112375764B (en) | Fruit low-acid regulatory gene MdMYB44 and application thereof | |
| CN106498088B (en) | CAPs molecular marker for identifying amaranthus rugosus based on SNP (single nucleotide polymorphism) sites and application of CAPs molecular marker | |
| CN113005139B (en) | Application of transcription factor PsMYB1 in regulation and control of synthesis of peony petal anthocyanin | |
| CN109081865B (en) | Phyllostachys pubescens PeVQ28 protein and coding gene and application thereof | |
| CN108949925B (en) | Molecular detection method for rapidly and accurately identifying weedy rice and cultivated rice | |
| CN107119048B (en) | Pseudocercospora mori rDNA and application thereof in molecular detection of pseudocercospora mori | |
| CN106978504B (en) | Preparation method and application of spinach SSR (simple sequence repeat) marker | |
| CN114182032B (en) | SNP molecular marker for detecting muskmelon seed coat color and application thereof | |
| CN107586866B (en) | Characteristic sequence, labeled primer and identification method of apocarya variety Moore | |
| KR20100021834A (en) | Method of discriminating ginseng cultivars using sts primers | |
| CN112695125B (en) | Katelia SSR molecular marker primer composition and application thereof | |
| CN111909942B (en) | Clone of cyclocarya paliurus gene CpSE1-like coding sequence and application thereof | |
| CN107354162A (en) | Paddy gene ORYsa;SIZ2 genetic engineering application | |
| CN102268429B (en) | Rapid cloning method of plant flowering related gene LFY/FLO homologous fragment and special primer thereof | |
| CN108330164B (en) | Characteristic sequence, primer and identification method of apocarya variety Moore | |
| CN110760530B (en) | Lycoris longituba LlDFRa gene, protein expressed by same and application of gene | |
| CN109082425B (en) | Rape boron high-efficiency gene BnA3NIP5; transposon insertion fragment TEQ of 1Q and primer and application thereof | |
| CN108949779B (en) | Gene SaWRKY expressed by drought stress of wild species of stipa arundinacea | |
| US6933374B2 (en) | Farnesyl pyrophosphate synthase protein, nucleic acid and promoter region thereof | |
| CN114395566B (en) | Application of sweet potato ERF transcription factor IbERF4 in promoting synthesis of plant chlorogenic acid substances | |
| CN108841842B (en) | Festuca arundinacea gene EfWRKY62 expressed by wild species of the saccharum arundinacea under low temperature stress | |
| CN114478730B (en) | Wheat TaVQ14 protein, and coding gene and application thereof | |
| Tao et al. | Identification and characterization of transcripts differentially expressed during embryogenesis in Capsella bursa-pastoris |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB03 | Change of inventor or designer information |
Inventor after: Lactus fragrans Inventor after: Chen Xiaoling Inventor after: Chen Biqin Inventor after: Fang Shengzuo Inventor after: Shang Xulan Inventor before: Lactus fragrans Inventor before: Chen Xiaolin Inventor before: Chen Biqin Inventor before: Fang Shengzuo Inventor before: Shang Xulan |
|
| CB03 | Change of inventor or designer information | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |