CN112795631A - Fluorescent quantitative reference gene under abiotic stress of cryptomeria fortunei, and special primer and application thereof - Google Patents
Fluorescent quantitative reference gene under abiotic stress of cryptomeria fortunei, and special primer and application thereof Download PDFInfo
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Abstract
The invention discloses a fluorescent quantitative reference gene under abiotic stress of cryptomeria fortunei, a special primer and application thereof, belonging to the technical field of plant molecular biology. The invention screens and designs 15 candidate internal reference genes, evaluates the stability of the candidate genes by 5 algorithms (delta-CT, geonorm, NormFinder, BestKeeper and RefFinder), obtains fluorescent quantitative internal reference genes suitable for various abiotic stresses of cryptomeria fortunei, designs real-time fluorescent quantitative PCR primers of the internal reference genes, has strong specificity and high amplification efficiency, can greatly improve the detection efficiency when the real-time fluorescent quantitative detection is adopted for the cryptomeria fortunei genes, and improves the reliability of detection results.
Description
Technical Field
The invention belongs to the technical field of plant molecular biology, and particularly relates to a fluorescent quantitative reference gene under abiotic stress of cryptomeria fortunei, and a special primer and application thereof, wherein the abiotic stress comprises drought stress, heat damage stress, low temperature stress and salt stress.
Background
Cryptomeria fortunei Hooibrenk, also known as Pinus longifola, is an arbor of genus Cryptomeria of family Cunninghamiae. When abiotic stress factors such as drought, heat damage, low temperature, salt damage and the like act on plants, a series of physiological metabolic reactions occur in the plants. However, no research on the gene of the cryptomeria fortunei under the abiotic stress has been reported. In the research process of the gene expression difference under the condition of the cryptomeria fortunei abiotic stress, the analysis and verification of the gene expression level by using a real-time fluorescence quantitative technology are required, and a stable and reliable reference gene is required, so that the screening of the stable-expression reference gene under the condition of the cryptomeria fortunei abiotic stress is a key factor for accurate real-time fluorescence quantitative result.
Disclosure of Invention
Aiming at the problems in the prior art, the first technical problem to be solved by the invention is to provide a fluorescent quantitative reference gene under abiotic stress of cryptomeria fortunei; the second technical problem to be solved by the invention is to provide a special primer for fluorescent quantitative reference genes under abiotic stress of cryptomeria fortunei; the third technical problem to be solved by the invention is to provide the application of the internal reference gene or the special primer in the fluorescence quantification of the cedar.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
fluorescent quantitative reference genes are obtained under abiotic stress of the cryptomeria fortunei, wherein the abiotic stress comprises drought stress, heat damage stress, salt stress and low temperature stress.
Further, fluorescent quantitative reference genes under drought stress of cryptomeria fortunei are CYP and HSP70 genes, and the nucleotide sequence of the CYP gene is shown in SEQ ID NO. 1; the nucleotide sequence of the HSP70 gene is shown in SEQ ID NO. 2.
The special primer for fluorescent quantitative reference gene under drought stress of cryptomeria fortunei,
the primer sequences of the CYP gene are as follows:
CYP forward primer: 5'-TCTCGGGCAGCATTTCACGC-3', respectively;
CYP reverse primer: 5'-AGCCGAAACTGGCGCCAACA-3', respectively;
the primer sequences of the HSP70 gene are as follows:
HSP70 forward primer: 5'-AACGCAAGGGCTTTGAGAA-3', respectively;
HSP70 reverse primer: 5'-ACCTGGCACGGGTTATGGT-3' are provided.
The fluorescent quantitative reference gene under the drought stress of the cryptomeria fortunei is applied to fluorescent quantitative of the cryptomeria fortunei.
The special primer of the fluorescent quantitative reference gene under the drought stress of the cedar is applied to fluorescent quantification of the cedar.
Further, fluorescent quantitative reference genes under the stress of cryptomeria fortunei heat damage are CYP and UBC genes, and the nucleotide sequence of the CYP gene is shown as SEQ ID NO. 1; the nucleotide sequence of the UBC gene is shown as SEQ D NO. 3.
The special primer for fluorescent quantitative reference gene under the stress of cryptomeria fortunei heat damage,
the primer sequences of the CYP gene are as follows:
CYP forward primer: 5'-TCTCGGGCAGCATTTCACGC-3', respectively;
CYP reverse primer: 5'-AGCCGAAACTGGCGCCAACA-3', respectively;
the primer sequences for the UBC gene are as follows:
UBC forward primer: 5'-CTCGCAGAATCATAAAGGAAACAC-3', respectively;
UBC reverse primer: 5'-CCATTGGATACTCTTCAGGCAAA-3' are provided.
The fluorescent quantitative reference gene under the stress of cryptomeria fortunei heat damage is applied to the fluorescent quantitative determination of cryptomeria fortunei.
The special primer of the fluorescence quantitative reference gene under the stress of cryptomeria fortunei heat damage is applied to fluorescence quantification of cryptomeria fortunei.
Further, the fluorescent quantitative reference genes of the cryptomeria fortunei under low-temperature stress are Rbcl, CYP and Actin genes, and the nucleotide sequence of the Rbcl gene is shown as SEQ ID NO. 4; the nucleotide sequence of the CYP gene is shown in SEQ ID NO. 1; the nucleotide sequence of the Actin gene is shown in SEQ ID NO. 6.
The special primer for fluorescent quantitative reference gene of cryptomeria fortunei under low temperature stress,
the primer sequences for the Rbcl gene are as follows:
rbcl forward primer: 5'-CGTATTACAGTTCGGTGGAGGG-3' the flow of the air in the air conditioner,
rbcl reverse primer: 5'-CACAAGCGGCAGCTAGTTCA-3', respectively;
the primer sequences of the CYP gene are as follows:
CYP forward primer: 5'-TCTCGGGCAGCATTTCACGC-3' the flow of the air in the air conditioner,
CYP reverse primer: 5'-AGCCGAAACTGGCGCCAACA-3', respectively;
primer sequences of the Actin gene are as follows:
actin forward primer: 5'-GTTGCCATTCAAGCCGTTCT-3' the flow of the air in the air conditioner,
an Actin reverse primer: 5'-AACAATTTCACGCTCAGCAGTAG-3' are provided.
The fluorescent quantitative reference gene under the low-temperature stress of the cryptomeria fortunei is applied to fluorescent quantitative determination of the cryptomeria fortunei.
The special primer of the fluorescence quantitative reference gene of the cryptomeria fortunei under low-temperature stress is applied to fluorescence quantification of the cryptomeria fortunei.
Further, the fluorescent quantitative reference genes under the stress of the cedar salt are Rbcl and 18SrRNA genes, and the nucleotide sequence of the Rbcl gene is shown as SEQ ID NO. 4; the nucleotide sequence of the 18S rRNA gene is shown in SEQ ID NO. 5.
The special primer for fluorescent quantitative reference gene under the stress of cedar salt,
the primer sequences for the Rbcl gene are as follows:
rbcl forward primer: 5'-CGTATTACAGTTCGGTGGAGGG-3', respectively;
rbcl reverse primer: 5'-CACAAGCGGCAGCTAGTTCA-3', respectively;
the primer sequences of the 18SrRNA gene are as follows:
18S rRNA forward primer: 5'-TCTGGTCCTGTTCCGTTGG-3', respectively;
18S rRNA reverse primer: 5'-GCTTTCGCAGTGGTTCGTC-3', respectively;
the fluorescent quantitative reference gene under the stress of cedar salt is applied to the fluorescent quantitative determination of cedar.
The special primer of the fluorescent quantitative reference gene under the stress of cedar salt is applied to the fluorescent quantitative determination of cedar.
Has the advantages that: compared with the prior art, the invention has the advantages that:
the method comprises the steps of carrying out local blast comparison (blast Ver: 2.4.0+) on internal reference genes reported to be used for other species and cryptomeria fortunei RNAseq data, finding out genes with the highest homology, carrying out online analysis on conserved structural domains and ORFs of the genes through blastx on an NCBI website, finally selecting and designing 15 candidate internal reference genes required by the method, evaluating the stability of the candidate genes through 5 algorithms (delta-CT, gem, NormFinder, BestKeeper and RefFinder), obtaining the fluorescent quantitative internal reference genes suitable for various abiotic stresses (drought stress, heat stress, low-temperature stress and salt stress) of cryptomeria fortunei, designing real-time fluorescent quantitative PCR primers of the internal reference genes, having strong specificity and high amplification efficiency, greatly improving the detection efficiency when the cryptomeria fortune genes are detected by adopting real-time fluorescence, and improving the reliability of detection results.
Drawings
FIG. 1 is a graph of the order of the expression stability (M) of 15 reference genes by the geNorm software for each abiotic stress treatment; in the figure, A is drought stress, B is heat damage stress, C is low temperature stress, and D is salt stress;
FIG. 2 is a graph of geNorm determining the optimal number of reference genes for accurate quantification under various abiotic stress treatments; in the figure, A is drought stress, B is heat damage stress, C is low temperature stress, and D is salt stress;
FIG. 3 shows the expression levels of stable and unstable genes, MAKP1 genes, as reference genes, under abiotic stress treatment; in the figure, A is drought stress, B is heat damage stress, C is low temperature stress, and D is salt stress;
FIG. 4 is a CQ value graph of 15 reference genes screened under each abiotic stress treatment; in the figure, A is drought stress, B is heat stress, C is low temperature stress, and D is salt stress.
Detailed Description
The invention is further described with reference to specific examples. These examples are intended to illustrate the invention and are not intended to limit the scope of the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention. In the following examples, unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
The following examples used test materials: selecting a good-growth-state disease-and-pest-free cryptomeria fortunei from Jiangxi Dagang mountain as a mother tree, cutting a semi-lignified branch which grows in the same year and has 2-3 lateral buds as a cutting slip (12-16cm) in 6 months of 2019, performing upper incision flat cutting, performing lower incision 45-degree oblique cutting, soaking in distilled water for 12 hours, using 1% sodium hypochlorite for 10 minutes, washing with distilled water for 3 times, and using 0.1 g.L-1And soaking the GGR rooting powder for 4 hours. The cedar cutting slips are cut in a white horse-based ground greenhouse of Nanjing forestry university, the volume ratio of soil matrix of grass carbon, perlite, vermiculite and yellow sand is 1: 1, and water is sprayed twice per week. 18 cryptomeria fortunei seedlings with consistent growth vigor are respectively selected for each stress experiment in 9 months in 2020. Irrigating Cryptomeria fortunei seedlings (200 mL per plant) with 15% PEG 6000 to simulate drought; irrigating cedar seedlings (200 mL per plant) in 1/4 Hoagland solution of 200mM sodium chloride to simulate salt stress; exposing the seedling of the cryptomeria fortunei to a simulated heat damage stress at 42 ℃; exposing the seedling of the cryptomeria fortunei to a simulated low temperature stress at 4 ℃; culturing the seedlings except the seedlings stressed by low temperature and heat damage at 25 ℃; all the seedlings were cultivated in a light incubator with the same photoperiod (12h light/12 h dark) and humidity (60%). Three biological replicates were made. Samples were taken at 0, 2, 6, 12, 24 and 48h after stress, respectively. At the time of sample collection, the samples were snap frozen with liquid nitrogen and then stored at-80 ℃ for further analysis.
Example 1
1. Cryptomeria fortunei total RNA extraction and cDNA synthesis
Total RNA of each sample was extracted according to the instructions of the Total RNA extraction kit of the Biotech Co., Ltd. Integrity, purity and concentration were checked by 1% agarose gel electrophoresis and uv spectrophotometer (NanoDrop2000, Thermo scientific, Wilmington, DE, USA).
Using total RNA as template and RT-qPCR special premix reagent kit (
III RT SuperMix for qPCR (+ gDNA wiper) R323-01 Nanjing Nodezam Biotech Co., Ltd.) cDNA was synthesized using 0.8. mu.gRNA each time for first strand cDNA synthesis and stored in a freezer at-20 ℃ until use.
2. Selection of reference genes and design of primers therefor
The internal reference genes reported for other species are subjected to local blast comparison (blastVer: 2.4.0+) with the data of cryptomeria fortunei RNAseq to find out the gene with the highest homology, and the conserved domain and ORF of the gene are subjected to online analysis through blastx on an NCBI website, so that 15 candidate internal reference genes required by the application are finally selected and designed, wherein the candidate internal reference genes are CYP, HSP70, Actin, beta-Actin, UBC, 18S rRNA, TUB, RPL2, UBQ, PBL, Rbcl, PP2A, TUA2, PGK1 and HIS4 respectively. And designing a CDS region of the internal reference Primer by using Primer software, wherein the parameters are as follows: the length of the PCR product is 70-250bp, the melting temperature is 58-62 ℃, and the CG content is 40-60%. The 15 candidate reference genes and primer sequences are shown in table 1.
TABLE 115 candidate reference genes and primer sequences
Remarking: e, amplifying the efficiency; r2Correlation coefficient
3. qPCR quantification
qRT-PCR was amplified using the StepOneNus RealTime PCR System (Applied Biosystems) as follows: after 30s at 95 ℃ 40 cycles with parameters of 10s at 95 ℃ and 30s at 60 ℃ a melting curve was generated at 60 ℃ to 95 ℃. Inverse directionThe system comprises the following steps: 10 μ L of 2 × ChamQ SYBR qPCR Master Mix (Low Rox premix); mu.L of 5-fold diluted cDNA and 0.4. mu.L of primers (10. mu.M) plus ddH2O to 20. mu.L. Each qRT-PCR analysis was performed in 3 replicates, each gene including a non-template control. And (3) obtaining a CT value through qRT-PCR, wherein the CT value is inversely proportional to the expression quantity of the gene, the larger the CT value is, the lower the expression quantity of the gene is, and conversely, the smaller the CT value is, the higher the expression quantity of the representative gene is. As shown in FIG. 4, in the abiotic stress, the average value of CT values of 15 reference genes in drought stress treatment is 6.24-28.32, the 18S rRNA expression level is the highest, and the beta-Actin expression level is lower. The average value of CT values of 15 reference genes in heat damage stress treatment is 6.29-29.17, the 18S rRNA expression level is the highest, and the TUB expression level is lower. The average value of CT values of 15 reference genes in low-temperature stress treatment is 6.35-28.27, the 18S expression level is the highest, and the beta-Actin expression level is lower. The average value of CT values of 15 reference genes in salt stress treatment is 6.21-28.68, wherein the 18S rRNA expression level is the highest, and the beta-Actin expression level is lower.
4. Stability evaluation
And analyzing the stability of the reference gene, and comprehensively analyzing the expression stability of the reference gene by utilizing five algorithms of delta-CT, geonorm, NormFinder, BestKeeper, RefFinder and the like. And (4) screening out stable reference genes.
5. Results
delta-CT analysis: obtaining a standard deviation average value through delta-CT analysis, wherein the higher the standard deviation average value is, the worse the gene stability is, and conversely, the lower the standard deviation average value is, the higher the gene stability is, and the result is shown in Table 2, and CYP is the most stable gene during drought and heat damage stress treatment; rbcl is the most stable gene under low temperature and salt stress treatment. TUA2 is the least stable gene during drought, low temperature and salt stress treatments; whereas the least stable gene during heat stress treatment was TUB.
TABLE 2 Delta-CT analysis results
Analysis by using the GeNorm software: calculating the expression stability M value of each candidate reference gene through the geonorm software, wherein the larger the M value is, the lower the stability is; conversely, the smaller the value of M, the higher the stability, where M ═ 1.5 is the upper limit (fig. 1). Furthermore, the pairwise variation value V according to geNormn/Vn+1Analysis to determine the appropriate number of reference genes due to pairwise variation values V of the examples of Cryptomeria fortunei under drought, heat and salt stress2/V3< 0.15 (FIGS. 2A, B and D), so that only 2 reference genes (CYP and HSP70) were required under drought stress, only 2 reference genes (CYP and UBC) were required under heat stress, and only 2 reference genes (Rbcl and 18SrRNA) were required under salt stress for relative expression analysis of the genes. And the pair-wise variation value V of the cryptomeria fortunei under low-temperature stress3/V4< 0.15 (FIG. 2C), therefore only 3 reference genes (Rbcl, CYP and Actin) were required for the relative expression analysis of the genes.
Analysis by NormFinder software: and calculating the stability value of the candidate reference gene by NormFinder software, wherein the stability is poorer when the stability value is higher, and the stability is better when the stability value is lower, namely the gene with the minimum stability value is the most stable gene. The results are shown in table 3, CYP being the most stable gene during drought and heat stress treatment; rbcl is the most stable gene under low temperature and salt stress treatment. TUA2 is the least stable gene during drought, low temperature and salt stress treatments; whereas the least stable gene during heat stress treatment was TUB.
TABLE 3 analysis results of the NormFinder software
Bestkoeper software analysis: standard Deviation (SD) of candidate reference genes was calculated by BestKeeper software, the smaller the SD value, the more stable the expression, the default threshold value of the program was 1, and when the SD value was greater than 1, the gene expression was considered unstable. The results are shown in Table 4, where Actin is the most stable expression under drought stress, PP2A is the most stable expression under heat stress, CYP is the most stable expression under low temperature stress, and 18S rRNA is the most stable expression under salt stress.
TABLE 4 BestKeeper software analysis results
RefFinder website analysis: calculating a geometric mean value of the stability ranks obtained by the analysis through a RefFinder website, wherein the smaller the geometric mean value is, the more stable the expression is; conversely, the larger the geometric mean, the more unstable the expression. The results are shown in Table 5, the stability of the gene in drought stress is CYP > HSP70 > Actin > beta-Actin > UBC > 18S rRNA > TUB > UBQ > PBL > RPL2 > Rbcl > PP2A > TUA2 > PGK1 > HIS4, wherein the geometric mean value of the gene CYP is 1.410, and the gene is the most stable gene; the geometric mean value of gene HIS4 was 14.240, which was the most unstable gene; the stability of the gene in the heat damage stress is CYP > UBC > HSP70 > PP2A > PBL > Actin > 18S rRNA > HIS4 > RPL2 > UBQ > PGK1 > beta-Actin > Rbcl > TUA2 > TUB, wherein the geometric mean value of the gene CYP is 1.19, and the gene is the most stable gene; the least stable gene was TUB, which had a geometric mean of 15; the stability of the gene in low temperature stress is Rbcl > CYP > Actin > UBC > 18S > beta-Actin > PBL > RPL2 > PP2A > HSP70 > PGK1 > UBQ > HIS4 > TUB > TUA2, wherein the geometric mean value of the gene Rbcl is 1.41, and the gene is the most stable gene; the geometric mean of gene TUA2 was 14.74, the most unstable gene; the stability of the gene in the salt stress is that Rbcl is more than 18S rRNA > Actin > HSP70 > CYP > TUB > UBC > HIS4 > beta-Actin > RPL2 > PP2A > PBL > TUA2 > UBQ > PGK1, wherein the geometric mean value of the gene Rbcl is 1.19, and the gene is the most stable gene; the geometric mean value of the gene PGK1 was 14.24, which was the least stable gene.
TABLE 5 RefFinder Web site analysis results
6. Verification of stability of reference gene
MAKP based on high throughput sequencing results1 as a target gene, and the stable gene and unstable gene in each abiotic stress as standards were calculated to confirm the applicability of the candidate genes evaluated in the present application. By 2-ΔΔCtThe method calculates the expression of the target gene. When an unstable gene PGK1 is used as an internal reference gene in the expression quantity of the MAKP1 gene (figure 3), the MAKP1 gene is highly expressed at the 24 th hour under drought stress and salt stress treatment; when the unstable gene TUA2 is used as an internal reference gene, the MAKP1 gene is highly expressed at 48h under heat damage stress and low-temperature stress treatment. Therefore, selection of an appropriate gene has a large influence on the expression level of the target gene.
Sequence listing
<110> Nanjing university of forestry
<120> fluorescent quantitative reference gene under abiotic stress of cryptomeria fortunei, and special primer and application thereof
<130> 1
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atctggtggt atttatagct taaataggtt ttctgagatg catgatgttc tgattgtctt 900
gcagacgcca agaggttgat tggaaggcgc tttactgatc cttccgttca gagtgatata 960
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tacaagggtg aggaaaagca tttttctgca gaagagattt catcgatggt tttgatgaag 1080
atgaaggaaa ttgctgaggc ttatctaggg tcaacaatta aaaatgctgt tgtcactgtg 1140
cctgcttatt tcaatgattc ccagagacag gctaccaagg atgcaggtgt catctctggt 1200
atgaatgtta tgagaatcat taatgagcca actgctgctg ccatcgctta tggcttagat 1260
aaaaaagcca caagtgtggg tgagaaaaat gtgttgattt ttgatttagg cgggggtact 1320
tttgatgttt ctcttttgac aatcgaagaa ggtatttttg aagtcaaggc cactgctgga 1380
gacacccatt tgggtggaga agattttgac aataggatgg ttaatcactt tgttcaggaa 1440
ttcaagagaa agtataagaa ggatatctct ggcaacgcaa gggctttgag aagattgaga 1500
acttcgtgtg agagggcaaa gagaaccctt tcatccactg cccagacgac cattgagatt 1560
gattctttat atgaaggtat cgacttctat tcaaccataa cccgtgccag gtttgaggaa 1620
ttgaacatgg atctcttcag aaagtgtatg gaacctgtgg agaaatgttt gagggatgcc 1680
aagatggaca agagcacaat tcacgatgtt gtcctcgtcg ggggctcaac tagaattccc 1740
aaggttcagc agctgttgca ggacttcttt aacgggaagg agctgtgcaa aagtatcaat 1800
cctgacgagg ccgttgcata tggagctgca gtgcaagctg caattctgag cggagaaggc 1860
aatgagaagg tgcaagattt gttgctgctt gacgtcactc ctctttctct tggtctggaa 1920
actgctggag gtgttatgac tgtcctcatt ccaaggaaca ccacaattcc cacaaagaag 1980
gaacaagtct tctctacata ctcagacaat cagcctggtg tgcttatcca ggtatacgag 2040
ggagagagaa ctcgaactcg cgataacaat ctgttgggca agtttgaact ctctggaatc 2100
cctcctgccc ctaggggtgt tcctcaaatc actgtttgct tcgacattga tgccaatggt 2160
attctgaatg tgtcagccga ggacaagact actggtcaga agaacaagat cactatcacc 2220
aatgataagg gtaggttaag taaggaggaa attgagaaga tggttcagga tgcagagaaa 2280
tataaatccg aggatgagga gcataagaaa aaggttgagt ccaaaaactc gttagaaaat 2340
tatgcataca acatgagaaa tactataaag gacgacaaga ttgcagccaa gctggatgca 2400
tcggacaaga agaagattga agatgccatt gagcaggcca ttcagtggct cgataataac 2460
cagcttgctg aatctgacga gtttgaggac aagatgaaag agcttgagag catttgtaac 2520
cccatcattg caaagatgta ccagggtgcg ggtggtgaaa tgcctggtgg tggttttggc 2580
gctcccgagg atggtccttc aaccggtgga ggcccaggtc ctaagatcga ggaagtcgat 2640
<210> 3
<211> 486
<212> DNA
<213> UBC
<400> 3
ttcacttgga tcgatacccg cgcagctatg tcgaacagta atctgcctcg cagaatcata 60
aaggaaacac agcgtttgct cactgagcca gcacctggta taagtgcttc tgcatccgag 120
gataatttac gatatttcaa tgtgatgatc cttggccctg ctcagtctcc ttatgaaggg 180
ggttgtttca agttggaatt atttttgcct gaagagtatc caatggcagc gccaaaggtt 240
cgctttctga cgaagattta ccaccccaac attgacaagc ttggtcgcat atgcctggat 300
attctgaagg acaagtggag tcctgctctt caaattcgaa ctgttcttct gagcatacaa 360
gcattgttga gtgcaccaaa cccagacgat ccactctctg agaacattgc aaagcactgg 420
aaatcaaatg aaactgaggc tgtggaaaca gcaaaagaat ggacaaggct gtatgcaagt 480
ggggct 486
<210> 4
<211> 1425
<212> DNA
<213> Rbcl
<400> 4
atgtcaccaa aaacagagac taaagcaagt gtcggattca aagctggtgt taaagattac 60
agattaactt attatactcc ggaatatcag accaaagata ctgatatctt agcagcattc 120
cgagtcactc ctcaacctgg agtacccccc gaagaagcgg gagcagcagt agccgccgaa 180
tcttccactg gtacgtggac gactgtttgg accgatggac ttaccagtct tgatcgttac 240
aaggggcgat gctatgatat tgaacccgtt cctggagagg aaagtcaatt tattgcctat 300
gtggcttacc ctttagatct ttttgaagaa ggttctgtta ctaacctgtt cacttctatt 360
gtaggtaatg tatttggatt caaagcctta cgggctctac gtctggaaga tttacggatt 420
cctcctgctt attcaaaaac tttccaaggc ccaccacatg gtattcaagt agaaagagat 480
aaattaaaca aatatggtcg tcctttgttg ggatgtacta taaaaccaaa attgggtcta 540
tctgccaaga attatggtag agcggtttat gaatgtctcc gtggtggact tgattttacc 600
aaggatgatg aaaacgtgaa ttcccaacca tttatgcgct ggagagatcg tttctgcttt 660
tgtgcagaag cactttataa agctcaggct gagacgggtg agattaaggg acattacctg 720
aatgctactg caggtacatg tgaagaaatg atgaaaagag cagtattcgc cagagaattg 780
ggagttccta tagtcatgca tgactatctg actggaggtt ttacggcaaa tacttcgttg 840
gctcattatt gccgagataa cggcctactt cttcacattc accgcgcaat gcatgcagtt 900
attgacagac aaagaaatca tggtatgcac ttccgtgtac tggctaaagc actacgtatg 960
tctggtggag atcatattca cgctggtact gtagtaggta aacttgaagg agaacgggaa 1020
gtgactttgg gttttgttga tctattgcgt gatgatttta ttgaaaaaga ccgaagtcgt 1080
ggtatttatt tcactcaaga ttgggtctct atgccgggtg ttctgcctgt agcttcagga 1140
ggtattcacg tttggcatat gcctgctctg accgagatct ttggggatga ttccgtatta 1200
cagttcggtg gagggacttt ggggcaccct tggggaaatg cacctggtgc agtggctaat 1260
cgggtcgctt tagaagcttg tgtacaagct cgtaatgaag gacgtgatct tgcgcgtgaa 1320
ggtaatcaag tgatccgcga agctactaaa tggagccctg aactagctgc cgcttgtgaa 1380
gtatggaaag aaatcaaatt tgaatttgat acgattgatc gcttg 1425
<210> 5
<211> 516
<212> DNA
<213> 18S rRNA
<400> 5
ggtgtgcact ggccctcacg tcccttctgc cggcggcgtg ttcctggcct taattggctg 60
ggtcgcggtt ccggcgccgt tactttgaaa aaattagagt gctcaaagca agcctacgct 120
ctgaatacat tagcatggaa taacgcgata ggagtctggt cctgttccgt tggccttcgg 180
gaccggagta atgattaata gggactgtcg ggggcattcg tatttcattg tcagaggtga 240
aattcttgga tttatggaag acgaaccact gcgaaagcat ttgccaagga tgttttcatt 300
aatcaagaac gaaagttggg ggctcgaaga cgatcagata ccgtcctagt ctcaaccata 360
aacgatgccg accagggatc ggcggatgtt gctctaagga ctccgccagc accttctgag 420
aaatcagagt gtttgggttc cggggggagt atggtcgcaa ggctgaaact taaaggaatt 480
gacggaaggg caccaccagg agtggagcct gcggct 516
<210> 6
<211> 1131
<212> DNA
<213> Actin
<400> 6
atggccgatg cagaggacat tcagcccctt gtctgcgaca atggaacagg aatggttaag 60
gctggttttg ctggagacga tgctcccagg gcagtgtttc ctagcattgt gggaagacct 120
cgtcacactg gagtcatggt ggggatggga cagaaggatg catatgtagg tgatgaggct 180
caatctaaaa gaggtattct cacattgaag taccctattg agcacggaat tgtatccaac 240
tgggatgata tggagaagat ctggcatcac actttttata atgagcttcg agttgctcca 300
gaagaacatc ctgttctcct cactgaggca cctcttaacc caaaagcaaa cagggagaaa 360
atgactcaaa ttatgtttga gaccttcaat acaccagcaa tgtatgttgc cattcaagcc 420
gttctctctc tatatgcaag tggtcgtact accggaattg ttcttgattc tggggatggt 480
gtcagccaca ctgttccaat ttatgaagga tatgcacttc cccatgctat ccttaggttg 540
gatcttgctg gtcgtgattt gacagatgct ttaatgaaga tcttgactga gagagggtac 600
tcattcacca ctactgctga gcgtgaaatt gttcgtgata tgaaagagaa gcttgcttat 660
gtagcactgg actttgaaca ggagcttgag acaagcaaga ctagttcttc ccttgagaag 720
agttatgagc ttcctgatgg acaggtaatt accattggtg cagagcggtt tagatgccct 780
gaagtcttgt tccagccatc tatgattgga atggaagctg caggtatcca tgagacaact 840
tacaactcca tcatgaagtg tgatgtggat atcagaaagg atctctatgg aaacattgtt 900
ctcagtggag gatctacaat gtttcctggc attgctgatc gtatgagcaa ggaaattact 960
gctcttgccc ctagcagcat gaagattaag gttgttgcac cgcctgagag gaagtatagt 1020
gtctggattg gagggtcaat cttggcgtct ctcagcacct tccaacagat gtggatagcc 1080
aagtccgagt atgatgagtc tggtccgtca attgttcaca gaaagtgctt c 1131
<210> 7
<211> 20
<212> DNA
<213> CYP Forward primer (artificial)
<400> 7
<210> 8
<211> 20
<212> DNA
<213> CYP reverse primer (artificial)
<400> 8
<210> 9
<211> 19
<212> DNA
<213> HSP70 Forward primer (artificial)
<400> 9
aacgcaaggg ctttgagaa 19
<210> 10
<211> 19
<212> DNA
<213> HSP70 reverse primer (artificial)
<400> 10
acctggcacg ggttatggt 19
<210> 11
<211> 24
<212> DNA
<213> UBC Forward primer (artificial)
<400> 11
ctcgcagaat cataaaggaa acac 24
<210> 12
<211> 23
<212> DNA
<213> UBC reverse primer (artificial)
<400> 12
ccattggata ctcttcaggc aaa 23
<210> 13
<211> 22
<212> DNA
<213> Rbcl forward primer (artificial)
<400> 13
cgtattacag ttcggtggag gg 22
<210> 14
<211> 20
<212> DNA
<213> Rbcl reverse primer (artificial)
<400> 14
<210> 15
<211> 19
<212> DNA
<213> 18S rRNA forward primer (artificial)
<400> 15
tctggtcctg ttccgttgg 19
<210> 16
<211> 19
<212> DNA
<213> 18S rRNA reverse primer (artificial)
<400> 16
gctttcgcag tggttcgtc 19
<210> 17
<211> 20
<212> DNA
<213> Actin Forward primer (artificial)
<400> 17
gttgccattc aagccgttct 20
<210> 18
<211> 23
<212> DNA
<213> Actin reverse primer (artificial)
<400> 18
aacaatttca cgctcagcag tag 23
Claims (6)
1. The fluorescent quantitative reference gene under the abiotic stress of the cryptomeria fortunei is characterized in that the abiotic stress comprises drought stress, heat damage stress, salt stress and low-temperature stress; fluorescent quantitative internal reference genes under drought stress of cryptomeria fortunei are CYP and HSP70 genes; the fluorescent quantitative internal reference genes are CYP and UBC genes under the stress of cryptomeria fortunei heat damage; fluorescent quantitative reference genes under the stress of cryptomeria salmonides are Rbcl and 18S rRNA genes; the fluorescent quantitative internal reference genes under the low-temperature stress of the cryptomeria fortunei are Rbcl, CYP and Actin genes;
the nucleotide sequence of the CYP gene is shown in SEQ ID NO. 1; the nucleotide sequence of the HSP70 gene is shown in SEQ ID NO. 2; the nucleotide sequence of the UBC gene is shown as SEQ ID NO. 3; the nucleotide sequence of the Rbcl gene is shown in SEQ ID NO. 4; the nucleotide sequence of the 18S rRNA gene is shown as SEQ ID NO. 5; the nucleotide sequence of the Actin gene is shown in SEQ ID NO. 6.
2. The special primer for fluorescent quantitative reference gene under the abiotic stress of cryptomeria fortunei of claim 1, which is characterized in that,
the primer sequences of the CYP gene are as follows:
CYP forward primer: 5'-TCTCGGGCAGCATTTCACGC-3', respectively;
CYP reverse primer: 5'-AGCCGAAACTGGCGCCAACA-3', respectively;
the primer sequences of the HSP70 gene are as follows:
HSP70 forward primer: 5'-AACGCAAGGGCTTTGAGAA-3', respectively;
HSP70 reverse primer: 5'-ACCTGGCACGGGTTATGGT-3', respectively;
the primer sequences for the UBC gene are as follows:
UBC forward primer: 5'-CTCGCAGAATCATAAAGGAAACAC-3', respectively;
UBC reverse primer: 5'-CCATTGGATACTCTTCAGGCAAA-3', respectively;
the primer sequences for the Rbcl gene are as follows:
rbcl forward primer: 5'-CGTATTACAGTTCGGTGGAGGG-3', respectively;
rbcl reverse primer: 5'-CACAAGCGGCAGCTAGTTCA-3', respectively;
the primer sequences of the 18S rRNA gene were as follows:
18S rRNA forward primer: 5'-TCTGGTCCTGTTCCGTTGG-3', respectively;
18S rRNA reverse primer: 5'-GCTTTCGCAGTGGTTCGTC-3', respectively;
primer sequences of the Actin gene are as follows:
actin forward primer: 5'-GTTGCCATTCAAGCCGTTCT-3' the flow of the air in the air conditioner,
an Actin reverse primer: 5'-AACAATTTCACGCTCAGCAGTAG-3' are provided.
3. The use of the fluorescent quantitative reference gene of cryptomeria fortunei of claim 1 in fluorescent quantitation of cryptomeria fortunei.
4. The use of claim 3, wherein the CYP and HSP70 genes are used as reference genes for the fluorescent quantitation under the drought stress of Cryptomeria fortunei, the CYP and UBC genes are used as reference genes for the fluorescent quantitation under the heat damage stress of Cryptomeria fortunei, the Rbcl and 18SrRNA genes are used as reference genes for the fluorescent quantitation under the salt stress of Cryptomeria fortunei, and the Rbcl, CYP and Actin genes are used as reference genes for the fluorescent quantitation under the low temperature stress of Cryptomeria fortunei.
5. The application of the special primer of the fluorescent quantitative reference gene of the cryptomeria fortunei under the abiotic stress of the cryptomeria fortunei in the fluorescent quantitative determination of the cryptomeria fortunei in the claim 2.
6. The use of claim 3, wherein the primer sequences of CYP and HSP70 genes are applied to the fluorescent quantitation under the drought stress of Cryptomeria fortunei, the primer sequences of CYP and UBC genes are applied to the fluorescent quantitation under the heat damage stress of Cryptomeria fortunei, the primer sequences of Rbcl and 18SrRNA genes are applied to the fluorescent quantitation under the salt stress of Cryptomeria fortunei, and the primer sequences of Rbcl, CYP and Actin genes are applied to the fluorescent quantitation under the low temperature stress of Cryptomeria fortunei.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113416794A (en) * | 2021-07-06 | 2021-09-21 | 南京林业大学 | Reference gene suitable for fluorescent quantitation of male flowers of salix dustpan, and primer and application thereof |
| CN116144822A (en) * | 2022-12-02 | 2023-05-23 | 四川省草原科学研究院 | Reference gene under abiotic stress of eremochloa ophiuroides, and primers and application thereof |
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| CN111139253A (en) * | 2020-01-20 | 2020-05-12 | 南京林业大学 | Cryptomeria fortunei CfCCR gene and application thereof |
| CN111763683A (en) * | 2020-06-30 | 2020-10-13 | 南京林业大学 | Cryptomeria fortunei CfICE1 gene and application thereof |
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| CN111139253A (en) * | 2020-01-20 | 2020-05-12 | 南京林业大学 | Cryptomeria fortunei CfCCR gene and application thereof |
| CN111763683A (en) * | 2020-06-30 | 2020-10-13 | 南京林业大学 | Cryptomeria fortunei CfICE1 gene and application thereof |
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| 储文渊等: "盐和干旱胁迫下杨树新内参基因的筛选", 《林业科学》 * |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113416794A (en) * | 2021-07-06 | 2021-09-21 | 南京林业大学 | Reference gene suitable for fluorescent quantitation of male flowers of salix dustpan, and primer and application thereof |
| CN116144822A (en) * | 2022-12-02 | 2023-05-23 | 四川省草原科学研究院 | Reference gene under abiotic stress of eremochloa ophiuroides, and primers and application thereof |
| CN116144822B (en) * | 2022-12-02 | 2024-03-22 | 四川省草原科学研究院 | Reference gene under abiotic stress of eremochloa ophiuroides, and primers and application thereof |
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