CN112280887B - Internal reference gene for trichosanthes kirilowii Maxim seedling stage male and female plant gene expression research and application thereof - Google Patents
Internal reference gene for trichosanthes kirilowii Maxim seedling stage male and female plant gene expression research and application thereof Download PDFInfo
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Abstract
The invention discloses an internal reference gene for trichosanthes kirilowii Maxim seedling stage male and female plant gene expression research and application thereof, and belongs to the technical field of molecular biology. The internal reference genes used for the research of the gene expression of the male and female plants in the seedling stage of the trichosanthes kirilowii are the action genes and/or GAPDH genes; the nucleic acid sequence of the action gene is shown as SEQ ID NO. 1; the nucleic acid sequence of the GAPDH gene is shown in SEQ ID NO. 2. The reference gene can improve the accuracy and reliability of the research on the expression of the male and female plant genes in the seedling stage of the trichosanthes kirilowii Maxim; lays a foundation for digging the male and female plant differential marker genes of the trichosanthes kirilowii Maxim in the seedling stage.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to an internal reference gene for trichosanthes kirilowii Maxim seedling stage male and female plant gene expression research and application thereof.
Background
In plant studies, analysis of gene expression patterns is one of the important research pathways for solving gene functions. Real-time fluorescent quantitative PCR is the most commonly used nucleic acid quantitative technique in gene expression pattern research, and has higher sensitivity, better specificity and wider detection range. However, there may be some differences in RNA concentration, RNA quality, cDNA synthesis efficiency, etc. of different samples, which may affect the accuracy of the real-time fluorescent quantitative PCR results. In the fluorescent quantitative PCR experiment, the use of reference genes can effectively correct the differences, thereby obtaining the real differences of the specific expression of target genes. At present, proper reference genes are screened from a large number of species, and the genes are generally genes with high copy number and relatively stable expression, are generally basic component proteins forming a cytoskeleton, and also are proteins involved in the biochemical metabolic process of organisms. However, in recent years, a large number of studies have revealed that no absolutely stable gene is expressed, and that the so-called constant expression of any reference gene is constant only under the influence of a certain type of cells or experimental factors. Therefore, it is necessary to first screen stable reference genes under a new study material or new test conditions.
Fructus Trichosanthis (Trichosanthes kirilowii maxim.) is a perennial root climbing herb of the genus Trichosanthes of the family Cucurbitaceae. The genus Trichosanthes is a larger genus of Cucurbitaceae, and there are more than 80 species worldwide. Besides being used as a traditional Chinese medicine, the seeds of the trichosanthes kirilowii Maxim are developed into leisure and health-care food. The female plant is mainly harvested from the seeds of the trichosanthes kirilowii Maxim, and the male plant is mainly harvested from the roots of the trichosanthes kirilowii Maxim. Therefore, the planting purposes are different in production, and the planting proportion of the male and female trichosanthes plants is different. In order to improve the planting area and benefit of the trichosanthes kirilowii Maxim, seed propagation is an economical and practical method, but researches prove that the female plants only account for 10-30% during seed propagation, and the proportion of the male and female plants is seriously disordered and cannot be controlled. The stability index for identifying the male and female plants of the trichosanthes kirilowii Maxim in the seedling stage cannot be found by the former through morphological, physiological, molecular marker and other methods. Therefore, the penciler intends to dig out the male and female plant differential genes of trichosanthes kirilowii Maxim in seedling stage by transcriptome sequencing, and the expression level of the differential genes needs to be verified by fluorescent quantitative PCR, which requires stable reference genes. At present, no reference gene is reported for the research on the gene expression of female and male plants in the seedling stage of trichosanthes kirilowii Maxim.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to provide an internal reference gene which is expressed most stably between male and female plants in the seedling stage of the trichosanthes kirilowii Maxim, and lays a foundation for digging a male and female plant differential marker gene in the seedling stage of the trichosanthes kirilowii Maxim.
In order to achieve the above purpose, the invention adopts the following technical scheme:
the internal reference genes for the research of the male and female plant gene expression in the seedling stage of the trichosanthes kirilowii are Actin genes and/or GAPDH genes; the nucleic acid sequence of the action gene is shown as SEQ ID NO. 1; the nucleic acid sequence of the GAPDH gene is shown in SEQ ID NO. 2.
The primer sequence of the action gene is as follows:
forward primer: TCAATCCTAAAGCCAACAGAG;
reverse primer: ACCATCACCAGAGTCAAGCA.
The primer sequence of the GAPDH gene is as follows:
forward primer: TGTTCACTCAATGACTGCCACC;
reverse primer: GCACTTTGCCGACAGCCTTA.
The application of the internal reference gene for the research on the gene expression of male and female plants in the seedling stage of trichosanthes kirilowii Maxim in the fluorescent quantitative PCR of trichosanthes kirilowii Maxim.
The application of the primer sequence of the fluorescence quantitative PCR of the action gene in the fluorescence quantitative PCR of trichosanthes kirilowii.
The fluorescent quantitative PCR primer sequence of the GAPDH gene is applied to the fluorescent quantitative PCR of trichosanthes kirilowii.
The application of the reference gene in preparing a trichosanthes kirilowii Maxim fluorescent quantitative PCR kit.
The specific primer is applied to preparation of a trichosanthes kirilowii Maxim fluorescent quantitative PCR kit.
A fluorescent quantitative PCR kit for analyzing the gene expression difference of male and female plants in the seedling stage of trichosanthes kirilowii comprises at least one of the specific primers.
On the basis of the scheme, the fluorescent quantitative PCR kit also comprises reagents commonly used in fluorescent quantitative PCR technology.
The application of an action gene and/or GAPDH gene as fluorescent quantitative PCR reference genes for the research of the expression of male and female plants in the seedling stage of trichosanthes kirilowii Maxim.
The screening process of the reference gene for the trichosanthes kirilowii Maxim seedling stage male and female plant gene expression research comprises the following steps:
(1) By utilizing transcriptome sequencing data of male and female plant leaves of 3 varieties of trichosanthes kirilowii Maxim in seedling stage, 12 trichosanthes kirilowii Maxim candidate reference genes are selected, and 12 pairs of reference gene primers for real-time fluorescence quantitative PCR are designed by taking the selected 12 candidate reference gene sequences as templates. The nucleotide sequences of the 12 candidate internal reference genes are respectively shown as SEQ ID NO. 1-SEQ ID NO.12, and the designed 12 pairs of real-time fluorescent quantitative PCR primer sequences are respectively shown as SEQ ID NO. 13-SEQ ID NO. 36;
(2) And respectively selecting the leaves of 3 varieties of trichosanthes kirilowii Maxim at the seedling stage and female plants as experimental materials, and carrying out a real-time fluorescent quantitative PCR experiment. The fluorescent quantitative PCR amplification system is as follows: 2X TB Green premix Ex Taq. Mu.l, 10. Mu.M forward primer and 10. Mu.M sense primer each 0.4. Mu.l, template cDNA 2. Mu.l, ROX reference dye II 0.4. Mu.l and ddH 2 O6.8. Mu.l. Fluorescent quantitative PCR amplification procedure: pre-denaturation at 95℃for 10min; denaturation at 95℃for 10s, annealing at 60℃for 30s, extension at 72℃for 30s, 40 cycles of reaction;
(3) The data obtained by real-time fluorescence quantitative PCR are imported into gemum, normFinder and BestKeeper software for analysis, comprehensive sequencing is carried out, and the optimal internal reference genes and the combination of the internal reference genes are screened out;
the specific comprehensive sequencing method comprises the following steps: in the stability evaluation results of the candidate reference genes by the gem software and the Normfinder software, the stability index M value or the SV value of the 12 candidate reference genes is ranked from small to large, the candidate reference gene with the minimum M value or the SV value is marked as 12 points, the candidate reference gene with the minimum M value or the SV value is marked as 11 points, the candidate reference gene with the third minimum M value or the SV value is marked as 10 points, and the candidate reference gene with the maximum M value or the SV value is marked as 1 point by analogy.
In the stability evaluation result of the candidate reference genes by the BestKeeper software, the standard deviation (SD value) of the 12 candidate reference genes is ranked from small to large, the candidate reference gene with the smallest SD value is marked as 12 points, the candidate reference gene with the second small SD value is marked as 11 points, and the candidate reference gene with the largest SD value is marked as 1 point by analogy.
And adding the scores of the candidate genes obtained by the three software, and sorting the candidate reference genes according to the score addition from large to small. And taking 2 candidate reference genes with highest comprehensive scores as the optimal reference genes.
The technical scheme of the invention has the advantages that:
the invention screens out the reference genes Actin and GAPDH which are most stably expressed between the male and female plants in the seedling stage of the trichosanthes kirilowii from 12 candidate reference genes; make up for the blank of reference genes for trichosanthes kirilowii gene expression research; the reference gene can improve the accuracy and reliability of researching the expression of male and female plant genes in the seedling stage; lays a foundation for digging the male and female plant differential marker genes of the trichosanthes kirilowii Maxim in the seedling stage.
Drawings
FIG. 1 is a geometric mean (Cq value) of Ct values of all test samples of 12 candidate reference genes;
FIG. 2 is a graph of the ordering of optimal reference genes for a geNorm software analysis, wherein the left is the most unstable reference gene and the right is the most stable reference gene;
FIG. 3 is a graph showing the results of the analysis of the number of optimal reference genes by the gemum software;
FIG. 4 is a diagram of the ordering of optimal reference genes analyzed by NormFinder software, wherein the left is the most stable reference gene and the right is the least stable reference gene;
FIG. 5 is a diagram of the sequencing of the optimal reference genes analyzed by BestKeeper software, wherein the left is the most stable reference gene and the right is the least stable reference gene.
Detailed Description
The terms used in the present invention generally have meanings commonly understood by those of ordinary skill in the art unless otherwise indicated.
The invention will be described in further detail below in connection with specific embodiments and with reference to the data. The following examples are intended to illustrate the invention and are not intended to limit the scope of the invention in any way.
In the following examples, various processes and methods, which are not described in detail, are conventional methods well known in the art. The primers used are all indicated at the first occurrence, and the same primers used thereafter are all identical to the first indicated ones.
Example 1 fluorescent quantitative PCR primer design of candidate reference genes
The invention selects 12 candidate internal reference genes from transcriptome data of male and female plant leaves of three varieties of trichosanthes kirilowii Maxim (Anhui trichosanthes kirilowii Maxim No. 9, anhui trichosanthes kirilowii Maxim No.13 and Anhui trichosanthes kirilowii Maxim No. 17) at seedling stage, wherein the internal reference genes are 18S ribosomal RNA (18 SrRNA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ubiquitin protein (UBQ 10), elongation protein (EF 1 alpha 1, EF1 alpha 2, EF1 alpha 3), actin (action, action 7, action 97), tubulin (alpha-tubulin, alpha-tubulin 3, beta-tubulin), and the sequence of each gene is shown as SEQ ID No. 1-SEQ ID No.12 in sequence. The 12 candidate reference gene sequences are used as templates, and Primer 5 is used for designing fluorescent quantitative PCR specific primers of the reference genes, and the fluorescent quantitative PCR specific primers are sequentially shown as SEQ ID NO. 13-SEQ ID NO. 36.
Respectively taking leaves of male and female plants of three different varieties of trichosanthes kirilowii Maxim (No. 9, no.13 and No. 17) at seedling stage as experimental materials, extracting RNA, reversely transcribing into cDNA as a template, and carrying out a real-time fluorescent quantitative PCR experiment by using the designed fluorescent quantitative PCR specific primers. The specificity of the primers is judged by using a dissolution curve generated by real-time fluorescence quantitative PCR, and the amplification efficiency of 12 candidate internal reference gene primers is judged by making a standard curve. Preparation of Standard Curve cDNA obtained by RNA reverse transcription was used as a template after gradient dilution (comprising 10 1 、10 2 、10 3 、10 4 、10 5 Five template gradients) by fluorescent quantitative PCR Ct values.
TABLE 1 amplification efficiency of candidate reference Gene fluorescent quantitative PCR primers
As shown in Table 1, the length of each candidate reference gene PCR product is within the range of 80-150bp, and the annealing temperature is within the range of 58-62 ℃. The dissolution curve of each candidate reference gene only has obvious single peak, the electrophoresis detection of the PCR product only has single band, and the amplification efficiency of each candidate reference gene primer is 90% -110% (Table 1), which shows that the specificity of each candidate reference gene fluorescent quantitative PCR primer is good. The data prove that the fluorescent quantitative PCR primer of the reference gene designed by the invention meets the requirements, and can continue the subsequent fluorescent quantitative PCR experiment.
Example 2 real-time fluorescent quantitative PCR experiments
The experimental materials are leaves of male and female plants of three trichosanthes varieties (Wan Lou 9, wan Lou 13 and Wan Lou 17) in seedling stage. All samples were immediately snap frozen in liquid nitrogen after sampling and then stored in a-80 ℃ freezer.
RNA of each sample was extracted using Takara miniBEST Plant RNA Extraction kit kit, and reverse transcription was performed using Takara PrimerScript reverse transcriptase kit. And (3) carrying out a real-time fluorescent quantitative PCR experiment by using 10-fold dilutions of cDNA obtained by reverse transcription as templates and using fluorescent quantitative PCR specific primers of each candidate internal reference gene.
Amplifying cDNA of three varieties of trichosanthes kirilowii Maxim (female and male plants of Anhui Lou No. 9, anhui Lou No.13 and Anhui Lou No. 17) seedling stage male and female plant leaf samples by using fluorescent quantitative PCR primers of each candidate reference gene: the fluorescent quantitative PCR instrument is a Applied Biosystems QuantStudio 5 real-time fluorescent quantitative PCR instrument, the fluorescent quantitative PCR adopts a Takara TB Green kit, and the reaction system is as follows: 2X TB Green premix Ex Taq. Mu.l; 2 μl of cDNA; 10. Mu.M upstream primer 0.4. Mu.l; 10. Mu.M downstream primer 0.4. Mu.l; ROX reference dye II 0.4 μl and ddH 2 O6.8. Mu.l. The reaction procedure: pre-denaturation at 95 ℃ for 10min, melting at 95 ℃ for 15s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 30s, 40 cycles of reaction: the temperature is 60-95 ℃, the temperature is gradually increased to 95 ℃ for 30 seconds, fluorescent signals are collected every 0.5 ℃, and the Ct values of all reference genes in different samples are obtained through real-time fluorescent quantitative PCR.
FIG. 1 shows the geometric mean (Cq value) of Ct values of all detection samples of 12 candidate reference genes, and shows that the expression amounts of the 12 candidate reference genes are different, the UBQ10 gene expression abundance is highest (Cq mean is minimum), and the EF1 alpha 3 gene expression abundance is lowest (Cq mean is maximum). The variation amplitude of the expression quantity of 12 candidate reference genes in different samples is also different, the Cq value of the EF1 alpha 1 gene is between 20.20 and 24.40, and the variation amplitude is the largest; whereas the Cq value of the 18SrRNA gene is between 23.00 and 25.29, the variation range is minimal.
EXAMPLE 3 software evaluation of reference genes under all samples
GeNorm software
In the invention, the gemm software is gemm_3.4, and the steps and standards are as follows:
first, the Δct value is calculated: finding out the minimum Ct value of each candidate reference gene in all samples, and subtracting the minimum Ct value from the Ct value of each sample to obtain the delta Ct value. Then using the function in Excel to calculate 2 of corresponding gene of corresponding sample -ΔCt The value, which is the relative quantitative data for each candidate reference gene, is also the data used in the analysis by the gemm software. In the invention, gemum firstly realizes the expression stability sequencing of 12 candidate reference genes by calculating the expression stability average value (M) (figure 2), and the result shows that under all samples, the two most stable reference genes are EF1 alpha 1 and EF1 alpha 2, the least stable reference genes are action 7, and the stability sequencing of each candidate reference gene is as follows:
EF1 alpha 1> ef1 alpha 2> ef1 alpha 3>Actin>GAPDH>Actin 97> alpha TUB >18SrRNA > beta TUB > alpha TUB3> UBQ10> action 7; the paired coefficient of variation V (FIG. 3) was then calculated using gemm, and the results were all below 0.15. Therefore, in the trichosanthes kirilowii gene expression study, 2 reference genes completely meet the experimental requirements.
NormFinder software
In the present invention, the NormFinder software steps and criteria are as follows:
the data required for the NormFinder software is also 2 -ΔCt Values. The results of the stability of the reference genes from the normFinder analysis showed that the most stable reference gene was action and the least stable was action 7 for all samples, the stability ranking of each candidate reference gene was as follows (FIG. 4):
Actin>EF1α2>Actin97>GAPDH>18SrRNA>αTUB>EF1α1>EF1α3>αTUB3>UBQ10>βTUB>Actin7。
bestkeeper software
In the invention, the steps and standards of the BestKeeper software are as follows:
the Cq value of each gene of each sample is directly input into an Excel table and is imported into BestKeeper software for internal reference stability analysis. According to the bestdeeper results, the stability of each candidate reference gene was ranked as follows (fig. 5): 18SrRNA > GAPDH > alpha TUB > UBQ10> action 7> action 97> EF1 alpha 2> EF1 alpha 1> EF1 alpha 3> beta TUB > alpha TUB3.
Comprehensive analysis of three software results
From the results of the three software, it can be seen that the most stable reference genes obtained by each software are different. Thus, the result of the 3 pieces of software needs to be integrated. The specific comprehensive sequencing method comprises the following steps: in the stability evaluation results of the candidate reference genes by the GeNorm software and the NormFinder software, the stability index M value or the SV value of the 12 candidate reference genes is ordered from small to large, the candidate reference gene with the minimum M value or the SV value is marked as 12 points, the candidate reference gene with the second minimum M value or the SV value is marked as 11 points, and the candidate reference gene with the maximum M value or the SV value is marked as 1 point by analogy.
In the stability evaluation result of the candidate reference genes by the BestKeeper software, the standard deviation (SD value) of the 12 candidate reference genes is ranked from small to large, the candidate reference gene with the smallest SD value is marked as 12 points, the candidate reference gene with the second small SD value is marked as 11 points, and the candidate reference gene with the largest SD value is marked as 1 point by analogy.
The scores of the candidate reference genes obtained by the three software are added, and the optimal reference gene is determined according to the sum of the scores of the candidate reference genes (table 2). It can be seen that of the 12 candidate reference genes, the two reference genes, action and GAPDH, were most stable in all samples tested. Therefore, the 2 genes of the action and the GAPDH can be used as reference genes for the research of the gene expression of the male and female plants of the trichosanthes kirilowii in the seedling stage.
Table 2 is ranking of stability of candidate internal reference genes analyzed by GeNorm, normFinder, bestKeeper software
The above description is only a preferred embodiment of the present invention, and is not intended to limit the invention in any way, and any person skilled in the art may make modifications or alterations to the disclosed technical content to the equivalent embodiments. However, any simple modification, equivalent variation and variation of the above embodiments according to the technical substance of the present invention still fall within the protection scope of the technical solution of the present invention.
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<110> academy of agricultural sciences in Jiangsu province
<120> internal reference gene for trichosanthes kirilowii Maxim seedling stage male and female plant gene expression research and application thereof
<160> 36
<170> SIPOSequenceListing 1.0
<210> 1
<211> 474
<212> DNA
<213> trichosanthes kirilowii (Trichosanthes kirilowii maxim.)
<400> 1
agaggtattc ttaccttgaa atatcccatt gagcatggta ttgtgagcaa ctgggatgat 60
atggaaaaga tttggcatca cacattctac aatgagcttc gtgtcgcacc tgaagaacac 120
ccagtgcttc ttactgaagc tcctctcaat cctaaagcca acagagaaaa gatgacacaa 180
atcatgtttg agacttttaa tgtacctgcc atgtatgttg ccatccaggc tgttctatct 240
ctgtacgcca gcggtcgtac aacaggtatt gtgcttgact ctggtgatgg tgttagtcac 300
actgtgccaa tctatgaggg ttatgctcta ccccatgcta tcctccgtct ggatcttgct 360
ggccgtgatt taactgatgc tttgatgaag attctcactg agagaggata catgttcaca 420
accactgccg aacgggaaat tgtccgtgac atgaaagaga agcttgctta tgtt 474
<210> 2
<211> 517
<212> DNA
<213> trichosanthes kirilowii (Trichosanthes kirilowii maxim.)
<400> 2
cacagacaaa gacaaggctg ctgcccattt gaagggtggt gcaaagaagg ttatcatctc 60
agctccaagc aaggatgctc ccatgtttgt tgttggagtc aatgagaagg aatacacatc 120
agatcttaat gttatatcta atgctagctg cactaccaac tgtcttgctc ctcttgcaaa 180
ggtcattaat gatagatttg gcattgttga gggcctgatg accactgttc actcaatgac 240
tgccacccaa aagactgttg atggaccatc tagcaaagac tggagaggtg gaagagctgc 300
ttcattcaac atcatcccta gcagtactgg agctgctaag gctgtcggca aagtgctgcc 360
cgcattgaat ggaaaattga ccggaatggc tttccgtgtt cctactgttg atgtctccgt 420
agttgacctt acagtgaggt tggagaaggc agcatcttat gaagatatta aagctgccat 480
caaggaggag tctgaaggaa agttgaaagg aattttg 517
<210> 3
<211> 501
<212> DNA
<213> trichosanthes kirilowii (Trichosanthes kirilowii maxim.)
<400> 3
ggatttggaa tttgtttttc ctattgcgta gattttcttc cttgcttgaa tatgacatcg 60
agaccagagc tgcaagctcc tccagaaata ttctacaacg atgctgaggc tagaaaatat 120
acttcatcct ctcgtatcat tgaaattcag gccaagatct cagaaagggc attggagctt 180
cttgctttgc ctgatgacgg agtacccaga ttgctccttg atattggctg tggatcgggt 240
cttagtgggg aaacattgtc tgagcacgga caccaatgga ttggtttgga tatctctcaa 300
tcaatgctta atgttgcatt ggagcgggag actgatggtg accttttact tggtgacatg 360
ggtcagggct tgggaattcg tcctggagtt attgatgggg ctattagcat ctctgcagtt 420
cagtggttgt gcaacgctga caagtcctca cataatccgc gactaagatt gaagtacgtc 480
ttacaaactt ctcaccatct c 501
<210> 4
<211> 843
<212> DNA
<213> trichosanthes kirilowii (Trichosanthes kirilowii maxim.)
<400> 4
ggtggtatgc aaattttcgt gaagacactg accggcaaga ccattaccct tgaggtcgag 60
tcgtctgata ccatcgacaa cgtcaaggcg aagatccagg acaaggaagg aattcccccg 120
gaccagcaac gtctcatctt cgccggcaag caacttgagg atggccgtac cttggccgac 180
tacaacatcc agaaggagtc gaccctccac ctggtcctcc gtcttcgtgg tggtatgcaa 240
attttcgtga agacactgac cggcaagacc attacccttg aggtcgagtc gtctgatacc 300
atcgacaacg tcaaggcgaa gatccaggac aaggaaggaa ttcccccgga ccagcagcgt 360
cttattttcg ccggaaagca actcgaggat ggccgtacct tggccgacta caacatccag 420
aaggagtcga ccctccacct ggtcctccgt cttcgcggtg gtatgcaaat tttcgtgaag 480
acactgaccg gcaagaccat tacccttgag gtcgagtcgt ctgataccat cgacaacgtc 540
aaggcgaaga tccaggacaa ggaaggaatt cccccggacc agcagcgtct tattttcgcc 600
ggaaagcaac tcgaggacgg ccgtaccttg gccgactaca acatccagaa ggagtcgacc 660
ctccacctgg tcctccgtct tcgcggtggt atgcaaattt tcgtgaagac actgaccggc 720
aagaccatta cccttgaggt cgagtcgtct gataccatcg acaacgtcaa ggcgaagatc 780
caggacaagg aaggaattcc cccggaccag cagcgtctta ttttcgccgg aaagcaactc 840
gag 843
<210> 5
<211> 882
<212> DNA
<213> trichosanthes kirilowii (Trichosanthes kirilowii maxim.)
<400> 5
atggatgcca ccacccccaa atactcaaag gcaaggtatg atgaaatcgt gaaggaagtc 60
tcctcttatc tcaagaaggt cggatacaac ccagaaaaaa tccccttcgt tcccatctct 120
ggttttgagg gtgacaacat gattgagagg tccaccaacc tcgactggta caagggacca 180
accctccttg aggctcttga cttgatccag gagcccaaga ggccctcaga caagcccctc 240
cgtctcccac ttcaggatgt ttacaagatc ggtggtattg gaactgtccc tgtcggtcgt 300
gttgaaactg gtgtcctcaa gcctggtatg gttgtctcct tcggaccaac tggattgacc 360
actgaagtta agtctgttga gatgcaccat gaagctctcc aagaagctct ccctggtgac 420
aacgtggggt tcaatgtgaa gaatgttgcc gtgaaggatc tcaagcgtgg cttcgttgcc 480
tcaaattcca aggatgatcc cgccaaggaa gctgccaatt tcacctctca agtcatcatc 540
atgaaccatc ctggtcaaat tggaaatggt tatgccccag ttcttgactg ccatacctcg 600
catattgccg tcaagttttc tgaaatcttg accaagattg acaggcgatc tgggaaagaa 660
ctcgagaagg agcctaagtt cttgaaaaac ggcgacgctg gatttgttaa gatgatcccc 720
accaagccca tggtggtgaa aaacttcccc gagtaccctc cgcttgggcg attttccgtg 780
agggacatgc gtcatactgt ggccatcggt gtcatcaaga gcgagaagaa ggatccaagc 840
ggccccaagg ccacaaaatc tgctgctaag aaatctggta ag 882
<210> 6
<211> 366
<212> DNA
<213> trichosanthes kirilowii (Trichosanthes kirilowii maxim.)
<400> 6
gctcttgacc agatcaacga gcccaagagg ccatcagaca agcccctacg tcttcccctt 60
caggacgtgt acaagattgg cggcattgga actgtgccag tgggccgtgt tgagactgga 120
gtcatcaagc caggcatggt tgtcaccttt ggtcctactg gcctgaccac tgaggtgaag 180
tccgttgaga tgcaccatga gtctctcctg gaggcgcttc ctggtgacaa cgtcggcttc 240
aacgtcaaga acgtggctgt caaggatctc aagcgtgggt acgtcgcttc caactccaag 300
gacgaccctg ccaaggaggc tgccaacttc accgcccagg tcatcatcat gaaccaccct 360
ggtcag 366
<210> 7
<211> 414
<212> DNA
<213> trichosanthes kirilowii (Trichosanthes kirilowii maxim.)
<400> 7
cgtgggtacg tggcctctaa ctccaaggat gaccctgcca aggaggctgc cagcttcacc 60
tcccaggtga tcatcatgaa ccaccctggg cagatcggca atggctacgc cccagtgctg 120
gactgccaca cctcacacat tgctgtcaag tttgctgagc tggtgaccaa gattgacagg 180
cgatctggta aggagattga ggctgcgccc aagttcctga agaacggtga cgctggtatt 240
gtgaagatga ttcccaccaa gcccatggtt gtggagacct tcgccaccta ccctcctctt 300
ggccgcttcg ctgtgcgtga catgagacaa acggttgctg ttggtgtcat caagggcgtg 360
gagaagaagg accccaccgg cgccaaggtt accaaggctg ccatcaagaa gaaa 414
<210> 8
<211> 771
<212> DNA
<213> trichosanthes kirilowii (Trichosanthes kirilowii maxim.)
<400> 8
gaagtactgg atgctcttca ggagcaacac gaagctcgtt gtagaaggtg tgatgccaaa 60
tcttttccat gtcatcccag ttgcttacaa tgccatgctc aatggggtat tcttacattg 120
aaatacccaa ttgagcatgg cattgtaagc aactgggatg acatggaaaa gatttggcat 180
cacaccttct acaacgagct tcgtgttgct cctgaagagc atccagtact tcttactgaa 240
gcaccactca acccaaaggc aaacagggag aagatgactc agatcatgtt tgaaaccttt 300
gacgtccctg ctatgtatgt tgcaatccag gccgttctat ctctctatgc cagtggtcgg 360
acaacaggta ttgtgttgga ttctggtgat ggagtgagcc acacagtgcc aatatacgag 420
gggttttcgc tccctcatgc aatcctacgt cttgaccttg ccggtcgtga tctaactgat 480
gctttgatga aaattcttac cgaaagaggt tacatgttca cgacaacagc cgagcgggaa 540
attgtccgtg atgtgaagga gaagcttgca tatgtggccc ttgattatga gcaagaactg 600
gagacagcca ggactagttc ggccattgag aaaagctacg aactaccaga tgggcaggtg 660
attacgattg gagctgagag attccgttgc ccagaagtac ttttccagcc atctctgatt 720
ggcatggaat ctcctggaat ccatgaaacc acatacaact ccatcatgaa g 771
<210> 9
<211> 1242
<212> DNA
<213> trichosanthes kirilowii (Trichosanthes kirilowii maxim.)
<400> 9
gccgagaaaa aggacatagt tggtttgcag gaacaggttt ttgtaagaaa agtgattgag 60
cttcacgaca agtacttggc ttatgtgaat gattgtttcc aaaaccacac actttttcac 120
aaggctctca aggaagcttt tgaagtcttt tgcaataagg gtgttgctgg aagttctagt 180
gccgaattac ttgccacctt ttgtgataac attcttaaga aaggaggaag cgagaagttg 240
agtgatgaag caatagagga gacacttgag aaggtagtga agttgcttgc ttatatctgc 300
gacaaagatt tgtttgctga attttataga aaaaaacttg cccgaaggct tctctttgac 360
aagagtgcca atgatgatca cgagagaagt atattgacca aattgaagca acaatgtggt 420
ggtcagttca cctctaagat ggaggggatg gtcactgatt tgacattggc aagggagaac 480
caaactagtt tcgaggagta tctgagcaat aatccacaag ctagtccggg aatcgactta 540
actgttactg ttttgaccac tggtttttgg ccaagctaca agtcttttga cctcaatctg 600
ccggcagaga tggtaaagtg tgttgaagtt ttcagagaat tttatcaaac aaaaacaaag 660
cacagaaaac tcacatggat ttactcattg ggtacatgta acatcagtgg aaaatttgaa 720
ccgaaaacga tggagctgat tgtgacaacc tatcaggctt ctgcactgct gctatttaat 780
tcctcagata gattaagtta ctcagaaatc atgacacaat taaacttgag tgatgatgat 840
gttgttagac tactccactc attgtcatgt gccaagtaca aaattcttaa caaggagcca 900
aatacgaaga ccatctctcc caatgatcac ttcgagttca atgcgaaatt caccgacaaa 960
atgaggagaa taaagatccc tcttccacct gtggatgaga aaaagaaagt tattgaagat 1020
gtcgacaagg atcgaagata tgctattgat gcctcgatcg tgcgtatcat gaagagtcgt 1080
aaagttttgg gtcaccagca gttagtgatg gagtgcgtcg agcaattggg tcgtatgttc 1140
aagcccgact tcaaggcgat taagaagaga atcgaagatc tgatcacccg ggactatcta 1200
gagagagaca aagacaaccc ccacttgttt aggtacttgg ct 1242
<210> 10
<211> 432
<212> DNA
<213> trichosanthes kirilowii (Trichosanthes kirilowii maxim.)
<400> 10
aaacttctgt tttctttttt cagtgatacc tccgaaggtg ttgcacacga cgcttttaac 60
acattcttta gcgagaccgg ttcaggcaag catgtgcctc gggctatatt tgttgatttg 120
gaaccaactg tgattgatga agttagaacc ggagcctacc gacaactctt tcatccggag 180
caattgatat ctggaaaaga agatgctgcc aataattttg ctcgaggaca ttacacagtt 240
ggaaaggaaa ttgtagatct ctgtcttgac cgagtcagaa aattggctga taactgcact 300
gggttacaag gatttctagt ctttaatgct gttggtggtg gcactggttc tggtctgggt 360
tcgttgttgt tagagcgctt gtcggtggat tatgggaaaa aatcaaagct tggtttcacc 420
agctacccgt cc 432
<210> 11
<211> 1497
<212> DNA
<213> trichosanthes kirilowii (Trichosanthes kirilowii maxim.)
<400> 11
ttgattgata tttattcaat caattatctc tattttccct ctgcgagtcg gcaatcacaa 60
tcagcggcat cttccccttt ctcccatttc tctctctgtt ttctctcgat ccttcgcccc 120
ttttcgagag agatcggatt cggattcggg atgagggaaa tcatcagcct tcacattggt 180
caagctggga ttcaggtcgg caattcttgc tgggagcttt actgcctcga acatggcatc 240
cagcctgatg gtttgatgcc cagtgacacc tcggttggtg ttgcacacga tgccttcaat 300
acgtttttca gcgaaactgg ttcgggcaag catgttccta gagcaatatt tgtggatctc 360
gaaccaactg ttatcgatga agtgaggagt gggacttatc gacagctttt ccatccggaa 420
cagctcatat ctggtaaaga agatgctgca aataattttg ccagagggca ttatacagtg 480
ggtagggaaa tagttgatct atgccttgac cgagtgcgaa aactagccga caattgtact 540
ggtttgcaag ggttcttagt atttaatgct gttggtggtg gcactggatc tggtttgggg 600
tcattacttt tggaacgctt gtctgtggat tatggaaaga agtcaaagct tggattttcc 660
atatatcctt ctccacaggt atctactgct gtcgtggaac cgtacaacag tgtgctttcc 720
actcattctc tgctagaaca cacagatgtc gctgtgcttc tggacaacga agccatttac 780
gatatctgca ggagatcatt ggatattgag aggccaactt acaccaactt gaaccgcttg 840
atatcccaag ttatctcttc tttaaccact tccttgaggt ttgatggtgc tatcaacgtg 900
gatataaccg agtttcaaac taaccttgta ccttatccac gtattcattt tatgctctct 960
tcttatgctc ctgtgatctc ttctgcaaaa gcttaccatg agcagctatc tgtccccgaa 1020
atcaccaatg ccgtatttga tccctcaagc atgatggtca aatgcgaccc aaggcatgga 1080
aagtacatgg catgctgttt gatgtataga ggagatgttg tacccaagga tgtgaatgct 1140
gctgttgcta ccatcaaaac gaagcgaaca gttcaatttg ttgactggtg tcctactggc 1200
ttcaaatgtg gtattaacta tcaaccccca agcgttgttc ctgggggcga tcttgccaag 1260
gtgcagcgag ccgtttgtat gataagcaac agcacagccg tggccgaggt cttttcaaga 1320
atcgaccaca aatttgacct catgtatgct aagcgggcat tcgtccattg gtacgtgggt 1380
gagggcatgg aggaaggtga attctctgaa gcccgtgagg atcttgctgc tctagaaaag 1440
gactatgagg aagtcggtgc cgaagatgtg gatgaagagg aggaagggga agattat 1497
<210> 12
<211> 231
<212> DNA
<213> trichosanthes kirilowii (Trichosanthes kirilowii maxim.)
<400> 12
cgtggatctc agcaatacag agctcttaca gtacccgagc tcactcagca aatgtgggat 60
gctaagaaca tgatgtgtgc tgctgatcct cgacatggtc gctatcttac tgcctctgct 120
atgtttaggg gtaagatgag caccaaggaa gttgatgaac agatgatcaa tgtacagaac 180
aagaactctt catactttgt tgagtggatc ccaaacaatg tgaaatctac g 231
<210> 13
<211> 21
<212> DNA
<213> Artificial sequence (Trichosanthes kirilowii maxim.)
<400> 13
tcaatcctaa agccaacaga g 21
<210> 14
<211> 20
<212> DNA
<213> Artificial sequence (Trichosanthes kirilowii maxim.)
<400> 14
accatcacca gagtcaagca 20
<210> 15
<211> 22
<212> DNA
<213> Artificial sequence (Trichosanthes kirilowii maxim.)
<400> 15
tgttcactca atgactgcca cc 22
<210> 16
<211> 20
<212> DNA
<213> Artificial sequence (Trichosanthes kirilowii maxim.)
<400> 16
gcactttgcc gacagcctta 20
<210> 17
<211> 20
<212> DNA
<213> Artificial sequence (Trichosanthes kirilowii maxim.)
<400> 17
ttctacaacg atgctgaggc 20
<210> 18
<211> 20
<212> DNA
<213> Artificial sequence (Trichosanthes kirilowii maxim.)
<400> 18
atcaaggagc aatctgggta 20
<210> 19
<211> 19
<212> DNA
<213> Artificial sequence (Trichosanthes kirilowii maxim.)
<400> 19
ccggcaagca acttgagga 19
<210> 20
<211> 22
<212> DNA
<213> Artificial sequence (Trichosanthes kirilowii maxim.)
<400> 20
taccaccacg aagacggagg ac 22
<210> 21
<211> 20
<212> DNA
<213> Artificial sequence (Trichosanthes kirilowii maxim.)
<400> 21
cggcgacgct ggatttgtta 20
<210> 22
<211> 20
<212> DNA
<213> Artificial sequence (Trichosanthes kirilowii maxim.)
<400> 22
tatgacgcat gtccctcacg 20
<210> 23
<211> 20
<212> DNA
<213> Artificial sequence (Trichosanthes kirilowii maxim.)
<400> 23
attggcggca ttggaactgt 20
<210> 24
<211> 20
<212> DNA
<213> Artificial sequence (Trichosanthes kirilowii maxim.)
<400> 24
tcatggtgca tctcaacgga 20
<210> 25
<211> 20
<212> DNA
<213> Artificial sequence (Trichosanthes kirilowii maxim.)
<400> 25
cctgaagaac ggtgacgctg 20
<210> 26
<211> 20
<212> DNA
<213> Artificial sequence (Trichosanthes kirilowii maxim.)
<400> 26
tctccacgcc cttgatgaca 20
<210> 27
<211> 20
<212> DNA
<213> Artificial sequence (Trichosanthes kirilowii maxim.)
<400> 27
aaggcaaaca gggagaagat 20
<210> 28
<211> 20
<212> DNA
<213> Artificial sequence (Trichosanthes kirilowii maxim.)
<400> 28
gtccgaccac tggcatagag 20
<210> 29
<211> 20
<212> DNA
<213> Artificial sequence (Trichosanthes kirilowii maxim.)
<400> 29
ccgaattact tgccaccttt 20
<210> 30
<211> 23
<212> DNA
<213> Artificial sequence (Trichosanthes kirilowii maxim.)
<400> 30
tgtctcctct attgcttcat cac 23
<210> 31
<211> 20
<212> DNA
<213> Artificial sequence (Trichosanthes kirilowii maxim.)
<400> 31
tctggaaaag aagatgctgc 20
<210> 32
<211> 20
<212> DNA
<213> Artificial sequence (Trichosanthes kirilowii maxim.)
<400> 32
agccaatttt ctgactcggt 20
<210> 33
<211> 20
<212> DNA
<213> Artificial sequence (Trichosanthes kirilowii maxim.)
<400> 33
agtgcgaaaa ctagccgaca 20
<210> 34
<211> 20
<212> DNA
<213> Artificial sequence (Trichosanthes kirilowii maxim.)
<400> 34
ccaaaagtaa tgaccccaaa 20
<210> 35
<211> 21
<212> DNA
<213> Artificial sequence (Trichosanthes kirilowii maxim.)
<400> 35
caaatgtggg atgctaagaa c 21
<210> 36
<211> 21
<212> DNA
<213> Artificial sequence (Trichosanthes kirilowii maxim.)
<400> 36
cctaaacata gcagaggcag t 21
Claims (2)
1. The application of an internal reference gene for the analysis of the gene expression of male and female plants in the seedling stage of trichosanthes kirilowii in the analysis of the gene expression of trichosanthes kirilowii is characterized in that the internal reference gene isActinGenes and/orGAPDHA gene; the saidActinThe nucleic acid sequence of the gene is shown as SEQ ID NO. 1; the saidGAPDHThe nucleic acid sequence of the gene is shown as SEQ ID NO. 2.
2.ActinGenes and/orGAPDHGene table for analyzing male and female plants in trichosanthes seedling stageThe application of the differential fluorescent quantitative PCR reference gene is characterized in thatActinThe nucleic acid sequence of the gene is shown as SEQ ID NO. 1; the saidGAPDHThe nucleic acid sequence of the gene is shown as SEQ ID NO. 2.
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| CN202011222286.XA CN112280887B (en) | 2020-11-05 | 2020-11-05 | Internal reference gene for trichosanthes kirilowii Maxim seedling stage male and female plant gene expression research and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
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| CN202011222286.XA CN112280887B (en) | 2020-11-05 | 2020-11-05 | Internal reference gene for trichosanthes kirilowii Maxim seedling stage male and female plant gene expression research and application thereof |
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