CN112458099B - Reference gene of chive and screening method and application thereof - Google Patents

Reference gene of chive and screening method and application thereof Download PDF

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CN112458099B
CN112458099B CN202011448711.7A CN202011448711A CN112458099B CN 112458099 B CN112458099 B CN 112458099B CN 202011448711 A CN202011448711 A CN 202011448711A CN 112458099 B CN112458099 B CN 112458099B
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郭元元
陈振东
张力
陈琴
蒋月喜
李洋
宋焕忠
车江旅
秦龙妹
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Abstract

The invention provides a reference gene of chive and a screening method and application thereof, belonging to the field of molecular biology. Selecting 13 candidate reference genes Ac18S, Af18s, Af28s, AcActin, AfActin, AfTUA, AfTUB, AsSAND, AfMYH, AfGAPDH, AfUBQ, AfEF1a and AfCYC, detecting the expression level of the candidate reference genes in high-temperature stress treatment by using a real-time fluorescent quantitative RT-qPCR technology, evaluating the stability of the 13 reference genes by combining GeNorm, NormFinder and BestKeeper software, and finally carrying out comprehensive analysis to obtain the most suitable reference gene of the chive under the high-temperature stress as AfActin and provide a reference for the scientific research related to the expression of the subsequent chive genes.

Description

Reference gene of chive and screening method and application thereof
Technical Field
The invention relates to the field of molecular biology, in particular to a reference gene of chive and a screening method and application thereof.
Background
Chives, also known as: scallion, chive, scallion and scallion. Latin name: allium fistulosum l.var.caespitosum makino, liliaceae, Allium, bulb outgrowth, oblong ovoid, narrow ovoid or ovoid cylinder; the bulb has reddish brown, purple red to yellow white outer skin, membranous or thin and leathery texture, and does not break. The leaves are hollow cylinders, tapering towards the top, dark green, and often slightly whitish. Small plant, extremely fine leaf, tender texture, fragrant taste and slight spicy flavor, and is mainly used for seasoning and removing fishy smell. The cultivation is originally produced in the west of Asia, is widely carried out in the south of China, and is carried out in Europe and some places in Asia. The chive is an important spicy seasoning which is necessary in daily life, and is an important vegetable variety which participates in precise poverty alleviation, north transportation of south vegetables and export dehydration.
The chives are cool and pleasant in nature. Under the annual production condition of the chives in southern regions, high temperature is the biggest industrial bottleneck in the summer and autumn production of the chives. Under the high-temperature environment, phenomena of green receding of scallion tubes, dry and yellow scallion tips, rotten scallion stalk, tissue aging, fiber increase, frequent disease occurrence, even absolute harvest and the like are easily caused, and the yield, the quality and the benefit of the chives are seriously influenced. The method has very important theoretical and application values for developing the heat-resisting molecular mechanism research of the chives aiming at the industrial demand.
The function of a gene is performed as a process from DNA to mRNA to protein, and the level of gene expression is generally measured by the amount of mRNA transcribed from the gene. Therefore, accurate assessment of the expression level of the target gene is of great importance for the study of biological functions. The reference gene is an endogenous reference gene, and ideally, the reference gene can be stably expressed in all tissues and cells under different environments and conditions and is not influenced by any internal and external factors. In the actual research process, the expression of the common reference genes is not absolutely constant in different tissues and organs, under different treatment conditions, in different types of cells and at different development stages, so that when the fluorescent quantitative PCR is used for researching the expression of the target genes, the first step is to screen the appropriate and stable reference genes according to different test conditions and test samples. Common reference genes include beta-actin, EF-1 alpha, 18S rRNA, 28S rRNA and the like. So far, no report about related research of internal reference genes special for chives under high-temperature stress exists. The molecular breeding research process of the chives is severely restricted. Therefore, it is necessary to dig a highly reliable reference gene in chives.
Disclosure of Invention
The invention aims to provide a reference gene for chive aiming at the problems, and provides a reference for subsequent scientific research related to chive gene expression.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
an internal reference gene of chive is AfActin, and the nucleotide sequence of the internal reference gene AfActin is shown as SEQ ID NO. 1.
Also provided is a PCR primer for amplifying the reference gene of claim 1, the nucleotide sequence of the PCR primer is: the forward sequence is shown as SEQ ID NO. 2; the reverse sequence is shown as SEQ ID NO. 3.
Also provides a screening method of the reference gene of the chive, which is characterized by comprising the following steps:
s1, selecting 10 chive genes and 3 other variety reference genes as reference candidate genes, wherein the 10 chive genes are: af18s, Af28s, AfActin, AfTUA, AfTUB, AfMYH, AfGAPDH, AfUBQ, AfEF1a, and AfCYC; the reference genes in 3 other varieties are: onion Ac18s, onion AcActin, garlic AsSAND; and designing fluorescent quantitative PCR primers.
S2, extracting total RNA of 'cinnamon shallot No. 1' and 'AF 35' under high-temperature stress treatment, carrying out reverse transcription to obtain cDNA serving as a template of fluorescent quantitative PCR, carrying out a fluorescent quantitative PCR experiment by taking the primer designed in the step S1 as a fluorescent quantitative PCR primer, and obtaining fluorescent quantitative PCR data.
S3, performing expression stability analysis on the fluorescence quantitative PCR data obtained in the step S2 by utilizing GeNorm, NormFinder and BestKeeper software, and screening out a stably expressed reference gene.
Preferably, the high temperature stress treatment conditions are: day 6: 00-20: 00: humidity 70% at 38 ℃, and illumination 3; at night 20: 00-6: 00 the next day, 35 ℃, humidity 65% and illumination 0; the control group non-stressed conditions were: day 6: 00-20: 00: at 23 ℃, humidity is 70%, illumination is 3, at night, 20: 00-6: 00 the next day, at 20 ℃, humidity is 65%, illumination is 0. Collecting the chive leaves at 0h, 6h, 12h, 24h, 48h and 72h of treatment respectively.
Preferably, Primer premier3.0 is adopted to design the internal reference candidate gene primers according to the chive transcription group data, the length of the primers is 18-23 bp, the Tm value is 57-62 ℃, the size of the product is 100-200 bp, and the specificity of the primers is detected on NCBI.
Preferably, GeNorm, NormFinder and BestKeeper software are adopted to respectively calculate an expression stability M value, a stability SV value and a coefficient of variation CV/standard deviation SD value of the reference genes under different treatments, and the expression stability of the reference genes is respectively sequenced from small to large according to the M value, the SV value, the CV value and the SD value, wherein the smaller the M value, the SV value, the CV value and the SD value, the more stable the corresponding reference genes are.
And respectively assigning scores to the stability ranking obtained in each software, solving a geometric mean value to obtain a comprehensive index ranking, wherein the smaller the mean value is, the more the ranking is, and the more stable the expression of the internal reference genes is.
Also provides the application of the reference gene in the onion in the analysis of the gene expression of the onion.
Due to the adoption of the technical scheme, the invention has the following beneficial effects:
1. the reference gene of the chive is provided for the first time to use the AfActin gene as the reference gene for the gene expression of the chive aiming at the development of heat-resistant varieties of the chive and the requirements of molecular assisted breeding in the industry, and the current situation that the chive does not have the reference gene is solved.
2. According to the screening method of the reference genes of the chives, Genorm, NormFinder and BestKeeper software analysis stability is carried out on 13 candidate reference genes. And (3) assigning scores to the stability ranking obtained in each software, solving a geometric mean value to obtain a comprehensive index ranking, comprehensively analyzing the results of each software, and successfully screening the applicable stable reference genes.
3. The detection primer pair provided by the invention has specificity, optimizes a PCR amplification program, greatly improves the detection efficiency, shortens the detection time, and improves the reliability of a detection result and the stability, reliability and repeatability of the research on the expression analysis of the chive gene.
Drawings
FIG. 1 is a melting curve of 13 reference candidate genes in example 3 of the present invention;
FIG. 2 is a boxed graph showing the distribution of Ct values of 13 reference candidate genes in example 3 of the present invention;
FIG. 3 is a graph showing the mean expression stability M values of reference genes of 13 reference candidate genes in example 3 of the present invention;
FIG. 4 is a diagram illustrating the coefficient of variation analysis of the optimal number of combinations in example 3.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to the following embodiments and the accompanying drawings. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Example 1
An internal reference gene of chive is AfActin, and the nucleotide sequence of the internal reference gene AfActin is shown as SEQ ID NO. 1.
Example 2
A PCR amplification primer of an internal reference gene AfActin, the nucleotide sequence of the PCR amplification primer is as follows: the forward sequence is shown as SEQ ID NO. 2; the reverse sequence is shown as SEQ ID NO. 3.
Example 3
A method for screening internal reference genes of chives comprises the following steps:
materials and methods
1. Test materials
Heat-resistant Scirpus myrtle No.1 potted plant and heat-labile AF35 potted plant are used as research materials.
And (3) placing the chive pot plant in an illumination incubator. The control group conditions were: day 6: 00-20: 00: at 23 ℃, humidity is 70%, illumination is 3, at 20:00 night, 6:00 days for the next time, at 20 ℃, humidity is 65%, illumination is 0; the conditions of the high-temperature stress treatment group are as follows: day 6: 00-20: 00: at 38 ℃, humidity is 70%, illumination is 3, at 20:00 night, 6:00 days for the next time, at 35 ℃, humidity is 65%, illumination is 0; the chive leaves were collected at 0h, 6h, 12h, 24h, 48h, 72h of treatment, respectively, with 3 biological replicates per stress treatment per 10 plants treated. The material was snap frozen in liquid nitrogen and stored at-80 ℃ for future use.
2. Test method
2.1RNA extraction and cDNA Synthesis
Total RNA of each treatment material was extracted using an RNA extraction kit (kang century), subjected to DNA removal treatment using DNase I (NEB), and cDNA was synthesized using a reverse transcription kit (Thermo) and stored at-20 ℃. All manipulations were performed according to kit instructions.
2.2 primer Synthesis
According to the nucleotide sequences obtained by transcriptome sequencing and documents respectively, an internal reference gene Primer is designed by adopting online Primer design software Primer Premier3.0, the length of the Primer is 18-23 bp, the Tm value is 57-62 ℃, the size of a product is 100-200 bp, and the specificity of the Primer is detected on NCBI (national center of Biotechnology information for Biotechnology information), which is detailed in Table 1.
TABLE 1 reference candidate genes and their primer sequences
Figure BDA0002825859390000041
Figure BDA0002825859390000051
2.3 fluorescent quantitation
And carrying out PCR by using cDNA obtained by high-temperature stress reverse transcription of the chives as a template and using an ABI 7500qPCR instrument.
The Reaction system (10. mu.L) was 1 × Reaction Mix2 × SYBR Green PCR Master Mix (QIAGEN) 5. mu.L, 1 × QN ROX Reference Dye (Applied Biosystems instruments only) 0.05. mu.L, upstream primer 0.7. mu.L, downstream primer 0.7. mu.L, cDNA 1. mu.L, ddH2O 2.55.55. mu.L.
The real-time fluorescent quantitative PCR reaction program is as follows: denaturation at 95 ℃ for 5 s; 5s of extension at 95 ℃, 30s of annealing at 60 ℃ and 40 cycles; melting curve at 70-95 ℃: and 5s at 0.5 ℃, and collecting signals.
3. Data processing and reference gene stability evaluation
The CT value box chart is made by using Microsoft Excel 2016 to process the CT value of the gene, using SPSS19.0 software, using GeNorm and BestKeeper software to respectively calculate the expression stability (M value), the stability value (SV value) and the Coefficient of Variation (CV)/Standard Deviation (SD) value of the reference gene under different processes, and ranking the expression stability of the reference gene according to the M value, the SV value and the CV/SD value from small to large, wherein the smaller the value is, the more stable the corresponding reference gene is. And finally, calculating the comprehensive stability sequencing of each reference gene according to the sequencing results of the geNorm and BestKeeper software.
Second, results and analysis
1. Selection of candidate reference Gene primers
Since there are few known reference genes in chives, when selecting genes, genes with higher transcript numbers and stable expression during heat stress treatment are selected as candidate reference genes according to chive transcriptome and expression profile data, and the sequences of the candidate reference genes are searched for primer synthesis, including 18s ribosome (18s), 28s ribosome (28s), Actin (ACT), beta Tubulin (TUB), alpha Tubulin (TUA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), polyubiquitin enzyme (UBQ), transcription elongation factor 1-alpha (EF1 alpha), and cyclophilin (CYC). Meanwhile, other crops of Allium such as onion (Ac18S, AcActin) and garlic ginseng (AsSAND) are simultaneously selected and compared.
TABLE 2 Gene function List
Figure BDA0002825859390000061
2. Specificity analysis of candidate reference Gene primers
Melting curves obtained by qRT-PCR of all samples of the chives under different heat stress treatment are shown in figure 1, except for two onion internal references of Ac18S and AcActin, the other samples are single melting peaks, and the primer is verified to have strong specificity.
The qRT-PCR amplification efficiency E is between 90% and 100%, the correlation coefficient R2 is more than 0.99, and the result meets the requirement of qRT-PCR on the primer and can be used for the next expression stability analysis.
3. Analysis of candidate reference Gene expression
The Ct value is an important parameter reflecting the abundance of gene expression. The Ct values of the 13 candidate reference genes for each treatment were counted, see FIG. 2. Ct values of the 13 candidate reference genes are all between 8.19 and 31.24, and the average Ct values are as follows from high to low: AfGAPDH > Af18s > AsSAND > AfTUA > Af28s > AfMYH > AfTUB > AfActin > AfEF1a > AfCYC > AfUBQ > AcActin > Ac18S, so AfGAPDH has the largest average expression abundance and the smallest average expression abundance of Ac 18S. In terms of the variation range, the Ct value variation range of Af18s is the smallest, and the Ct value variation range of Ac18s is the largest. And the change range of the Ct value of the internal reference gene (Af series) selected from the chive transcriptome is smaller than that of the internal reference genes of other congeneric crops. Different reference genes have large differences in the transcription levels of different thermotolerant treatments, and therefore reference screening according to different treatments is necessary.
4. Expression stability of candidate reference genes under temperature stress
4.1 GeNorm software analysis
GeNorm software uses the value of M to evaluate the stability of each expression, the smaller the value of M, the more stable, and the higher the value of M, the less stable. Analysis results of geNorm software show that 13 candidate internal reference genes have different M values in different heat stress treatments of the chives as shown in figure 3, the rightmost AfEF1a and Af28s have the best stability, and the leftmost AcActin has the worst stability. According to the expression stability, the sequence is as follows: AfEF1a ═ AfF 28s < AfUBQ < AfActin < AfTUB < AsSAND < AfGAPDH < AfMYH < AfCYC < AfTUA < Af18s < Ac18S < AcActin.
In addition, a histogram of the pair variation (Vn/n +1) of the reference gene was generated by using GeNorm software, and the number of the most suitable reference genes was selected. The selection threshold is 0.15, and when Vn/n +1 is more than 0.15, the most suitable number of reference genes is n + 1. GeNorm histogram analysis showed that when the V3/4 value (0.161) was chosen to be greater than the program recommended value of 0.15 (FIG. 4), the most suitable number of genes for the reference set was 4. Therefore, the analysis result of GeNorm software shows that the most stable gene combinations are expressed under different heat stress conditions of the chives, namely 4: AfEF1 α, Af28s, AfUBQ, and AfActin.
4.2 NormFinder analysis
The calculation principle of the program is similar to that of geNorm, and the result of the NormFinder shows that the smaller the SV (stability value) value is, the more stable the expression of the reference gene is, and meanwhile, the program screens out the most suitable reference gene. NormFinder analysis showed (table 3) that the most suitable internal reference gene was AfActin, which is most stably expressed during heat stress (SV ═ 0.021); AfTUA expression is least stable (SV 0.381).
TABLE 3 NormFinder analysis of SV values
Name of Gene SV value Standard error of
Ac18S 0.089 0.027
Af18s 0.354 0.084
Af28s 0.049 0.023
AcActin 0.089 0.027
AfActin 0.021 0.030
AfTUA 0.381 0.091
AfTUB 0.073 0.025
AsSAND 0.130 0.035
AfMYH 0.102 0.029
AfGAPDH 0.140 0.037
AfUBQ 0.055 0.023
AfEF1a 0.045 0.023
AfCYC 0.064 0.024
4.3 BestKeeper software analysis
BestKeeper judges the stability of an internal reference gene mainly by comparing the Standard Deviation (SD) of the CT value of the gene with the Coefficient of Variation (CV). The larger the correlation coefficient (r), the smaller the Standard Deviation (SD) and the Coefficient of Variation (CV), and the better the stability of the reference gene. The default threshold for SD values is 1.0 below which expression is considered stable; when SD > 1, the expression of the reference gene is unstable. According to the BestKeeper analysis table, SD values of Af18s, AsSAND, AfMYH and AfGAPDH are all less than 1.0, and Af18s < AfGAPDH < AfMYH < AsSAND, which indicates that the expression of the Af18s internal reference gene is most stable under the heat stress treatment of chives.
4.4 Integrated analysis
Geometric mean values were calculated for the stability rankings analyzed by Ge Norm and bestkoeper software to obtain a composite index ranking (see table 4), with smaller indices indicating more stable expression of the reference gene.
TABLE 4. analysis by integration
Figure BDA0002825859390000091
As can be seen from the table, the highest gene expression stability of the 13 candidate reference genes after comprehensive sequencing in the heat stress process of the chives is AfActin, so that the AfActin is selected as the reference gene of the chives.
The above description is intended to describe in detail the preferred embodiments of the present invention, but the embodiments are not intended to limit the scope of the claims of the present invention, and all equivalent changes and modifications made within the technical spirit of the present invention should fall within the scope of the claims of the present invention.
Sequence listing
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gatgttcttg atgcaatgcc agagggtatc aagcagaaca aagcaaggac aatcctgcag 2280
catcttagtg aagcatggcg atgttggaaa gcaaatatcc cttggaaggt tcctggatta 2340
ccagtcccta ttgagaacat gattcttaga tatgtgaagt caaaagcaga ttggtggact 2400
aatgttgcac attacaatcg tgagcgtatt agaagaggtg caacagttga taaaactgtt 2460
tgtcgaaaaa atctaggaag attgactcgg ttgtggttga aggctgagca ggaaagacaa 2520
cataactatc tcaaagatgg tccatatgtc actcctgaag aagcggtagc catctataca 2580
actactgtac attggttgga gtcaagaaaa ttctctccta ttcctttccc tcctctttca 2640
tacaagcatg acaccaaatt actcattctt gccttggaga gactaaaaga atcttacagt 2700
gttgcagtga gacttaacca gctacaaaga gaagaactag gtctgatcga gcaagcttac 2760
gacaacccac acgaagcctt atctagaata aaacgtcact tgctcacaca aagagctttc 2820
aaagaagttg gcattgagtt catggatttg tatagctatc tgattccagt atatgaaatt 2880
gagcctcttg agaaaatcac ggatgcatac cttgatcaat atctttggta tgaaggtgat 2940
aaacggcacc ttttcccgaa ttggattaaa ccagctgatt cagaaccacc gcctcttctt 3000
gtatataaat ggtgtcaagg tatcaacaat ttacaaggaa tatgggatac tggtgatggg 3060
cagtgtgttg ttatgcttca aacaaaattc gaaaaattct ttgagaagat agatttgaca 3120
atgttaaaca ggcttctccg tttggttctt gaccacaaca ttgctgacta tgtgactgct 3180
aagaacaatg tggtgttatc ttacaaggat atgagtcata caaactctta tggtctcatt 3240
agaggtctgc aatttgcttc atttgtggtt cagtactttg gccttgtgct tgatctctta 3300
cttcttgggt taactcgagc tagtgaaata gcggggccac ctcagatgcc aaatgagttc 3360
attacctatg ctgacaccaa agttgaaact aggcatccaa tcagattgta ttcacgatac 3420
attgatcgga tccatatact tttccgcttc actcatgatg aagcacgtga tctcattcag 3480
agatatctgt ctgaacatcc tgatccaaat aatgagaata tggttggtta taataacaaa 3540
aagtgttggc ctagggatgc tagaatgagg ctcatgaaac atgatgtcaa ccttggccga 3600
agtgttttct gggatatgaa gaaccgactg cctcgtagta tcaccacttt ggagtgggaa 3660
aacagcttcg tctctgttta cagcaaggat aaccctaact tgttatttag catgtgtggt 3720
ttcgaggttc gtattttacc aaagatacgg atgactcagg aagcatttag caacacaaaa 3780
gatggtgttt ggaatctgca gaacgaacag accaaagaac gaacagcaat cgctttctta 3840
cgtgttgatg atgagcacat gaaggtgttt gagaaccgtg tgaggcagat acttatgtca 3900
tcagggtcaa ctacattcac aaagattgtt aacaaatgga acactgctct cattggcctg 3960
atgacatatt ttcgtgaagc cactgtacat actcaagaat tgttagatct gttagttaaa 4020
tgtgaaaaca agattcaaac tcgtatcaag attggtttga attcaaaaat gcctagcagg 4080
tttcctcctg ttatatttta tacacccaag gaaattggag gtcttggtat gttatcgatg 4140
ggccacattt taattccaca aagcgacctt cgttacagtc agcagacaga tgttggtgta 4200
acacacttca gaagtggtat gagtcatgaa gaagatcaac ttattcctaa tttgtatcgg 4260
tacattcagc catgggagag tgaatttatc gactcgcagc gtgtgtgggc tgaatacgca 4320
ttgaagaggc aggaagctca gtcacaaaat aggcgcctga ctcttgagga tcttgaggat 4380
tcgtgggata gaggtatacc aagaatcaat actttgttcc agaaagaccg tcacacactt 4440
gcttatgaca aggggtggag agtacgtacg gatttcaagc agtaccaagt tctcaaacaa 4500
aatccatttt ggtggaccca ccagcgtcat gatggcaagt tgtggaattt gaacaactac 4560
cgtactgatg tcatccaagc attaggaggg gttgaaggga ttttagagca taccttattc 4620
aagggcacat attttccaac ttgggaaggt ctcttctggg agaaagcttc tggttttgag 4680
gagtcgatga agtataaaaa gttgaccaat gctcaaaggt ccgggttgaa tcaaatccct 4740
aatcgtcgat tcacgctatg gtggtcacct accattaatc gtgcaaatgt ttatgttgga 4800
tttcaagttc aactggatct cacgggaata tttatgcatg gaaagatccc taccctgaaa 4860
atttcattga ttcagatatt ccgtgctcat ctttggcaga aagtacatga gagtgttgtt 4920
atggatcttt gtcaagtgtt ggatcaagag ttagatgcac tggaaataga gacggtgcaa 4980
aaggagacaa ttcatccaag aaagagttac aagatgaaca gttcttgtgc tgatattctt 5040
ttatttgctt ctcatagatg gccaatgtct aaacctagcc tagttgccga gtcaaaagac 5100
gtttttgatc aaaaagctag taataaatat tggatagatg tgcaactcag gtggggagat 5160
tatgattccc atgacataga acgttacaca agggccaaat ttatggatta cacgactgac 5220
aatatgtcta tatatccatc tccaaccggt gtgatgattg gacttgatct cgcatataat 5280
ctgcactctg cttttggtaa ctggttccca gggtcaaaac ctttactggc ccaggcaatg 5340
aacaaaatta tgaagtcaaa tcctgcttta tatgttttgc gagagaggat aaggaaaggt 5400
ttgcagctgt attcatctga gcctacagaa ccatatttgt catctcaaaa ttatggagag 5460
atcttctcta atcaaatcat atggtttgtg gacgacacaa atgtgtatcg tgttactatt 5520
cacaaaacat tcgaaggaaa tctgactact aaaccaatca atggtgccat ctttatattc 5580
aatccaagaa caggacaact ttttctgaag gtcatccaca ctagtgtgtg ggcagggcaa 5640
aagcgtcttg gtcaacttgc gaagtggaaa acagcagaag aggtggctgc tctagttcgc 5700
tctcttcctc ctgaagaaca acccaagcaa attattgtaa cccgaaaggg tatgttggat 5760
ccattagagg tccatttact tgattttcca aacattgtaa tcaaaggtag tgagttgcaa 5820
cttccatttc aagcttgttt aaagatcgag aaatttggcg acttgatctt gaaggcaact 5880
gaaccacaga tggttctttt caatatctac gatgattggt tgaagagtat ctcatcgtat 5940
actgcattct ctcgtctcat tcttatacta cgtgcactgc atgtgaataa cgaaaaggct 6000
aagatgttat tgaagccaga taagacaatc gtcaccgagc cacatcatat atggccttct 6060
cttactgatg atcaatggat gaaagtggaa gttgctctta gagatcttat attatctgat 6120
tatgccaaaa agaacaatgt caatacatca gcattaacac agtctgagat tagagatatt 6180
atacttggtg ctgagataac tccgccctca caacagaggc aacaaattgc tgaaattgag 6240
aaacaggcca aagaagcaag ccagttaact gcagttacaa cgagaacaac caatgtacat 6300
ggcgatgaac tcattgtgac gacaacaagt ccatacgagc aagcatcaca caaaggaaag 6360
gctgattggc gtacaagggc tttctcagca gcaaatctgt atcttcgagt taaccacatt 6420
tatgtcaact cagatgatat caaggaaacc ggttatactt acatcatgcc caaaaacgta 6480
ttgaagaaat tcatatgcgt agcagatcta aaaactcaga tagcaggtta tttgtacggt 6540
ttaagcccac aagacaatcc tcaagtgaag gagattagat gtattgtaat ggtacctcaa 6600
tgggggactc agcaacaagt ccatcttcca tcggctcttc ctgagcatga ctttctaaat 6660
gacttggagc cattgggttg gatgcataca caacaaaatg aacttcctca gctatctcca 6720
caggacgtta caacccatgc tcgaattctc gaaaacaata agaattggga tggggagaaa 6780
tgcatcatct taacctgcag tttcactcca ggatcatgtt ctatgattgc gtataagctc 6840
acaccaaccg gttatgaatg gggtcgagcc aacaaagaca caggcagcaa tccacatggt 6900
taccttccaa ctcactatga gaaggttcaa atgctactaa gtgatcgctt cttaggcttc 6960
tacatggtac cggataatgg gccatggagc tacaatttta tgggagtgaa gcatacagtg 7020
agtatgagat atggtgtaaa gcttggaacc ccaaaagatt tctaccatga ggaacatagg 7080
ccaacacatt ttctagaatt cagcaatttg gaggaaggag ataatgcaga ggtcgatcgg 7140
gaagatacat tctcttagtt ttgtgagaag cgatgttgca tatgttttgc ttatatgtaa 7200
tgcttctgta tactgcaatg tacgagtaac tagattttgt acagaatatt tgttgcaacg 7260
tgtattcaga gttctcatgt attaatatgc gctctagatt gttggatgat gagaggtggt 7320
acaaccttgc taatgttctt tgtttctttt cactacttta ttaactactc tgttgagcaa 7380
caatattgta taagcagaaa tttagcgcgt a 7411
<210> 2
<211> 20
<212> DNA
<213> Forward primer
<400> 2
cctccgaaat acgaaaaggc 20
<210> 3
<211> 20
<212> DNA
<213> Reverse primer
<400> 3
gtggtggcac ttcattacgg 20

Claims (3)

1. The allium fistulosum internal reference gene is characterized in that the internal reference gene is AfActin, and the nucleotide sequence of the internal reference gene AfActin is shown as SEQ ID No. 1.
2. The PCR primer for amplifying the reference gene of claim 1, wherein the nucleotide sequence of the PCR primer is: the forward sequence is shown as SEQ ID NO. 2; the reverse sequence is shown as SEQ ID NO. 3.
3. Use of an Allium fistulosum internal reference gene of claim 1 in an Allium fistulosum gene expression assay.
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