CN109609512A - Application of the iris PP2A gene as reference gene - Google Patents

Application of the iris PP2A gene as reference gene Download PDF

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CN109609512A
CN109609512A CN201811590095.1A CN201811590095A CN109609512A CN 109609512 A CN109609512 A CN 109609512A CN 201811590095 A CN201811590095 A CN 201811590095A CN 109609512 A CN109609512 A CN 109609512A
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leu
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CN109609512B (en
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梁芳
张燕
牛苏燕
郝平安
蒋素华
许申平
袁秀云
马杰
崔波
王墨霏
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Zhengzhou Normal University
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Abstract

The invention belongs to iris gene engineering technology fields, and in particular to an irisPP2AGene is as reference gene application patent application matters.It is describedPP2AGene cDNA sequence length is 2207 bp, ORF, coding phosphor pepsin 2A(PP2A comprising 1764 bp one complete, long).Further using Real-Time Fluorescent Quantitative PCR Technique to thisPP2AExpression of gene under the conditions of iris normal growth temperature and low temperature stress has studies have shown that the gene expression amount is relatively stable and is used as reference gene application potential.Further as reference gene for analyzingPhNAC1When expression conditions, show accurately to analyze result.Good analysis application foundation can be established for iris cold resistance genescreen and cold resistance Screening of Germplasm, therefore there is preferable practical value and research application meaning.

Description

Application of the iris PP2A gene as reference gene
Technical field
The invention belongs to iris gene engineering technology fields, and in particular to an irisPP2AGene is as internal reference base Because applying patent application matters.
Background technique
Protein Phosphatase 2A (protein phosphatase 2A, PP2A) is a kind of main silk ammonia in eucaryote body Acid/threonine protein phosphatases is made of, the master control signal in plant Structural subunits A, catalytic subunit C and adjusting subunit B Transduction and cell metabolism.In animal and yeast, PP2A plays a role in a plurality of signal pathway.However, it is in plant signal Effect in approach is known little about it, and has now been found that it is primarily involved in the signal of the hormones such as abscisic acid, plant production element and ethylene and turns It leads.
Iris (Phalaenopsis It spp. is) general designation of orchid family Phalaenopsis list stem epiphytic orchid, because its flower pattern is excellent Beauty, pattern is rich and varied, and the florescence is longer, becomes the smooth product in annual flower for many years with very high ornamental value.Originate in heat Band subtropical zone, property like warm chilly, and growth thermophilic is 18~28 DEG C, and 10 DEG C of winter or less will stop growing.Therefore, low temperature It is the important environmental factor for influencing iris growth.And the northern area of China winter-spring season temperature is lower, if you need to cultivate iris, then It need to be cultivated in modern greenhouse, and heat higher cost, huge energy consumption, thus cause production cost unprecedented soaring, it constrains The sound development of iris industry.Also therefore, cold resistance resource and gene excavating be iris breeding work important goal it One.
During iris cold resistance genescreen, real-time fluorescence quantitative PCR is one of more common technological means. And the application foundation of Real-Time Fluorescent Quantitative PCR Technique first is that the selection of suitable reference gene.In the prior art, for iris Have been reported that more reference gene.It is known that: reference gene is essentially the base of constant expression under the conditions of some Cause, to be the expression situation of testing goal gene under given conditions as reference；But in real work, at any The reference gene that expression can be stablized under part there's almost no.Also therefore, need to different situations be screened with most suitable reference gene.And For iris cold resistance genescreen, there is not yet preferably can be used as having for reference gene in cold resistance genescreen Close report.
Summary of the invention
Present invention is primarily aimed at provide an irisPP2AGene, studies have shown that the gene is in normal growth temperature Expression can be stablized in different butterfly orchid varieties and different time under the conditions of degree and low temperature stress, therefore have and be used as internal reference base Because of application potential, and it is based on the reference gene, can be the gene studies of iris correlation function and the sieve of low temperature resistant iris resource Certain technical foundation is established in choosing.
Details are as follows for the technical solution that the application is taken.
IrisPP2AGene, the gene express relatively stable one under the conditions of normal growth temperature and low temperature stress It causes, it is describedPP2AGene, cDNA sequence length are 2207 bp, the ORF comprising 1764 bp one complete, long, specifically CDNA base sequence is as shown in SEQ ID NO.1.
For the gene, when being expanded using PCR, it is as follows that primer sequence can refer to design:
PP2A-F:5 '-TTTGAGYGATTTGTGAAGGC-3 ',
PP2A-R:5 '-TGGAAAAAMATAACAGCAGG-3 '.
The irisPP2ACoded by said gene Phospoprotein enzyme 2A(PP2A), which includes 587 amino acid, amino Acid sequence is as shown in SEQ ID NO.2.
The irisPP2AApplication of the gene as reference gene is used for assay correlation function gene expression feelings Condition, specifically for example for the domain functional gene NAC protein gene (PhNAC1) expression analysis.
With irisPP2AReal-time fluorescence quantitative PCR determination method of the gene as reference gene, when detection and analysis, For irisPP2AGene, primer sequence design are as follows:
QPP2A-F:5'-TCTGTTGGCTGTGGAAGGAT-3',
QPP2A-R:5'-AAATCCGACCTGGTAGTTTCTG-3'.
The irisPP2AReal-time fluorescence quantitative PCR determination method of the gene as reference gene, in detection point AnalysisPhNAC1When expression, for the domain NAC protein gene (PhNAC1), primer sequence design is as follows:
QPhNAC1-F:5'-ATCTGAACAAGTGCGAGCCT-3',
QPhNAC1-R:5'-ATCCTTACCAGTTGCCTTCC-3'.
The application excavates in iris low temperature stress transcript profile database arrive for the first timePP2AGene cDNA divides gene Analysis shows that the cDNA sequence includes the ORF, coding phosphor pepsin 2A(PP2A of 1764 bp one complete, long).Further Using Real-Time Fluorescent Quantitative PCR Technique to thisPP2AExpression of gene under the conditions of iris normal growth temperature and low temperature stress Case study shows that the gene expression amount is relatively stable, has and is used as reference gene application potential.Further as internal reference Gene is for analyzingPhNAC1When expression conditions, show accurately to analyze result.
Generally, provided hereinPP2AGene, expresses relatively stable in low temperature stress, utilizes this Characteristic can establish good analysis using base for iris cold resistance genescreen and cold resistance iris Screening of Germplasm Plinth, therefore there is preferable practical value and research application meaning.
Detailed description of the invention
Fig. 1 is irisPP2AThe melting curve of gene real-time fluorescence quantitative PCR；
Fig. 2 is irisPP2AThe standard curve of gene real-time fluorescence quantitative PCR；
Fig. 3 isPP2AFluorescence threshold of the gene in different cultivars iris low temperature stress different time real-time fluorescence quantitative PCR；
(caption: D: butterfly orchid variety " big capsicum "；F: butterfly orchid variety " the Fuller setting sun "；CK1: normal growth temperature；CK2:16 DEG C/11 DEG C of 3 d of processing after；After T1:11 DEG C/6 DEG C are handled 1 day；After T2:11 DEG C/6 DEG C are handled 2 days；T3:11 DEG C/6 DEG C are handled 3 days Afterwards；After T5:11 DEG C/6 DEG C are handled 5 days；After T7:11 DEG C/6 DEG C are handled 7 days；0 h: butterfly orchid variety " big capsicum " normal growth temperature Degree；1 h, 2 h, 4 h, 12 h, 24 h, 48 h: 4 DEG C of 1 h of processing of butterfly orchid variety " big capsicum ", 2 h, 4 h, 12 h, 24 h, 48 h)；
Fig. 4 isPP2AIn the expression electrophoretogram of different cultivars iris low temperature stress different time；
Fig. 5 isPP2AAs reference gene in irisPhNAC1Application in gene quantification expression study；
Fig. 6 isActinAs reference gene in irisPhNAC1Application in gene quantification expression study.
Specific embodiment
Explanation is further explained to the application below with reference to embodiment.Before introducing specific embodiment, with regard to following realities It applies Experimental Background situation in part in example and briefly introduces and be described as follows.
Biomaterial:
Iris " big capsicum " (PhalaenenopsisBig Chili) and " the Fuller setting sun " (Phalaenenopsis Fuller ' s Sunset), both of which is common iris cultivar；Material employed in following embodiments is derived from Zhengzhou teacher In model institute orchid Industry Technology Center intelligence heliogreenhouse；
Relevant primer sequent synthesis and examining order are by the handsome biotech company's offer completion in Shanghai；
Experiment reagent:
Reverse Transcriptase M-MLV reagent (reverse transcription for PCR prepares cDNA), PrimeScript RT Reagent Kit With gDNA Eraser(for real-time fluorescence quantitative PCR reverse transcription prepare cDNA), SYBR Premix Ex TaqTM(being used for real-time fluorescence quantitative PCR) etc., is purchased from TaKaRA company；
RNAprep polysaccharide polyphenol plant total RNA extraction reagent box etc. is purchased from Tiangeng Reagent Company；
Laboratory apparatus:
Micro-spectrophotometer (RNA concentration mensuration use), U.S. Quawell Q5000；
Fluorescence quantitative PCR instrument Mastercycler ep realplex2, German Eppendorf Products；
Plant growth cabinet, U.S. PERCIVAL E-41HO2.
Embodiment 1
The present embodiment is mainly to iris reference genePP2AAcquisition process and sequence structure feature briefly introduce and be described as follows.
(1) low temperature stress processing and the preparation of cDNA template
In previous experiments design, obtained under the conditions of low temperature stress for screening and the relatively stable base of expression under normal growth Cause, inventor have carried out low temperature stress processing, specific processing mode to related iris material are as follows:
In greenhouse choose with growth period two kinds of biennial iris " big capsicum " (hereafter with D replacing representation) and " the Fuller setting sun " (hereafter with F replacing representation), 26 DEG C/22 DEG C 15 d(of preculture trainings of normal temperature in plant growth cabinet The condition of supporting: 60 μm of olm of illumination-2·s-1, 12 h/12 h of Light To Dark Ratio, relative humidity 70 ~ 90%), then low temperature stress is handled Different time, each 3 plants of processing, if biology repeats three times；
Low temperature stress processing method: simulation nature gradually falling temperature method is used, that is, 20 DEG C/16 DEG C of first diurnal temperature processing 3 D, then 16 DEG C/11 DEG C processing 3 d, last 11 DEG C/6 DEG C 7 d of processing；The variation of temperature each time be all made of per hour increase or Reduce by 1 DEG C of mode.
It is control 1(CK1 with the blade of normal growth temperature) it samples, it is taken after 16 DEG C/11 DEG C 3 d of processing for control 2(CK2) Sample, in 11 DEG C/6 DEG C processing after 1 d(T1), 2 d(T2), 3 d(T3), 5 d(T5) and 7 d(T7) sample.
Separately take one group of butterfly orchid variety " big capsicum ", 4 DEG C of low-temperature treatment different times: 1 h, 2 h, 4 h, 12 h, 24 h, 48 h sampling is control (0 h) sampling with the blade of normal growth temperature.
Samples taken is said with reference to kit specification using RNAprep polysaccharide polyphenol plant total RNA extraction reagent box Bright book, extract respectively different iris sample blades 21 total serum IgEs (DCK1, DCK2, DT1, DT2, DT3, DT5, DT7, FCK1, FCK2, FT1, FT2, FT3, FT5, FT7,0 h, 1 h, 2 h, 4 h, 12 h, 24 h, 48 h), and are coagulated using agarose Gel electrophoresis identifies the integrality of extracted total serum IgE；Meanwhile the A260/A280 value and A260/A230 value of total serum IgE are measured, and survey The concentration of fixed extracted total serum IgE.
Electrophoresis detection the result shows that: 28S, 18S and 5S band are clear, and the brightness of 28S band is about 2 times of 18S.Measurement The result shows that: A260/A280 is between 1.8~2.1, and total rna concentration is between 200~350 ng/ μ L.These result tables Bright, extracted total serum IgE quality is preferable, and subsequent reverse transcription cDNA preparation is used equally for use.
Further, reference book is tried using PrimeScript RT reagent Kit with gDNA Eraser The reverse transcription of agent box at the first chain of cDNA, as real-time fluorescence quantitative PCR reaction template, directly apply, or will prepared by CDNA -20 DEG C save backup.
(2) transcript profile sequencing andPP2AThe acquisition of gene
The blade for the two iris different disposal times cultivated under the conditions of normal growth temperature and low-temperature treatment is subjected to RNA- Seq sequencing, obtains from transcript profile result databasePP2AThe cDNA sequence of gene, sequencing and analysis the result shows that:PP2ABase The cDNA overall length of cause is 2207 bp, includes a complete ORF, 1764 bp of the ORF long, encodes Protein Phosphatase 2A (PP2A).
It is to be understood that inventor determines that iris " big capsicum " is to resist cold kind according to Physiological test results early period, And " the Fuller setting sun " be do not resist cold kind, be based on this phenotypic difference, to two kinds under low temperature stress the different disposal time Transcript profile be sequenced.It is sequenced in comparison process, discoveryPP2AGene is not influenced by kind and processing time, i.e., two A kind different time expression quantity is almost the same, therefore speculates that the gene can be used as reference gene use.In order to further determine The experiment speculates that inventor has carried out subsequent realtime fluorescent quantitative PCR experiment (referring to embodiment 2), further demonstrated this Estimation result.
It should be noted that further BLAST compare analysis shows, the irisPP2AGene order with it is listed small Lanyu irisPP2A(GenBank accession number: XM_020743358) gene order consistency is that the area 99%, ORF has 15 bases Difference, the amino acid sequence of coding and small Lanyu iris PP2A consistency are 99%, there is 1 amino acid difference, but the prior art In for small Lanyu iris PP2A expression characterization and its particular use be a lack of further study and inquire into.
Embodiment 2
On the basis of embodiment 1, the present embodiment is mainly with regard to irisPP2AGene is in normal growth temperature and low temperature stress item The stability of transcriptional expression is researched and analysed under part, and specific experiment situation is briefly introduced and is described as follows.
(1) real-time fluorescence quantitative PCR design of primers
Based on the sequencing result of embodiment 1, designPP2AGene real-time fluorescence quantitative PCR primer sequence is as follows:
QPP2A-F:5'-TCTGTTGGCTGTGGAAGGAT-3',
QPP2A-R:5'-AAATCCGACCTGGTAGTTTCTG-3'；
It should be noted that the amplified production length based on the primer sequence is 186 bp.
(2) real-time fluorescence quantitative PCR reacts
Take the 21 of iris different cultivars prepared in the embodiment 1 of equivalent, Different hypothermia treatment conditions and the blade of time A cDNA sample is uniformly mixed and is used as standard items, dilutes 10 times, 100 times, 1000 times and 10000 times and undiluted sample respectively As 5 gradients, respectively using the cDNA of 5 gradients as template, it is fixed that real-time fluorescence is carried out using primer designed in step (1) Measure PCR reaction (3 repetitions)；
When quantitative fluorescent PCR reacts, the design of 20.0 μ L reaction systems is as follows:
2 × SYBR Premix Ex Taq II, 10.0 μ L；
Primers F and R(are 10 mmol/L), each 0.8 μ L；
CDNA template, 2.0 μ L；
ddH2O, 6.4 μ L；
Reaction condition are as follows: 95 DEG C of initial denaturations 30 s, 95 DEG C of denaturation 15 s, 58 DEG C of annealing 15 s, 72 DEG C of 15 s of extension carry out 40 Circulation.
Melting curve and standard curve are automatically generated by real-time fluorescence quantitative PCR instrument.Melting curve is as shown in Figure 1, analysis As can be seen thatPP2AThe melting curve of gene real-time fluorescence quantitative PCR be it is unimodal, it is special to show that designed primer has Property, product is single.Standard curve as shown in Fig. 2, analysis it is found thatPP2AThe standard curve of gene real-time fluorescence quantitative PCR is y =-3.299x+20.17, amplification efficiency 101%, related coefficient 0.999.
The sample cDNA handled using 21 iris different cultivars Different hypothermias in embodiment 1 is carried out glimmering in real time as template Fluorescent Quantitative PCR reaction, as a result as shown in figure 3, obtain the Ct cycle threshold in this 21 samples average value be 21.51~ 23.42 showing to usePP2APrimer progress real-time fluorescence quantitative PCR result is reliable,PP2AGene can be used for iris and normally give birth to Real-time fluorescence quantitative PCR research under the conditions of long temperature and low-temperature treatment.
Further reaction product is detected with 1.5% agarose gel electrophoresis, as a result as shown in Figure 4, it can be seen thatPP2ABrightness of the gene in 21 samples is almost the same, shows that expression is stablized in this 21 samples.
Embodiment 3
Result based on embodiment 2, it can be determined that irisPP2AGene has the potentiality applied as reference gene.Therefore, Inventor is with irisPP2AGene is as reference gene, with the domain NAC protein genePhNAC1For, to the gene in iris Expression characterization under the conditions of low temperature stress in two different cultivars different disposal time blades is researched and analysed, related experiment Process is briefly discussed below.
(1) design of primers
The real-time fluorescence quantitative PCR detection domain NAC protein gene (PhNAC1) when, design of primers is as follows:
QPhNAC1-F:5'-ATCTGAACAAGTGCGAGCCT-3',
QPhNAC1-R:5'-ATCCTTACCAGTTGCCTTCC-3'；
It should be noted that amplified production length is 155 bp when being expanded using the primer sequence.
(2) real-time fluorescence quantitative PCR detects
Using the 21 iris sample cDNA obtained in embodiment 1 as template, withPhNAC1The qPhNAC1-F of gene and QPhNAC1-R is primer, is carried out real-time fluorescence quantitative PCR detection (3 repetitions)；The design of 20.0 μ L reaction systems is as follows:
2 × SYBR Premix Ex Taq II, 10.0 μ L；
Primers F and R(are 10 mmol/L), each 0.8 μ L；
CDNA template, 2.0 μ L；
ddH2O, 6.4 μ L；
Reaction condition are as follows: 95 DEG C of initial denaturations 30 s, 95 DEG C of denaturation 15 s, 58 DEG C of annealing 15 s, 72 DEG C of 15 s of extension carry out 40 Circulation.
PhNAC1Gene relative expression quantity uses 2-ΔΔCtMethod calculates.
Experimental result is as shown in Figure 5.Analysis can be seen that two kinds of iris " big capsicum " and " the Fuller setting sun " 11 DEG C/6 DEG C of low-temperature treatments under the conditions of,PhNAC1Gene relative expression quantity obviously rises in CK2；When gradually temperature-fall period terminates (T1) expression quantity is declined after carrying out 11 DEG C/6 DEG C 1 d of processing, later and starts to gradually rise, (T5) highest when 5 d, " big capsicum " is in the 7th d(T7) again decline and " the Fuller setting sun " continues to increase." big capsicum " kind is in 4 DEG C of low-temperature treatment conditions Under, expression quantity is declined in 4 h, and 12 h, 24 h and 48 h are in gradually rise trend later.
Embodiment 4
Further to investigatePP2AThe accuracy of result when as the application of iris reference gene, the operation of reference implementation example 3, The application is with commonActinAs reference gene, to the domain the NAC protein gene of iris (PhNAC1) expression carry out Analysis, correlated process are briefly discussed below.
Used in real-time fluorescence quantitative PCR reactionActinDesign of primers is as follows:
QActin-F:5'-GTTCTTTCCCTATATGCTAGTGGC-3',
qActin-R: 5'-GAAGGATGGCATGAGGAAGTG-3'。
PCR reaction system, reaction condition and gene relative expression quantity calculation method are the same as embodiment 3.
Experimental result is as shown in Figure 6.Analysis as can be seen that withActinWhen for reference gene,PhNAC1Gene is in iris Relative expression levels under two kind Different hypothermias processing times, withPP2AWhen for reference genePhNAC1Gene expression amount It is almost the same (but also there is technicality in the respective growth stage) to change general trend.Generally, withPP2AAs reference gene into The genetic analysis of row correlation function is the result is that more reliable.
SEQUENCE LISTING
<110>Zhengzhou Normal University
<120>application of the iris PP2A gene as reference gene
<130> none
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 2207
<212> DNA
<213> Phalaenopsis spp.
<400> 1
ccgaaaccct cgtctgcttc acttcccatc taccggctcc tagtttcgct taaaagtatc 60
tccttcgaga ccaaaccctc ccaatacaat tgcgatcaga gataactggt aaggaaacgg 120
cgagatcgcg gtgtagagag acccgaaatt actatctttt tcctccttcc gctctgttac 180
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cgattgctat tctaattgat gaacttaaaa atgaagatat tcagctgagg ttgaactcca 300
tacgtaggct ttctactata gcaagggcac ttggtgaaga aagaactcgg aaggagttga 360
ttccttttct cagcgataac aatgacgatg atgatgaagt tcttcttgca atggcggagg 420
aactgggtgt attcattcct tatgttggag gcgtggaata tgctcatgtc ttgctccctc 480
ctttggagac tttgtgtact gtagaggaaa cttgtgtcag agataaagct gttgagtcct 540
tgtgtagaat tggatcacag atgaaggaaa gtgatatagt ggattggttt gttcctttgg 600
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agctttgtca agatgacatg cctatggtga gaagatctgc agcatccaac ttgggaaaat 780
ttgcaagtac tgttgaacaa agccacttga agacagatgt aatgtctatg tttgaggatc 840
ttactcaaga tgatcaagac tctgtacgtc tgttggctgt ggaaggatgc gcagcacttg 900
gaaaactgtt ggaaccccag gagtgtgtat ctcacattct tccagtaatt attaacttct 960
ctcaggataa atcatggcgc gttcgctata tggttgctaa tcagttgtat gaacttagtg 1020
aagctgttgg tccagaaact accaggtcgg atttagttcc agcttatgtt agacttctcc 1080
gtgataatga ggctgaagtt cgcatagcag ctgctggcaa agtaacaaag ttttgccaga 1140
ttttgggtcc tgaactagcc atccaacaca ttcttacttg tgtcaaggaa ttgtcatcag 1200
attcatccca acacgttcgc tcagctttgg cgtcagttat catgggaatg gcaccagttt 1260
taggaaaaga ggcaacaatt gaacagcttc ttcctatttt tctttctttg ttgaaagatg 1320
aattccctga tgttagacta aacatcatca gcaagcttga ccaagtaaat caggtgattg 1380
gaattgacct gttgtcccag tcattattgc ctgccattgt tgagcttgca gaggacagac 1440
attggcgtgt tcgattggcc atcattgagt acattccctt gttagcgagt cagttaggtg 1500
tgagattttt tgatgataag cttggtgctc tgtgcatgca gtggctggaa gacaaggtat 1560
tctcaattag agatgctgct gccaataatt tgaagcggct tgctgaggaa tttggaccag 1620
aatgggcaat gcagcacatt gttccgcagg tgctggataa gatgaataac ccgcactatt 1680
tgtatcgcat gactttcctg catgcaatag cccttttatc cccggtcatg ggtccagaca 1740
tcacatacca gcggctttta cctgtcgtca ttaatgcctc aaaagacagg gtgcctaaca 1800
tcaaattcaa tgtagcaaag gtgctgcaat ctctcctccc tatagtcgac tcatctgttg 1860
tcgagaaaac catcaggccc tgtcttgttg agcttagcga agacccggat gtcgatgtga 1920
ggtattttgc ccaccaagca atacaatctt gtgatcagtt aatgatttcg ggctagagga 1980
ttttatcatc gtgtgtgaaa ttctattcat cctcttgccg ttttgactgc ctgctgttat 2040
gtttttccat gtcttcattt atattgttgt ctagagctct gattaacacc attaggccta 2100
acaatgatat agtatttttg tattattctg aatttttatt tgctttcaaa attagaatcg 2160
ctgcctgtac aacttaattc tttatttagt tttcctcctt ggttttg 2207
<210> 2
<211> 587
<212> PRT
<213> Phalaenopsis spp.
<400> 2
Met Ala Met Leu Asp Glu Pro Leu Tyr Pro Ile Ala Ile Leu Ile Asp
1 5 10 15
Glu Leu Lys Asn Glu Asp Ile Gln Leu Arg Leu Asn Ser Ile Arg Arg
20 25 30
Leu Ser Thr Ile Ala Arg Ala Leu Gly Glu Glu Arg Thr Arg Lys Glu
35 40 45
Leu Ile Pro Phe Leu Ser Asp Asn Asn Asp Asp Asp Asp Glu Val Leu
50 55 60
Leu Ala Met Ala Glu Glu Leu Gly Val Phe Ile Pro Tyr Val Gly Gly
65 70 75 80
Val Glu Tyr Ala His Val Leu Leu Pro Pro Leu Glu Thr Leu Cys Thr
85 90 95
Val Glu Glu Thr Cys Val Arg Asp Lys Ala Val Glu Ser Leu Cys Arg
100 105 110
Ile Gly Ser Gln Met Lys Glu Ser Asp Ile Val Asp Trp Phe Val Pro
115 120 125
Leu Val Lys Arg Leu Ala Ala Gly Glu Trp Phe Thr Ala Arg Val Ser
130 135 140
Ser Cys Gly Leu Phe His Ile Ala Tyr Pro Ser Ser Pro Asp Met Leu
145 150 155 160
Lys Ala Glu Leu Arg Ser Ile Tyr Gly Gln Leu Cys Gln Asp Asp Met
165 170 175
Pro Met Val Arg Arg Ser Ala Ala Ser Asn Leu Gly Lys Phe Ala Ser
180 185 190
Thr Val Glu Gln Ser His Leu Lys Thr Asp Val Met Ser Met Phe Glu
195 200 205
Asp Leu Thr Gln Asp Asp Gln Asp Ser Val Arg Leu Leu Ala Val Glu
210 215 220
Gly Cys Ala Ala Leu Gly Lys Leu Leu Glu Pro Gln Glu Cys Val Ser
225 230 235 240
His Ile Leu Pro Val Ile Ile Asn Phe Ser Gln Asp Lys Ser Trp Arg
245 250 255
Val Arg Tyr Met Val Ala Asn Gln Leu Tyr Glu Leu Ser Glu Ala Val
260 265 270
Gly Pro Glu Thr Thr Arg Ser Asp Leu Val Pro Ala Tyr Val Arg Leu
275 280 285
Leu Arg Asp Asn Glu Ala Glu Val Arg Ile Ala Ala Ala Gly Lys Val
290 295 300
Thr Lys Phe Cys Gln Ile Leu Gly Pro Glu Leu Ala Ile Gln His Ile
305 310 315 320
Leu Thr Cys Val Lys Glu Leu Ser Ser Asp Ser Ser Gln His Val Arg
325 330 335
Ser Ala Leu Ala Ser Val Ile Met Gly Met Ala Pro Val Leu Gly Lys
340 345 350
Glu Ala Thr Ile Glu Gln Leu Leu Pro Ile Phe Leu Ser Leu Leu Lys
355 360 365
Asp Glu Phe Pro Asp Val Arg Leu Asn Ile Ile Ser Lys Leu Asp Gln
370 375 380
Val Asn Gln Val Ile Gly Ile Asp Leu Leu Ser Gln Ser Leu Leu Pro
385 390 395 400
Ala Ile Val Glu Leu Ala Glu Asp Arg His Trp Arg Val Arg Leu Ala
405 410 415
Ile Ile Glu Tyr Ile Pro Leu Leu Ala Ser Gln Leu Gly Val Arg Phe
420 425 430
Phe Asp Asp Lys Leu Gly Ala Leu Cys Met Gln Trp Leu Glu Asp Lys
435 440 445
Val Phe Ser Ile Arg Asp Ala Ala Ala Asn Asn Leu Lys Arg Leu Ala
450 455 460
Glu Glu Phe Gly Pro Glu Trp Ala Met Gln His Ile Val Pro Gln Val
465 470 475 480
Leu Asp Lys Met Asn Asn Pro His Tyr Leu Tyr Arg Met Thr Phe Leu
485 490 495
His Ala Ile Ala Leu Leu Ser Pro Val Met Gly Pro Asp Ile Thr Tyr
500 505 510
Gln Arg Leu Leu Pro Val Val Ile Asn Ala Ser Lys Asp Arg Val Pro
515 520 525
Asn Ile Lys Phe Asn Val Ala Lys Val Leu Gln Ser Leu Leu Pro Ile
530 535 540
Val Asp Ser Ser Val Val Glu Lys Thr Ile Arg Pro Cys Leu Val Glu
545 550 555 560
Leu Ser Glu Asp Pro Asp Val Asp Val Arg Tyr Phe Ala His Gln Ala
565 570 575
Ile Gln Ser Cys Asp Gln Leu Met Ile Ser Gly
580 585

Claims (5)

1. irisPP2AApplication of the gene as reference gene, which is characterized in that the gene is in normal growth temperature and low temperature Equal stable and consistent is expressed under stress conditions, can be used for correlation function gene table under the conditions of assay normal growth or low temperature stress Up to situation；It is describedPP2AGene, cDNA sequence length are 2207 bp, the ORF comprising 1764 bp one complete, long, tool Body cDNA base sequence is as shown in SEQ ID NO.1.
2. iris as described in claim 1PP2AApplication of the gene as reference gene, which is characterized in that the functional gene For the domain NAC protein genePhNAC1。
3. a kind of utilize irisPP2AReal-time fluorescence quantitative PCR determination method of the gene as reference gene, feature It is, when detection and analysis, for irisPP2AGene, primer sequence design are as follows:
QPP2A-F:5'-TCTGTTGGCTGTGGAAGGAT-3',
QPP2A-R:5'-AAATCCGACCTGGTAGTTTCTG-3'.
4. real-time fluorescence quantitative PCR determination method as claimed in claim 2, which is characterized in that test and analyzePhNAC1Table When up to situation, for the domain NAC protein genePhNAC1, primer sequence designs as follows:
QPhNAC1-F:5'-ATCTGAACAAGTGCGAGCCT-3',
QPhNAC1-R:5'-ATCCTTACCAGTTGCCTTCC-3'.
5. a kind of prepare irisPP2AThe PCR amplification method of gene, which is characterized in that when PCR amplification, primer sequence is designed such as Under:
PP2A-F:5 '-TTTGAGYGATTTGTGAAGGC-3 ',
PP2A-R:5 '-TGGAAAAAMATAACAGCAGG-3 '.
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