CN106874707A - A kind of screening technique of the reference gene related to willow adversity gene expression regulation - Google Patents
A kind of screening technique of the reference gene related to willow adversity gene expression regulation Download PDFInfo
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Abstract
The invention discloses a kind of screening technique of the reference gene related to willow adversity gene expression regulation, step includes：It is GSE13990 from numbering, GSE15242, the genetic chip of GSE21171 and GSE23637, probe in genetic chip is normalized, obtain candidate's reference gene, recycling InterproScan softwares carries out function prediction, and it is reference gene that selection maintains the related gene of willow Life Base function.The screening technique is obtained in that the new reference gene beyond prior art report, enrich the selection of willow reference gene, approach is provided to filter out more stable, more preferably reference gene, research to the expression of willow anti contravariance related gene, and be systematically carried out willow degeneration-resistant Journal of Sex Research it is significant, there is important directive function to the degeneration-resistant elite plant of further seed selection.
Description
Technical field
Adjusted the present invention relates to the technical field of genetic engineering, more particularly to a kind of expression with willow adversity gene
Control the screening technique of related reference gene.
Background technology
Willow (Populus trichocarpa) is important greening energy seeds, Poplar Industry is greatly developed, for gloomy
Woods resource it is abundant, the improvement of natural ecological environment is of great advantage.Willow China be distributed mainly on North China, northwest etc. arid and
Semiarid zone, arid can not only influence photosynthetic rate, shoot growth speed and the dry-matter accumulation of blade, and willow can also done
Mitigate again, the volume of timber is reduced, so as to govern the fast growing of willow.Arid is often relevant with the osmotic stress that salinity high causes,
Influence growth and the yield of willow.Willow as important woody mode crop, in the anti-environment stress decile such as drought resisting, high salt tolerance
Sub- genetic improvement aspect development is notable.With continuing to develop for technique for gene engineering, the research of adversity gene is also increasingly subject to
Everybody favor.Therefore, the expression of research anti contravariance related gene not only can be with the resistance mechanism of systematic research willow, also
The seed selection of the degeneration-resistant elite plant of willow can preferably be instructed.
Real-time fluorescence quantitative PCR (Quantitative real-time PCR) is the most frequently used, most quick, simplest base
Because of the method for expression quantitative analysis.Used as the method for strong detection gene expression profile, it can analyze gene not
With the differential expression in growth period and different tissues organ, and between sample under the conditions of varying environment gene differential expression
It is mainly manifested in sensitivity and specificity higher Deng, its advantage, and had wider compared with former molecular engineering
General dynamic range.Also exactly these advantages cause that its selection to reference gene is very harsh.Due to each during PCR
Species diversity, correction as a result and standardization are still the problem of most challenge.And the use of reference gene is considered as most to close
Suitable standard method because its can delicately modified R NA initial amounts and reverse transcription efficiency etc. difference, and then obtain target gene
Specifically expressing it is very poor.
The expression that preferable reference gene can should be stablized under any circumstance.In real-time fluorescence quantitative PCR expression
In analysis, house-keeping gene is the most commonly used reference gene.Because house-keeping gene is the important component of cell, and expression stabilization,
Therefore its applicability need not be verified when in use, but there are some researches show house-keeping gene is expressed under varying experimental conditions
Have very big difference (Suzuki et al., 2000；Thellin et al., 1999；Vandesompele et al.,
2002).Some conventional reference genes are not suitable anymore for as reference gene due to instable expression under certain condition,
And gradually replaced by new reference gene.With the development of biochip technology and transcript profile sequencing technologies, using gene table
A kind of effective ways of the gene studies person with reference to selection are had become up to the new reference gene of data mining.However, this at present
The method extensive use in xylophyta not yet so that the traditional reference gene for being not appropriate for all experiment conditions is still much ground
The person of studying carefully continues to use, and this turns into a drawback of gene expression analysis research.
At present, Triose phosphate dehydrogenase (glyceraldehyde-3-phosphate, GAPDH), actin
(actin), ubiquitin (ubiquitin, UBQ), ubiquitin binding enzyme (ubiquitin conjugating enzyme, UBC), 18S
RRNA (18S rRNA), 25S rRNAs (25SrRNA), transcriptional elongation factor (elongation factor 1
Alpha, EF1 α), tubulin (tubulin beta, TUB) and translation elongation factor (translation elongation
Factor, TEF) etc. house-keeping gene be more conventional reference gene (Kim et al., 2003).However, due to development of plants
Stage and different experiment conditions, the transcriptional level of most of house-keeping genes are also different.Up to the present one is not found yet to fit
It is the reference gene of gene expression analysis under all conditions to cooperate.For example, it was discovered by researchers that in red clover (Trifolium
Pratense) in blade, UBC2 and UBQ10 expression is most stable；In stem, UBC2 and YLS8 expression is relatively stable；In root,
EIF-4a and UBC2 expression stabilizations；And in each tissue, the stability of GAPDH and SAND is relatively low.Additionally, Actin,
The tradition house-keeping gene such as ubiquitin, 18S rRNA, EF1 α, TUB and TUA has been applied to the analysis of willow real-time fluorescence quantitative PCR
In, however, in terms of screening system, these genes still have many not enough (Pettengill et al., 2012).Therefore, in order to
The accuracy of real-time fluorescence quantitative PCR data is improved, suitable reference gene is screened and is just particularly important.
The content of the invention
It is an object of the invention to overcome the deficiencies in the prior art, there is provided one kind and willow adversity gene expression regulation phase
The screening technique of the reference gene of pass, with screen obtain it is more stable, more preferably with the various adversity gene expression regulation phases of willow
The reference gene of pass.
The present invention is achieved by the following technical solutions：
The invention provides a kind of screening technique of the reference gene related to willow adversity gene expression regulation, including with
Lower step：
(1) genetic chip related to willow adversity gene expression regulation is collected, selection numbering is GSE13990,
The genetic chip of GSE15242, GSE21171 and GSE23637 is specificity screening chip；
(2) probe included in 4 genetic chips of step (1) is normalized, step includes：
Step one：The normalization microarray data treatment software Expression developed using Affymetrix company of the U.S.
The MAS5 algorithms carried in Console are calculated the P-Value values of each probe in genetic chip, screen P-Value values<
0.05 probe；
Step 2：Using the probe after the RMA algorithm calculating siftings in Expression Console softwares in the different sides of body
CV values under the conditions of compeling, the order by probe according to CV values from small to large is ranked up, the smaller data for representing probe of CV values from
The degree of dissipating is smaller；CV values represent the coefficient of variation, and CV values=SD standard deviations/MV average values, standard deviation reflects the discreteness of data,
Data are bigger with average value, and standard deviation is bigger, and coefficient of variation reflection be data discrete degree absolute value, by flat
The influence of average and standard deviation, the coefficient of variation can compare two groups of data for having different average values and standard deviation.
(3) the willow gene in selection step (2) corresponding to the probe of sequence preceding 50 is candidate gene, is utilized
InterproScan softwares carry out function prediction to 50 candidate genes, and selection maintains the related gene of willow Life Base function
It is the reference gene.
Further, in the step 2, Different stress condition includes salt stress and drought stress.
Further, in the step (3), the related gene of willow Life Base function is maintained to include participating in energetic supersession
With the related gene of Amino acid synthesis.
The present invention has advantages below compared to existing technology：The invention provides a kind of and willow adversity gene expression regulation
The screening technique of related reference gene, the method is obtained in that the new reference gene beyond prior art report, enriches
The selection of willow reference gene, approach is provided to filter out more stable, more preferably reference gene.It is anti contravariance related to willow
The research of the expression of cause, and be systematically carried out willow degeneration-resistant Journal of Sex Research it is significant, it is anti-to further seed selection
Inverse elite plant has important directive function.
Brief description of the drawings
Fig. 1 is the expression stability result figure of reference gene under geNorm analysis salt stresses；
Fig. 2 is the expression stability result figure of reference gene under geNorm analysis drought stresses.
Specific embodiment
Embodiment 1
1st, material
The present embodiment method therefor is known to those skilled in the art the conventional method of dawn unless otherwise instructed, used
The material such as reagent, unless otherwise instructed, be commercially available purchase product.
2nd, method
The screening technique of 2.1 reference genes
(1) genetic chip related to the expression of willow adversity gene is collected, it is GSE13990, GSE15242 to select numbering,
The genetic chip of GSE21171 and GSE23637 is specificity screening chip；GSE13990, GSE15242, GSE21171 and
The microarray data of GSE23637 is from https://www.ncbi.nlm.nih.gov/gds/Term=websites directly download
Obtain.
(2) using the normalization microarray data treatment software Expression Consle of Affymetrix company of U.S. exploitation,
Probe to being included in 4 genetic chips of step (1) is normalized, and step includes：
Step one：The MAS5 algorithms carried using Expression Consle softwares are calculated and obtain each in genetic chip
The P-Value values of probe, screen P-Value values<0.05 probe；
Step 2：Using the probe after the RMA algorithm calculating siftings that Expression Consle softwares are carried in salt stress
With the signal representation value and CV values under drought stress conditions, the order by probe according to CV values from small to large is ranked up, CV values
The smaller data discrete degree for representing probe is smaller；
(3) the willow gene corresponding to the probe of selection step (2) sequence preceding 50 is candidate gene, is utilized
InterproScan softwares carry out function prediction to 50 candidate genes, and selection participates in the related gene of willow Life Base function
It is the reference gene, such as, selection participates in energetic supersession, the related gene of Amino acid synthesis, but not limited to this, these genes
Cell base metabolism synthesis is participated in, its function is guarded, thus it is speculated that it expresses stabilization in vivo.
2.2 the selection results
Screening altogether obtains the new reference gene do not reported of 6 prior arts, respectively PtRG1, PtRG2, PtRG3,
PtRG4, PtRG5, PtRG6, its nucleotide sequence is successively as shown in SEQ ID NO.1-6.
The functional verification of 2.3 reference genes
2.3.1 the extraction and reverse transcription of poplar leaf total serum IgE
3cm Nanlin 95 clone tree tissue culture rooted seedlings high are taken, is gathered after carrying out salt stress and drought stress treatment respectively to it
Blade, obtains testing sample, and Trizol methods extract testing sample total serum IgE, using TAKARA companies reverse transcription reagent box
The RNA reverse transcriptions that PrimeScriptTM RT Master Mix (R036A) will be extracted are standby as masterplate cDNA into cDNA.
The method of salt stress treatment is：Sprinkling treatment is carried out with the NaCl of 200mmol/L, then respectively at 0, Isosorbide-5-Nitrae,
8,12,24 hours collection blades, as testing sample.
The method of drought stress treatment is：Sprinkling treatment is carried out with the polyethylene glycol (PEG) that volume ratio is 25%, so
Blade was gathered respectively at 0,1,4,8,12,24 hours afterwards, as testing sample.
Control group is set simultaneously, and control group does not do any Stress treatment.
2.3.2 design of primers
House-keeping gene is often used to above-mentioned 6 newfound reference genes and other 5 willows using Primer5.0 softwares
TUA8, EF1 α, 18S rRNA, actin, PtUBQ carry out design of primers, and the primer sequence of acquisition is as shown in table 1 below：
Table 1：The primer sequence table of reference gene
2.3.3 real-time fluorescence quantitative PCR analysis
Using the SYBRR Premix Ex TaqTMII real-time quantitative PCR kits of Dalian treasured bioengineering Co., Ltd
Carry out quantitative analysis.Use Bio-Radi Cycler IQ real-time PCRs and 96 orifice plates.Each hole adds according to 20 μ L systems
Sample：1 μ L templates cDNA, 1 μ L sense primers (10 μm of ol/L), 1 μ L anti-sense primers (10 μm of ol/L), 10 μ L SYBRR Premix
Ex TaqTM II, 7 μ L sterilized waters.Each sample is repeated 4 times.Amplified reaction program is：95 DEG C of predegenerations 30 seconds；95 DEG C of denaturation 5
Second, 60 DEG C are annealed 18 seconds, and 72 DEG C extend 15 seconds, and 39 circulations gather the fluorescence signal of solubility curve, and experimental data is using relative
Sizing technique is analyzed.
Using Bio-Rad iQ5 analysis softwares, derived in Nanlin 95 clone tree tissue culture seedling leaf by not according to solubility curve
With the Ct values of the testing sample for the treatment of, as the basis of subsequent data analysis.
2.3.4 data process＆analysis
1) geNorm analyses
The Ct values that real-time fluorescence quantitative PCR is obtained are converted to relative quantification data, and step is：Minimum Ct is found first
Value minCt, then subtracts minCt with the Ct values of other genes, obtains △ Ct, and △ Ct >=0, then uses Excel software meters
Calculate each gene under different experimental conditions relative to highest expression quantity gene relative expression quantity E=2-△Ct, finally,
The data that will be calculated save as Excel file, are stored in geNorm software I nputData files, soft using geNorm
Part calculates the M values of each gene.Result is as shown in Figure 1, 2.
M values in geNorm softwares refer to the average expression index of stability of gene, and M values are smaller, and the stability of gene is higher, instead
It is then lower, most stable of house-keeping gene can be thereby determined that.Generally, with M=1.5 as the upper limit, the base only less than 1.5
Stablize relatively because being just considered as expression.As can be seen that under salt stress in Fig. 1,2,11 stability of reference gene from it is low to
Height is ordered as PtRG2=PtRG3<PtRG4<PtRG1<PtRG5<1.5<PtRG6<TUA8<EF1α<actin<PtUBQ<18S
RRNA, wherein, the M values of PtRG2, PtRG3, PtRG4, PtRG1, PtRG5 are less than 1.5, show that they stablize relatively.Coerced in arid
Under compeling, the stability of 11 reference genes is ordered as PtRG3=PtRG5 from low to high<PtRG1<PtRG6<TUA8<PtRG4<
PtRG2<1.5<EF1α<PtUBQ<actin<18S rRNA, wherein, PtRG3, PtRG5, PtRG1, PtRG6, TUA8, PtRG4,
The M values of PtRG2 are less than 1.5, show that their expression is stablized relatively.GeNorm analysis results can be seen that the application screening is obtained
Gene PtRG1-6, it is more stable compared to more traditional house-keeping gene expression, with obvious advantage, be more suitable for and willow
The related reference gene of adversity gene expression regulation.
2) NormFinder analyses
The Ct values that real-time fluorescence quantitative PCR is obtained are converted to relative quantification data, and step is：Minimum Ct is found first
Value minCt, then subtracts minCt with the Ct values of other genes, obtains △ Ct, and △ Ct >=0, then uses Excel software meters
Calculate each gene under different experimental conditions relative to highest expression quantity gene relative expression quantity E=2-△Ct, finally,
The data that will be calculated save as Excel file, imported into NormFinder programs, are calculated the M of candidate's reference gene
Value.Result is as shown in table 2 below, 3：
Table 2：The expression stability of reference gene under NormFinder software analysis salt stresses
Table 3：The expression stability of reference gene under NormFinder software analysis drought stresses
M values in NormFinder analyses refer to the stable expression value of gene, and M values are lower, then the expression of the gene is more stable.
Be can be seen that in table 2,3, under salt stress, in 11 reference genes, the reference gene PtRG1-5 that present invention screening is obtained is arranged as
2-6, illustrate that it expresses the willow house-keeping gene (in addition to TUA8) that compares more stable.Under drought stress, 11 internal reference bases
Because in, reference gene PtRG1, PtRG2 other house-keeping genes that compare that present invention screening is obtained are more stable.NormFinder points
Analysis also demonstrate that the gene that present invention screening is obtained is better than conventional house-keeping gene.
3) BestKeeper analyses
BestKeeper programs are to be calculated to obtain standard deviation SD and dependency number through EXCEL softwares according to the Ct values of reference gene
According to as a result as shown in table 4 below, 5：
Table 4：The expression stability of reference gene under BestKeeper software analysis salt stresses
Table 5：The expression stability of reference gene under BestKeeper software analysis drought stresses
In the analysis of BestKeeper programs, SD values are smaller, and the stability of gene is better.Therefore, be can be seen that from table 4,5,
Under salt stress, PtRG1-6 sequences in the gene 1-6, better than other 5 house-keeping genes.Under drought stress, PtRG1-6 genes
Arrangement 1-7, better than other house-keeping genes in addition to TUA8, and PtRG1, PtRG3, PtRG5, PtRG6 arrangement are first four, say
Expression stability of the bright PtRG1-6 genes under salt and drought stress is integrally better than conventional house-keeping gene.
3 conclusions
The present invention utilizes real time fluorescence quantifying PCR method, soft with geNorm, NormFinder and BestKeeper this 3
6 reference genes and 5 house-keeping gene different time sections under salt and drought stress conditions that part is obtained to present invention screening
Gene expression amount carries out statistical comparative analysis, as a result shows：The new internal reference base for obtaining is screened by biochip technology
Because more more stable than traditional house-keeping gene, wherein geNorm software analysis show that PtRG3 is showed under salt stress and drought stress
For most stable, optimum does reference gene；NormFinder software analysis show that under salt stress TUA8 stability is best, most
Reference gene is suitably done, and under drought stress, preferably, optimum does reference gene to the stability of PtRG1；BestKeeper journeys
Sequence analysis show that under salt stress and drought stress preferably, optimum does reference gene to PtRG5 stability.Comprehensive this three sections soft
The analysis result of part, we can draw to draw a conclusion：Under salt stress and drought stress, filtered out by biochip technology
New reference gene than traditional house-keeping gene stabilization, wherein, the expression of PtRG1, PtRG3, PtRG5 these three genes is relatively all
It is very stable, be suitable as the reference gene of willow real-time fluorescence quantitative PCR under salt and drought stress, due to PtRG1,
PtRG3, PtRG5 these three genes expression quantity in willow are different, so we should select in suitable as the case may be
Ginseng gene, the gene that selection is closer to destination gene expression level is used as reference gene.
It is above a kind of detailed implementation method of the invention and specific operating process, is with technical solution of the present invention as preceding
Put and implemented, but protection scope of the present invention is not limited to the above embodiments.
SEQUENCE LISTING
<110>Agricultural University Of Anhui
<120>A kind of screening technique of the reference gene related to willow adversity gene expression regulation
<130> /
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 948
<212> DNA
<213> populus trichocarpa
<400> 1
atgaagtcca ttggagtcct aatgacttgt ccgatgcaca aatacctaga acaacagcta 60
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gcggaactaa ttgcgtcatt gccaagtttg gaaattgttg ccagttacag tgtaggattg 240
gataaaattg atttgagaaa gtgtgaagag aaagggatta gagttgctaa tacccctgat 300
gttttgactg atgatgttgc tgacttagca attgggttga tattgggggt tttgaggggg 360
atttgtgctt ctgatgctta cgtcaggatt ggcaagtgga aggatgctga ttttggattg 420
gcaacaaagt ttagtggtaa atcagttggc atcgttgggt tgggcagaat tggtacagct 480
attgcgaaga gagctgaagc ttttggttgc tccataagtt acttctccag gtcacaaaaa 540
ccatttgcaa actacaaatt ctactcgaac attattgact tggccaccag ttgccaaatc 600
ctcatcgtgg cttgtgcttt gacagaggaa acccgccata ttatcaatcg cgaagtcatt 660
gatgctctgg gtccaaaggg cattctaata aacattggac ggggtgctca cgtggatgaa 720
actgagcttg tgtctgcatt gcttgaaggt cggctaggtg gcgcagggct tgatgtgtat 780
gaaaatgagc ctgatgtacc tgaagaattg ctcggtcttg gcaatgttgt cctccagcct 840
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aagattgacc tggccaagtg tagagagaga ggtatcaggg tcactaatac ccctgatgtt 300
ttgaccgatg atgttgctga tttggctatt gggttgatgt tggctgtctt gaggaggctt 360
tgtccgagtg atcgctatgt taggagtggt cagtggaaaa ggggtgatta caagttgacc 420
actaagttta ctgggaagtc agttggcatt attgggttgg gcaggattgg cctggcgatt 480
gccaagagag ctgaagcatt tagttgccca attagttacc acactagagc agagaaatca 540
gatgtaaaat acaagtacta tcctagtgtt gttgagttgg ctgccaactg tcaaattctc 600
gttgttgcat gtgcattaac tgaagaaact cgccacatta tcaatcgtga agtcatcaat 660
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gtagggagtg gcaccatgga aaccaggaaa gaaatggctg accttgttgt tggcaatttg 900
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ttggaaccca tcaaagaaca attccccatc ttgtcttatg ctgacttcta ccaattagct 300
ggtgttgttg ctgttgaagt tacaggaggg cctgagattc cttttcaccc tgggagacca 360
gacaagtctg atccacctcc agaaggtcgc ttgcctgatg caaccaaagg ttcagaccat 420
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gttctggatt cgagggaccc tggaccccca acccacttgt tttcgacaac tcctatttca 660
aggaactcct cagtggagag aaagaaggtc ttatccagct tccatcagac aaaactcttc 720
tggaggatcc agtcttccgt ccccttgttg aaaactatgc tgaagatgag gatgcattct 780
ttgcagatta ttcagaagct catttga 807
<210> 4
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<212> DNA
<213> populus trichocarpa
<400> 4
atgacgaaga actatccaac tgtaagcgaa gagtacagca aagccgttga aaaggccaag 60
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tggcactcag ctggaacctt cgatgttaag acaaaaaccg gtgggccctt tggtaccatg 180
aggtactcgg cagagctggc acatggcgct aacaatggtc tcgacattgc tgtcagactc 240
cttgagtcca tcaaggagca gtttcccatc ctctcctatg ccgatttcta ccaacttgct 300
ggtgttgttg gtgttgaaat cactggtggt cctgaggttc ctttccaccc tgggagagag 360
gacaagcctg agccacctcc agaaggccgt ctgcctgatg ctactaaggg ttctgatcac 420
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aaccctctca tctttgataa ctcttacttc aaggagctct tgagtgggga gaaggaaggg 600
cttctacaac ttccatctga caaggctctt ctatctgacc ctatcttccg cccatatgtt 660
gacaaatatg ctgctgtatg taccaaaaat atcatctgtt gtgcttcagg cgcttgccag 720
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<210> 5
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<213> populus trichocarpa
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atgttacctt ctacactaat aagccctgta tttcaaagtt tctcaaccac aaaggcgtac 60
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agcgaattag ggtttggcct aatcgcctct ctttctgccc cagagccgca ccaactcttc 180
gtctcagaga tggccaagtc aaagaatcac acagcacaca accagtcaca caaagctcac 240
caaaatggca tcaagaaacc caagaggcat cgccacacct ccacgaaagg gatggacccc 300
aagtttttga ggaaccagag gtacgcaagg aagcataaca agaagtgtga tgagacagcc 360
accgaggaag agtag 375
<210> 6
<211> 753
<212> DNA
<213> populus trichocarpa
<400> 6
atgggcaagt gctaccctac agtgagcgaa gagtatcaga aggcagtgga gaaatgcaag 60
aggaagctca gaggactcat cgctgaaaaa cactgcgctc ccctcatgct ccgattagca 120
tggcactctg ctggtacttt tgatgtgaat accaagacag gagggccatt tgggacgatc 180
agacacccag atgagcttgc tcatggagct aacaatggtc ttgatattgc tgtcaggctt 240
ttggaacccc tcaaagagca gttccccaac ttgtcttatg ctgacttcta ccaattagct 300
ggtgttgttg ctgttgaaat tactggaggg cctgaggttc cttttcaccc tgggagacca 360
gacaagtctg atccacctcc agaaggtcgc ttgcctgatg caaccaaagg ttcagaccat 420
ttaagggatg tctttggcca tatgggtctt agtgacaaag atattgttgc cctatccggt 480
ggtcacactc tgggaaggtg ccacaaggag cgttctggat ttgagggacc ctggaccccc 540
aaccctcttg ttttcgacaa ctcctatttc aaggaactcc tcagtggaga gaaagaaggt 600
cttatccagc tccctacaga taaaaccctt ctggaggatc cagtcttccg cccccttgtt 660
gaaaaatatg ctgcagatga ggatgccttc tttgcagatt atgctgaagc tcatatgaag 720
ctctcagagc ttgggtttgc cgaggcatat tga 753
Claims (3)
1. a kind of screening of the reference gene related to willow adversity gene expression regulation, it is characterised in that comprise the following steps：
(1) genetic chip related to willow adversity gene expression regulation is collected, it is GSE13990, GSE15242 to select numbering,
The genetic chip of GSE21171 and GSE23637 is specificity screening chip；
(2) probe included in 4 genetic chips of step (1) is normalized, step includes：
Step one：Each probe in genetic chip is calculated using the MAS5 algorithms in Expression Console softwares
P-Value values, screen P-Value values<0.05 probe；
Step 2：Using the probe after the RMA algorithm calculating siftings in Expression Console softwares in Different stress bar
CV values under part, the order by probe according to CV values from small to large is ranked up；
(3) the willow gene in selection step (2) corresponding to the probe of sequence preceding 50 is candidate gene, using InterproScan
Software carries out function prediction to 50 candidate genes, and it is the internal reference base that selection maintains the related gene of willow Life Base function
Cause.
2. the screening technique of a kind of reference gene related to willow adversity gene expression regulation according to claim 1,
Characterized in that, in the step 2, Different stress condition includes salt stress and drought stress.
3. the screening technique of a kind of reference gene related to willow adversity gene expression regulation according to claim 1,
Characterized in that, in the step (3), maintaining the related gene of willow Life Base function to include participating in energetic supersession and amino
The related gene of acid synthesis.
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