CN113416794B - Reference gene suitable for fluorescent quantitation of male flowers of salix dustpan, and primer and application thereof - Google Patents

Abstract

The invention discloses a fluorescent quantitative reference gene of male flowers of salix dustpan, and a special primer and application thereof. The invention selects dustpan willow male flowers as experimental materials, and the male flowers cover 4 different development stages: differentiation period, dormancy period, initial flowering period and full flowering period of male flowers. 2 internal reference genes (DnaJ and ACT) which accord with the fluorescent quantitative PCR applicable to the male flowers of the dustpan are screened out through fluorescent quantitative PCR analysis, amplification curve and melting curve analysis, LinRegPCR amplification efficiency analysis and gene expression stability analysis of geNorm, NormFinder and BestKeeper in sequence, wherein the gene sequence of the DnaJ gene is shown as SEQ ID No.1, and the gene sequence of the ACT gene is shown as SEQ ID No. 2. The method fills the current situation that the male flowers of the salix dustpan have no reference genes in different development periods, provides powerful support for accurate quantification of the genes related to the development of the male flowers of the salix dustpan, and can improve the stability and accuracy of research.

Description

Reference gene suitable for fluorescent quantitation of male flowers of salix dustpan, and primer and application thereof

Technical Field

The invention belongs to the technical field of willow fluorescence quantification, and particularly relates to a fluorescent quantitative reference gene of male flowers of salix dustpan and primers and application thereof.

Background

Willow (Salix) is widely distributed in northern hemisphere, and is often used as afforestation tree species due to its fast growth speed and strong environmental adaptability. The flowers of willows are unisexual flowers, male and female isoplants, and the mosla inflorescence, leaves of the flowers are opened first, and the male flowers generate a large amount of pollen after being mature, so that the willows not only cause environmental pollution, but also are important allergens, the pollen is small and light, a large amount of pollen floats in the air when blown by wind, and allergy is easily caused after people contact and inhale the pollen. Therefore, the research on the molecular mechanism of willow male flower development becomes a research hotspot in recent years.

The Salix dustpan (Salix suchowensis) belongs to Salix genus of Salicaceae family, is a shrub which is native in China and can bloom in the current year after cuttage, is short in individual and short in generation period, is easy to carry out large-scale field test, is a willow tree which analyzes a whole genome sequence at the earliest in Salix genus, belongs to a diploid shrub, can bloom in the current year, can be used as a mode species for forest functional gene excavation, but at present, no reference gene for the research related to the development of Salix dustpan male flowers exists, and the commonly used reference gene can show unstable characters under different plants and experimental conditions, so that the accuracy of target gene detection is influenced, and therefore, the screening of the reference gene which is stably expressed in different periods of the development of Salix dustpan male flowers is a key factor for real-time fluorescent quantitative result accuracy.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide the fluorescent quantitative reference gene suitable for the male flowers of the salix dustpan, the gene can meet the requirement of real-time fluorescent quantitative detection on the transcriptional expression level of the male flowers of the salix dustpan, and the stability and reliability of gene expression analysis and research of the male flowers of the salix dustpan are improved. Another objective of the invention is to provide a primer special for the reference gene. The invention also aims to provide application of the reference gene or the special primer.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

the fluorescent quantitative reference genes suitable for the male flowers of the salix dustpan are DnaJ genes and ACT genes, wherein the gene sequence of the DnaJ gene is shown as SEQ ID NO.1, and the gene sequence of the ACT gene is shown as SEQ ID NO. 2.

The special primer suitable for the fluorescent quantitative reference gene of the male flowers of the salix dustpan has the following primer sequence of DnaJ gene:

DnaJ Forward primer 5'-GAGCCTATTTATGCCCTTTC-3' (SEQ ID NO.3)

DnaJ reverse primer 5'-GACGTAGTCCTTATCCACCTT-3' (SEQ ID NO. 4).

The special primer suitable for the fluorescent quantitative reference gene of the male flowers of the salix dustpan has the following primer sequences of the ACT gene:

ACT Forward primer 5'-CCAAGGAGGCTGCTGGTAAT-3' (SEQ ID NO.5)

ACT reverse primer 5'-CCAAGGCCACCATGTCTGTC-3' (SEQ ID NO. 6).

The application of the fluorescent quantitative reference gene suitable for the male flowers of the salix dustpan to fluorescent quantitation.

The application of the primer sequence of the DnaJ gene in the fluorescence quantification of the male flowers of the salix dustpan.

The application of the primer sequence of the ACT gene in the fluorescence quantification of the male flowers of the salix dustpan.

Compared with the prior art, the invention has the advantages that:

according to the application, gene stability evaluation is carried out on 9 internal references through GeNorm software, NormFinder software and BestKeeper software, and the internal reference genes stably expressed in different development periods of male flowers of the willows are screened out through analyzing results of the software. The method comprises the steps of obtaining two stably expressed reference genes which are DnaJ and ACT genes respectively, determining homologous genes in the salix dustpan by comparing with the whole genome of the salix dustpan, designing quantitative primers for each gene, screening the two reference genes suitable for the salix dustpan in different development periods by taking cDNA (complementary deoxyribonucleic acid) of the male flowers of the salix dustpan in 4 different development periods as templates, simultaneously disclosing the sequences of the two reference genes, and designing real-time fluorescent quantitative primers based on the sequences. The method solves the problem that no internal reference gene exists in the existing dustpan willow male flower detection, and the designed real-time fluorescent quantitative primer has strong primer specificity when being used for gene expression analysis in the dustpan willow male flower development process, so that the detection efficiency when good genes in the dustpan willow male flower are detected in a real-time fluorescent quantitative mode can be greatly improved, and the reliability of a detection result is improved.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments will be briefly described below.

FIG. 1 is a drawing showing a sample collected from a male flower of Salix dustpan, in which FIG. 1A shows a differentiation period of the male flower, FIG. 1B shows a resting period of the male flower, FIG. 1C shows an initial flowering period of the male flower, and FIG. 1D shows a full flowering period of the male flower;

FIG. 2 is a gel diagram of agarose gel electrophoresis for detecting RNA quality, wherein the leftmost side is Maker, and A, D, C, D sequentially corresponds to four samples in different periods in FIG. 1;

FIG. 3 is a boxed graph of Ct value distributions for 9 candidate reference genes;

FIG. 4 is a graph of the results of determination of the optimal number of reference genes for accurate quantitative analysis by geNorm;

FIG. 5 is a graph of expression stability of 9 candidate reference genes calculated by geNorm software;

FIG. 6 is a result chart of the selected reference gene by SmCAT assay.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to the accompanying drawings. The following examples are only for illustrating the technical solutions of the present invention more clearly, and therefore are only examples, and the protection scope of the present invention is not limited thereby. It is to be noted that, unless otherwise specified, technical or scientific terms used herein shall have the ordinary meaning as understood by those skilled in the art to which the invention pertains.

The main test materials used in the following examples were: the tested material was willow planted in white horse base of Nanjing forestry university. Randomly selecting 3 plants which grow well and have consistent height and growth vigor as samples, selecting four stages of male flower development (male flower differentiation period, dormancy period, early flowering period and full flowering period) from 2020, 9 to 2021, 3, collecting 3 male flowers from each tree in each period, quickly freezing by liquid nitrogen, and storing at-80 ℃ until use.

Examples

1. Total RNA extraction and cDNA synthesis of plant tissue

Total RNA was isolated from male flowers at different stages of development using the RNAprep Pure Polysaccharide Plant Total RNA ExtrACTion Kit (TIANGEN, NanJing, China), and RNA concentration and quality were determined using a spectrophotometer (METTLER TOLEDO, Switzeridand). RNA was detected by electrophoresis on 1.0% agar gel. Preparation of cDNA reverse transcription reaction System for real-time fluorescence relative quantitative PCR analysis A20. mu.L cDNA reverse transcription reaction system was constructed using 1. mu.g total RNA, according to One-Step gDNA Removal and cDNA Synthesis SuperMix (Trans, NanJeng, China)) instructions. After the reaction is finished, the reaction solution is stored at-20 ℃ for later use.

2. Selection of reference genes and design of primers

The major reference genes in other plants were queried according to literature and the 9 reference genes needed for the experiment were finally designed, DnaJ (SEQ ID NO.1), ACT (SEQ ID NO.2), α -TUB2(SEQ ID NO.7), H2A (SEQ ID NO.8), α -TUB1(SEQ ID NO.9), CDC2(SEQ ID NO.10), GAPDH (SEQ ID NO.11), TIP41(SEQ ID NO.12), β -TUB (SEQ ID NO.13), respectively. Firstly, the applicant compares the sequence with the genome sequence of the salix dustpan to determine the full-length sequence and the full-length transcript sequence of each candidate gene, designs a primer by using PrimerPremier 5.0 according to the transcribed full-length sequence of the gene, and identifies the specificity of the primer by using a BLAST tool. The primers were all synthesized by Biotechnology, Inc. (Table 1), and the specificity of the products was examined by initially screening the primers by ordinary PCR, observing the bands of the PCR products using agarose gel and gel imaging systems. Selecting a primer with correct band size, good band specificity and no primer dimer, further detecting the primer specificity by qRT-PCR, and selecting a primer with a single peak image, no miscellaneous peak and no peak in negative control as a final primer.

TABLE 1 reference genes and their corresponding primers

3. Establishment of reference gene primer standard curve

And (3) preparing respective standard curves for the primers of each pair of reference genes, and calculating the amplification efficiency of the corresponding primers. The reverse transcribed cDNA was diluted 5-fold into 5 gradients (1, 1/5, 1/25, 1/125, 1/625) as template for the establishment of the standard curve. At the same time, by dd H₂And O is used as a negative control template to detect reagents or artificial pollution in the experimental process. All samples were replicated 3 times to ensure confidence in the experimental data. qRT-PCR was performed using Applied Biosystems StepOne (Thermo Fisher Scientific, USA), and the amplification efficiency and slope of each candidate gene were determined using the obtained data results, and the amplification efficiency of fluorescent quantitative PCR required that the amplification efficiency of the selected primers was between 90% and 110%.

4. Fluorescence real time quantitation

qRT-PCR was performed using an Applied Biosystems StepOne (Thermo Fisher Scientific, USA), each reaction containing forward and reverse primersEach 4pM, 10-fold diluted template cDNA2ul, 10ul PowerUp^TMSYBR^TMGreen MasterMix (Thermo Fisher, USA), plus ddH₂O to 20ul reaction system. Each reaction was performed in triplicate. The reaction was performed on a 7500Fast Real-Time PCR System (Applied Biosystems, USA). The qRT-PCR reaction conditions were: the melt curve analysis was performed at 95 ℃ for 3 minutes, followed by 40 cycles of 95 ℃ for 15 seconds, 60 ℃ for 15 seconds, 72 ℃ for 30 seconds, at the end of each experiment, using default parameters at 55-95 ℃ for a period of 60 seconds in 0.3 ℃ increments, all analyses being set to three replicates.

5. Data processing

(1) The geNorm software calculates the value of gene expression stability M by using a Pairwise comparison method (Pairwise comparison approach). The judgment criterion is that when the M value is less than 1.5, the stability is higher the lower the M value is, and conversely, the stability is worse. The software can also calculate the average paired variation V value of the normalization factor after introducing 1 new reference gene, and determine the number of the required optimal reference genes according to the Vn/Vn +1 value. When the pair variation value Vn/Vn +1 is less than 0.15, the most suitable genes of the reference gene combination are the top n of the high stability ranking. On the other hand, if Vn/Vn +1>0.15, the number of optimum reference gene combinations is n + 1.

(2) The NormFinder software adopts a model-based method to obtain an expression stability value of an internal reference gene, then screens out the most appropriate internal reference gene according to the stability value, and the internal reference gene with the minimum expression stability value is the most appropriate internal reference gene according to the judgment standard.

(3) The BestKeeper calculates the Standard Deviation (SD) and the Coefficient of Variation (CV) of the Ct value by adopting repeated pairing correlation analysis, correlation analysis and regression analysis, and finally determines the reference gene with better stability by comparing the values.

6. Results

(1) RNA extraction quality and primer specificity detection

OD260/OD280 and OD260/OD230 of each sample meet the requirements, and detection of an agarose gel electrophoresis image shows that 28S and 18S bands are clear, the degradation phenomenon does not occur, and the requirements are met (figure 2). The melting curves of the 9 pairs of primers in the fluorescence real-time quantification are only provided with an obvious single peak, and the electrophoresis detection is also only provided with a single strip diagram, which shows that the primers can specifically amplify the corresponding products of the reference genes, no primer dimer exists, the repeatability of the amplification curve of each sample to be detected is good, the template can specifically amplify, and the real-time fluorescence quantification result is accurate and reliable.

(2) Ct value analysis of reference Gene

The Ct value is inversely proportional to the expression level of the gene, the larger the Ct value is, the lower the expression level of the gene is, and conversely, the smaller the Ct value is, the higher the expression level of the gene is represented. The mean value of Ct values of 9 reference genes was 16.59-29.79, with higher DnaJ and ACT expression levels and lower beta-TUB expression levels (FIG. 3).

(3) Software analysis

Analysis by using the GeNorm software: the geNorm software measures the stability of the gene according to the average variation M value, the default cut-off value of the software is 1.5, the gene higher than 1.5 is not suitable for being used as an internal reference, and the lower the M value is, the more stable the gene is. In addition, the geNorm software also determines the appropriate number of the reference genes according to the pairing difference value Vn/Vn +1 of the candidate reference genes, and when the Vn/Vn +1 is less than 1.5, the number of the n reference genes is adopted. According to the software, it is calculated that V2/V3 is 0.147 < 1.5, so two reference genes (FIG. 4), DnaJ and ACT (FIG. 5) should be selected in different development stages of male flowers.

Analysis by NormFinder software: the NormFinder software is evaluated by calculating the stability value of the candidate reference gene, and the gene with the minimum stability value is the most stable gene when the stability value is higher and the stability is worse.

The value of DnaJ was 0.072, the most stable gene, and the value of CDC2 was 0.930, the most unstable gene (Table 2).

TABLE 2 analysis results of the NormFinder software

Ranking	1	2	3	4	5
						Gene	DnaJ	ACT	H2A	α-TUB2	α-TUB1
Stable value	0.072	0.194	0.387	0.445	0.453
						Ranking	6	7	8	9
Gene	TIP41	β-TUB	GAPDH	CDC2
						Stable value	0.628	0.662	0.902	0.930

Bestkoeper software analysis: the BesterKeeper software judges the expression stability of genes based on the Standard Deviation (SD) and the Coefficient of Variation (CV) of the Ct value of an internal reference gene, and directly analyzes the Ct value of gene expression. The smaller the SD value, the more stable the expression, the program default threshold value is 1, when SD value is greater than 1, the gene expression is considered unstable. The results of the analysis showed that α -TUB1, TIP41, H2A and β -TUB all had SD values greater than 1, the remaining genes had SD values less than 1, DnaJ was the most stable expression and β -TUB was the most unstable expression (Table 3).

TABLE 3 BestKeeper software analysis results

Ranking	1	2	3	4	5
						Gene	DnaJ	ACT	α-TUB2	CDC2	GAPDH
SD	0.44	0.56	0.58	0.61	0.65
						CV	2.18	2.59	2.74	2.84	3.13
Ranking	6	7	8	9
						Gene	α-TUB1	TIP41	H2A	β-TUB
SD	1.10	1.27	1.82	2,32
						CV	5.07	6.26	8.19	11.44

(4) Verification of stability of reference gene

The results of analysis by combining BestKeeper, geNorm and Normfinder 3 software show that DnaJ and ACT are suitable reference genes for the development of male flowers of Salix dustpan (Table 4).

TABLE 4 composite ranking

Ranking	1	2	3	4	5
						BestKeeper	DnaJ	ACT	α-TUB2	CDC2	GAPDH
NormFinder	DnaJ/ACT		α-TUB2	α-TUB1	H2A
						geNorm	DnaJ	ACT	H2A	α-TUB2	α-TUB1
Comprehensive ranking	DnaJ	ACT	α-TUB2	H2A	α-TUB1
						Ranking
	6	7	8	9
						BestKeeper	α-TUB1	TIP41	H2A	β-TUB
NormFinder	β-TUB	TIP41	GAPDH	CDC2
						geNorm	TIP41	β-TUB	GAPDH	CDC2
Comprehensive ranking	GAPDH	TIP41	β-TUB	CDC2

SmCAT (SEQ ID NO.14) is selected to verify the stability of the screened internal reference genes DnaJ and ACT, and CDC2 genes with unstable expression are used for comparison, so that the situation that SmCAT has similar variation trend when the screened internal reference genes are used as internal references is found, but the expression conditions of SmCAT genes are different when CDC2 genes are used as internal references, and the screened internal reference genes are accurate and reliable (figure 6).

Unless specifically stated otherwise, the numerical values set forth in these examples do not limit the scope of the invention. In all examples shown and described herein, unless otherwise specified, any particular value should be construed as merely illustrative, and not as a limitation, and thus other examples of example embodiments may have different values.

Sequence listing

<110> Nanjing university of forestry

<120> reference gene suitable for fluorescence quantification of male flowers of salix dustpan, and primer and application thereof

<130> 2021

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aagcgcgctt tcgttcactg gtatgttggt gagggcatgg aggagggtga gttctccgag 1260

gcgcgtgagg atcttgctgc actcgagaag gattatgaag aggtaggcgc ggaatcagcc 1320

gagggtgagg atgaagacgg tgaggagtac atgtga 1356

<210> 10

<211> 885

<212> DNA

<213> dustpan willow (Salix suchowensis)

<400> 10

atggatcagt atgagaaagt ggaaaaaatt ggtgaaggaa cctacggagt ggtctacaaa 60

gctcgtgatc gtgtcaccaa tgagaccatt gctttgaaga agatccgttt ggagcaggaa 120

gacgagggcg tacccagcac tgctatccga gaaatctctc tcttgaaaga gatgcagcat 180

ggtaacattg tcagactgca ggatgtggtg cacagtgaga agcgccttta cttggttttt 240

gagtatctag acttggattt gaagaagcac atggattctt cacctgaatt tgctaatgat 300

ccacgcctgg ttaaaacatt tctttatcaa attctccgtg gcattgcata ctgccattct 360

catagagttc tgcatcgaga tttgaaacct cagaatttgc tcattgatcg ccgtaccaac 420

gcgctgaagc ttgcagattt tggactggct agagcatttg gtatacctgt taggacattt 480

acacatgagg ttgttaccct gtggtataga gcccctgaaa ttctgcttgg atctcgccat 540

tactcgactc cagttgatgt atggtcagtg ggatgtatat ttgctgagat ggtgaaccag 600

aagccactgt tcccagggga ttccgagata gatgaactat tcaaaatttt cagaatcttg 660

ggtactccaa atgaggacac atggcccgga gttacttctt tgcctgactt caagagtgca 720

ttccctaagt ggccttctaa ggatttggca actgtagttc caactcttga taaagctggc 780

gtggatcttc tcgctaaaat gctttgcttg gatcccacta aaagaattac tgccaggagt 840

gctttggagc atgaatactt caaggatatt ggttttgtac cttaa 885

<210> 11

<211> 1362

<212> DNA

<213> dustpan willow (Salix suchovensis)

<400> 11

atggctaccc acgcagctct tgcctcttca agaatccctg ccaatacaag acttccctca 60

aagatcaacc actctttccc cactcaatgc tccttaaaga ggctagaagt ggctgagttt 120

tctgggcttc gagccagttc atgtgtaacc tatgccaaga acgctggtga gggatccttc 180

tttgatgtgg tggcttccca acttgctcca aaggttgcag tttcaactcc tgtcagggca 240

gaaactgtgg ccaaattaaa ggttgctatc aacggatttg gacgcattgg caggaacttc 300

ctgcgatgct ggcatggtcg caaagactct ccccttgatg taattgttgt caatgacagt 360

ggtggtgtca agaacgcttc ccacttgttg aaatatgatt caatgcttgg aactttcaaa 420

gcagaggtga aaattgtgga caacgagacc atcagtgttg atggcaagcc cattaaggtt 480

gtttccagca gagaccctct caagcttcct tgggctgagc tcggaataga cattgttatt 540

gagggaaccg gagtttttgt ggatggtcct ggtgctggga aacatattca agctggtgcc 600

aagaaagtta tcatcactgc tccagccaaa ggtgccgata ttccaaccta tgttgttggt 660

gttaacgaaa aggactacgg ccatgaggtt gccgacatta taagtaatgc ttcctgcacc 720

acaaattgtc tggctccctt tgtgaaaatc ctggatgaag aattcggcat tgtcaaggga 780

acaatgacaa caactcactc ctacactgga gatcagaggc tcttggatgc ttcacaccga 840

gacttgagga gagccagggc tgcagcattg aacatagtcc caacaagcac tggtgcagcc 900

aaggctgtat ctcttgtgct gccccagctc aagggcaagc tcaatggcat cgcactccgt 960

gtcccgacac ccaatgtttc agttgttgac cttgttgtga atgttgcgaa gaagggcatt 1020

acagcagaag atgtcaatgg agccttcaga aaggccgctg gggggccatt gaagggtgta 1080

ttggacgtgt gtgatgttcc tcttgtgtct gttgacttcc gatgctctga tgtttcctca 1140

accattgact cttcattgac catggtcatg ggagatgata tggtcaaggt tgtcgcctgg 1200

tatgacaatg aatggggata cagccaaagg gtcgtcgatt tagcacatct tgtagcggaa 1260

agtggccagg agtggctgca gcaggaagtg gagacccatt ggaggatttc tgcaagacaa 1320

acccagctga tgaggaatgc aaagtttatg aagcttagat ga 1362

<210> 12

<211> 870

<212> DNA

<213> dustpan willow (Salix suchovensis)

<400> 12

atggacgtgg aagtagatga caaagaccta aaagccgccg gcgccgaggt tttaaccgac 60

gaccgtcatg gactccgcat ccatggctgg gaaatcgtat cttgcaacgg ctccattctc 120

aactcctcct ctctgatcac atgggaagaa aagttaaaaa cttctcattt accagagatg 180

gtttttggag aaagttgttt agtacttaaa catgcgacca gtggtaccaa aattcatttc 240

aacgcgtttg atgccctaac tggctggaaa caagaggctt tacctccggt cgaagtccct 300

gctgctgcgc aatggaaatt tagaagcaaa ccattccagc aggtgatatt ggattatgat 360

tatacattca cgacgcctta ttgtggtagt gaaacaatgg aatttgacac agagaagaaa 420

aacagcgagg aatttctgga ggctagctgc agcccctgtt gggaagactg tgaagagcaa 480

attgatgtgg ttgcacttgc atcaaaagag cctattcttt tctatgatga ggtagtcttg 540

tatgaagatg aactggctga taatggggtg tcgcttttaa ctgttaaagt gagagtcatg 600

ccgagttgct ggtttcttct tttgcgattc tggcttagag ttgatggagt gctaatgaga 660

ttaagggaca ctcgtatgca ttgtattttt agtgacagcg caaatcctat tgttcttcga 720

gaaagctgct ggagagaagc cacctttgaa gctctggctg ctaaaggata tcctgctgac 780

tctgcttcat atagtgatcc aagcatcatc agccaaaggc ttcccatcat catgcacaag 840

actcaaaagc ttagggtcgg tccactgtaa 870

<210> 13

<211> 1341

<212> DNA

<213> dustpan willow (Salix suchowensis)

<400> 13

atgagagaaa tccttcacat ccaagcaggc caatgcggca accaaatagg agcaaagttt 60

tgggaagtag tatgtgcaga acacgggatt gactccaccg gtcggtacaa tggtgactcg 120

gctctccaac tcgagcgagt taatgtttac tataatgaag ccagctgtgg aagatttgtc 180

cctcgtgctg ttctagtgga tcttgaaccg ggtactatgg acagtcttag atccggcccg 240

tacgggcaga tttttagacc ggataatttt gtgtttggcc aatctggtgc tggtaataac 300

tgggctaaag gacattatac ggagggcgcg gagctgattg attctgttct tgatgttgtt 360

aggaaggagg ctgagaactg tgactgcctg caaggatttc aggtatgcca ctcactgggg 420

ggtggtacag ggtctggaat gggaacactt ttgatctcga aaataagaga ggaatacccg 480

gaccggatga tgctaacatt ctctgttttc ccatctccaa aggtctcaga cactgtggtc 540

gagccttaca atgcaactct ctctgttcac cagcttgttg aaaatgctga tgagtgtatg 600

gttcttgata atgaggctct ttatgatatc tgcttccgta ctctcaagct cgcaactccc 660

agctttgggg atctgaacca cctgatttca gccaccatga gtggtgttac atgctgcctt 720

cgtttccctg gtcagctcaa ttcagacctt cgcaaacttg ctgtgaacct catcccattc 780

ccccgtcttc actttttcat ggttggcttt gcacctctca cttcccgtgg ctcgcagcag 840

taccgttccc taactgtacc tgaactcacc caacaaatgt gggattccaa gaatatgatg 900

tgtgctgctg atccccgcca tggcagatat ctcacagcct ctgccatgtt tcgtgggaaa 960

atgagcacaa aggaagttga tgagcagatg atcaacgttc aaaacaagaa ctcatcctac 1020

tttgtcgaat ggatccccaa caatgtcaag tctactgtct gtgacattcc tcctacaggc 1080

ttgacaatgg cttccacttt cattggcaac tccacatcaa tccaagagat gttccgaaga 1140

gttagcgagc agttcactgc catgttccgc aggaaggctt tcttgcattg gtacacggga 1200

gagggaatgg atgagatgga atttacagag gctgagagca acatgaacga tttggtctca 1260

gagtaccaac aataccagga cgcaactgcc gacgaggaag gcgagtatga agatgaggaa 1320

gaataccagg atgaggccta a 1341

<210> 14

<211> 1479

<212> DNA

<213> dustpan willow (Salix suchovensis)

<400> 14

atggatccct acaagcaccg tccgtcaagc gctttcagca ctccatactg gactacaaat 60

tctggagctc cggtttggaa caacaactcg tctctgaccg ttggatctag aggtccaatc 120

ctcctcgagg attaccatct ggtggagaag attgccaatt ttgacaggga gaggattcca 180

gagcgtgtcg tccatgctag gggagccagt gcaaagggtt tctttgaggt tacccatgat 240

atctctggtc tcacatgtgc tgattttctc cgggcccctg gagttcagac acctgtcatt 300

gtccgcttct ccacagttat ccatgagcgt ggcagccctg aaaccctgag ggatccacgt 360

ggatttgcag tgaagtttta caccagagag ggtaactttg atcttgtggg aaacaatttc 420

cctgtcttct tcatccgtga tgggatgaaa ttcccagaca tggtgcatgc ccttaagccc 480

aaccccaagt ctcatattca ggagaactgg aggattcttg acttcttctc ccaccatcct 540

gaaagtttgc acatgttctc cttcctattt gatgatgtgg gtgtgccaca agattataga 600

catatggaag gctctggtgt taacacctac acgttgatca acaaggctgg aaaagcccat 660

tatgtgaaat ttcattggaa acctacttgt ggtgtgaaat gtttgttgga ggacgaggca 720

attaaagtag gaggcacaaa tcacagccat gctactcagg atctatacga ctccattgcg 780

gctggcaact atcctgagtg gaaacttttc attcagacaa ttgatcctga ccatgaagcc 840

aggtttgatt ttgacccact tgatgtaacg aagacctggc ctgaggatat cttgcccctg 900

cagccagttg gtcgcttggt cttgaataag aacatcgaca acttctttgc tgaaaatgag 960

cagcttgctt tctgccctgc tattgtggtt cctggtatct actattcaga tgacaagcta 1020

ctccagactc gaatcttctc ctattctgat acccagaggc accgtcttgg accaaactat 1080

ctgcagctcc ctgctaatgc tcccaagtgt gctcatcata acaatcacca tgaaggtttc 1140

atgaatttca tgcataggga cgaggaggtc aactatttcc catcaaggta cgatcctgtt 1200

cgccatgctg agagtttccc cattcctcct gctgtctgca gtggaaagcg tgagaagtgc 1260

atcattgaga aggagaacaa cttcaagcaa cctggagaga gataccgatc ctgggcacca 1320

gacaggcaag aacgatttat ttgccgatgg gttgatgcct tatctgaccc acgcgccaca 1380

tatgagattc gcagcatctg gatctcatac tggtctcagg ctgataaatc tttgggtcag 1440

aagctagcat ctcgtctcag cgtgagacca agcatttga 1479

Claims (4)

1. The application of the combination of the DnaJ gene and the ACT gene as reference genes in the fluorescent quantitation of the male flowers of the salix dustpan in the differentiation period, the resting period, the initial flowering period and the full flowering period is characterized in that the sequence of the DnaJ gene is shown as SEQ ID NO.1, and the sequence of the ACT gene is shown as SEQ ID NO. 2.
2. 2. The use of claim 1 wherein the primer sequences of the DnaJ gene are as follows:

   DnaJ Forward primer 5'-GAGCCTATTTATGCCCTTTC-3'

   DnaJ reverse primer 5'-GACGTAGTCCTTATCCACCTT-3'.
3. 3. Use according to claim 1, wherein the primer sequences of the ACT gene are as follows:

   ACT Forward primer 5'-CCAAGGAGGCTGCTGGTAAT-3'

   ACT reverse primer 5'-CCAAGGCCACCATGTCTGTC-3'.
4. 4. The application of the primer combination for detecting the DnaJ gene and the ACT gene in the fluorescence quantification of the male flowers of the salix dustpan in the differentiation period, the dormancy period, the initial flowering period and the full flowering period is characterized in that the primer combination consists of the following primers:

   DnaJ Forward primer 5'-GAGCCTATTTATGCCCTTTC-3'

   DnaJ reverse primer 5'-GACGTAGTCCTTATCCACCTT-3'

   ACT Forward primer 5'-CCAAGGAGGCTGCTGGTAAT-3'

   ACT reverse primer 5'-CCAAGGCCACCATGTCTGTC-3'.

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