CN113046460B

CN113046460B - Leek reference gene under gray mold stress condition, primer of reference gene and application

Info

Publication number: CN113046460B
Application number: CN202110249053.7A
Authority: CN
Inventors: 宋海娜; 贺军利; 程世平; 谢丽华; 姚鹏强; 张志录
Original assignee: Pingdingshan University
Current assignee: Pingdingshan University
Priority date: 2021-03-08
Filing date: 2021-03-08
Publication date: 2023-01-31
Anticipated expiration: 2041-03-08
Also published as: CN113046460A

Abstract

The invention belongs to the technical field of genetic engineering breeding, and particularly relates to a leek reference gene under the condition of gray mold stress, a primer of the reference gene and application of the reference gene. The invention provides reference genes UBC1 and UBC2 of leeks under the condition of gray mold stress, wherein the nucleotide sequence of UBC1 is shown as SEQ ID NO. 1, and the nucleotide sequence of UBC2 is shown as SEQ ID NO. 2. In the internal reference genes provided by the invention, UBC1 and UBC2 genes have extremely high expression stability, and the internal reference genes provided by the invention can be used for screening high-botrytis-resistance excellent germplasm resources in the existing Chinese chives, developing and utilizing key regulatory factors with induced resistance to botrytis cinerea, preventing botrytis cinerea from invading into host cells from the source, preventing botrytis cinerea from invading into Chinese chive cells by means of biological induction control, reducing the use of related pesticides for botrytis cinerea and enhancing the edibility and commodity of the Chinese chives.

Description

Leek reference gene under gray mold stress condition, primer of reference gene and application

Technical Field

The invention belongs to the technical field of genetic engineering breeding, and particularly relates to a leek reference gene under the condition of gray mold stress, a primer of the reference gene and application of the reference gene.

Background

The leek is a perennial root herbaceous plant in the genus of Allium in the family of Liliaceae, has the same source of food and medicine, contains rich nutrient substances such as carotene, protein, various vitamins, and more than 20 trace elements such as calcium, phosphorus, iron, and the like, and is cultivated all over the country in China. One of the diseases which are high in morbidity, wide in distribution and difficult to solve in the cultivation of the Chinese chives is gray mold caused by botrytis, pathogenic bacteria are facultative parasitic bacteria, generated conidia are spread along with wind, water, agricultural operations and the like, and plants are easy to rot after being infected.

The cultivation of high-quality and high-gray mold resistance germplasm resources is a main way for improving gray mold resistance of the Chinese chives, the Chinese chives have the germplasm resources with high gray mold resistance, but the Chinese chives have specific apomixis characteristics, most of filial generations are consistent with female parent characters, and the cultivation of a new excellent and high gray mold resistance variety by a traditional crossbreeding method is difficult.

Disclosure of Invention

In order to solve the problems, the invention provides an internal reference gene of leeks under the condition of gray mold stress, a primer of the internal reference gene and application of the internal reference gene. In the application of the internal reference gene provided by the invention, the UBC1 and UBC2 genes have extremely high expression stability, lay a foundation for screening the expression of the key candidate gene of the anti-gray mold Chinese chives in Chinese chive breeding, and can improve the commodity and the edibility of the Chinese chives.

In order to achieve the purpose, the invention provides the following technical scheme:

the invention provides a leek reference gene under the condition of gray mold stress, wherein the reference gene comprises UBC1 and UBC2 genes; the nucleotide sequence of UBC1 is shown as SEQ ID NO. 1, and the nucleotide sequence of UBC2 is shown as SEQ ID NO. 2.

The invention also provides a group of amplification primers of leek internal reference genes UBC1 and UBC2, wherein the amplification primers are designed to obtain UBC1 and UBC2 amplification primers based on UBC1 and UBC2 gene sequences in the internal reference genes according to the principle of real-time fluorescent quantitative PCR primer design.

Preferably, the nucleotide sequence of the amplification primer of UBC1 is shown as SEQ ID NO. 3 and SEQ ID NO. 4; the nucleotide sequence of the amplification primer of UBC2 is shown as SEQ ID NO. 5 and SEQ ID NO. 6.

The invention also provides the application of the reference gene or the amplification primer in fluorescent quantitative PCR detection.

The invention also provides the application of the reference gene or the amplification primer in quantitative detection of the leek gene.

The invention also provides the application of the reference gene or the amplification primer in screening the related gene for resisting gray mold in leeks.

The invention also provides application of the reference gene or the amplification primer in screening gray mold resistant leek varieties caused by the botrytis cinerea.

The invention provides reference genes UBC1 and UBC2 of leeks under the condition of gray mold stress, wherein the nucleotide sequence of UBC1 is shown as SEQ ID NO. 1, and the nucleotide sequence of UBC2 is shown as SEQ ID NO. 2. In the internal reference genes provided by the invention, UBC1 and UBC2 genes have extremely high expression stability, and the internal reference genes provided by the invention can be used for screening high-botrytis-resistance excellent germplasm resources in the existing Chinese chives, developing and utilizing key regulatory factors with induced resistance to botrytis cinerea, preventing botrytis cinerea from invading into host cells from the source, preventing botrytis cinerea from invading into Chinese chive cells by means of biological induction control, reducing the use of related pesticides for botrytis cinerea and enhancing the edibility and commodity of the Chinese chives.

Drawings

FIG. 1 is a melting curve of EF-1. Alpha. Gene;

FIG. 2 is a melting curve of GAPDH3 gene;

FIG. 3 is a melting curve of GAPDH4 gene;

FIG. 4 is a melting curve of the TUB gene;

FIG. 5 is a melting curve of UBC1 gene;

FIG. 6 is a melting curve of UBC2 gene;

FIG. 7 is a melting curve of UBQ1 gene;

FIG. 8 is a melting curve of UBQ2 gene;

FIG. 9 is CT value analysis of candidate reference genes at different time when Allium fistulosum infects leeks;

figure 10 is the results of analysis by the geonorm software.

Detailed Description

The invention provides a leek internal reference gene under the condition of gray mold stress, which comprises UBC1 and UBC2 genes, wherein the nucleotide sequence of the UBC1 is shown as SEQ ID NO. 1, and the nucleotide sequence of the UBC2 is shown as SEQ ID NO. 2.

The reference genes UBC1 and UBC2 provided by the invention have extremely high expression stability, and when leeks are infected with gray mold, the UBC1 and UBC2 genes cannot be affected by exogenous or endogenous genesThe influence of the factors can still keep stable expression, and a stable reference gene is provided for the expression and analysis of the leek gene under the condition of botrytis cinerea stress as a reference. The UBC1 and UBC2 genes are stably expressed in folium Allii tuberosi leaf under the stress of Botrytis cinerea-Leptospermum scoparium and normal conditions, and can be relatively quantified by fluorescent quantitative PCR (2) ^－△△CT Method), determining the expression level of the candidate gene related to the gray mold resistance of the Chinese chives, and further screening the related gene of the Chinese chives, which is resistant to the gray mold (gray mold caused by botrytis cinerea) according to the change of the expression level of the candidate gene under stress and normal conditions. The relative quantification of the expression level of the target gene is analyzed by comparing CT (Cycle threshold) values, and the relative expression level of the target gene in each sample is 2 ^–ΔΔCT . Δ Δ CT = (CT) here _{Target gene} -CT _{Internal reference gene} ) _Time,X -(CT _{Target gene} -CT _{Internal reference gene} ) _Time,0 。

The invention also provides a group of amplification primers of leek internal reference genes UBC1 and UBC2, wherein the amplification primers are designed to obtain UBC1 and UBC2 amplification primers based on the UBC1 and UBC2 gene sequences in the application according to the principle of real-time fluorescent quantitative PCR primer design. In the present invention, the nucleotide sequence of the amplification primer of UBC1 is preferably as shown in SEQ ID NO. 3 and SEQ ID NO. 4; the nucleotide sequence of the amplification primer of UBC2 is preferably shown as SEQ ID NO. 5 and SEQ ID NO. 6. The amplification primer provided by the invention is designed based on UBC1 and UBC2 genes, and can be used for stably amplifying to obtain a target sequence.

The invention also provides the application of the internal reference gene or the amplification primer in quantitative detection of the leek gene.

The invention also provides application of the reference gene or the amplification primer in screening gray mold resistant leek varieties caused by botrytis cinerea. The UBC1 and UBC2 genes or the amplification sequences can screen the gray mold Chinese chives variety caused by the high-resistance staphylococcus scallion through the marker gene of the Chinese chives high-resistance staphylococcus scallion (the expression quantity of the marker gene is in direct proportion to the gray mold resistance caused by the staphylococcus scallion) according to the change of the expression quantity of the marker gene.

In order to further illustrate the present invention, the following detailed description will be given with reference to the accompanying drawings and examples for a set of leek reference genes and primers and applications of the reference genes provided in the present invention, but they should not be construed as limiting the scope of the present invention.

Examples

1. Preparation of the experiment

(1) Preparation of Experimental materials

1) Culture of botrytis squamosa

Preparing a fresh PDA culture medium, weighing 200g of peeled potatoes, 20g of cane sugar, 20g of agar powder and 1000ml of pure water. Sterilizing at 121 deg.C for 20min in autoclave, cooling, and pouring into plate in clean bench. And (3) picking the purified staphylococcus scallion botrytis hyphae to a new PDA culture medium by using an inoculating loop, streaking, and culturing in an incubator at 20 ℃ for 3d for later use.

2) Inoculation and culture of leek leaves

Taking the healthy and disease-free gray mold resistant leek leaves which are proved to have vigorous growth by experiment Tian Zhongqu and are sterilized ddH in a super clean workbench ₂ Cleaning for 2-3 times, sucking excess water with sterilizing filter paper, cutting into folium Allii tuberosi segments with length of about 5cm with scissors, and puncturing the epidermis of the middle part of folium Allii tuberosi with dissecting needle to form wound.

Experimental groups: taking the botrytis squamosa fungus cake with the diameter of 5mm, provided by the step 1), and inversely inoculating the botrytis squamosa fungus cake to the wound of the leaf;

control group: the wound of folium Allii tuberosi leaf is inoculated with sterile PDA culture medium with diameter of 5mm and cultured at 20 deg.C for 3 days.

The leek leaves of the control group and the experimental group are respectively treated for 20 sections, placed in a culture dish paved with sterile water-soaked sterile filter paper, covered on the culture dish by a sterile preservative film, cultured in a thermostat with the temperature of 20 ℃, the humidity of 95 percent and the illumination of 12 h/darkness of 12h for 0h,24h,48h and 72h, and then sampled. Taking 3 leaves from the experimental group and the control group at each culture stage, and recording the samples of the control group of 0h as CK0-1, CK0-2 and CK0-3; samples of the experimental group 24h are marked as T24-1, T24-2 and T24-3, and samples of the corresponding control group are marked as CK24-1, CK24-2 and CK24-3; samples of the experimental group of 48h are marked as T48-1, T48-2 and T48-3, and samples of the corresponding control group are marked as CK48-1, CK48-2 and CK48-3; samples of the experimental group 72h were designated as T72-1, T72-2, T72-3, and samples of the corresponding control group as CK72-1, CK72-2, CK72-3; and (4) quickly freezing the sample by liquid nitrogen, and storing the sample in an ultra-low temperature refrigerator at-80 ℃ for later use.

(2) Extraction and reverse transcription of total RNA of leek leaves

The extraction of the total RNA of the leek leaf sample adopts an RNAscope total RNA extraction kit (DP 419) of Tiangen Biochemical technology (Beijing) Co., ltd, and the operation steps are carried out according to the standard steps of the kit. After the leek RNA sample is qualified by agarose gel electrophoresis, cDNA is synthesized by using a PrimeScript (TM) first strand cDNA synthesis kit (6110A) of Takara company, and the operation steps refer to the instruction book, and the cDNAs (CK 0-1, CK0-2, CK0-3, T24-1, T24-2, T24-3, CK24-1, CK24-2, CK24-3, T48-1, T48-2, T48-3, CK48-2, CK48-3, T72-1, T72-2, T72-3, CK72-1, CK72-2 and CK 72-3) synthesized by different groups in the step (1) are stored in a refrigerator at the temperature of-20 ℃ for later use.

(3) Primer design of candidate reference genes

The homologous sequences of leek beta-Tubulin (Tubulin, TUB), ubiquitin-like extension protein (UBQ), polyubiquitin-like enzyme gene (UBC), glucose-3-phosphate dehydrogenase (Glycerebrade-3-phosphated-gene-Nase, GAPDH) and transcription elongation factors (EF-1 alpha) are screened according to sequencing annotation results of leek leaf transcriptome induced by Botrytis schoenoprashii at different times and full-length transcriptome sequencing sequence information by reference to a reference plant common internal reference gene.

Wherein the nucleotide sequence of GAPDH3 is shown as SEQ ID NO. 7 in the sequence table,

the nucleotide sequence of GAPDH4 is shown as SEQ ID NO 8 in the sequence table;

wherein the nucleotide sequence of UBQ1 is shown as SEQ ID NO. 9 in the sequence table,

the nucleotide sequence of UBQ2 is shown as SEQ ID NO 10 in the sequence table;

wherein the nucleotide sequence of TUB is shown as SEQ ID NO. 11 in the sequence table,

the nucleotide sequence of EF-1 alpha is shown as SEQ ID NO. 12 in the sequence table.

Corresponding primer sequences were designed and the primer sequence information is shown in table 1.

TABLE 1 sequence information of candidate internal reference gene primers for Chinese chives

(4) Preliminary detection of reference Gene

And (3) mixing 21 parts of cDNA (CK 0-1, CK0-2, CK0-3, T24-1, T24-2, T24-3, CK24-1, CK24-2, CK24-3, T48-1, T48-2, T48-3, CK48-2, CK48-3, T72-1, T72-2, T72-3, CK72-1, CK72-2 and CK 72-3) with initial concentration obtained by reverse transcription provided in the step (2) to obtain a sample M1, diluting the cDNA of the M1 by 20 times, and performing fluorescence quantitative PCR amplification by using the diluted cDNA of the M1 as a template and the primers provided in the table 1.

The fluorescence quantitative PCR kit adopts the method of Saimerfi corporation

SelectMasterMix (REF: 4472908), 20. Mu.L of the reaction system: masterMix 10. Mu.L, 2. Mu.L each of upstream and downstream primers (1 pmol), 1. Mu.L of cDNA template, 5. Mu.L of cDNA ₂ And O. The fluorescent quantitative PCR amplification procedure is as follows: pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 15s, extension at 60 ℃ for 30s, reaction for 40 cycles, and collection of fluorescence signals at 60 ℃. Melting curve program: 95 ℃ for 15s; at 65 ℃ for 1min;95 ℃,20s, and stepping 0.2 ℃ every 20 s; 30 ℃ for 1min. Each one of which isThe sample is repeated for three times, the Cycle Threshold (CT) of the sample to be measured is derived according to the melting curve, the specificity of each pair of primers is judged, and the melting curve can be used for subsequent experiments when the melting curve is a single peak. The reaction is carried out in a ViiA fluorescent quantitative PCR instrument of ABI in America ^TM 7 model real-time fluorescent quantitative PCR system (Applied Biosystems), and the data analysis adopts Quant Studio ^TM Real-time PCR software.

(5) Standard curve construction and primer amplification efficiency analysis

Diluting the cDNA of the M1 obtained in the step (4), and sequentially diluting by 5 times to obtain cDNA samples of which the M1 is diluted by 5 times, 25 times, 125 times and 625 times; fluorescence quantitative PCR was performed using the cDNA of M1 diluted in a gradient as a template, and the primers were those provided in Table 1. Analyzing the linear relation between the mass concentration (logarithm value) and the CT value of the cDNA template by using Excel software to obtain a correlation coefficient (R) ² ) And slope (S) using the formula E = (5) ^-1/S -1) × 100% the amplification efficiency of the primers was calculated.

(6) Analysis of expression stability of reference Gene

And respectively diluting the cDNA of the leek leaves in the experimental group and the cDNA of the leek leaves in the control group at the corresponding time point by 20 times, and performing fluorescent quantitative PCR amplification by using the selected candidate reference gene primer pair with the melting curve being a single peak, wherein the preparation and amplification procedures of the reaction system are the same as those described above. The amplification results were analyzed for expression stability of candidate reference genes using GeNorm, normfinder and Bestkeeper software. The GeNorm sorts the candidate reference genes according to the expression stability (M value), wherein the smaller the M value is, the more stable the gene expression is; paired difference analysis according to normalization factor (V) _n/n+1 ) Determination of the best set of reference genes, V _n/n+1 < 0.15 represents that the optimum number of reference genes under this condition is n. The NormFinder software can screen out the optimal reference gene according to the expression stability of the reference gene; according to CT values of different candidate internal reference genes under different conditions, the Bestkeeper software judges the stability of the internal reference genes by comparing the magnitude of a correlation coefficient (r), a Standard Deviation (SD), a Coefficient of Variation (CV) and a P value of pairing generated among the genes. When SD>1 or P>0.05, the gene is judged to be unsuitable as an internal reference gene; correlationThe larger the coefficient is, the smaller the standard deviation and the coefficient of variation are, and the better the stability of the reference gene is.

2. Test and conclusion

(1) Primer specificity and amplification efficiency

21 initial concentrations of cDNAs (CK 0-1, CK0-2, CK0-3, T24-1, T24-2, T24-3, CK24-1, CK24-2, CK24-3, T48-1, T48-2, T48-3, CK48-2, CK48-3, T72-1, T72-2, T72-3, CK72-1, CK72-2 and CK 72-3) obtained in the example step (2) were mixed to obtain a sample M1, the cDNAs of M1 were diluted 20 times, and the specificity of the primers was verified by quantitative fluorescence PCR using 8 candidate internal reference genes in Table 1 using the diluted mixed cDNAs as templates, and the results are shown in FIGS. 1 to 8, wherein FIG. 1 is a melting curve of the EF-1. Alpha. Gene, FIG. 2 is a melting curve of the GAPDH3 gene, FIG. 3 is a melting curve of the GAPDH4 gene, FIG. 4 is a melting curve of the UBB gene, FIG. 5 is a melting curve of the EF-1. Gene, and FIG. 2 is a melting curve of UBQ-2 is a melting curve of the UBQ-2. The results show that the melting curves of the 8 pairs of primers are all unimodal, indicating that the primers provided in table 1 are specific.

The mixed cDNA diluted 20 times was diluted by 5 times to obtain 5-, 25-, 125-and 625-times diluted cDNA samples, and the obtained 1-, 5-, 25-, 125-and 625-times diluted cDNA samples were used as templates for the fluorescent quantitative PCR amplification using the primers provided in Table 1, and the amplification efficiency is shown in Table 2.

TABLE 2 fluorescent quantitative PCR amplification efficiency analysis of candidate internal reference genes of Chinese chives

As can be seen from Table 2, the amplification efficiency of the amplification primers provided by the invention is 101.12% -113.00%, R ² Is 0.9912-0.9971, and the result shows that the mass concentration of cDNAAnd a clear linear relation exists between the PCR primer and the CT value, and the amplification efficiency of the primer meets the fluorescent quantitative requirement.

(2) Analysis of expression level of reference Gene

The cDNA of the leek leaves of the experimental group was diluted 20 times, respectively, for 0h,24h,48h and 72h and the leek leaves of the control group for the corresponding time, and then subjected to fluorescent quantitative PCR amplification, and the results are shown in FIG. 9 and Table 3. FIG. 9 is a CT value analysis of candidate reference genes at different times of infection of leeks by Botrytis schoenoprashii, wherein the horizontal lines in the boxes represent the mean values.

TABLE 3 CT values of candidate internal reference genes at different time of infection of leek by Botrytis schoenoprashii

As can be seen from fig. 9 and table 3, the CT values of the candidate reference genes are 24.07 to 31.99, and the CT values of the different candidate reference genes are different, wherein the average CT values of UBC2, UBQ2 and GAPDH4 are smaller and about 25; the average Ct value of UBQ1 is the largest and is 31.498; average CT value of GAPDH3 is 26.95; the average CT value of EF-1 alpha, TUB and UBC1 is about 28. The difference of CT values of different candidate reference genes indicates that the expression levels of the candidate reference genes are different, wherein the expression levels of UBC2, UBQ2 and GAPDH4 are the highest.

(3) Analysis of expression stability of reference Gene

1) GeNorm software calculates the stability of gene expression (M value), and in general, genes with an M value less than 1.5 are considered to express relatively stable genes, with the smaller the M value, the more stable the gene expression. The results of measurement of 8 candidate reference genes provided in examples are shown in fig. 10, table 10, and table 11. Wherein A in FIG. 10 is the analysis of the expression stability of the candidate reference gene and B in FIG. 10 is the determination of the normalized reference gene.

TABLE 10 stability of Gene expression (M value)

TABLE 11 paired difference analysis of normalized reference genes

As can be seen from A in FIG. 10 and Table 10, the M values of the 8 candidate reference genes were all less than 1.5 at different time periods when leek was infected with Botrytis schoenopsis fistulosi, and the expression of both UBC1 and UBC2 genes was most stable, followed by UBQ1 > GAPDH4 > UBQ2 > GAPDH 3> TUB > EF-1 α.

As can be seen from B in FIG. 10 and Table 11, the optimal number of the reference genes was determined according to (Vn/n + 1), and from B in FIG. 10, all Vn/n +1 were less than 0.15 during the different time periods when the leeks were infected with Botrytis schoenopsis scallion, indicating that there were 2 reference genes with the most stable expression in leeks.

2) The NormFinder software evaluates the stability of genes by comparing the intra-and inter-group variation of samples and the results of the software analysis are shown in table 12.

TABLE 12NormFinder software analysis of the expression stability of the internal reference genes of leek induced by Botrytis schlengensis

From Table 12, the most stable genes are UBQ1, followed by UBC2 and UBC1.

3) According to CT values of different candidate internal reference genes under different conditions, the Bestkeeper software judges the stability of the internal reference genes by comparing the magnitude of a correlation coefficient (r), a Standard Deviation (SD), a Coefficient of Variation (CV) and a P value of pairing generated among the genes. When SD >1 or P >0.05, the gene is judged to be unsuitable as an internal reference gene; the larger the correlation coefficient is, the smaller the standard deviation and the variation coefficient are, and the better the stability of the internal reference gene is.

TABLE 13BestKeeper software analysis of expression stability of candidate reference genes

It can be seen from table 13 that the SD values of 8 candidate reference genes are all less than 1, but the P values of GAPDH3, EF-1 α, UBQ2 and TUB are greater than 0.05, so these four genes are not suitable as reference genes under the present experimental conditions, the P values of the remaining 4 genes UBQ1, UBC2 and GAPDH4 are less than 0.05, and the correlation coefficients of these four genes are large and have relatively high stability.

The analysis results of three software combined show that the expression stability of four genes of UBQ1, UBC1, UBC2 and GAPDH4 has different ranks, the geNorm software analyzes that the gene with the most stable expression has two genes of UBC1 and UBC2, then UBQ1 and GAPDH4, and the NormFinder software analyzes that the gene with the most stable expression has UBQ1, then UBC2, UBC1 and GAPDH4. The BestKeeper software analysis shows that UBQ1, UBC1, UBC2 and GAPDH4 have higher stability, but the CT value of the UBQ1 is higher relative to other 3 genes, and the Standard Deviation (SD) and the variation Coefficient (CV) of the GAPDH4 are higher relative to other 3 genes, so that the UBC1 and the UBC2 are reference genes which are stably expressed in the leek leaves under the condition of botrytis cinerea-schoenophora fistulosa infection.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Sequence listing

<110> Flat-topped mountain college

<120> reference gene of leek under gray mold stress condition, primer of reference gene and application

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gaggagaagt ccgaagttgc ggcggcacag cagaggttga tctataaagg acggatcttg 180

aaggatgatc aaaccctagc tagctacggt gtggaagctg accataccat tcacttggtt 240

cgtggttttg cacagtcagt gtcttctaac acaggagcaa caaatgttaa tgcatctgga 300

actgcaaact ccagtcctgg agttacaaga ggtgtcggtg tcactcctga aggtttcagc 360

ccattaccat ttccaaatct tagctctata ggtgcggcta atctatctgc aactgggttc 420

ccagaacttg atagaatgca gcaacaaatt actcagaatc caaacatgat gagagaatta 480

atgaatttac ctgtgattca gaacatgatg aacaaccctg atttaatgcg caacttaatt 540

atgagcaatc cacagatgag agaaatcata gaccgcaacc ctgagcttgg gcatgtgcta 600

aatgatccta gcactcttag gcagacacta gaagctgcaa ggaatcctga aataatgaga 660

gagatgatgc gtaacactga cagggcaatg agcaatattg aatcgtctcc agaaggcttt 720

aacatgctta ggcgaatgta tgaaactgtt caggaaccat ttttgaatgc cacaaccaca 780

gcaggggaga gtggtggcgc aggatcaaat ccttttgctg ctcttctggg taacacaggc 840

accacaaggg ctacaaaccc ttcaaccact gctggttctg aaaccactaa tgattctcct 900

gttccaaata ccaacccact acctaatcca tggggcagtg ctgcgccggg tacaccatca 960

agcaacacaa gatctactgc tgctaattcg gaaacaatgg ctcctggaat tgctgggtta 1020

ggcgggcttg ggattcctgg attggggagt atggctagcg gtgtacctga ccctagcatg 1080

cttaatcagg ttatgcaaaa cccggccatg tcacagatga tgcagtctct tctttccaat 1140

cctcaatata tgaaccagat tcttagtttg aacccccaac tgcggactgc tatggatgca 1200

aatcctcaac tgagagagat gatgcagaat cccgaattta ttcaacagct agccaaccct 1260

gaaacaatgc agcgccttat gtcattgcag tcactatttt cacagtttgc tggccagcaa 1320

cccagccaag gacaaagccc gttggtccct ggtgctgcag gaggtgcagg aataacaaac 1380

aatagcatgg actttttaac gaacatgctt ggtggtctcg gtcttggtgg cctcggtgct 1440

ccaaatcctt caaatttacc accagaagaa ttatatgcga cacaactaac gcagctccaa 1500

gaaatgggtt tctttgatac ccaagagaat attcgtgctc ttagcgccac tgctggaaat 1560

gttagtgctg cggtcgaaag gttattgggt aatcttggtc agtaa 1605

<210> 10

<211> 918

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

atgcaaatct ttgtgaaaac ccttaccgga aagaccatca ccctcgaggt tgagagctcc 60

gatacgattg acaatgtcaa agccaaaatc caagacaagg aaggaatccc cccagaccaa 120

cagcgtctca tttttgctgg caagcaactt gaagatggta gaacactagc agattacaat 180

attcaaaaag aatcgaccct gcacttggtc ctccgactaa gaggaggcat gcaaatattt 240

gtaaaaactc ttactggcaa gaccatcaca cttgaggtag aaagctcgga tacaatagac 300

aatgtaaagg cgaagatcca agataaggaa ggtattcctc ctgatcagca gaggttgatc 360

ttcgcaggaa agcaattgga agatgggaga actttagccg attataacat tcaaaaagag 420

tccacacttc atctggttct tagattgaga ggagggatgc aaatatttgt gaagaccctg 480

acagggaaaa caattacgtt agaagttgaa agctcggata caatagataa cgtaaaagcg 540

aagatccagg ataaagaagg aattcctcca gatcagcaga ggttgatctt tgcaggaaag 600

caattggaag atgggagaac ccttgcagat tataacatcc aaaaggagtc gaccctgcat 660

cttgtactga ggttgcgtgg tggaatgcag atatttgtaa agacactgac tgggaaaaca 720

attacattgg aagtggagag ctctgataca atcgacaacg tgaaggcaaa gatccaggac 780

aaggagggga ttccaccaga tcagcagagg ctgatatttg cagggaagca gctggaggat 840

ggaaggaccc ttgctgatta taatatccag aaggagtcca ctcttcacct tgtgctccgt 900

cttcgtggtg gtatttga 918

<210> 11

<211> 1356

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

atgagggaat gcatctcgat ccacatcgga caggctggta ttcaagtcgg aaacgcctgc 60

tgggagcttt actgtcttga gcacgggatt cagcctgatg gccaaatgcc aagtgacaag 120

actgtaggcg gaggtgatga tgctttcaac acttttttca gtgaaactgg tgctggaaag 180

catgtgcccc gtgctatctt tgttgatctt gagcccactg tcattgatga agtcaggaca 240

ggaacttacc gccagctgtt tcatcctgaa cagctcatta gtggcaagga ggatgctgcc 300

aacaactttg cccgtggcca ctatactatt ggcaaggaga ttgttgacct ctgcttagac 360

cgaatccgaa agctggcaga caactgcacg gggcttcaag gcttccttgt attcaacgca 420

gttggtggag gcactggttc tggtttgggc tctctacttc ttgagaggct ttctgtcgac 480

tacggaaaga aatccaagct tggattcact gtatacccat ctcctcaagt ctcaacctca 540

gttgtcgagc cctacaacag cgtcctctcc acccactcgc ttctggaaca cactgatgtc 600

gctgttcttc ttgacaacga agctatttac gacatttgca ggaggtctct tgacattgaa 660

cgcccaacct acactaactt gaatcgtctt gtgtctcagg tcatatcttc actgacggcc 720

tcacttcgtt tcgacggagc cctaaacgtg gacgtaacag agttccaaac caacctggtc 780

ccataccccc gaatccactt catgctctcc tcctacgctc ccgtaatctc cgcggagaag 840

gcctaccacg agcagctgtc ggtggcggag atcacgaact cagcattcga gccgtcctct 900

atgatggcca agtgcgaccc gaggcatgga aagtacatgg cctgctgcct catgtaccgc 960

ggcgacgtcg tccccaagga tgtgaacgct gcggtcgcca ccataaagac gaagcgcacg 1020

atccagttcg tggactggtg ccctacgggc ttcaagtgcg ggatcaacta ccagcccccg 1080

accgtcgtgc cgggcgggga tctggccaag gtgcagaggg ctgtgtgcat gatttcgaac 1140

agcacgagcg tggcggaggt gtttggtagg attgatcata agtttgatct gatgtacgcg 1200

aagagggcgt ttgtgcattg gtatgttggg gaagggatgg aggaagggga gttcagcgag 1260

gcgagggagg atttggcggc gctggagaag gattatgagg aggttggtgc tgagtctgct 1320

gaaggcgacg agggggatga gggtgaggag tattga 1356

<210> 12

<211> 1260

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

atggctttgg tgttgcatgc tactagcaat aacaaaaatg cttggaaagg acttattgct 60

gcagaataca gtggagtcca ggtgcaactt gtccagaatt ttcagatgga tgtttcaaat 120

aagactcctg aatttttgaa gatgaatcca cttggaaagg tgcctgtatt ggaaactcct 180

gatggtccag tttttgaaag caatgccata gcccgctatg taactcgctt aaagccagat 240

aattctctat atggatcttc cttgatcgaa tatgccttaa ttgagcaatg gatggacttt 300

gctgcaactg aagtcgaccc aaatatcgca tggtggtttt acccaaggct tggatacaga 360

ccatttatcc aacaggccga agaagtcgca attgcaaatt taaagagggc ctttgaagct 420

ttgaacactc atcttgcttc aaacacttac cttgtgggcg actccataac tttagctgat 480

attgtcatga catgcaattt atatacagga tttgctggtg tcctcactaa aagcttcaca 540

accgaatatc ctcacattga gaggtacttc tggactatgg taaatcaacc aaatgttcgt 600

aaaataatgg gagaggttaa gcagaccgag tccattcctc ctgttccatc aaagaagcca 660

gtgtcgaagc ccaaagaagc taagaaggag cctaaagaag ctaagaagga agctcctaag 720

cctaaagtag aggcaccacc tgctccagtt gaagaggagg cacctaaacc caaaccaaaa 780

aatccccttg atttattacc gccaagcaag atggtactgg atgactggaa gaggttgtac 840

tcgaacacta agaccaactt ccgtgaagtt gccattaaag gattctggga tatgtacgac 900

ccagaagggt actctctatg gttctgcgac tacaaataca acgacgagaa cactgtgtcc 960

ttcgtgaccc tgaacaaggt cggaggtttc ctgcagcgta tggacctcgc tcgcaaatat 1020

gcatttggca aaatgcttgt gatcggttcg gaaccgccct acaaagttaa gggactgtgg 1080

ctcttccgtg ggcctgaaat ccctaaattc gtgatggaag agtgctacga catggagctc 1140

tacgagtgga ccaaggttga tattaatgat gagacgcaaa aggagagggt taatgcgatg 1200

attgaagacc aggagccttt tgaaggcgag gctctgctcg atgctaagtg cttcaaataa 1260

<210> 13

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

gacaagccag ttgctgtatt cg 22

<210> 14

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

ctttgcacca cccttaatgt ga 22

<210> 15

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

aatcactgcc acccagaaga ct 22

<210> 16

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

acggaatgac atgccagtaa gc 22

<210> 17

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

gtcaactccg tcaccgtaaa ca 22

<210> 18

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

gcaacttcgg acttctcctc ta 22

<210> 19

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

accttgtgct ccgtcttcgt g 21

<210> 20

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

aacatctggc ttaccgtcgt ct 22

<210> 21

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

cccatctcct caagtctcaa cct 23

<210> 22

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

gacctcctgc aaatgtcgta aat 23

<210> 23

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

accgagtcca ttcctcctgt tc 22

<210> 24

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

gggtttaggt gcctcctctt ca 22

Claims

1. The leek reference gene under the condition of gray mold stress is characterized by comprising UBC1 and UBC2 genes; the nucleotide sequence of UBC1 is shown as SEQ ID NO. 1, and the nucleotide sequence of UBC2 is shown as SEQ ID NO. 2.

2. The use of the reagent for detecting the leek reference gene of claim 1 in the quantitative detection of the leek reference gene.

3. A group of leek reference gene UBC1 and UBC2 amplification primers, wherein the amplification primers are designed to obtain UBC1 and UBC2 amplification primers based on UBC1 and UBC2 gene sequences in the reference gene of claim 1 according to the principle of real-time fluorescent quantitative PCR primer design.

4. The amplification primer of claim 3, wherein the nucleotide sequence of the amplification primer of UBC1 is shown as SEQ ID NO. 3 and SEQ ID NO. 4; the nucleotide sequence of the amplification primer of UBC2 is shown as SEQ ID NO. 5 and SEQ ID NO. 6.

5. The use of the amplification primer of claim 3 or 4 in quantitative detection of leek internal reference genes.