Ribes nobile fluorescent quantitative reference gene applicable to drought stress as well as primer and application of Ribes nobile fluorescent quantitative reference gene

Abstract

The invention provides a fluorescent quantitative reference gene of Ribes nobile applicable to drought stress as well as a primer and application of the fluorescent quantitative reference gene. The reference gene provided by the invention is polynucleotide as shown in SEQ ID NO: 1. The reference gene can avoid detection result deviation caused by instability of gene expression due to drought stress, provides powerful support for accurate quantification of drought tolerance / drought resistance related functional genes of Ribes nobile, and can improve the stability and accuracy of research.

Description

Technical Field

[0001] The invention relates to the technical field of plant molecular biology, and in particular to a ribes odorifera fluorescence quantitative internal reference gene suitable for use under drought stress, a primer thereof and an application thereof. Background Art

[0002] Ribes odoratum HLWendl is a deciduous shrub of the Ribes genus of the Ribes family. It has important economic and ornamental value and strong ecological adaptability. Ribes odoratum fruit contains a large amount of nutrients, including high levels of vitamin C, vitamin E and total sugar, and rich amino acid species. Ribes odoratum is native to North America and grows along mountain rivers. It has been introduced and cultivated in Liaoning, Heilongjiang, Qinghai and other places in my country.

[0003] There is currently no reference gene for fluorescence quantification of Ribes odoratus under drought stress. Conventional universal reference genes are currently commonly used in research on Ribes odoratus. However, the expression of conventional universal reference genes in different plants, at different developmental stages, and under different experimental conditions may be unstable, thus affecting the accuracy of target gene expression detection. Therefore, screening for reference genes that are stably expressed in Ribes odoratus under drought stress is a key factor in ensuring accurate fluorescence quantification results. Summary of the invention

[0004] In order to solve the above technical problems, the inventors of the present application obtained a set of stably expressed internal reference genes from Ribes odoratus after a series of explorations and innovations.

[0005] In a first aspect, the present invention provides any of the following genes or gene fragments or a combination thereof:

[0006] (1) A gene or gene fragment as shown in (a) or (b) or (c) below:

[0007] (a): a gene having a cDNA sequence as shown in SEQ ID NO:1, or a gene having a sequence with 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more similarity to the cDNA sequence as shown in SEQ ID NO:1, or a gene derived from the genus Ribes and being a natural allele of the gene having a cDNA sequence as shown in SEQ ID NO:1, or a gene having a sequence with 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more similarity to the cDNA sequence as shown in SEQ ID NO:1 and derived from the genus Ribes and being a natural allele of the gene having a cDNA sequence as shown in SEQ ID NO:1;

[0008] (b): a fragment of the gene described in (a) which can be specifically amplified;

[0009] (c) a gene fragment having a cDNA sequence as shown in nucleotides 2114 to 2452 of SEQ ID NO:1, or a gene fragment having a sequence with 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more similarity to the sequence of the gene fragment having a cDNA sequence as shown in nucleotides 2114 to 2452 of SEQ ID NO:1, or a gene fragment derived from the genus Ribes and being a natural allele of the gene fragment having a cDNA sequence as shown in nucleotides 2114 to 2452 of SEQ ID NO:1, or a gene fragment having a sequence with 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more similarity to the sequence of the gene fragment having a cDNA sequence as shown in nucleotides 2114 to 2452 of SEQ ID NO:1 and being derived from the genus Ribes and being a natural allele of the gene fragment having a cDNA sequence as shown in nucleotides 2114 to 2452 of SEQ ID NO:1;

[0010] (2) A gene or gene fragment as shown in (d), (e) or (f) below:

[0011] (d) A gene having a cDNA sequence as shown in SEQ ID NO:2, or a gene having a sequence with 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more similarity to the cDNA sequence as shown in SEQ ID NO:2, or a gene derived from the genus Ribes and being a natural allele of the gene having a cDNA sequence as shown in SEQ ID NO:2, or a gene having a sequence with 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more similarity to the cDNA sequence as shown in SEQ ID NO:2 and derived from the genus Ribes and being a natural allele of the gene having a cDNA sequence as shown in SEQ ID NO:2;

[0012] (e): A fragment of the gene described in (d) that can be specifically amplified;

[0013] (f) a gene fragment having a cDNA sequence as shown in nucleotides 1047 to 1378 of SEQ ID NO:2, or a gene fragment having a sequence with 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more similarity to the sequence of the gene fragment having a cDNA sequence as shown in nucleotides 1047 to 1378 of SEQ ID NO:2, or a gene fragment derived from the genus Ribes and being a natural allele of the gene fragment having a cDNA sequence as shown in nucleotides 1047 to 1378 of SEQ ID NO:2, or a gene fragment having a sequence with 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more similarity to the sequence of the gene fragment having a cDNA sequence as shown in nucleotides 1047 to 1378 of SEQ ID NO:2 and being derived from the genus Ribes and being a natural allele of the gene fragment having a cDNA sequence as shown in nucleotides 1047 to 1378 of SEQ ID NO:2;

[0014] (3) A gene or gene fragment as shown in (g), (h) or (i) below:

[0015] (g): a gene having a cDNA sequence as shown in SEQ ID NO:3, or a gene having a sequence with 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more similarity to the sequence of the gene having a cDNA sequence as shown in SEQ ID NO:3, or a gene derived from the genus Ribes and being a natural allele of the gene having a cDNA sequence as shown in SEQ ID NO:3, or a gene having a sequence with 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more similarity to the sequence of the gene having a cDNA sequence as shown in SEQ ID NO:3 and being derived from the genus Ribes and being a natural allele of the gene having a cDNA sequence as shown in SEQ ID NO:3;

[0016] (h): a fragment of the gene described in (g) and the fragment can be specifically amplified;

[0017] (i) a gene fragment having a cDNA sequence as shown in nucleotides 512 to 851 of SEQ ID NO:3, or a gene fragment having a sequence with 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more similarity to the gene fragment having a cDNA sequence as shown in nucleotides 512 to 851 of SEQ ID NO:3, or a gene fragment derived from the genus Ribes and being a natural allele of the gene fragment having a cDNA sequence as shown in nucleotides 512 to 851 of SEQ ID NO:3, or a gene fragment having a sequence with 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more similarity to the gene fragment having a cDNA sequence as shown in nucleotides 512 to 851 of SEQ ID NO:3 and being derived from the genus Ribes and being a natural allele of the gene fragment having a cDNA sequence as shown in nucleotides 512 to 851 of SEQ ID NO:3;

[0018] (4) A combination of genes or gene fragments: consisting of the gene or gene fragment described in (1) and the gene or gene fragment described in (2) above;

[0019] (5) Gene or gene fragment composition: composed of the gene or gene fragment described in (1) and the gene or gene fragment described in (3) above;

[0020] (6) Gene or gene fragment composition: composed of the gene or gene fragment shown in (2) and the gene or gene fragment shown in (3) above;

[0021] (7) Gene or gene fragment composition: composed of the gene or gene fragment shown in (1), the internal reference gene shown in (2) above, and the gene or gene fragment shown in (3) above.

[0022] The sequence of the gene or gene fragment may be a cDNA sequence or a genomic sequence corresponding to the cDNA sequence; the gene or gene fragment may be obtained by amplification or by artificial synthesis.

[0023] Natural alleles: Due to the existence of natural variation, the same gene may have sequence differences between different individuals or homologous genes between different species. Such genes are called natural alleles. The sequence similarity between natural alleles may be more than 95% or more than 96% or more than 97% or more than 98% or more than 99%, but the functions of these natural alleles are the same or similar, that is, these natural alleles all have the function of serving as internal reference genes.

[0024] The above-mentioned natural alleles may be derived from the same species of Ribes odoratum, specifically from different natural individuals of Ribes odoratum, i.e., wild resources, or from different varieties of Ribes odoratum, i.e., artificially modified resources. The above-mentioned natural alleles may also be derived from plants of other species within the genus Ribes, or from natural individuals thereof, i.e., wild resources, or from different varieties thereof, i.e., artificially modified resources.

[0025] Specific amplification refers to using DNA or RNA of Ribes genus or Ribes odoratum as template, using primers to perform PCR amplification, and the amplified product is a target specific fragment (such as the intercepted fragment), and theoretically does not contain other fragments. Of course, in practical operation, due to various uncontrollable reasons, some other fragments may also be contained, but in the cognition of those skilled in the art, it is acceptable and does not affect the identification and use of specific fragments.

[0026] Wherein, the gene or gene fragment is an internal reference gene.

[0027] The gene or gene fragment is used as an internal reference gene to detect the expression level of the plant gene under drought stress conditions or under no stress conditions; further preferably, the expression level of the gene in the plant leaves is detected.

[0028] The plant for detecting the gene expression level of the plant is a plant of the genus Ribes, and more preferably Ribes odoratum.

[0029] In a second aspect, the present invention provides any of the following primer pairs or primer pair combinations:

[0030] (1) a primer pair for amplifying the gene or gene fragment described in (1) above;

[0031] (2) a primer pair for amplifying the gene or gene fragment described in (2) above;

[0032] (3) a primer pair for amplifying the gene or gene fragment described in (3) above;

[0033] (4) a combination of the primer pair described in (1) above and the primer pair described in (2) above;

[0034] (5) a combination of the primer pair described in (1) above and the primer pair described in (3) above;

[0035] (6) a combination of the primer pair described in (2) above and the primer pair described in (3) above;

[0036] (7) A combination of the primer pair described in (1) above, the primer pair described in (2) above, and the primer pair described in (3) above.

[0037] Further preferably, the primer pair is: a primer pair for amplifying the gene fragment described in (c) above, and the upstream primer sequence has a similarity of more than 98%, more than 99%, or more than 99.5% to SEQ ID NO:4, and the downstream primer sequence has a similarity of more than 98%, more than 99%, or more than 99.5% to SEQ ID NO:5; or a primer pair for amplifying the gene fragment described in (f) above, and the upstream primer sequence has a similarity of more than 98%, more than 99%, or more than 99.5% to SEQ ID NO:6, and the downstream primer sequence has a similarity of more than 98%, more than 99%, or more than 99.5% to SEQ ID NO:7; or a primer pair for amplifying the gene fragment described in (i) above, and the upstream primer sequence has a similarity of more than 98%, more than 99%, or more than 99.5% to SEQ ID NO:8, and the downstream primer sequence has a similarity of more than 98%, more than 99%, or more than 99.5% to SEQ ID NO:9.

[0038] More specifically, the primer pair is: a primer pair as shown in SEQ ID NO:4 and SEQ ID NO:5, or a primer pair as shown in SEQ ID NO:6 and SEQ ID NO:7, or a primer pair as shown in SEQ ID NO:8 and SEQ ID NO:9.

[0039] Wherein, the primer pair is a primer pair used to amplify an internal reference gene when detecting the expression level of a plant gene;

[0040] Preferably, the plant is a plant of the genus Ribes; further preferably, the plant of the genus Ribes is Ribes odoratum.

[0041] The internal reference gene is an internal reference gene suitable for detecting the gene expression level of plants under drought stress conditions or under no stress conditions.

[0042] The detecting of the gene expression level of the plant is detecting the gene expression level of the plant under drought stress conditions or under no stress conditions.

[0043] The method for detecting gene expression level is real-time fluorescence quantitative PCR;

[0044] The detecting of the plant gene expression level is detecting the gene expression level in plant leaves.

[0045] In a third aspect, the present invention provides any of the following applications:

[0046] Use of any of the above genes or gene fragments as internal reference genes in detecting plant gene expression levels;

[0047] Use of any of the above-mentioned primer pairs or primer pair combinations in detecting plant gene expression levels.

[0048] Wherein, the detection of plant gene expression level is to detect the gene expression level of the plant under drought stress conditions or without any stress conditions.

[0049] The detecting of the plant gene expression level is detecting the gene expression level in plant leaves.

[0050] The plant used for detecting the plant gene expression level is a plant of the genus Ribes; preferably, the plant is Ribes odoratum.

[0051] The method for detecting the plant gene expression level is real-time fluorescence quantitative PCR.

[0052] In a fourth aspect, the present invention provides a method for detecting the expression level of a plant gene, comprising the following steps (a) or (b):

[0053] (a) using any of the above genes or gene fragments as an internal reference gene;

[0054] (b) using any of the primer pairs or primer pair combinations described above as primers for amplifying an internal reference gene.

[0055] Wherein, the detection of plant gene expression level is to detect the gene expression level of the plant under drought stress conditions or without any stress conditions.

[0056] The detecting of the plant gene expression level is detecting the gene expression level in plant leaves.

[0057] The plant used for detecting the plant gene expression level is a plant of the genus Ribes; preferably, the plant is Ribes odoratum.

[0058] The method for detecting the plant gene expression level is real-time fluorescence quantitative PCR.

[0059] Any of the above-mentioned internal reference genes of the present invention is applicable to the detection of gene expression levels of any species in the genus Ribes. The detection can be performed within the range of one or several species, or within the range of the entire genus Ribes. As a specific example, it is used for the detection of gene expression levels of Ribes odoratum.

[0060] The above-mentioned internal reference genes can be used for detecting gene expression levels in plants under normal conditions (ie, without any stress conditions), and can also be used for detecting gene expression levels in plants under different degrees of drought stress conditions.

[0061] The "drought stress conditions" described in any part of the present invention may refer to natural drought conditions or any artificial drought conditions, without limitation, and all fall within the scope of protection claimed in the present invention. In addition, there is no limitation on the degree of drought, and any drought condition considered by those skilled in the art to be a drought condition falls within the "drought stress conditions" described in the present invention.

[0062] Detection of gene expression level: Genes include genes encoding proteins or functional RNA molecules, genes acting as regulatory elements, and any other genes. It can refer to detection at the DNA level or detection at the RNA level. Expression level generally refers to expression amount, relative expression amount or absolute expression amount, which can be determined by technicians in this field according to actual needs without limitation.

[0063] The invention performs FPKM calculation of gene expression on transcriptome data of Ribes odoratus seedlings under drought stress, screens out genes with gene sequencing length greater than 500 bp and expression coefficient of variation less than 0.05 in six drought stress treatments, compares expression differences of these genes at different stages through CT values ​​after a fluorescence quantitative test, screens out internal reference genes stably expressed in Ribes odoratus leaves under drought stress by using Genorm, Normfinder and BestKeeper analysis software, designs real-time fluorescence quantitative PCR primers based on the sequences of these genes, fills the current situation that Ribes odoratus has no internal reference genes under drought stress, the internal reference genes can avoid the deviation of detection results caused by the instability of gene expression due to drought stress, provides strong support for the accurate quantification of functional genes related to drought tolerance / drought resistance of Ribes odoratus, and can improve the stability and accuracy of research. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 Agarose gel electrophoresis is used to detect primer specificity.

[0065] Figure 2 is the melting curve of the candidate reference gene.

[0066] Figure 3 is the amplification curve of the candidate reference gene.

[0067] Figure 4 The Venn diagram of candidate genes. DETAILED DESCRIPTION

[0068] The following are some specific embodiments of the present invention, wherein the technical means or reagents used are conventional methods and reagents known in the art unless otherwise specified.

[0069] Example

[0070] Experimental methods

[0071] Two-year-old Ribes odorata seedlings with good growth were selected and five treatment groups were set up. Starting from July 5, the Ribes odorata seedlings in the six treatment groups were watered with 500 ml of water at the same time, and then the treatment was carried out according to the rules of normal watering treatment (control, CK, keep the surface soil moist), watering once after 3d drought treatment (T1, 500 ml / 3d), watering once after 6d drought treatment (T2, 500 ml / 6d), watering once after 9d drought treatment (T3, 500 ml / 9d), watering once after 12d drought treatment (T4, 500 ml / 12d) and watering once after 15d drought treatment (T5, 500 ml / 15d). Samples (i.e. leaves) were collected on the second day after the third round of watering in each treatment group. Cryopreservation tubes and tin foil were prepared before sampling, and they were marked. After the samples were collected, they were immediately put into liquid nitrogen and brought back to the laboratory for use. Three biological replicates were taken for each treatment group, and a total of 18 samples were collected.

[0072] Total RNA was extracted from leaf tissues using the TRIzol method. The concentration and purity of the extracted total RNA were determined and tested using an Agilent 2100 bioanalyzer (Agilent Technologies, CA, USA). The integrity of the RNA was detected using 1.0% agarose gel electrophoresis.

[0073] The starting RNA for library construction is total RNA. Oligo(dT) magnetic beads are used to enrich mRNA with polyA tails, and then divalent cations are used to randomly interrupt the obtained mRNA in Fragmentation Buffer. The fragmented mRNA is used as a template and random oligonucleotides are used as primers to synthesize the first chain of cDNA in the M-MuLV reverse transcriptase system. The RNA chain is then degraded with RNaseH, and the second chain of cDNA is synthesized with dNTPs as raw materials in the DNApolymerase I system. The purified double-stranded cDNA is end-repaired, A-tailed, and connected to sequencing adapters. AMPure XP beads are used to screen cDNA of 370-420bp, PCR amplification is performed, and AMPure XP beads are used to purify the PCR product again to finally obtain the library.

[0074] After the library construction is completed, Qubit2.0 Fluorometer is used for preliminary quantification, and the library is diluted to 1.5 ng / ul. Then, the insert size of the library is detected using Agilent 2100 bioanalyzer. After the insert size meets expectations, qRT-PCR is used to accurately quantify the effective concentration of the library (the effective concentration of the library is higher than 1.5 nM) to ensure the quality of the library.

[0075] After library construction, sequencing was performed on the illimina sequencing platform.

[0076] RSEM software was used to quantitatively analyze the gene expression level of each sample. The bowtie2 parameter in RSEM was set to mismatch 0, the readcount was converted to FPKM, and the gene expression level was estimated using the FPKM value. The average, standard deviation (SD) and coefficient of variation (CV) of FPKM for different genes in different drought treatment groups were calculated, and genes were selected according to the criteria of gene sequencing fragments greater than 500bp, CV<0.55 and SD<3.6 of gene expression (FPKM) in the six treatments.

[0077] Table 1 Mean, standard deviation and coefficient of variation of FPKM of candidate genes in different experimental groups

[0078]

[0079]

[0080] Genes were screened from the transcriptome data of the six treatments, and the sequences of the genes were obtained from the original fasta files obtained from the transcriptome sequencing. Primers were then designed using the online primer 3plus (https: / / www.primer3plus.com / index.html) with a length of 20–25 bp and a CG content of 43%–55%, which were synthesized by Shanghai Jierui Bioengineering Co., Ltd.

[0081] First, the specificity of the primers was verified by ordinary PCR, and PCR amplification was performed in a 20 μL system: 10 μL 2×PremixTaq (Takara, Japan), 1 μL primer, 1 μL cDNA and 8 μL ddH2O. The PCR amplification program was: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 15 s, 56℃ annealing for 35 s, 72℃ extension for 32 s, 72℃ extension for 2 min, 48 cycles; stored at 4℃. The PCR amplified products were subjected to 1.5% agarose gel electrophoresis, and specific primers that amplified a single band, amplified products without primer dimers and non-specific amplification were screened for subsequent RT-qPCR. A total of 17 pairs of primers were obtained.

[0082] According to TB RT-qPCR amplification was performed according to the instructions of Premix Ex TaqTMII (Tli RNaseH Plus), and the final system was 20μl: 0.4μl cDNA, 10μl TB Green Premix Ex TaqTMⅡ (Tli RNaseH Plus) (2×), 0.3μL forward primer (10μM), 0.3μL reverse primer (10μM). Amplification was performed using the CFX Connect Real-Time system (BIO-RAD), and the reaction program was: 95℃30s; 95℃5s, 60℃30s, and the number of cycles was 50 times.

[0083] The cycle threshold (Ct) in the qRT-PCR results was sorted using Microsoft Excel 2010, and the stability of 17 candidate genes was calculated and analyzed using the RefFinder platform based on GeNorm, Normfinder and BestKeeper software, and the analysis results and comprehensive rankings of different software were obtained. The RefFinder platform automatically calculates the geometric mean of the weight of each gene in the four algorithms. Subsequently, it reorders these candidate reference genes according to the geometric mean of each gene to generate a comprehensive ranking. The gene with the lowest geometric mean is considered to be a more stable reference gene, while the gene with a higher geometric mean is considered to be a less stable gene to evaluate whether it is suitable for use as an internal reference gene for fluorescent quantitative PCR reaction. The Venn statistical diagram of the top ten genes in stability was drawn using online software (https: / / jvenn.toulouse.inrae.fr / app / example.html), and the most accurate and appropriate internal reference gene was obtained by combining various methods.

[0084] Results and Analysis

[0085] Primer specificity verification:

[0086] The results of 1% agarose gel electrophoresis were as follows Figure 1 , screen out specific primers for further research.

[0087] Synthesis of the first strand of cDNA: cDNA was synthesized using the FastKing gDNA Dispelling RT SuperMix (TIANGENBIOTECH, Beijing, China) kit, and the thermal cycle conditions were set according to the instructions: 42°C for 15 min, 95°C for 3 min. The reverse transcribed cDNA was placed in a -20°C refrigerator.

[0088] Analysis of candidate gene expression: Fluorescence quantitative PCR analysis was performed using the selected specific primers. The melting curves of 17 candidate internal reference genes were single peaks ( Figure 2 ), the primer specificity is good; the amplification curve is S-shaped ( Figure 3 ).

[0089] The cycle threshold (Ct) is an important criterion for selecting internal reference genes. The stability of gene expression can be judged by the change trend of the Ct value. The Ct value analysis results of 17 candidate internal reference genes in Ribes odoratus under six drought treatment conditions are shown in Table 2.

[0090] Table 2 Analysis of cT value of fluorescence quantitative PCR results

[0091]

[0092] Expression stability analysis of candidate reference genes

[0093] When the Genorm software is analyzed, the default stability value below 1.5 is the numerical range of stable expression of the internal reference gene. In the data obtained by the NormFinder software, the expression stability value is negatively correlated with the gene stability, that is, the smaller the value, the more stable the gene expression. The BestKeeper software uses the Ct value obtained by the qRT-PCR reaction to judge the stability of the internal reference gene expression. The lower the standard deviation (SD) and coefficient of variation (CV), the better the stability of gene expression.

[0094] In the Genorm analysis, the expression stability values ​​of the 17 candidate reference genes ranged from 0.389 to 3.599. The lowest expression stability values ​​were Cluster-37.23244, Cluster-37.24426, Cluster-37.24001, and Cluster-37.22646. These four genes had the highest expression stability. The three genes with the least stable expression stability were Cluster-37.24007 (2.177), Cluster-37.23838 (2.499), and Cluster-37.32890 (3.599).

[0095] In the NormFinder algorithm, the results showed that the top three stability genes were Custer-37.22228 (0.277), Cluster-37.22897 (0.407) and Cluster-37.22646 (0.734), while Cluster-37.23838 (4.218), Cluster-37.24007 (4.369) and Cluster-37.32890 (11.74) were the three most unstable genes.

[0096] In BestKeeper, the expression stability values ​​ranged from 0.53 to 8.01, among which Cluster-37.22897 (0.53), Cluster-37.22066 (0.64) and Cluster-37.22273 (0.85) were the three genes with the highest expression stability, and Cluster-37.24007 (2.43), Cluster-37.23838 (2.77) and Cluster-37.32890 (8.01) were the three genes with the lowest expression stability.

[0097] Table 3 Analysis of the expression stability of candidate reference genes by three softwares

[0098]

[0099]

[0100] The top three genes in the comprehensive ranking are Cluster-37.22228, Cluster-37.22646, and Cluster-37.22897, and the gene with the worst expression stability is Cluster-37.32890.

[0101] Table 4 Comprehensive comparison of gene expression stability using several analytical methods

[0102]

[0103] The top ten genes involved in Delta CT, GeNorm, Normfinder and BestKeeper and ranked in expression stability are Cluster-37.22228, Cluster-37.22646, Cluster-37.22897, Cluster-37.24001, Cluster-37.22273 and Cluster-37.24280.

[0104]

[0105]

Claims

1. A gene or gene fragment, any of the following: (1) A gene or gene fragment as shown in (a) or (b) or (c) below: (a): a gene having a cDNA sequence as shown in SEQ ID NO:1, or a gene having a sequence with 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more similarity to the cDNA sequence as shown in SEQ ID NO:1, or a gene derived from the genus Ribes and being a natural allele of the gene having a cDNA sequence as shown in SEQ ID NO:1, or a gene having a sequence with 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more similarity to the cDNA sequence as shown in SEQ ID NO:1 and derived from the genus Ribes and being a natural allele of the gene having a cDNA sequence as shown in SEQ ID NO:1; (b): a fragment of the gene described in (a) which can be specifically amplified; (c) a gene fragment having a cDNA sequence as shown in nucleotides 2114 to 2452 of SEQ ID NO:1, or a gene fragment having a sequence with 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more similarity to the sequence of the gene fragment having a cDNA sequence as shown in nucleotides 2114 to 2452 of SEQ ID NO:1, or a gene fragment derived from the genus Ribes and being a natural allele of the gene fragment having a cDNA sequence as shown in nucleotides 2114 to 2452 of SEQ ID NO:1, or a gene fragment having a sequence with 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more similarity to the sequence of the gene fragment having a cDNA sequence as shown in nucleotides 2114 to 2452 of SEQ ID NO:1 and being derived from the genus Ribes and being a natural allele of the gene fragment having a cDNA sequence as shown in nucleotides 2114 to 2452 of SEQ ID NO:1; (2) A gene or gene fragment as shown in (d), (e) or (f) below: (d) A gene having a cDNA sequence as shown in SEQ ID NO:2, or a gene having a sequence with 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more similarity to the cDNA sequence as shown in SEQ ID NO:2, or a gene derived from the genus Ribes and being a natural allele of the gene having a cDNA sequence as shown in SEQ ID NO:2, or a gene having a sequence with 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more similarity to the cDNA sequence as shown in SEQ ID NO:2 and derived from the genus Ribes and being a natural allele of the gene having a cDNA sequence as shown in SEQ ID NO:2; (e): A fragment of the gene described in (d) that can be specifically amplified; (f) a gene fragment having a cDNA sequence as shown in nucleotides 1047 to 1378 of SEQ ID NO:2, or a gene fragment having a sequence with 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more similarity to the sequence of the gene fragment having a cDNA sequence as shown in nucleotides 1047 to 1378 of SEQ ID NO:2, or a gene fragment derived from the genus Ribes and being a natural allele of the gene fragment having a cDNA sequence as shown in nucleotides 1047 to 1378 of SEQ ID NO:2, or a gene fragment having a sequence with 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more similarity to the sequence of the gene fragment having a cDNA sequence as shown in nucleotides 1047 to 1378 of SEQ ID NO:2 and being derived from the genus Ribes and being a natural allele of the gene fragment having a cDNA sequence as shown in nucleotides 1047 to 1378 of SEQ ID NO:2; (3) A gene or gene fragment as shown in (g), (h) or (i) below: (g): a gene having a cDNA sequence as shown in SEQ ID NO:3, or a gene having a sequence with 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more similarity to the sequence of the gene having a cDNA sequence as shown in SEQ ID NO:3, or a gene derived from the genus Ribes and being a natural allele of the gene having a cDNA sequence as shown in SEQ ID NO:3, or a gene having a sequence with 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more similarity to the sequence of the gene having a cDNA sequence as shown in SEQ ID NO:3 and being derived from the genus Ribes and being a natural allele of the gene having a cDNA sequence as shown in SEQ ID NO:3; (h): a fragment of the gene described in (g) and the fragment can be specifically amplified; (i) a gene fragment having a cDNA sequence as shown in nucleotides 512 to 851 of SEQ ID NO:3, or a gene fragment having a sequence with 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more similarity to the gene fragment having a cDNA sequence as shown in nucleotides 512 to 851 of SEQ ID NO:3, or a gene fragment derived from the genus Ribes and being a natural allele of the gene fragment having a cDNA sequence as shown in nucleotides 512 to 851 of SEQ ID NO:3, or a gene fragment having a sequence with 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more similarity to the gene fragment having a cDNA sequence as shown in nucleotides 512 to 851 of SEQ ID NO:3 and being derived from the genus Ribes and being a natural allele of the gene fragment having a cDNA sequence as shown in nucleotides 512 to 851 of SEQ ID NO:3; (4) A combination of genes or gene fragments: consisting of the gene or gene fragment described in (1) and the gene or gene fragment described in (2) above; (5) Gene or gene fragment composition: composed of the gene or gene fragment described in (1) and the gene or gene fragment described in (3) above; (6) Gene or gene fragment composition: composed of the gene or gene fragment shown in (2) and the gene or gene fragment shown in (3) above; (7) Gene or gene fragment composition: composed of the gene or gene fragment shown in (1), the internal reference gene shown in (2) above, and the gene or gene fragment shown in (3) above.

2. The gene or gene fragment according to claim 1, characterized in that: The gene or gene fragment is an internal reference gene; Preferably, the gene or gene fragment is used as an internal reference gene to detect the expression level of a plant gene under drought stress conditions or under no stress conditions; further preferably, the expression level of the gene in plant leaves is detected.

3. The gene or gene fragment according to claim 1 or 2, characterized in that: The plant for detecting the gene expression level of the plant is a plant of the genus Ribes, and more preferably Ribes odoratum.

4. Any of the following primer pairs or primer pair combinations: (1) a primer pair for amplifying the gene or gene fragment described in claim 1 (1); (2) a primer pair for amplifying the gene or gene fragment described in (2) of claim 1; (3) a primer pair for amplifying the gene or gene fragment described in (3) of claim 1; (4) a combination of the primer pair described in (1) above and the primer pair described in (2) above; (5) a combination of the primer pair described in (1) above and the primer pair described in (3) above; (6) a combination of the primer pair described in (2) above and the primer pair described in (3) above; (7) A combination of the primer pair described in (1) above, the primer pair described in (2) above, and the primer pair described in (3) above.

5. The primer pair or primer pair combination according to claim 4, characterized in that: The primer pair for amplifying the gene fragment described in claim 1 (c) is: a primer pair as shown in SEQ ID NO: 4 and SEQ ID NO: 5, or a primer pair for amplifying the gene fragment described in claim 1 (c), wherein the upstream primer sequence has a similarity of more than 98%, more than 99%, or more than 99.5% to SEQ ID NO: 4, and the downstream primer sequence has a similarity of more than 98%, more than 99%, or more than 99.5% to SEQ ID NO: 5; The primer pair for amplifying the gene fragment described in claim 1 (f) is: a primer pair as shown in SEQ ID NO: 6 and SEQ ID NO: 7, or a primer pair for amplifying the gene fragment described in claim 1 (f), wherein the upstream primer sequence has a similarity of more than 98%, more than 99%, or more than 99.5% to SEQ ID NO: 6, and the downstream primer sequence has a similarity of more than 98%, more than 99%, or more than 99.5% to SEQ ID NO: 7; The primer pair for amplifying the gene fragment described in (i) of claim 1 is: a primer pair as shown in SEQ ID NO:8 and SEQ ID NO:9; or, a primer pair for amplifying the gene fragment described in (i) of claim 1, and the upstream primer sequence has a similarity of more than 98%, more than 99%, or more than 99.5% with SEQ ID NO:8, and the downstream primer sequence has a similarity of more than 98%, more than 99%, or more than 99.5% with SEQ ID NO:

9.

6. The primer pair or primer pair combination according to claim 4 or 5, characterized in that: The primer pair is a primer pair used to amplify an internal reference gene when detecting the expression level of a plant gene; Preferably, the plant is a plant of the genus Ribes; More preferably, the Ribes plant is Ribes odoratum.

7. The primer pair or primer pair combination according to any one of claims 4 to 6, characterized in that: The internal reference gene is an internal reference gene used to detect the gene expression level of plants under drought stress conditions or under no stress conditions; And / or, the detecting of the plant gene expression level is detecting the gene expression level of the plant under drought stress conditions or without any stress conditions; and / or, the method for detecting the gene expression level is real-time fluorescence quantitative PCR; And / or, the detecting of the gene expression level in the plant is detecting the gene expression level in plant leaves.

8. Any of the following applications: Use of the gene or gene fragment described in any one of claims 1 to 3 as an internal reference gene in detecting plant gene expression levels; Use of the primer pair or primer pair combination described in any one of claims 4 to 7 in detecting plant gene expression levels.

9. A method for detecting the expression level of a plant gene, comprising the steps shown in (a) or (b): (a) using the gene or gene fragment described in any one of claims 1 to 3 as an internal reference gene; (b) using the primer pair or primer pair combination described in any one of claims 4 to 7 as a primer for amplifying an internal reference gene.

10. The use according to claim 8 or the method according to claim 9, characterized in that: The detecting of the gene expression level of the plant is detecting the gene expression level of the plant under drought stress conditions or under no stress conditions; And / or, the detecting of the gene expression level in the plant is detecting the gene expression level in plant leaves; And / or, the plant used in the detection of plant gene expression level is a plant of the genus Ribes; preferably, the plant is Ribes odoratum; Preferably, the method for detecting the expression level of plant genes is real-time fluorescence quantitative PCR.

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