Kiwi fruit fluorescent quantitative reference gene suitable for abiotic stress and primer and application thereof

Abstract

The invention provides a kiwi fruit fluorescent quantitative reference gene applicable to abiotic stress as well as a primer and application of the kiwi fruit fluorescent quantitative reference gene. The reference gene provided by the invention is a gene with a cDNA sequence as shown in SEQ ID NO: 1. The reference gene disclosed by the invention is of great significance to research on the gene expression quantity of actinidia kolomikta.

Description

Technical Field

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

[0002] Actinidia kolomikta, also known as dog date fruit, is a deciduous vine plant of the genus Actinidia in the family Actinidia. It is a species with strong cold resistance in the genus Actinidia. Its fruit is soft and juicy, sweet and sour, rich in nutritional value, and contains a large amount of vitamin C. It has extremely high utilization value in medicine, food and health care. The leaves often change from white to pink during the growth process. It is a good foliage plant with good development and utilization value.

[0003] Reverse transcription real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) is one of the most widely used technologies for measuring gene expression levels, with the characteristics of high sensitivity, strong specificity, good reproducibility, low cost, fast reaction speed and wide application range. When performing qRT-PCR, a gene with stable expression is required as an internal reference gene to calibrate the relative expression of other genes.

[0004] The expression of internal reference genes will change to a certain extent in different tissues and organs of plants and under different stress conditions. In order to make the results of qRT-PCR more accurate, it is particularly important to use appropriate methods to screen out internal reference genes with higher stability. Summary of the invention

[0005] The inventors of the present invention obtained an internal reference gene suitable for Actinidia plants through research and developed corresponding primers accordingly.

[0006] Based on the above invention, the present invention requests protection for the following technical solutions:

[0007] The present invention provides any of the following genes or gene fragments or a combination thereof:

[0008] (1) A gene or gene fragment as shown in (a) or (b) below:

[0009] (a) A gene having a cDNA sequence as shown in SEQ ID NO: 1, or a gene having a sequence having 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: 1, or a gene derived from a plant of the genus Actinidia or Actinidia kolomikta 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 having 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: 1 and derived from a plant of the genus Actinidia or Actinidia kolomikta and being a natural allele of the gene having a cDNA sequence as shown in SEQ ID NO: 1;

[0010] (b) a gene fragment having a cDNA sequence as shown in nucleotides 714 to 861 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 as shown in nucleotides 714 to 861 of SEQ ID NO:1, or a gene fragment derived from a plant of the genus Actinidia and being a natural allele of the gene fragment as shown in the cDNA sequence as shown in nucleotides 714 to 861 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 as shown in nucleotides 714 to 861 of SEQ ID NO:1 and being derived from a plant of the genus Actinidia and being a natural allele of the gene fragment as shown in the cDNA sequence as shown in nucleotides 714 to 861 of SEQ ID NO:1;

[0011] (2) A combination of a gene or gene fragment as described in (a) or (b) and a gene or gene fragment as described in (c) or (d) below:

[0012] (c) A gene having a cDNA sequence as shown in SEQ ID NO: 2, or a gene having a sequence having 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: 2, or a gene derived from a plant of the genus Actinidia or Actinidia kolomikta 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 having 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: 2 and derived from a plant of the genus Actinidia or Actinidia kolomikta and being a natural allele of the gene having a cDNA sequence as shown in SEQ ID NO: 2;

[0013] (d) A gene fragment having a cDNA sequence as shown in nucleotides 325 to 473 of SEQ ID NO:2, or a gene fragment having a sequence having 95% or more, 96% or more, 97% or more, 98% or more, or 99% similarity to the sequence of the gene fragment having a cDNA sequence as shown in nucleotides 325 to 473 of SEQ ID NO:2, or a gene fragment derived from a plant of the genus Actinidia and being a natural allele of the gene fragment having a cDNA sequence as shown in nucleotides 325 to 473 of SEQ ID NO:2, or a gene fragment having 95% or more, 96% or more, 97% or more, 98% or more, or 99% similarity to the sequence of the gene fragment having a cDNA sequence as shown in nucleotides 325 to 473 of SEQ ID NO:2 and being derived from a plant of the genus Actinidia and being a natural allele of the gene fragment having a cDNA sequence as shown in nucleotides 325 to 473 of SEQ ID NO:2.

[0014] Any of the above-mentioned genes or gene fragments or their combinations are suitable as internal reference genes for plant roots under cold stress, plant roots under waterlogging stress, plant stems under salt stress, plant leaves under cold stress, different tissues of plants under no stress, plant roots under salt stress, plant stems under cold stress, plant stems under waterlogging stress, plant leaves under salt stress or plant leaves under waterlogging stress. The plant is an Actinidia plant, specifically Actinidia kolomikta, and the different tissues of the plant are roots, stems and leaves.

[0015] In addition to the gene or gene fragment shown in (a) or (b) and the combination of the gene or gene fragment shown in (a) or (b) and the gene or gene fragment shown in (c) or (d), the internal reference gene suitable for plant roots under salt stress can also be a combination of the gene or gene fragment shown in (a) or (b) and the gene or gene fragment shown in (e) or (f) below;

[0016] In addition to the gene or gene fragment shown in (a) or (b) and the combination of the gene or gene fragment shown in (a) or (b) and the gene or gene fragment shown in (c) or (d), the internal reference gene suitable for plant stems under cold stress or flooding stress can also be a combination of the gene or gene fragment shown in (a) or (b) and the gene or gene fragment shown in (g) or (h) below;

[0017] In addition to the gene or gene fragment shown in (a) or (b) and the combination of the gene or gene fragment shown in (a) or (b) and the gene or gene fragment shown in (c) or (d), the internal reference gene suitable for plant leaves under salt stress or waterlogging stress can also be a combination of the gene or gene fragment shown in (a) or (b) and the gene or gene fragment shown in (i) or (j) below;

[0018] In addition to the gene or gene fragment shown in (a) or (b) and the combination of the gene or gene fragment shown in (a) or (b) and the gene or gene fragment shown in (c) or (d), the internal reference gene suitable for plant leaves under cold stress can also be a combination of the gene or gene fragment shown in (a) or (b) and the gene or gene fragment shown in (k) or (l) below;

[0019] In addition to the gene or gene fragment shown in (a) or (b) and the combination of the gene or gene fragment shown in (a) or (b) and the gene or gene fragment shown in (c) or (d), the internal reference gene suitable for different tissues of plants without any stress may also be the gene or gene fragment shown in (e) or (f) and the combination of the gene or gene fragment shown in (c) or (d) below;

[0020] (e) A gene having a cDNA sequence as shown in SEQ ID NO: 13, or a gene having a sequence having 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: 13, or a gene derived from a plant of the genus Actinidia or Actinidia kolomikta and being a natural allele of the gene having a cDNA sequence as shown in SEQ ID NO: 13, or a gene having a sequence having 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: 13 and derived from a plant of the genus Actinidia or Actinidia kolomikta and being a natural allele of the gene having a cDNA sequence as shown in SEQ ID NO: 13;

[0021] (f) a gene fragment having a cDNA sequence as shown in nucleotides 360 to 480 of SEQ ID NO: 13, 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 as shown in nucleotides 360 to 480 of SEQ ID NO: 13, or a gene fragment derived from a plant of the genus Actinidia and being a natural allele of the gene fragment as shown in nucleotides 360 to 480 of SEQ ID NO: 13, 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 as shown in nucleotides 360 to 480 of SEQ ID NO: 13 and being derived from a plant of the genus Actinidia and being a natural allele of the gene fragment as shown in nucleotides 360 to 480 of SEQ ID NO: 13;

[0022] (g) a gene having a cDNA sequence as shown in SEQ ID NO: 14, or a gene having a sequence having 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: 14, or a gene derived from a plant of the genus Actinidia or Actinidia kolomikta and being a natural allele of the gene having a cDNA sequence as shown in SEQ ID NO: 14, or a gene having a sequence having 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: 14 and derived from a plant of the genus Actinidia or Actinidia kolomikta and being a natural allele of the gene having a cDNA sequence as shown in SEQ ID NO: 14;

[0023] (h): a gene fragment having a cDNA sequence as shown in nucleotides 1133 to 1277 of SEQ ID NO: 14, 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 1133 to 1277 of SEQ ID NO: 14, or a gene fragment derived from a plant of the genus Actinidia and being a natural allele of the gene fragment having a cDNA sequence as shown in nucleotides 1133 to 1277 of SEQ ID NO: 14, 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 1133 to 1277 of SEQ ID NO: 14 and being derived from a plant of the genus Actinidia and being a natural allele of the gene fragment having a cDNA sequence as shown in nucleotides 1133 to 1277 of SEQ ID NO: 14. The gene segment indicated by nucleotides 1133 to 1277 in NO:14 is the gene segment of the natural allele;

[0024] (i) A gene having a cDNA sequence as shown in SEQ ID NO: 15, or a gene having a sequence having 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: 15, or a gene derived from a plant of the genus Actinidia or Actinidia kolomikta and being a natural allele of the gene having a cDNA sequence as shown in SEQ ID NO: 15, or a gene having a sequence having 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: 15 and derived from a plant of the genus Actinidia or Actinidia kolomikta and being a natural allele of the gene having a cDNA sequence as shown in SEQ ID NO: 15;

[0025] (j) a gene fragment having a cDNA sequence as shown in nucleotides 373 to 483 of SEQ ID NO: 15, 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 as shown in nucleotides 373 to 483 of SEQ ID NO: 15, or a gene fragment derived from a plant of the genus Actinidia and being a natural allele of the gene fragment as shown in the cDNA sequence as shown in nucleotides 373 to 483 of SEQ ID NO: 15, 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 as shown in nucleotides 373 to 483 of SEQ ID NO: 15 and being derived from a plant of the genus Actinidia and being a natural allele of the gene fragment as shown in the cDNA sequence as shown in nucleotides 373 to 483 of SEQ ID NO: 15;

[0026] (k) a gene having a cDNA sequence as shown in SEQ ID NO: 16, or a gene having a sequence having 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: 16, or a gene derived from a plant of the genus Actinidia or Actinidia kolomikta and being a natural allele of the gene having a cDNA sequence as shown in SEQ ID NO: 16, or a gene having a sequence having 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: 16 and derived from a plant of the genus Actinidia or Actinidia kolomikta and being a natural allele of the gene having a cDNA sequence as shown in SEQ ID NO: 16;

[0027] (l) A gene fragment having a cDNA sequence as shown in nucleotides 157 to 277 of SEQ ID NO: 16, or a gene fragment having a sequence having 95% or more, 96% or more, 97% or more, 98% or more, or 99% similarity to the sequence of the gene fragment having a cDNA sequence as shown in nucleotides 157 to 277 of SEQ ID NO: 16, or a gene fragment derived from the genus Actinidia and being a natural allele of the gene fragment having a cDNA sequence as shown in nucleotides 157 to 277 of SEQ ID NO: 16, or a gene fragment having 95% or more, 96% or more, 97% or more, 98% or more, or 99% similarity to the sequence of the gene fragment having a cDNA sequence as shown in nucleotides 157 to 277 of SEQ ID NO: 16 and being derived from a plant of the genus Actinidia and being a natural allele of the gene fragment having a cDNA sequence as shown in nucleotides 157 to 277 of SEQ ID NO: 16.

[0028] The present invention also provides any of the following primer pairs or combinations of primer pairs:

[0029] (1) a primer pair for amplifying the gene or gene fragment described in (a) or (b) above;

[0030] (2) a combination of the following primer pairs: a primer pair for amplifying the gene or gene fragment described in (a) or (b) above, and a primer pair for amplifying the gene or gene fragment described in (c) or (d) above;

[0031] (3) a combination of the following primer pairs: a primer pair for amplifying the gene or gene fragment described in (a) or (b) above, and a primer pair for amplifying the gene or gene fragment described in (e) or (f) above;

[0032] (4) a combination of the following primer pairs: a primer pair for amplifying the gene or gene fragment described in (a) or (b) above, and a primer pair for amplifying the gene or gene fragment described in (g) or (h) above;

[0033] (5) a combination of the following primer pairs: a primer pair for amplifying the gene or gene fragment described in (a) or (b) above, and a primer pair for amplifying the gene or gene fragment described in (i) or (j) above;

[0034] (6) A combination of the following primer pairs: a primer pair for amplifying the gene or gene fragment described in (a) or (b) above, and a primer pair for amplifying the gene or gene fragment described in (k) or (l) above.

[0035] (7) A combination of the following primer pairs: a primer pair for amplifying the gene or gene fragment described in (c) or (d) above, and a primer pair for amplifying the gene or gene fragment described in (e) or (f) above.

[0036] In the above primer pair or combination of primer pairs, the primer pair for amplifying the gene or gene fragment described in (b) is: a primer pair as shown in SEQ ID NO: 3 and SEQ ID NO: 4, or a primer pair for amplifying the gene fragment described in (b) in claim 1, wherein the upstream primer sequence has a similarity of more than 98%, more than 99%, or more than 99.5% to SEQ ID NO: 3, and the downstream primer sequence has a similarity of more than 98%, more than 99%, or more than 99.5% to SEQ ID NO: 4;

[0037] In the above primer pair or combination of primer pairs, the primer pair for amplifying the gene or gene fragment described in (d) is: a primer pair as shown in SEQ ID NO: 5 and SEQ ID NO: 6, or a primer pair for amplifying the gene fragment described in (d) in claim 1, wherein the upstream primer sequence has a similarity of more than 98%, more than 99%, or more than 99.5% to SEQ ID NO: 5, and the downstream primer sequence has a similarity of more than 98%, more than 99%, or more than 99.5% to SEQ ID NO: 6;

[0038] In the above primer pair or combination of primer pairs, the primer pair for amplifying the gene or gene fragment described in (j) is: a primer pair as shown in SEQ ID NO: 7 and SEQ ID NO: 8, or a primer pair for amplifying the gene fragment described in (j) in claim 3, wherein the upstream primer sequence has a similarity of more than 98%, more than 99%, or more than 99.5% to SEQ ID NO: 7, and the downstream primer sequence has a similarity of more than 98%, more than 99%, or more than 99.5% to SEQ ID NO: 8;

[0039] In the above primer pair or combination of primer pairs, the primer pair for amplifying the gene or gene fragment described in (l) is: a primer pair as shown in SEQ ID NO:9 and SEQ ID NO:10, or a primer pair for amplifying the gene fragment described in (l) in claim 3, wherein the upstream primer sequence has a similarity of more than 98%, more than 99%, or more than 99.5% to SEQ ID NO:9, and the downstream primer sequence has a similarity of more than 98%, more than 99%, or more than 99.5% to SEQ ID NO:10;

[0040] In the above primer pair or combination of primer pairs, the primer pair for amplifying the gene or gene fragment described in (f) is: a primer pair as shown in SEQ ID NO: 11 and SEQ ID NO: 12, or a primer pair for amplifying the gene fragment described in (f) in claim 3, wherein the upstream primer sequence has a similarity of more than 98%, more than 99%, or more than 99.5% to SEQ ID NO: 11, and the downstream primer sequence has a similarity of more than 98%, more than 99%, or more than 99.5% to SEQ ID NO: 12;

[0041] Among the above-mentioned primer pairs or combinations of primer pairs, the primer pair for amplifying the gene or gene fragment described in (h) is: a primer pair as shown in SEQ ID NO: 17 and SEQ ID NO: 18, or a primer pair for amplifying the gene fragment described in (h) in claim 3, and the upstream primer sequence has a similarity of more than 98%, more than 99%, or more than 99.5% to SEQ ID NO: 17, and the downstream primer sequence has a similarity of more than 98%, more than 99%, or more than 99.5% to SEQ ID NO: 18.

[0042] In the above primer pair or combination of primer pairs, the primers are primers for amplifying internal reference genes when detecting the expression level of plant genes;

[0043] The primer pair or primer pair combination described in (1) or (2) is a primer for amplifying an internal reference gene when detecting gene expression in plant roots under cold stress, plant roots under waterlogging stress, plant stems under salt stress, plant leaves under cold stress, different tissues of plants without any stress, plant roots under salt stress, plant stems under cold stress, plant stems under waterlogging stress, plant leaves under salt stress, or plant leaves under waterlogging stress;

[0044] When used to detect gene expression in plant roots under salt stress, the primers for amplifying the internal reference gene are the primers or primer combinations described in (1) or (2) or (3);

[0045] When used to detect gene expression in plant stems under cold stress or waterlogging stress, the primers for amplifying the internal reference gene are the primers or primer combinations described in (1) or (2) or (4);

[0046] When used to detect gene expression in plant leaves under salt stress or waterlogging stress, the primers for amplifying the internal reference gene are the primers or primer combinations described in (1) or (2) or (5);

[0047] When used to detect gene expression in plant leaves under cold stress, the primers for amplifying the internal reference gene are the primers or primer combinations described in (1) or (2) or (6);

[0048] When used to detect gene expression in different tissues of plants without any stress, the primers for amplifying the internal reference gene are the primers or primer combinations described in (1) or (2) or (7);

[0049] The plant is a plant of the genus Actinidia, specifically Actinidia serrata, and the different tissues of the plant are roots, stems and leaves.

[0050] The method for detecting gene expression level is real-time fluorescence quantitative PCR.

[0051] The present invention also provides any of the following applications:

[0052] Use of any of the above genes or gene fragments or a combination thereof as an internal reference gene for Actinidia plants;

[0053] Use of any of the above primer pairs or primer pair combinations as primers for amplifying internal reference genes of Actinidia plants.

[0054] In the above application, the internal reference gene is a reference gene of plant roots under cold stress, plant roots under flooding stress, plant stems under salt stress, plant leaves under cold stress, different tissues of plants under no stress, plant roots under salt stress, plant stems under cold stress, plant stems under flooding stress, plant leaves under salt stress, or plant leaves under flooding stress;

[0055] The Actinidia plant may specifically be Actinidia kolomikta;

[0056] The different tissues of the plant are roots, stems and leaves.

[0057] In any of the above applications, when used to detect gene expression in plant roots under cold stress, plant roots under waterlogging stress, plant stems under salt stress, plant leaves under cold stress, different tissues of plants without any stress, plant roots under salt stress, plant stems under cold stress, plant stems under waterlogging stress, plant leaves under salt stress or plant leaves under waterlogging stress, internal reference genes (a) or (b), or a combination of (a) or (b) and (c) or (d) can be used; and primers for amplifying the corresponding internal reference genes are used accordingly.

[0058] In any of the above applications, when used to detect gene expression in plant roots under salt stress, the internal reference gene (a) or (b) can be used alone, or a combination of (a) or (b) and (c) or (d) can be used, or a combination of (a) or (b) and (e) or (f) can be used;

[0059] In any of the above applications, when used to detect gene expression in plant stems under cold stress or waterlogging stress, the internal reference gene (a) or (b) can be used alone, or a combination of (a) or (b) and (c) or (d) can be used, or a combination of (a) or (b) and (g) or (h) can be used;

[0060] In any of the above applications, when used to detect gene expression in plant leaves under salt stress or waterlogging stress, the internal reference gene (a) or (b) can be used alone, or a combination of (a) or (b) and (c) or (d) can be used, or a combination of (a) or (b) and (i) or (j) can be used;

[0061] In any of the above applications, when used to detect gene expression in plant leaves under cold stress, the internal reference gene (a) or (b) can be used alone, or a combination of (a) or (b) and (c) or (d) can be used, or a combination of (a) or (b) and (k) or (l) can be used;

[0062] In any of the above applications, when used to detect gene expression in different tissues of a plant without any stress, the internal reference gene (a) or (b) can be used alone, or a combination of (a) or (b) and (c) or (d) can be used, or a combination of (c) or (d) and (e) or (f) can be used.

[0063] The present invention also provides a method for detecting the expression level of a plant gene, comprising the following steps (a) or (b):

[0064] (a) using any of the above genes or gene fragments or a combination thereof as an internal reference gene;

[0065] (b) using any of the primer pairs or primer pair combinations described above as primers for amplifying an internal reference gene.

[0066] The plant is a plant of the genus Actinidia, and specifically may be Actinidiakolomikta;

[0067] The different tissues of the plant are roots, stems and leaves;

[0068] The method for detecting the plant gene expression level is real-time fluorescence quantitative PCR.

[0069] In the above-mentioned method for detecting plant gene expression levels, when used to detect gene expression in plant roots under cold stress, plant roots under waterlogging stress, plant stems under salt stress, plant leaves under cold stress, different tissues of plants without any stress, plant roots under salt stress, plant stems under cold stress, plant stems under waterlogging stress, plant leaves under salt stress or plant leaves under waterlogging stress, internal reference genes (a) or (b), or a combination of (a) or (b) and (c) or (d) can be used; and primers for amplifying the corresponding internal reference genes are used accordingly.

[0070] In the above method for detecting the gene expression level of a plant, when used to detect the gene expression of a plant root under salt stress, the internal reference gene (a) or (b) can be used alone, or a combination of (a) or (b) and (c) or (d) can be used, or a combination of (a) or (b) and (e) or (f) can be used;

[0071] In the above method for detecting gene expression levels in plants, when used to detect gene expression in plant stems under cold stress or waterlogging stress, the internal reference gene (a) or (b) can be used alone, or a combination of (a) or (b) and (c) or (d) can be used, or a combination of (a) or (b) and (g) or (h) can be used;

[0072] In the above method for detecting the gene expression level of a plant, when used to detect the gene expression of plant leaves under salt stress or waterlogging stress, the internal reference gene (a) or (b) can be used alone, or a combination of (a) or (b) and (c) or (d) can be used, or a combination of (a) or (b) and (i) or (j) can be used;

[0073] In the above method for detecting the gene expression level of a plant, when used to detect the gene expression of plant leaves under cold stress, the internal reference gene (a) or (b) can be used alone, or a combination of (a) or (b) and (c) or (d) can be used, or a combination of (a) or (b) and (k) or (l) can be used;

[0074] In the above method for detecting plant gene expression levels, when used to detect gene expression in different tissues of plants without any stress, the internal reference gene (a) or (b) can be used alone, or a combination of (a) or (b) and (c) or (d) can be used, or a combination of (c) or (d) and (e) or (f) can be used.

[0075] Stable reference genes are the prerequisite for the application of qRT-PCR technology. The stability of reference genes directly affects the calculation results of gene expression. Ideal reference genes should be stably expressed in different tissues and under different conditions. The expression stability of most reference genes will change in different tissues or under different environmental conditions.

[0076] A single internal reference gene can simplify the experimental process, facilitate data analysis and reduce experimental costs. Multiple internal reference genes can improve the accuracy of the results, enhance the stability of the experiment and be applicable to complex experimental conditions.

[0077] Based on the genome data of Actinidia chinensis, this study screened out 8 candidate reference genes, used qRT-PCR technology to detect and analyze the reference genes in different organs of Actinidia chinensis and under different abiotic stress conditions, and used geNorm, NormFinder, BestKeeper software and RefFinder tools to comprehensively evaluate the stability of the candidate reference genes, aiming to determine the reference genes that are stable under various conditions. The present invention found through research that the combination of AkGAPDH and AkGAPDH+AkEF-1α is suitable for kiwifruit to express stably under various stresses and combinations of different tissues, and is suitable as a reference gene in various situations. In addition, some gene combinations that are stably expressed under specific stresses and combinations of specific tissues were obtained, providing a basis for the development of related experiments on Actinidia chinensis. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] Figure 1 The RT-PCR results of candidate internal reference genes of Actinidia zedoaria.

[0079] Figure 2 is the melting curve of the candidate internal reference gene.

[0080] Figure 3 The Ct value distribution of candidate internal reference genes in Actinidia zedoaria under different conditions.

[0081] Figure 4 Reliability verification of the internal reference genes selected for Actinidia chinensis. DETAILED DESCRIPTION

[0082] 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.

[0083] The plant materials used in this study were from Qilidi, Hengdaohezi, Mudanjiang City, Heilongjiang Province. The clones were obtained by tissue culture and preserved in the tissue culture room of Mudanjiang Normal College (129.561293E, 44.589029N).

[0084] The roots and stems of 15 cm tall Actinidia kiwifruit seedlings and the leaves of 25 cm tall Actinidia kiwifruit seedlings were selected for subsequent experiments. The culture conditions were 25°C, 16 h light / 8 h dark, 1500 Lx light intensity, and each sample was repeated 3 times.

[0085] Simulated salt stress: Actinidia dogwood was irrigated with 500 mL of 200 mmol / L NaCl solution, and the roots, stems and leaves of the plants were taken as materials at 0 h (control), 12 h, 24 h and 48 h of treatment, with 3 biological replicates for each treatment.

[0086] Simulated low temperature stress: Actinidia dogwood was subjected to a low temperature treatment of 4°C, and the roots, stems and leaves of the plants at 0 h (control), 8 h, 24 h and 48 h of treatment were used as materials, with 3 biological replicates for each treatment.

[0087] Simulated flooding stress: The roots of Actinidia dogwood were completely immersed in water. The liquid level was higher than the root area and was set 5 cm above the soil surface. The roots, stems and leaves of the plants at 0 h (control), 6 h, 12 h, 24 h and 48 h were used as materials. Three biological replicates were taken for each treatment.

[0088] All the above plant samples were quickly frozen with liquid nitrogen immediately after collection and stored in a -80°C refrigerator for future use.

[0089] The total RNA of the material was extracted using an RNA extraction kit (FastPure Universal Plant Total RNA Isolation Kit, Nanjing Novozymes), and the concentration and integrity of the RNA were detected using an ultramicrospectrophotometer and 1% agarose gel electrophoresis, respectively. Using 1 μg of RNA as a template, reverse transcriptase (M-MLV(H-)Reverse Transcriptase, Nanjing Novozymes) was used to reverse transcribe and synthesize cDNA, which was then stored at -20°C for future use.

[0090] AkGAPDH, AkEF-1α, AkTUA, AkCYP, AkRPL19, AkTCTP, AkF-box and AkUBE3 were screened as candidate reference genes from the genome data of Actinidia chinensis, and primers were designed using Primer Premier 6.0 (Table 1) and synthesized by Harbin Ruiboxingke.

[0091] Table 1 Primer information of candidate internal reference genes of Actinidia serrata

[0092]

[0093] The slope of the standard curve was generated by diluting the cDNA template 10 times, and E(%) = (10 -1 / slope –1)×100 to calculate the amplification efficiency of each candidate internal reference gene, expressed as R 2The reliability of the standard curve regression equation was predicted and repeated three times. This experiment used Applied Biosystems real-time fluorescence PCR instrument and 2x SYBR Green qPCR MasterMixII for qRT-PCR. The reaction system (total volume 20μL) contained the following components: SYBR Green Master Mix 10μL, cDNA template 2μL, upstream and downstream primers 0.4μL each, and ddH2O 7.2μL. The amplification program was: 95℃ pre-denaturation for 30s; 95℃ denaturation for 15s, 59℃ annealing for 30s, a total of 40 cycles, and 3 biological replicates were set for each treatment.

[0094] The Ct values ​​(Cyclethreshold, Ct) of different samples were analyzed and evaluated using geNorm, NormFinder and BestKeeper software respectively. The above results were comprehensively analyzed using the RefFinder tool to determine the internal reference genes suitable for qRT-PCR in different tissues and under different stress conditions of Actinidia zedoaria.

[0095] Results and Analysis

[0096] Analysis of candidate internal reference gene primer specificity: The specificity of candidate internal reference gene primers was analyzed using PCR technology. The electrophoresis results showed that the amplification products of each primer were all single bands with the expected band size ( Figure 1 ), and qRT-PCR was performed using cDNA from Actinidia chinensis leaves as a template. The results showed that the melting curves of all candidate reference genes showed a single peak ( Figure 2 ), indicating that the primers used have good specificity and can be used for subsequent experiments.

[0097] Analysis of the expression abundance of candidate reference genes: The expression abundance of 8 candidate reference genes was analyzed. The results showed that in the comprehensive analysis of different tissues and abiotic stresses, AkGAPDH had the smallest average Ct value and the highest expression abundance; AkF-box had the largest average Ct value and the lowest expression abundance. AkGAPDH had the smallest fluctuation range of Ct value, ranging from 21.8 to 25.9, and the expression was the most stable. AkTUA had the largest fluctuation range, ranging from 21.8 to 32.8, and the expression was the most unstable ( Figure 3 )

[0098] Analysis of expression stability of candidate reference genes

[0099] geNorm analysis results: The geNorm software was used to calculate the stability value (M) of the candidate reference genes. The smaller the M value, the more stable the gene expression. The results showed that under salt stress, AkGAPDH was recommended as the reference gene in roots, stems and leaves; under cold stress, AkGAPDH was recommended as the reference gene in roots and stems, and AkEF-1α was recommended as the reference gene in leaves; under waterlogging stress, AkGAPDH was recommended as the reference gene in roots, stems and leaves; AkGAPDH was recommended as the reference gene in the comprehensive analysis of different tissues and abiotic stresses (Table 2).

[0100] In addition, this experiment used the average pairwise coefficient of variation (Vn / Vn+1) to determine the number of internal reference genes for qRT-PCR in Actinidia chinensis. The V2 / 3 values ​​of the experimental data were all below the critical threshold of 0.15 ( Figure 3 ), which indicates that the top two genes are sufficient for qRT-PCR normalization in each experimental group. Under salt stress, it is necessary to normalize the combination of roots and leaves (AkGAPDH and AkTCTP) and stems (AkGAPDH and AkEF-1α); under cold stress, it is necessary to normalize the combination of roots and stems (AkGAPDH and AkEF-1α) and leaves (AkEF-1α and AkRPL19); under flooding stress, it is necessary to normalize the combination of roots (AkGAPDH and AkTCTP), stems (AkGAPDH and AkCYP), and leaves (AkGAPDH and AkUBE3); in different tissues (AkGAPDH and AkEF-1α) normalization; the comprehensive analysis V2 / 3 is 0.149, which is lower than 0.15, and it is necessary to normalize the combination of (AkGAPDH and AkEF-1α).

[0101] Table 2 GeNorm analysis results of 8 candidate internal reference genes of Actinidia chinensis

[0102]

[0103]

[0104] Figure 3 Analysis of reference gene pair variation (Vn / Vn+1) based on geNorm

[0105]

[0106] NormFinder analysis results: NormFinde software was used to calculate the stability value (S) of the candidate reference genes. The smaller the S value, the more stable the gene expression. The results showed that under salt stress, AkGAPDH had the best stability in roots, stems and leaves; under cold stress, AkEF-1α had the best stability in roots and leaves, and AkGAPDH and AkTUA had the best stability in stems; under flooding stress, AkF-box had the best stability in roots, and AkGAPDH had the best stability in stems and leaves; in different tissues, AkEF-1α and AkTCTP had the highest stability, followed by AkGAPDH; comprehensive analysis results showed that AkGAPDH had the best stability under different tissues and abiotic stresses, followed by AkEF-1α (Table 4).

[0107] Table 4 NormFinder analysis results of 8 candidate internal reference genes of Actinidia kenezaki

[0108]

[0109] BestKeeper analysis results: Bestkeeper was used to analyze the stability of candidate reference genes. The lower the standard deviation (SD) and the higher the Pearson correlation coefficient (r), the more stable the expression. If the SD is greater than 1.0, it is not suitable as a reference gene. The results show that under salt stress, the SD of the six candidate reference genes in the root is less than 1, and the order of r is AkTCTP>AkRPL19>AkGAPDH>AkEF-1α>AkF-box>AkCYP, indicating that AkTCTP has the best stability. The SD of the five candidate reference genes in the stem is less than 1, and the order of r is AkGAPDH>AkTUA>AkEF-1α>AkCYP>AkTCTP, indicating that AkGAPDH has the best stability. The SD of the five candidate reference genes in the leaf is less than 1, and the order of r is AkCYP>AkGAPDH>AkTCTP. TP>AkEF-1α>AkF-box, indicating that AkCYP has the best stability. Under cold stress, the SD of the three candidate internal reference genes in the root was less than 1, and the size of r was ranked as AkEF-1α>AkGAPDH>AkTCTP, indicating that AkEF-1α has the best stability. The SD of the two candidate internal reference genes in the stem was less than 1, and the size of r was ranked as AkGAPDH=AkTUA, indicating that AkGAPDH and AkTUA have the best stability. The SD of the four candidate internal reference genes in the leaf was less than 1, and the size of r was ranked as AkEF-1α>AkGAPDH>AkRPL19>AkF -box, indicating that AkEF-1α is the most stable. Under waterlogging stress, the SD of the four candidate internal reference genes in the root was less than 1, and the size of their r was ranked as AkF-box>AkTCTP>AkEF-1α>AkGAPDH, indicating that AkF-box is the most stable. The SD of the three candidate internal reference genes in the stem was less than 1, and the size of their r was ranked as AkGAPDH>AkCYP>AkEF-1α, indicating that AkGAPDH is the most stable. The SD of the six internal reference genes in the leaf was less than 1, and the size of their r was ranked as AkEF-1α>AkGAPDH>AkUBE3>AkCYP>A AkRPL19>AkTUA, indicating that AkEF-1α has the best stability; the SD of the five candidate internal reference genes in different tissues is less than 1, and the size of their r is ranked as AkEF-1α>AkTCTP>AkF-box>AkRPL19>AkGAPDH, indicating that AkEF-1α has the best stability; the comprehensive analysis results show that the SD of AkGAPDH and AkEF-1α are both less than 1, and the size of their r is ranked as AkGAPDH>AkEF-1α, so the comprehensive analysis results show that AkGAPDH has the best stability and is suitable as an internal reference gene in different tissues and under different stress conditions (Table 5).

[0110] Table 5 Bestkeeper analysis results of 8 candidate internal reference genes of Actinidia kenezaki

[0111]

[0112] RefFinder analysis results: In order to more comprehensively evaluate the stability of the eight candidate reference genes, the RefFinder software was used to conduct a comprehensive analysis of different tissues and abiotic stresses of Actinidia zedoaria. The results showed that under salt stress, AkGAPDH was the most stable in the root, followed by AkTCTP, AkF-box, AkEF-1α, AkCYP, AkRPL19, AkUBE3, and AkTUA. AkGAPDH was the most stable in the stem, followed by AkEF-1α, AkTUA, AkCYP, AkTCTP, AkUBE3, and AkTUA. F-box, AkRPL19, AkGAPDH was the most stable in leaves, followed by AkCYP, AkEF-1α, AkTCTP, AkUBE3, AkF-box, AkRPL19, and AkTUA; under cold stress conditions, AkGAPDH was the most stable in roots, followed by AkEF-1α, AkTCTP, AkCYP, AkUBE3, AkRPL19, AkTUA, and AkF-box; AkGAPDH was the most stable in stems, followed by AkTUA, AkUBE3, AkTCTP, AkEF-1α, and AkCYP , AkRPL19, and AkF-box. In leaves, AkGAPDH was the most stable, followed by AkRPL19, AkEF-1α, AkF-box, AkTUA, AkUBE3, AkCYP, and AkTCTP. Under waterlogging stress, AkGAPDH was the most stable in roots, followed by AkEF-1α, AkF-box, AkTCTP, AkTUA, AkCYP, AkRPL19, and AkUBE3. In stems, AkGAPDH was the most stable, followed by AkTUA, AkF-box, AkUBE3, AkEF-1α, AkCYP, AkTCTP, AkRPL19, and AkGAPDH in leaves were the most stable, followed by AkCYP, AkEF-1α, AkTCTP, AkUBE3, AkTUA, AkRPL19, and AkF-box; AkTCTP and AkEF-1α had the highest stability in different tissues, followed by AkGAPDH. The comprehensive analysis results showed that AkGAPDH had the best stability, followed by AkEF-1α, AkTCTP, AkRPL19, AkF-box, AkUBE3, AkCYP, and AkTUA (Table 6).

[0113] Table 6 RefFinder analysis results of 8 candidate internal reference genes of Actinidia kenezaki

[0114]

[0115] The comprehensive analysis results of the above experiments and four softwares show that under different tissues and abiotic stresses, the AkGAPDH gene of Actinidia dogwood has a small Ct fluctuation range, high expression level and good stability, making it the most suitable internal reference gene.

[0116] Reliability verification of selected internal reference genes

[0117] To further verify the reliability of the selected reference genes, the most stable AkGAPDH, AkGAPDH+AkEF-1α combination and the least stable AkTUA were used as reference genes, and the relative expression levels of AkMYB73, AkNAC2, AkbHLH92 and AkWRKY65 in Actinidia chinensis were analyzed. The transcriptome data of Actinidia chinensis in different tissues (roots, stems and leaves) and under 4°C stress were used as references. The results are shown in Figure 2. Figure 4As shown. The samples of different tissues were roots, stems and leaves, and the samples under 4℃ stress were leaves. In different tissues, when AkGAPDH and AkGAPDH+AkEF-1α were used as reference genes, AkMYB73 and AkbHLH92 were expressed at the highest level in stems and at the lowest level in roots; AkNAC2 was expressed at the highest level in leaves and at the lowest level in stems; AkWRKY65 was expressed at the highest level in roots and at the lowest level in stems, and the relative expression levels of the four genes were highly consistent with the transcriptome data; when AkTUA was used as the reference gene, AkMYB73, AkNAC2 and AkbHLH92 were expressed at the highest level in leaves and at the lowest level in roots, and AkWRKY65 was expressed at the highest level in stems and at the lowest level in leaves, and the relative expression levels of the four genes were significantly different from the transcriptome data. Under 4℃ low temperature stress conditions, when AkGAPDH and AkGAPDH+AkEF-1α combination were used as reference genes, the expression levels of AkMYB73, AkNAC2 and AkWRKY65 increased significantly at 8h and decreased slightly at 24h, and the expression level of AkbHLH92 gradually increased with the extension of treatment time. The expression trends of the four genes were highly consistent with the transcriptome data. When AkTUA was used as the reference gene, the expression levels of AkNAC2 and AkWRKY65 increased significantly at 8h and showed a downward trend at 24h, which was consistent with the expression trend compared with the transcriptome data, but the gene expression fluctuated more. The expression levels of AkMYB73 and AkbHLH92 genes increased significantly at 8h and increased with the extension of treatment time at 24h. This change of AkMYB73 gene was different from the trend of the transcriptome. When the unstable AkTUA gene was used as the internal reference gene, the expression level showed obvious differences, which further verified the accuracy of the selected internal reference gene. The verification results of AkGAPDH and AkGAPDH+AkEF-1α were the same, and the expression trend was highly consistent with the transcriptome data. It had a good standardization effect on the relative expression of the target gene and could truly reflect the expression trend of the gene. It can be inferred that AkGAPDH is sufficient as an internal reference gene to measure the gene stability of Actinidia kiwifruit.

Claims

1. Gene or gene fragment or their combination: (1) A gene or gene fragment as shown in (a) or (b) 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 of the gene as shown in SEQ ID NO: 1, or a gene derived from a plant of the genus Actinidia or Actinidia kolomikta 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 of the gene as shown in SEQ ID NO: 1 and derived from a plant of the genus Actinidia or Actinidia kolomikta and being a natural allele of the gene having a cDNA sequence as shown in SEQ ID NO: 1; (b) a gene fragment having a cDNA sequence as shown in nucleotides 714 to 861 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 as shown in nucleotides 714 to 861 of SEQ ID NO:1, or a gene fragment derived from a plant of the genus Actinidia and being a natural allele of the gene fragment as shown in the cDNA sequence as shown in nucleotides 714 to 861 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 as shown in nucleotides 714 to 861 of SEQ ID NO:1 and being derived from a plant of the genus Actinidia and being a natural allele of the gene fragment as shown in the cDNA sequence as shown in nucleotides 714 to 861 of SEQ ID NO:1; (2) A combination of a gene or gene fragment as described in (a) or (b) and a gene or gene fragment as described in (c) or (d) below: (c) A gene having a cDNA sequence as shown in SEQ ID NO: 2, or a gene having a sequence having 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: 2, or a gene derived from a plant of the genus Actinidia or Actinidia kolomikta 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 having 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: 2 and derived from a plant of the genus Actinidia or Actinidia kolomikta and being a natural allele of the gene having a cDNA sequence as shown in SEQ ID NO: 2; (d) A gene fragment having a cDNA sequence as shown in nucleotides 325 to 473 of SEQ ID NO:2, or a gene fragment having a sequence having 95% or more, 96% or more, 97% or more, 98% or more, or 99% similarity to the sequence of the gene fragment having a cDNA sequence as shown in nucleotides 325 to 473 of SEQ ID NO:2, or a gene fragment derived from a plant of the genus Actinidia and being a natural allele of the gene fragment having a cDNA sequence as shown in nucleotides 325 to 473 of SEQ ID NO:2, or a gene fragment having 95% or more, 96% or more, 97% or more, 98% or more, or 99% similarity to the sequence of the gene fragment having a cDNA sequence as shown in nucleotides 325 to 473 of SEQ ID NO:2 and being derived from a plant of the genus Actinidia and being a natural allele of the gene fragment having a cDNA sequence as shown in nucleotides 325 to 473 of SEQ ID NO:

2.

2. The gene or gene fragment or a combination thereof according to claim 1, characterized in that: The gene or gene fragment or a combination thereof is suitable as an internal reference gene for plant roots under cold stress, plant roots under waterlogging stress, plant stems under salt stress, plant leaves under cold stress, different tissues of plants under no stress, plant roots under salt stress, plant stems under cold stress, plant stems under waterlogging stress, plant leaves under salt stress or plant leaves under waterlogging stress. The plant is an Actinidia plant, specifically Actinidia kolomikta, and the different tissues of the plant are roots, stems and leaves.

3. The gene or gene fragment or a combination thereof according to claim 2, characterized in that: The internal reference gene suitable for plant roots under salt stress may also be a combination of the gene or gene fragment shown in (a) or (b) and the gene or gene fragment shown in (e) or (f) below; The internal reference gene suitable for plant stems under cold stress or flooding stress may also be a combination of the gene or gene fragment shown in (a) or (b) and the gene or gene fragment shown in (g) or (h) below; The internal reference gene suitable for plant leaves under salt stress or waterlogging stress may also be a combination of the gene or gene fragment shown in (a) or (b) and the gene or gene fragment shown in (i) or (j) below; The internal reference gene suitable for plant leaves under cold stress may also be a combination of the gene or gene fragment shown in (a) or (b) and the gene or gene fragment shown in (k) or (l) below; The internal reference gene suitable for different tissues of plants without any stress may also be a combination of the gene or gene fragment shown in (e) or (f) and the gene or gene fragment shown in (c) or (d) below; (e) A gene having a cDNA sequence as shown in SEQ ID NO: 13, or a gene having a sequence having 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: 13, or a gene derived from a plant of the genus Actinidia or Actinidia kolomikta and being a natural allele of the gene having a cDNA sequence as shown in SEQ ID NO: 13, or a gene having a sequence having 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: 13 and derived from a plant of the genus Actinidia or Actinidia kolomikta and being a natural allele of the gene having a cDNA sequence as shown in SEQ ID NO: 13; (f) a gene fragment having a cDNA sequence as shown in nucleotides 360 to 480 of SEQ ID NO: 13, or a gene fragment having a sequence having 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 360 to 480 of SEQ ID NO: 13, or a gene fragment derived from a plant of the genus Actinidia and being a natural allele of the gene fragment having a cDNA sequence as shown in nucleotides 360 to 480 of SEQ ID NO: 13, or a gene fragment having a sequence having 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 360 to 480 of SEQ ID NO: 13 and being derived from a plant of the genus Actinidia and being a natural allele of the gene fragment having a cDNA sequence as shown in nucleotides 360 to 480 of SEQ ID NO: 13; (g) a gene having a cDNA sequence as shown in SEQ ID NO: 14, or a gene having a sequence having 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: 14, or a gene derived from a plant of the genus Actinidia or Actinidia kolomikta and being a natural allele of the gene having a cDNA sequence as shown in SEQ ID NO: 14, or a gene having a sequence having 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: 14 and derived from a plant of the genus Actinidia or Actinidia kolomikta and being a natural allele of the gene having a cDNA sequence as shown in SEQ ID NO: 14; (h): a gene fragment having a cDNA sequence as shown in nucleotides 1133 to 1277 of SEQ ID NO: 14, 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 1133 to 1277 of SEQ ID NO: 14, or a gene fragment derived from a plant of the genus Actinidia and being a natural allele of the gene fragment having a cDNA sequence as shown in nucleotides 1133 to 1277 of SEQ ID NO: 14, 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 1133 to 1277 of SEQ ID NO: 14 and being derived from a plant of the genus Actinidia and being a natural allele of the gene fragment having a cDNA sequence as shown in nucleotides 1133 to 1277 of SEQ ID NO:

14. The gene segment indicated by nucleotides 1133 to 1277 in NO:14 is the gene segment of the natural allele; (i) A gene having a cDNA sequence as shown in SEQ ID NO: 15, or a gene having a sequence having 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: 15, or a gene derived from a plant of the genus Actinidia or Actinidia kolomikta and being a natural allele of the gene having a cDNA sequence as shown in SEQ ID NO: 15, or a gene having a sequence having 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: 15 and derived from a plant of the genus Actinidia or Actinidia kolomikta and being a natural allele of the gene having a cDNA sequence as shown in SEQ ID NO: 15; (j) a gene fragment having a cDNA sequence as shown in nucleotides 373 to 483 of SEQ ID NO: 15, 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 as shown in nucleotides 373 to 483 of SEQ ID NO: 15, or a gene fragment derived from a plant of the genus Actinidia and being a natural allele of the gene fragment as shown in the cDNA sequence as shown in nucleotides 373 to 483 of SEQ ID NO: 15, 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 as shown in nucleotides 373 to 483 of SEQ ID NO: 15 and being derived from a plant of the genus Actinidia and being a natural allele of the gene fragment as shown in the cDNA sequence as shown in nucleotides 373 to 483 of SEQ ID NO: 15; (k) a gene having a cDNA sequence as shown in SEQ ID NO: 16, or a gene having a sequence having 95% or more, 96% or more, 97% or more, 98% or more, or 99% similarity to the sequence of the gene having a cDNA sequence as shown in SEQ ID NO: 16, or a gene derived from a plant of the genus Actinidia or Actinidia kolomikta and being a natural allele of the gene having a cDNA sequence as shown in SEQ ID NO: 16, or a gene having a sequence having 95% or more, 96% or more, 97% or more, 98% or more, or 99% similarity to the sequence of the gene having a cDNA sequence as shown in SEQ ID NO: 16 and derived from a plant of the genus Actinidia or Actinidia kolomikta and being a natural allele of the gene having a cDNA sequence as shown in SEQ ID NO: 16; (l) A gene fragment having a cDNA sequence as shown in nucleotides 157 to 277 of SEQ ID NO: 16, or a gene fragment having a sequence having 95% or more, 96% or more, 97% or more, 98% or more, or 99% similarity to the sequence of the gene fragment having a cDNA sequence as shown in nucleotides 157 to 277 of SEQ ID NO: 16, or a gene fragment derived from the genus Actinidia and being a natural allele of the gene fragment having a cDNA sequence as shown in nucleotides 157 to 277 of SEQ ID NO: 16, or a gene fragment having 95% or more, 96% or more, 97% or more, 98% or more, or 99% similarity to the sequence of the gene fragment having a cDNA sequence as shown in nucleotides 157 to 277 of SEQ ID NO: 16 and being derived from a plant of the genus Actinidia and being a natural allele of the gene fragment having a cDNA sequence as shown in nucleotides 157 to 277 of SEQ ID NO:

16.

4. Any of the following primer pairs or combinations of primer pairs: (1) a primer pair for amplifying the gene or gene fragment described in (a) or (b) of claim 1; (2) A combination of the following primer pairs: a primer pair for amplifying the gene or gene fragment described in (a) or (b) of claim 1, and a primer pair for amplifying the gene or gene fragment described in (c) or (d) of claim 1; (3) A combination of the following primer pairs: a primer pair for amplifying the gene or gene fragment described in (a) or (b) of claim 1, and a primer pair for amplifying the gene or gene fragment described in (e) or (f) of claim 3; (4) A combination of the following primer pairs: a primer pair for amplifying the gene or gene fragment described in (a) or (b) of claim 1, and a primer pair for amplifying the gene or gene fragment described in (g) or (h) of claim 3; (5) A combination of the following primer pairs: a primer pair for amplifying the gene or gene fragment described in (a) or (b) of claim 1, and a primer pair for amplifying the gene or gene fragment described in (i) or (j) of claim 3; (6) A combination of the following primer pairs: a primer pair for amplifying the gene or gene fragment described in (a) or (b) of claim 1, and a primer pair for amplifying the gene or gene fragment described in (k) or (l) of claim 3. (7) A combination of the following primer pairs: a primer pair for amplifying the gene or gene fragment described in (c) or (d) of claim 1, and a primer pair for amplifying the gene or gene fragment described in (e) or (f) of claim 3.

5. The primer pair or the combination of primer pairs according to claim 4, characterized in that: The primer pair for amplifying the gene or gene fragment described in (b) is: the primer pair shown in SEQ ID NO:3 and SEQ ID NO:4, or, a primer pair for amplifying the gene fragment described in (b) in claim 1, wherein the upstream primer sequence has a similarity of more than 98%, more than 99%, or more than 99.5% to SEQ ID NO:3, and the downstream primer sequence has a similarity of more than 98%, more than 99%, or more than 99.5% to SEQ ID NO:4; The primer pair for amplifying the gene or gene fragment described in (d) is: the primer pair shown in SEQ ID NO:5 and SEQ ID NO:6, or, a primer pair for amplifying the gene fragment described in (d) in claim 1, wherein the upstream primer sequence has a similarity of more than 98%, more than 99%, or more than 99.5% to SEQ ID NO:5, and the downstream primer sequence has a similarity of more than 98%, more than 99%, or more than 99.5% to SEQ ID NO:6; The primer pair for amplifying the gene or gene fragment described in (j) is: the primer pair shown in SEQ ID NO: 7 and SEQ ID NO: 8, or, a primer pair for amplifying the gene fragment described in (j) in claim 3, wherein the upstream primer sequence has a similarity of more than 98%, more than 99%, or more than 99.5% to SEQ ID NO: 7, and the downstream primer sequence has a similarity of more than 98%, more than 99%, or more than 99.5% to SEQ ID NO: 8; The primer pair for amplifying the gene or gene fragment described in (l) is: the primer pair shown in SEQ ID NO:9 and SEQ ID NO:10, or, a primer pair for amplifying the gene fragment described in (l) in claim 3, wherein the upstream primer sequence has a similarity of more than 98%, more than 99%, or more than 99.5% to SEQ ID NO:9, and the downstream primer sequence has a similarity of more than 98%, more than 99%, or more than 99.5% to SEQ ID NO:10; The primer pair for amplifying the gene or gene fragment described in (f) is: the primer pair shown in SEQ ID NO: 11 and SEQ ID NO: 12, or, a primer pair for amplifying the gene fragment described in (f) in claim 3, wherein the upstream primer sequence has a similarity of more than 98%, more than 99%, or more than 99.5% to SEQ ID NO: 11, and the downstream primer sequence has a similarity of more than 98%, more than 99%, or more than 99.5% to SEQ ID NO: 12; The primer pair for amplifying the gene or gene fragment described in (h) is: a primer pair as shown in SEQ ID NO: 17 and SEQ ID NO: 18, or a primer pair for amplifying the gene fragment described in (h) in claim 3, and the upstream primer sequence has a similarity of more than 98%, more than 99%, or more than 99.5% to SEQ ID NO: 17, and the downstream primer sequence has a similarity of more than 98%, more than 99%, or more than 99.5% to SEQ ID NO:

18.

6. The primer pair or primer pair combination according to claim 4 or 5, characterized in that: The primers are primers used to amplify internal reference genes when detecting the expression level of plant genes; The primer pair or primer pair combination described in (1) or (2) is a primer for amplifying an internal reference gene when detecting gene expression in plant roots under cold stress, plant roots under waterlogging stress, plant stems under salt stress, plant leaves under cold stress, different tissues of plants without any stress, plant roots under salt stress, plant stems under cold stress, plant stems under waterlogging stress, plant leaves under salt stress, or plant leaves under waterlogging stress; When used to detect gene expression in plant roots under salt stress, the primers for amplifying the internal reference gene are the primers or primer combinations described in (1) or (2) or (3); When used to detect gene expression in plant stems under cold stress or waterlogging stress, the primers for amplifying the internal reference gene are the primers or primer combinations described in (1) or (2) or (4); When used to detect gene expression in plant leaves under salt stress or waterlogging stress, the primers for amplifying the internal reference gene are the primers or primer combinations described in (1) or (2) or (5); When used to detect gene expression in plant leaves under cold stress, the primers for amplifying the internal reference gene are the primers or primer combinations described in (1) or (2) or (6); When used to detect gene expression in different tissues of plants without any stress, the primers for amplifying the internal reference gene are the primers or primer combinations described in (1) or (2) or (7); The plant is a plant of the genus Actinidia, specifically Actinidia serrata, and the different tissues of the plant are roots, stems and leaves.

7. Any of the following applications: Use of the gene or gene fragment or a combination thereof as claimed in any one of claims 1 to 3 as an internal reference gene of an Actinidia plant; Use of the primer pair or primer pair combination described in any one of claims 4 to 6 as amplification primers for an internal reference gene of an Actinidia plant.

8. The use according to claim 7, characterized in that: The internal reference gene is an internal reference gene of plant roots under cold stress, plant roots under flooding stress, plant stems under salt stress, plant leaves under cold stress, different tissues of plants under no stress, plant roots under salt stress, plant stems under cold stress, plant stems under flooding stress, plant leaves under salt stress, or plant leaves under flooding stress; The Actinidia plant may specifically be Actinidia kolomikta; The different tissues of the plant are roots, stems and leaves.

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 or a combination thereof as 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 6 as a primer for amplifying an internal reference gene.

10. The method according to claim 9, characterized in that: The detecting of the plant gene expression level is detecting the gene expression level of plant roots under cold stress, plant roots under flooding stress, plant stems under salt stress, plant leaves under cold stress, different tissues of plants under no stress, plant roots under salt stress, plant stems under cold stress, plant stems under flooding stress, plant leaves under salt stress or plant leaves under flooding stress; The plant is a plant of the genus Actinidia, and specifically may be Actinidiakolomikta; The different tissues of the plant are roots, stems and leaves; The method for detecting the plant gene expression level is real-time fluorescence quantitative PCR.

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