Fluorescent quantitative PCR internal reference gene of pinus kesiya var. lasiocarpa and primer and application thereof

Abstract

The application discloses a fluorescence quantitative PCR internal reference gene of Pinus kesiya var. lasiocarpa, primers and application thereof, and belongs to the field of molecular biology. The present application takes different organ tissues and different proliferation stage calli of Pinus kesiya var. lasiocarpa as plant materials, and preliminarily determines eight candidate internal reference genes by using transcriptome data. Primers are designed according to the nucleotide sequences of the candidate internal reference genes, the specificity and amplification efficiency of the primers are verified, the stability of the candidate internal reference genes is analyzed by using geNorm, NormFinder and BestKeeper software and RefFinder online tools, and finally four target genes are selected to verify the internal reference gene of the present application. The internal reference gene of the present application provides a theoretical basis and an important platform for subsequent important gene mining, gene expression level research and other breeding work of Pinus kesiya var. lasiocarpa, and especially provides a stable internal reference gene for related research in the process of callus differentiation of Pinus kesiya var. lasiocarpa.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, specifically to a fluorescence quantitative PCR internal reference gene of *Pinus kesiao*, its primers, and their applications, particularly to an internal reference gene, primers, and their applications for callus tissues of *Pinus kesiao* in different tissues, organs, or different stages of proliferation. Background Technology

[0002] *Pinus kesiya* var. *langbianensis* is an evergreen tree belonging to the genus *Pinus* in the family Pinaceae. It is a geographical variety of *Pinus cassia*, mainly distributed in mountainous areas at altitudes of 700-1800 m. It is an important timber and resin-producing tree species in Yunnan Province. It is characterized by rapid growth, high yield, high resin production, and strong environmental adaptability, playing a vital role in forestry production and ecological construction in Yunnan Province. Currently, researchers mainly utilize conventional breeding methods to conduct breeding research on *Pinus kesiya*. However, with the rapid development of molecular biology, molecular breeding technology has been widely applied in forest tree breeding, and *Pinus kesiya* has also entered the field of molecular breeding. Targeted regulation of gene expression controlling a specific trait can greatly improve breeding efficiency, and identifying the genes regulating the target trait is the primary prerequisite for realizing this technology.

[0003] Real-time quantitative reverse transcription PCR (qRT-PCR) is characterized by high sensitivity, strong specificity, and good reproducibility, making it an important tool for studying the transcriptional expression levels of target genes. When analyzing qRT-PCR data, a stable internal reference gene is usually required to correct experimental errors and verify experimental quality. Ideally, the internal reference gene should exhibit relatively stable and consistent expression levels across different organs, tissues, growth stages, and treatment conditions. However, numerous studies have shown that internal reference genes are not universally applicable across different species and organs. The expression stability of internal reference genes varies across different developmental stages, tissues, organs, and experimental conditions, making them unsuitable for simultaneous application to multiple experimental conditions. Therefore, to ensure more accurate and reliable results in analyzing target gene expression levels, it is essential to select appropriate internal reference genes tailored to different experimental requirements.

[0004] Currently, although there are studies on reference genes for other pine species, the significant genomic differences between different pine varieties, including substantial variations in genome size and specific gene sequences, mean that reference genes screened from other pine species are insufficient for analyzing qRT-PCR results of *Pinus simonii*. To date, no suitable reference genes for *Pinus simonii* qRT-PCR research have been reported; therefore, this invention is proposed. Summary of the Invention

[0005] In order to overcome the shortcomings and deficiencies of the prior art, the purpose of this invention is to provide a fluorescence quantitative PCR internal reference gene of Pinus sylvestris, its primers and applications.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] This invention provides a fluorescence quantitative PCR internal reference gene for Pinus koraiensis, wherein the internal reference gene includes at least one of SAND, β-TUB, UBQ13, PP2A, ACT7, and α-TUB;

[0008] Furthermore, the internal reference genes for real-time PCR of different organs and tissues of Pinus sylvestris var. sapiens include at least one of SAND, β-TUB, UBQ13, and PP2A;

[0009] Preferably, the different organs and tissues include roots, pine needles, terminal buds, stem segments, male cones, seeds, and immature cones;

[0010] Furthermore, the internal reference genes for real-time PCR of callus tissue at different proliferation stages of Pinus kesiao are included, wherein the internal reference genes include at least one of β-TUB, UBQ13, ACT7, and α-TUB.

[0011] Preferably, the callus tissue at different proliferation stages includes callus tissue at 0d, 6d, 9d, 12d, 15d, 18d, and 21d stages.

[0012] Preferably, the nucleotide sequence of the SAND is as shown in SEQ ID NO.4; the nucleotide sequence of β-TUB is as shown in SEQ ID NO.6; the nucleotide sequence of UBQ13 is as shown in SEQ ID NO.8; the nucleotide sequence of PP2A is as shown in SEQ ID NO.7; the nucleotide sequence of ACT7 is as shown in SEQ ID NO.2; and the nucleotide sequence of α-TUB is as shown in SEQ ID NO.5.

[0013] The present invention also provides primers for amplifying the above-mentioned fluorescence quantitative PCR internal reference gene of Pinus kesiao, the primer sequences of which are as follows:

[0014] The primer sequences for amplifying SAND are shown in SEQ ID NO. 15-16; the primer sequences for amplifying β-TUB are shown in SEQ ID NO. 19-20; the primer sequences for UBQ13 are shown in SEQ ID NO. 23-24; the primer sequences for PP2A are shown in SEQ ID NO. 21-22; the primer sequences for ACT7 are shown in SEQ ID NO. 11-12; and the primer sequences for α-TUB are shown in SEQ ID NO. 17-18.

[0015] The present invention also provides the application of the above-mentioned fluorescence quantitative PCR internal reference gene or primer of Pinus kesiao in the detection of Pinus kesiao gene.

[0016] Preferably, the gene detection is to detect the transcriptional expression level of the *Pinus yew* gene; more preferably, the gene detection is to detect the transcriptional expression level of the *Pinus yew* gene by quantitative real-time PCR.

[0017] Furthermore, the application of the above-mentioned fluorescence quantitative PCR internal reference genes or primers in different organs and tissues of *Pinus kesao-ko* in gene detection of different organs and tissues of *Pinus kesao-ko*.

[0018] Preferably, the gene detection is to detect the gene transcriptional expression level in different organs and tissues of *Pinus kesiao*; more preferably, the gene detection is to detect the gene transcriptional expression level in different organs and tissues of *Pinus kesiao* by quantitative real-time PCR.

[0019] Furthermore, the application of the aforementioned fluorescent quantitative PCR internal reference genes or primers from callus tissues at different proliferation stages of *Pinus kesiao* in gene detection of callus tissues at different proliferation stages of *Pinus kesiao*.

[0020] Preferably, the gene detection is to detect the gene transcriptional expression level of callus tissue at different proliferation stages of Pinus kesiya; more preferably, the gene detection is to detect the gene transcriptional expression level of callus tissue at different proliferation stages of Pinus kesiya by quantitative real-time PCR.

[0021] The present invention also provides a real-time PCR detection kit, wherein the detection kit includes the primers described above.

[0022] The kit also includes other reagents used in quantitative real-time PCR.

[0023] The present invention also provides the application of the above primers or detection kits in screening internal reference genes stably expressed in different organs and tissues of Pinus kesao-yomi and / or in verifying the expression stability of internal reference genes in different organs and tissues of Pinus kesao-yomi.

[0024] The stable expression of the internal reference gene includes at least one of SAND, β-TUB, UBQ13, and PP2A.

[0025] The present invention also provides the application of the above primers or detection kits in screening internal reference genes stably expressed in callus tissues at different proliferation stages of Pinus kesiao and / or in verifying the expression stability of internal reference genes in callus tissues at different proliferation stages of Pinus kesiao.

[0026] The stable expression of the internal reference gene includes at least one of β-TUB, UBQ13, ACT7, and α-TUB.

[0027] The present invention has the following advantages and effects compared with the prior art:

[0028] This invention uses callus tissue from different organs and tissues of *Pinus kesawati* (roots, needles, terminal buds, stem segments, male cones, seeds, and immature cones) and at different proliferation stages (0d, 6d, 9d, 12d, 15d, 18d, and 21d) as plant materials. Transcriptome data was used to initially identify eight candidate internal reference genes. Primers were designed based on the nucleotide sequences of these candidate genes. Primer specificity and amplification efficiency were verified by gel electrophoresis and qRT-PCR. The stability of the candidate internal reference genes was analyzed using geNorm, NormFinder, BestKeeper software, and the RefFinder online tool. Finally, four target genes were selected to validate the internal reference genes of this invention. The internal reference genes of this invention provide a theoretical basis and an important platform for subsequent breeding work such as important gene discovery and gene expression level research in *Pinus kesawati*, especially providing stable internal reference genes for related research on callus differentiation in *Pinus kesawati*. Attached Figure Description

[0029] Figure 1 It is a technical flowchart.

[0030] Figure 2 This is an electrophoresis diagram of eight candidate internal reference genes.

[0031] Figure 3 These are the melting curves of eight candidate internal reference genes.

[0032] Figure 4 This shows the distribution of Ct values ​​for the eight internal reference genes across all samples.

[0033] Figure 5 The graph shows the results of the geNorm software analysis on the expression stability of eight candidate internal reference genes and the optimal number of internal reference genes. Among them, A: Analysis results of gene expression stability in different organs and tissues of *Pinus kesiya*; B: Analysis results of gene expression stability in different proliferation stages of *Pinus kesiya* callus; C: Analysis results of the number of internal reference genes in different organs and tissues of *Pinus kesiya*; D: Analysis results of the number of internal reference genes in different proliferation stages of *Pinus kesiya* callus.

[0034] Figure 6 This is a graph showing the results of Bestkeeper software analysis on the expression stability of eight candidate internal reference genes; where A: analysis results of different organs and tissues of *Pinus kesiao*; B: analysis results of different proliferation stages of *Pinus kesiao* callus.

[0035] Figure 7 This is a bar chart showing the expression levels of the target gene in different organs and tissues of *Pinus kesiaoensis* with the SAND gene as an internal reference gene; where A: Pkes07g03380; B: Pkes01g10760; C: Pkes05g13910; D: Pkes09g17350.

[0036] Figure 8 This is a bar chart showing the expression levels of the target gene in different organs and tissues of *Pinus kesiaoensis* with β-TUB as the internal reference gene; where A: Pkes07g03380; B: Pkes01g10760; C: Pkes05g13910; D: Pkes09g17350.

[0037] Figure 9 This is a bar chart showing the expression levels of the target gene in different organs and tissues of *Pinus kesiaoensis* with UBQ13 as the internal reference gene; where A: Pkes07g03380; B: Pkes01g10760; C: Pkes05g13910; D: Pkes09g17350.

[0038] Figure 10 This is a bar chart showing the expression levels of the target gene in different organs and tissues of *Pinus kesao-ko* with PP2A as the internal reference gene; where A: Pkes07g03380; B: Pkes01g10760; C: Pkes05g13910; D: Pkes09g17350.

[0039] Figure 11 This is a bar chart showing the expression levels of target genes (Pkes07g03380, Pkes01g10760, Pkes05g13910 and Pkes09g17350) in different proliferation stages of *Pinus koraiensis* callus with β-TUB as the internal reference gene.

[0040] Figure 12 This is a bar chart showing the expression levels of the target gene in different proliferation stages of *Pinus kesaoensis* callus with UBQ13 as the internal reference gene; where A: Pkes07g03380; B: Pkes01g10760; C: Pkes05g13910; D: Pkes09g17350.

[0041] Figure 13 This is a bar chart showing the expression levels of the target gene in different proliferation stages of *Pinus kesaoensis* callus with ACT7 as the internal reference gene; where A: Pkes07g03380; B: Pkes01g10760; C: Pkes05g13910; D: Pkes09g17350.

[0042] Figure 14 This is a bar chart showing the expression levels of the target gene in different proliferation stages of *Pinus kesiao* callus with α-TUB as the internal reference gene; where A: Pkes07g03380; B: Pkes01g10760; C: Pkes05g13910; D: Pkes09g17350.

[0043] Figure 15This is a bar chart showing the expression levels of target genes (Pkes07g03380, Pkes01g10760, Pkes05g13910 and Pkes09g17350) in Pinus sylvestris var. mongolica, with TPI11 as the internal reference gene. Detailed Implementation

[0044] The present invention will be further described in detail below with reference to embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto. Test methods in the following embodiments that do not specify specific experimental conditions are generally performed under conventional experimental conditions or according to the manufacturer's recommended experimental conditions. Unless otherwise specified, the materials and reagents used are commercially available.

[0045] The technical flowchart of this invention is as follows: Figure 1 As shown.

[0046] Example 1

[0047] 1. Experimental Materials

[0048] The test materials were callus tissues from different tissues and organs (roots, needles, terminal buds, stem segments, male cones, seeds, and immature cones) and different proliferation stages (0d, 6d, 9d, 12d, 15d, 18d, and 21d) of *Pinus simonii* seed orchards in Pu'er, Yunnan. Three well-grown *Pinus simonii* trees of uniform height and vigor were randomly selected as sample collection trees for the collection of different tissues and organs. Roots, needles, terminal buds, stem segments, male cones, seeds, and immature cones were collected, with three batches of samples collected from each tissue and organ from each tree (three replicates). After collection, the samples were placed in centrifuge tubes, rapidly frozen in liquid nitrogen, and then stored at -80℃ for later use.

[0049] Callus materials at different proliferation stages were collected in the tissue culture laboratory of the Tropical Forestry Research Institute, Chinese Academy of Forestry. Callus materials at different proliferation stages (0d, 6d, 9d, 12d, 15d, 18d, 21d) were collected. Three bottles of healthy and uniform callus materials were selected from each stage for sampling. The samples were placed in centrifuge tubes, quickly frozen with liquid nitrogen, and then stored at -80℃ for later use.

[0050] 2. Experimental Methods

[0051] 2.1 Total RNA extraction and cDNA synthesis from plant tissues

[0052] Total RNA was isolated from callus samples from different tissues and organs of *Pinus kesoniaensis* and at different stages of proliferation using an RNA extraction kit (RNAprep Pure Plant Kit, Tiangen). The RNA was then reverse transcribed into cDNA for subsequent experiments. The reverse transcription reaction system was prepared according to the instructions of the reverse transcription kit (HiScript IV 1st strand cDNA synthesis kit, Novizan), using 1 μg of total RNA to establish a 20 μL cDNA reverse transcription reaction system. The cDNA product was stored at -20°C for later use.

[0053] 2.2 Selection of candidate internal reference genes and primer design

[0054] Using the FPKM values ​​of all genes obtained from the transcriptome sequencing of *Pinus sylvestris*, combined with gene annotation, the most stably expressed genes were initially selected as candidate internal control genes. Subsequently, referring to common internal control gene types reported in the literature, eight candidate internal control genes were initially identified, including TPI11, ACT7, UBC10, SAND, α-TUB, β-TUB, PP2A, and UBQ13 genes, whose nucleotide sequences are shown in SEQ ID NO. 1–8. Suitable quantitative primers were designed using an online website. PCR product lengths are typically 80–300 bp, and primer lengths of 18–24 bp are preferred. The product lengths designed in this technique were between 123–200 bp, and the primer lengths were all 20 bp. Detailed primer information is shown in Table 1.

[0055] Table 1 Primer information for 8 candidate internal reference genes

[0056]

[0057] 2.3 Primer-specific detection and amplification efficiency

[0058] The length of each gene amplification product was verified using conventional PCR, and primer specificity was detected. Using equal volumes of cDNA samples from different organs and tissues of *Pinus koraiensis* and callus at different proliferation stages as templates, PCR amplification was performed using a high-fidelity enzyme (2×Phanta MaxMaster Mix, Novizan). The reaction products were analyzed for primer specificity using 1–1.2% agarose gel electrophoresis. Amplification efficiency was determined by quantitative real-time PCR. After mixing equal volumes of cDNA samples, the mixture was serially diluted 5-fold using ddH2O to obtain 5... 0 5 -1 5 -2 5 -3 5 -4 cDNA templates of varying concentrations. Using the above-mentioned samples at different concentrations as templates, Roche LightCycler was used. The 480 II real-time PCR instrument was used for qPCR amplification according to the instructions for the real-time PCR reagent (SupRealQPurple Universal SYBR qPCR Master Mix, Novizan). Each sample was set up in triplicate. A standard curve was plotted with the logarithm of the template dilution factor on the x-axis and the average Ct value at the corresponding dilution concentration on the y-axis. The slope (K), correlation coefficient (R2), and corresponding amplification efficiency (E) were calculated.

[0059] 2.4 Analysis of expression stability of candidate internal reference genes

[0060] The expression stability of eight candidate reference genes in different organs and tissues of *Pinus kesoniaensis* at different proliferation stages was analyzed using three commonly used computational programs: geNorm, NormFinder, and BestKeeper, as well as the online tool RefFinder (https: / / blooge.cn / Ref-Finder). geNorm determined stable expression by calculating the stability value (M) of candidate reference genes, typically using M=1.5 as a threshold. A smaller M value indicates more stable gene expression; when M>1.5, the gene is considered unsuitable as a reference gene. Furthermore, the optimal number of reference genes was determined based on the ratio of the variance value (V) to the number of reference genes (n) (i.e., Vn / Vn+1). NormFinder evaluated the stability of reference genes by calculating their expression stability value (SV). There is no absolute threshold for SV; the evaluation criterion is that a smaller SV value indicates better stability. Typically, the gene with the smallest SV value is selected as the optimal single reference gene. Bestkeeper primarily evaluates gene stability by comparing the standard deviation (SD) and coefficient of variation (CV) of the Ct values ​​of candidate internal reference genes. A smaller SD indicates greater gene stability, and a SD < 1.0 indicates relatively stable gene expression. The RefFinder (https: / / blooge.cn / Ref-Finder) online tool provides a comprehensive ranking of internal reference gene stability results.

[0061] 2.5 Validation of the stability of the internal reference gene

[0062] Genes were randomly selected as target genes. The expression patterns of the target genes were analyzed using both the most stable and the least stable candidate internal control genes, with three biological replicates for each group. Two [replication methods were employed]. -ΔΔCt The relative expression levels of genes were calculated using a method, and graphs were plotted using Graph-Pad Prism 9 software.

[0063] 3. Results and Analysis

[0064] 3.1 Analysis of primer specificity and amplification efficiency of candidate internal reference genes

[0065] Using cDNA from equal amounts of mixed Pinus sylvestris dauricum organs and callus tissues at different proliferation stages as templates, conventional PCR amplification was performed. The results showed that the amplification products of all candidate internal reference gene primers were single bands, with no non-specific amplification, and the size was as expected. Figure 2 qPCR analysis showed that the melting curves of all candidate internal reference genes exhibited a single signal peak. Figure 3 This indicates that the primers used can specifically amplify the corresponding products of each internal reference gene without primer dimer formation. The amplification curves for each test sample showed good repeatability, indicating high specificity of the qRT-PCR reaction and accurate and reliable quantitative results. Amplification efficiency (E) calculations showed that the E values ​​for the candidate internal reference genes ranged from 91.289% to 110.681%, and the linear correlation coefficient R of the standard curve was [value missing]. 2 The values ​​ranged from 0.9868 to 0.9943 (Table 2). These results indicate that the primers for each candidate gene exhibited good specificity and amplification efficiency, meeting the requirements for subsequent experiments and suitable for further analysis.

[0066] Table 2. Amplification efficiency of 8 candidate internal reference genes

[0067]

[0068] 3.2 Analysis of expression abundance of candidate internal reference genes

[0069] The cycle threshold (Ct) value refers to the number of cycles required for the fluorescence signal intensity to first reach a set threshold during a PCR reaction. A smaller Ct value indicates a higher level of gene expression. The Ct values ​​of the eight candidate internal control genes showed significant variations in expression levels across different organs and tissues and at different stages of callus proliferation. The overall Ct values ​​ranged from 14.456 to 28.603. SAND showed the highest expression abundance, with Ct values ​​ranging from 14.456 to 19.020, while TPI11 showed the lowest, with Ct values ​​ranging from 17.980 to 28.603. Preliminary analysis of box curve fluctuations indicated that SAND was relatively stable, while TPI11 showed poor stability. Figure 4 ).

[0070] 3.3 Analysis of candidate internal reference gene expression stability using geNorm software

[0071] The geNorm analysis results show that ( Figure 5In the A section, the M values ​​of six candidate genes from different organs and tissues of *Pinus kesiaoensis* were less than 1.5, and their expression stability, from strongest to weakest, was SAND, β-TUB, UBQ13, PP2A, UBC10, and α-TUB. Among them, SAND had the lowest M value (0.570), indicating the highest expression stability and suitability as an internal reference gene. The paired difference (Vn / Vn+1) analysis of the normalized factors of the candidate internal reference genes showed... Figure 5 In the C section, when n=3, the V3 / V4 value (0.1062) is initially lower than the threshold (0.15), indicating that three internal reference genes are needed to correct the expression levels of the target gene in different organs and tissues of *Pinus kesonianica*. When n=4, the Vn / Vn+1 ratio is the smallest, representing the optimal number of internal reference genes. However, when n=6 or 7, the difference values ​​are both greater than 0.15, indicating that using multiple reference genes increases the instability and complexity of the experiment.

[0072] The geNorm analysis results show that ( Figure 5 In the study of *Pinus kesiaoensis* callus tissue at different proliferation stages, seven candidate genes had M values ​​less than 1.5, with expression stability ranging from strong to weak: β-TUB, α-TUB, SAND, UBQ13, ACT7, PP2A, and UBC10. Among these, β-TUB had the lowest M value (0.698), indicating the highest expression stability and suitability as an internal reference gene. The paired variance (Vn / Vn+1) analysis of the normalized factors of the candidate internal reference genes showed... Figure 5 In the D section, when n=3, the V3 / V4 ratio is 0.1244, significantly lower than the threshold of 0.15. When n=4, the Vn / Vn+1 ratio is the smallest, at 0.1017, which is a relatively ideal number of internal reference genes. However, when n=5, 6, or 7, the difference values ​​are all greater than 0.15, indicating that using multiple reference genes is not suitable.

[0073] In summary, based on the analysis results of geNorm software, SAND is the most suitable internal reference gene in different organs and tissues of Pinus kesiya; and β-TUB is the most suitable internal reference gene in different proliferation stages of Pinus kesiya callus.

[0074] 3.4 NormFinder software analysis of candidate internal reference gene expression stability

[0075] In different organ tissue samples from *Pinus simonii*, Normfinder analysis (Table 3) showed that the three most stable genes were the candidate internal control genes β-TUB, SAND, and UBQ13. Among them, β-TUB had the lowest SV value (0.487), indicating the highest expression stability; SAND was second with an SV value of 0.723; and UBQ13 had an SV value of 0.887. Furthermore, the candidate internal control genes α-TUB, ACT7, and TPI11 all had SV values ​​greater than 1.5, indicating low expression stability. TPI11 had the worst expression stability with an SV value of 2.238.

[0076] Normfinder analysis (Table 3) showed that the three most stable genes expressed at different stages of *Pinus kesiao* callus proliferation were the candidate internal control genes β-TUB, UBQ13, and ACT7. Among them, β-TUB had the lowest SV value (0.118), indicating the highest expression stability; UBQ13 was next with an SV value of 0.221; and ACT7 had an SV value of 0.532. TPI11, with an SV value of 2.423, significantly exceeded the threshold of 1.5, indicating the lowest expression stability and unsuitability as an internal control gene for different stages of *Pinus kesiao* callus proliferation.

[0077] In summary, based on the analysis results from Normfinder software, the suitable internal reference genes for different organs and tissues of *Pinus kesiao* are β-TUB, SAND, and UBQ13; and the suitable internal reference genes for different stages of callus proliferation in *Pinus kesiao* are β-TUB, UBQ13, and ACT7.

[0078] Table 3. Expression stability of 8 candidate internal reference genes analyzed using NormFinder software.

[0079]

[0080] 3.5 BestKeeper software analysis of candidate internal reference gene expression stability

[0081] BestKeeper software analysis results showed that in different organ and tissue samples of Pinus simonii ( Figure 6 In the A group, the top three genes with good expression stability were the candidate internal reference genes SAND, β-TUB, and UBQ13, with SD values ​​of 0.209, 0.256, and 0.398, respectively; the gene with the worst expression stability was ACT7, with an SD value of 1.801. At different stages of callus proliferation in *Pinus kesiaoensis* (A group), the expression stability was significantly improved. Figure 6Among the B-type genes, the top three with good expression stability were the candidate internal reference genes ACT7, UBQ13, and β-TUB, with SD values ​​of 0.212, 0.456, and 0.497, respectively; the gene with the worst expression stability was TPI11, with an SD value of 1.364.

[0082] In summary, based on the analysis results of BestKeeper software, the suitable internal reference genes for different organs and tissues of *Pinus kesiya* are SAND, β-TUB, and UBQ13; and the suitable internal reference genes for different stages of callus proliferation in *Pinus kesiya* are ACT7, UBQ13, and β-TUB.

[0083] 3.6 RefFinder online website analysis of candidate internal reference gene expression stability

[0084] The stability results of internal reference genes were comprehensively ranked using the RefFinder online tool. Table 4 shows that, among different organ tissue samples from *Pinus kesawii*, the final comprehensive ranking indicates that SAND has the best expression stability, significantly better than other genes, making it suitable as an internal reference gene. Table 5 shows that, at different stages of callus proliferation in *Pinus kesawii*, the final comprehensive ranking shows that β-TUB ranks first, indicating that it has the best expression stability and is suitable as an internal reference gene. Furthermore, TPI11 is the least stable gene and is not suitable as an internal reference gene.

[0085] Table 4. Stability of candidate reference genes in different organs and tissues of *Pinus simonii* analyzed by RefFinder.

[0086]

[0087] Table 5. Stability of candidate reference genes at different proliferation stages of *Pinus kesiao* callus in RefFinder analysis.

[0088]

[0089] 3.7 Validation of the target gene

[0090] Based on the above analysis, this invention uses the SAND gene, which is most stably expressed in different organs and tissues of *Pinus kesiao*, as the internal reference gene, and the β-TUB gene, which is most stably expressed in callus tissue at different proliferation stages of *Pinus kesiao*, as the internal reference gene. Four genes, Pkes07g03380, Pkes01g10760, Pkes05g13910, and Pkes09g17350, were randomly selected as target genes, and their nucleotide sequences are shown in SEQ ID NO. 25–28. Suitable quantitative primers were designed using an online website; detailed information on the qRT-PCR primers is shown in Table 6.

[0091] Table 6 Primer information for the four target genes

[0092]

[0093] In different organ tissues of *Pinus kesawii*, quantitative experiments were performed using the top-ranked genes SAND, β-TUB, UBQ13, PP2A, and the least unstable gene TPI11 as internal reference genes. In callus tissues at different proliferation stages of *Pinus kesawii*, quantitative experiments were performed using the top-ranked genes β-TUB, UBQ13, ACT7, α-TUB, and the least unstable gene TPI11 as internal reference genes. The results showed that when qRT-PCR was performed using the genes SAND, β-TUB, UBQ13, and PP2A of this invention as internal reference genes in different organ tissues of *Pinus kesawii*, the expression levels obtained were basically consistent with the transcriptome data. Figures 7-10 This indicates that the SAND, β-TUB, UBQ13, and PP2A genes are all suitable as internal reference genes for different organs and tissues of *Pinus koraiensis*. In callus tissues at different proliferation stages of *Pinus koraiensis*, when qRT-PCR was performed using the β-TUB, UBQ13, ACT7, and α-TUB genes of this invention as internal reference genes, the expression levels obtained were basically consistent with the transcriptome data. Figures 11-14 This indicates that β-TUB, UBQ13, ACT7, and α-TUB genes are all suitable as internal control genes for callus tissue at different proliferation stages of *Pinus koraiensis*, and these genes have significant advantages compared to other candidate internal control genes. When the least stable TPI11 gene was used as the internal control gene for validation, the expression trends of all four target genes changed significantly, and the qRT-PCR results were inconsistent with the transcriptome data, leading to completely different results. This indicates that this gene is not suitable as an internal control gene for callus tissue at different organ tissues and different proliferation stages of *Pinus koraiensis*. Figure 15 ).

[0094] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A real-time PCR internal reference gene for Pinus kesao-sinensis, characterized by: The internal reference gene includes at least one of SAND, β-TUB, UBQ13, PP2A, ACT7, and α-TUB; For different organs and tissues of Pinus kesao-yomi, the internal reference genes for real-time PCR include at least one of SAND, β-TUB, UBQ13, and PP2A; And / or, for callus tissues at different proliferation stages of Pinus koraiensis, the internal reference genes for quantitative real-time PCR include at least one of β-TUB, UBQ13, ACT7, and α-TUB; The nucleotide sequences of the SAND are shown in SEQ ID NO.4; the nucleotide sequence of β-TUB is shown in SEQ ID NO.6; the nucleotide sequence of UBQ13 is shown in SEQ ID NO.8; the nucleotide sequence of PP2A is shown in SEQ ID NO.7; the nucleotide sequence of ACT7 is shown in SEQ ID NO.2; and the nucleotide sequence of α-TUB is shown in SEQ ID NO.

5.

2. The fluorescence quantitative PCR internal reference gene of *Pinus kesiao* according to claim 1, characterized in that: The different organs and tissues include roots, pine needles, terminal buds, stem segments, male cones, seeds, and immature cones; And / or, the callus tissue at different proliferation stages includes callus tissue at 0d, 6d, 9d, 12d, 15d, 18d, and 21d stages.

3. Primers for amplifying the fluorescence quantitative PCR internal reference gene of *Pinus kesiao* as described in claim 1 or 2, characterized in that: The primer sequences for amplifying SAND are shown in SEQ ID NO. 15-16; the primer sequences for amplifying β-TUB are shown in SEQ ID NO. 19-20; the primer sequences for UBQ13 are shown in SEQ ID NO. 23-24; the primer sequences for PP2A are shown in SEQ ID NO. 21-22; the primer sequences for ACT7 are shown in SEQ ID NO. 11-12; and the primer sequences for α-TUB are shown in SEQ ID NO. 17-18.

4. The application of the fluorescence quantitative PCR internal reference gene of *Pinus sapiens* according to any one of claims 1 to 2 or the primers according to claim 3 in the detection of *Pinus sapiens* genes, characterized in that: For one of the following applications: (1) Application in gene detection of different organs and tissues of Pinus simonii; (2) Application in gene detection of callus tissue at different proliferation stages of Pinus simonii.

5. The application according to claim 4, characterized in that: The gene detection refers to the detection of the transcriptional expression level of the *Pinus kesiao* gene.

6. The application according to claim 5, characterized in that: The gene detection was performed by detecting the transcriptional expression level of the *Pinus sylvestris* gene using quantitative real-time PCR.

7. A real-time PCR detection kit, characterized in that: The detection kit includes the primers as described in claim 3.

8. The application of the primers of claim 3 or the detection kit of claim 7, characterized in that, For one of the following applications: (a) In screening for internal reference genes stably expressed in different organs and tissues of Pinus simonii and / or in verifying the stability of internal reference gene expression in different organs and tissues of Pinus simonii; (b) Application of reference genes stably expressed in callus tissues at different proliferation stages of Pinus kesiao in screening and / or in verifying the stability of reference gene expression in callus tissues at different proliferation stages of Pinus kesiao.

9. The application according to claim 8, characterized in that: In application (a), the stable expression of the internal reference gene includes at least one of SAND, β-TUB, UBQ13, and PP2A.

10. The application according to claim 8, characterized in that: In application (b), the stable expression of the internal reference gene includes at least one of β-TUB, UBQ13, ACT7, and α-TUB.

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