Fluorescent quantitative PCR reference gene under salt stress of hippophae rhamnoides and application thereof

Abstract

The invention discloses a fluorescent quantitative PCR reference gene under salt stress of sea-buckthorn and application thereof, the reference gene stably expressed under salt stress is screened from different tissue parts of sea-buckthorn, and the screening range is more comprehensive and more accurate: in 20 candidate genes, 18S gene is most stably expressed in sea-buckthorn leaves under the stress of salt with different concentrations; the CAC gene can be most stably expressed in the sea-buckthorn root and the sea-buckthorn stem under the stress of salts with different concentrations, so that the 18S gene and the CAC gene are used as reference genes, and an effective correction tool can be provided for screening and researching the sea-buckthorn salt-tolerant gene; and the influence of inconsistent expression stability of reference genes in different tissue parts on quantitative analysis and salt-tolerant gene screening research can be avoided.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, and particularly to a reference gene for fluorescence quantitative PCR under salt stress in Hippophae rhamnoides and its application. Background Art

[0002] Hippophae rhamnoides L. is a plant of the genus Hippophae in the family Elaeagnaceae, and is a deciduous shrub.

[0003] Hippophae rhamnoides is rich in polyphenols, flavonoids, carotenoids, polysaccharides, vitamins and other substances, and thus has prominent biological activities, such as antioxidant, anti-inflammatory, antibacterial, anti-tumor, anti-cancer, immune regulation and tissue regeneration. Since ancient times, it has been considered a medicinal and edible plant, so the development and utilization of Hippophae rhamnoides are relatively extensive. As a drought-resistant and nitrogen-fixing tree species, Hippophae rhamnoides is widely distributed in various regions of Qinghai. Growing in extreme environments for a long time makes the Hippophae rhamnoides in this area often have strong cold resistance, drought resistance and salt tolerance.

[0004] Soil salinization, combined with unreasonable irrigation measures, will cause the phenomenon of secondary salinization of the soil to become more and more serious and common. The growth and development of plants in a high-salt environment will be delayed, and in severe cases, it will lead to water loss, withering and death, resulting in a large reduction in crop yields, thus seriously hindering the sustainable development of agriculture and animal husbandry. Therefore, the research on the salt tolerance mechanism of plants has attracted much attention from scholars at home and abroad. As an important ecological and economic plant, the discovery of its salt tolerance genes is of great significance for the cultivation of transgenic plants with stress resistance, and stable reference genes are crucial for the functional research of salt tolerance genes in Hippophae rhamnoides.

[0005] Fluorescence quantitative PCR (quantitative Real-time PCR, qRT-PCR) is one of the most commonly used techniques for quantifying gene expression levels, and is widely used in gene expression research due to its high accuracy, strong specificity and high throughput. According to a large number of previous studies, the expression stability of the same reference gene is not as stable as expected in different plants or different tissues, growth and development stages of the same plant under different stresses. That is to say, the expression stability of reference genes is inconsistent in different plants, different tissues, growth and development stages of the same plant, and different stress conditions. Selecting an inappropriate reference gene will cause deviation in the relative expression level of the target gene, thus affecting the results of scientific research experiments. Therefore, reference genes must be screened and experimentally verified for different species, tissues and experimental conditions. However, so far, there have been few reports on the screening of reference genes in Hippophae rhamnoides. Summary of the Invention

[0006] Therefore, based on the above background, the present invention has screened out reference genes suitable for salt stress conditions in Hippophae rhamnoides from different tissues of Hippophae rhamnoides, providing an effective calibration tool for the screening of salt tolerance genes in Hippophae rhamnoides.

[0007] Therefore, the present invention provides the following technical solutions:

[0008] One of the objectives of the present invention is to provide:

[0009] Reference genes for fluorescence quantitative PCR under salt stress in seabuckthorn, wherein the reference genes are 18S gene and / or CAC gene; the sequence of the 18S gene is as shown in SEQ ID.NO.1 or a complementary nucleotide sequence; the sequence of the CAC gene is as shown in SEQID.NO.2 or a complementary nucleotide sequence.

[0010] Furthermore, the reference genes are 18S gene and CAC gene.

[0011] Another objective of the present invention is to provide:

[0012] A method for screening reference genes for fluorescence quantitative PCR under salt stress in seabuckthorn, which comprises the following steps:

[0013] S1, Salt stress treatment of different tissues of seabuckthorn: Randomly group seabuckthorn seedlings, subject seabuckthorn to salt stress treatment at different concentrations, water the salt stress control group (CK) normally, and collect the roots, stems, and leaves of seabuckthorn under control treatment and salt stress treatment respectively;

[0014] S2, RNA extraction and cDNA template preparation of different tissues of seabuckthorn: Grind the roots, stems, and leaves of seabuckthorn into fine powder in liquid nitrogen respectively, use a plant RNA rapid extraction kit to extract the total RNA in the roots, stems, and leaves of seabuckthorn, and store it in a -80°C refrigerator for later use;

[0015] Use II 1 st Strand cDNA Synthesis Kit to reverse-transcribe the total RNA of seabuckthorn, and store the obtained reverse transcription product in a -20°C refrigerator;

[0016] S3, Select candidate reference genes and synthesize primers:

[0017] According to the seabuckthorn transcriptome sequencing results obtained in step S2, search for the candidate reference gene sequences for fluorescence quantitative PCR, design and synthesize fluorescence quantitative PCR amplification primers, and use PCR amplification to obtain the amplification annealing temperature of the candidate reference genes;

[0018] S4, Perform real-time fluorescence quantitative PCR analysis on the cDNA products of the roots, stems, and leaves of seabuckthorn obtained in step 2;

[0019] S5, Statistically analyze the Ct values obtained by real-time fluorescence quantitative PCR of each tissue part under different salt stresses;

[0020] S6. The expression stability of different reference genes in various tissue parts of seabuckthorn under different degrees of salt stress was evaluated using the reference gene stability analysis software geNorm, BestKeeper, and NormFinder, respectively.

[0021] S7. Comprehensive analysis was carried out using RefFinder, and the stability ranking values of the obtained candidate reference genes in different tissue parts were statistically analyzed. Combining the results of the reference gene stability analysis software geNorm, BestKeeper, and NormFinder, the most stably expressed reference genes in different tissue parts were established.

[0022] Furthermore, the fluorescence quantitative PCR amplification reaction system in step S4 is as follows: pre-denaturation at 95°C for 30 s; denaturation at 95°C for 5 s, annealing at 49 - 60°C for 30 s according to the annealing temperature corresponding to the fluorescence quantitative PCR amplification primers of the 20 candidate reference genes described in step 3, extension at 72°C for 30 s, for 40 - 45 cycles.

[0023] The third object of the present invention provides:

[0024] Special primers for reference genes in fluorescence quantitative PCR of seabuckthorn under salt stress. The forward primer sequence of the 18S gene is as shown in SEQ ID.NO.3, and its reverse primer is as shown in SEQ ID.NO.4, specifically as follows:

[0025] F: 5,-CAAATGTACGCATACCAAGGACA SEQ ID.NO.3;

[0026] R: 5,-GGGTTTCTAATGACACGCAAAAG SEQ ID.NO.4;

[0027] The forward primer sequence of the CAC gene is as shown in SEQ ID.NO.5, and its reverse primer is as shown in SEQ ID.NO.6, specifically as follows:

[0028] F: 5,-CAAATGTACGCATACCAAGGACA SEQ ID.NO.5;

[0029] R: 5,-GGGTTTCTAATGACACGCAAAAG SEQ ID.NO.6.

[0030] The fourth object of the present invention provides the application of reference genes in fluorescence quantitative PCR of seabuckthorn under salt stress in fluorescence quantitative PCR of seabuckthorn.

[0031] The fifth object of the present invention is to provide the application of special primers for reference genes in fluorescence quantitative PCR of seabuckthorn under salt stress in fluorescence quantitative PCR of seabuckthorn.

[0032] A sixth object of the present invention is to provide the application of the reference gene for fluorescence quantitative PCR under salt stress in seabuckthorn in the screening or research of salt-tolerant genes in seabuckthorn.

[0033] A seventh object of the present invention is to provide the application of the specific primer of the reference gene for fluorescence quantitative PCR under salt stress in seabuckthorn in the screening or research of salt-tolerant genes in seabuckthorn.

[0034] The advantages of the present invention are as follows:

[0035] The present invention provides an 18S gene that can be most stably expressed in seabuckthorn leaves under different concentrations of salt stress; a CAC gene that can be most stably expressed in seabuckthorn roots and seabuckthorn stems under different concentrations of salt stress. Therefore, the reference gene composed of its 18S gene and CAC gene can not only provide an effective calibration tool for the screening and research of salt-tolerant genes in seabuckthorn, but also avoid the influence of inconsistent expression stability of reference genes in different tissue parts on quantitative analysis and the screening and research of salt-tolerant genes. Description of the Drawings

[0036] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0037] Figure 1 Electrophoresis diagram of 20 candidate reference genes in Example 1 of the present invention;

[0038] Figure 2 Dissolution curve of the reference gene CAC in Example 1 of the present invention;

[0039] Figure 3 Dissolution curve of the stable reference gene 18S in Example 1 of the present invention;

[0040] Figure 4 Amplification curve of the reference gene CAC in Example 1 of the present invention;

[0041] Figure 5 Amplification curve of the reference gene 18S in Example 1 of the present invention. Detailed Embodiments

[0042] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Apparently, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0043] Example 1: A method for screening reference genes for fluorescence quantitative PCR under salt stress in seabuckthorn, comprising the following steps:

[0044] S1. Salt stress treatment of different tissues of seabuckthorn: Randomly group seabuckthorn seedlings, perform salt stress treatments on seabuckthorn at different concentrations, water the salt stress control group (CK) normally, and collect the roots, stems, and leaves of seabuckthorn under the control treatment and salt stress treatment respectively.

[0045] S2. RNA extraction and cDNA template preparation of different tissues of seabuckthorn: Grind the roots, stems, and leaves of seabuckthorn into fine powders respectively in liquid nitrogen, use a plant RNA rapid extraction kit to extract the total RNA in the roots, stems, and leaves of seabuckthorn, and store it in a -80°C refrigerator for later use;

[0046] Use II 1 st the Strand cDNA Synthesis Kit to reverse transcribe the total RNA of seabuckthorn, and store the obtained reverse transcription product in a -20°C refrigerator;

[0047] S3. Select candidate reference genes and synthesize primers:

[0048] According to the seabuckthorn transcriptome sequencing results obtained in step S2, search for the candidate reference gene sequences for fluorescence quantitative PCR, design and synthesize the amplification primers for fluorescence quantitative PCR, and use PCR amplification to obtain the amplification annealing temperature of the candidate reference genes;

[0049] S4. Perform real-time fluorescence quantitative PCR analysis on the cDNA products of the roots, stems, and leaves of seabuckthorn obtained in step 2;

[0050] S5. Statistically analyze the Ct values obtained by real-time fluorescence quantitative PCR of each tissue part under different salt stresses;

[0051] S6. Use the geNorm, BestKeeper, and NormFinder reference gene stability analysis software to evaluate the expression stability of different reference genes in each tissue part of seabuckthorn under different salt stress degrees;

[0052] S7. Use RefFinder for comprehensive analysis, count the stability ranking values of the obtained candidate reference genes in different tissue parts, and combine the results of the reference gene stability analysis software such as geNorm, BestKeeper, and NormFinder to establish the most stably expressed reference genes in different tissue parts.

[0053] The specific operation process is as follows:

[0054] S1: Salt stress treatment of different tissues of Hippophae rhamnoides

[0055] In October 2018, randomly collect the seeds of wild Hippophae rhamnoides in Huangyuan County, Qinghai Province. In late March 2019, surface disinfect the seeds with potassium permanganate, then soak them in warm water at 40 °C in an incubator for 48 h for germination. During this period, rinse the seeds with clean water every 10 - 12 h to prevent mildew. Subsequently, sow them in flower pots with a size of 12 cm (height) × 15 cm (diameter) (peat: garden soil: perlite = 40:30:30 (volume%)), cultivate them in a light incubator and cover with a plastic film. Remove the plastic film after more than 1 / 3 of the seeds germinate. Water the seedlings once every two to three days (100 mL / pot) during the seedling stage, and keep the relative soil moisture at about 80% of the field water holding capacity (the field water holding capacity is 36%) by the weighing method, and apply Hoagland nutrient solution once every 2 weeks.

[0056] When the seedlings grow to about 10 cm, conduct salt stress treatment. Randomly divide the seedlings into groups, and conduct salt stress treatment on Hippophae rhamnoides at different concentrations. The salt stress control group (CK) is watered normally, and the salt stress treatment groups (SS) are treated with salt solutions at concentrations of 200 mmol / L (SS 1 ), 400 mmol / L (SS 2 ), 600 mmol / L (SS3), 800 mmol / L (SS4), and 1000 mmol / L (SS5) on Hippophae rhamnoides seedlings. Collect the roots, stems, and leaves of the control and stressed Hippophae rhamnoides respectively. After washing them with ultrapure water and blotting the surface moisture with filter paper, immediately put them into cryotubes. Each sample of different tissue parts is collected three times repeatedly. After the collected samples are bagged and numbered, they can be stored in a -80 °C ultra-low temperature refrigerator after quick-freezing with liquid nitrogen for subsequent experiments.

[0057] S2: RNA extraction from different tissues of Hippophae rhamnoides and preparation of cDNA templates

[0058] The roots, stems, and leaves of seabuckthorn were ground into fine powders separately in liquid nitrogen, and the total RNA in the roots, stems, and leaves of seabuckthorn was extracted according to the instructions of the RN33-EASYspinplus Plant RNA Rapid Extraction Kit of Beijing Aidlab Biotechnologies Co., Ltd., and stored in a -80°C refrigerator for later use. 1% agarose gel electrophoresis; quality and concentration detection: 1 μL of the detection sample was used, and the OD260 / 280 value and concentration were detected with a micro nucleic acid and protein detector, and the value between 1.8 - 2.2 and a single peak was considered a qualified sample. Use II 1 st the instructions of the Strand cDNA Synthesis Kit to reverse transcribe the total RNA of seabuckthorn. The obtained reverse transcription products were stored in a -20°C refrigerator.

[0059] S3: Select candidate reference genes and synthesize PCR amplification primers

[0060] In this example, according to the gene sequences related to seabuckthorn transcriptome sequencing, after Blast alignment with the NCBI database, 20 gene sequences were determined as the sequences of seabuckthorn reference candidate genes. Subsequently, specific quantitative primers were designed based on these sequences, and the most stable reference genes in different tissue parts of seabuckthorn under different salt stresses were screened out using fluorescence quantitative technology. Among them, the primer sequences, annealing temperatures, amplification lengths, gene accession numbers, nucleotide sequence numbers, etc. of 20 seabuckthorn fluorescence quantitative PCR candidate reference genes are shown in Table 1:

[0061]

[0062] The gene sequence of the above candidate gene 18S is as shown in SEQ ID.NO.1 or its complementary nucleotide sequence, and its primer sequence can be seen in Table 1.

[0063] The gene sequence of the above candidate gene CAC is as shown in SEQ ID.NO.2 or its complementary nucleotide sequence, and its primer sequence can be seen in Table 1.

[0064] The gene sequence of the above candidate gene CYP2 is as shown in SEQ ID.NO.7 or its complementary nucleotide sequence, and its primer sequence can be seen in Table 1.

[0065] The gene sequence of the above candidate gene RPL13 is as shown in SEQ ID.NO.8 or its complementary nucleotide sequence, and its primer sequence can be seen in Table 1.

[0066] The gene sequence of the above candidate gene HIS3 is as shown in SEQ ID.NO.9 or its complementary nucleotide sequence, and its primer sequence can be seen in Table 1.

[0067] The gene sequence of the above candidate gene TATA is as shown in SEQ ID.NO.10 or its complementary nucleotide sequence, and its primer sequence can be seen in Table 1.

[0068] The above-mentioned candidate gene UBI-lp gene sequence is as shown in SEQ ID.NO.11 or its complementary nucleotide sequence, and its primer sequence can be seen in Table 1.

[0069] The above-mentioned candidate gene DnaJ-pl gene sequence is as shown in SEQ ID.NO.12 or its complementary nucleotide sequence, and its primer sequence can be seen in Table 1.

[0070] The above-mentioned candidate gene TUB gene sequence is as shown in SEQ ID.NO.13 or its complementary nucleotide sequence, and its primer sequence can be seen in Table 1.

[0071] The above-mentioned candidate gene TUBβC gene sequence is as shown in SEQ ID.NO.14 or its complementary nucleotide sequence, and its primer sequence can be seen in Table 1.

[0072] The above-mentioned candidate gene 28S gene sequence is as shown in SEQ ID.NO.15 or its complementary nucleotide sequence, and its primer sequence can be seen in Table 1.

[0073] The above-mentioned candidate gene UBQ10 gene sequence is as shown in SEQ ID.NO.16 or its complementary nucleotide sequence, and its primer sequence can be seen in Table 1.

[0074] The above-mentioned candidate gene TUA gene sequence is as shown in SEQ ID.NO.17 or its complementary nucleotide sequence, and its primer sequence can be seen in Table 1.

[0075] The above-mentioned candidate gene CAP1 gene sequence is as shown in SEQ ID.NO.18 or its complementary nucleotide sequence, and its primer sequence can be seen in Table 1.

[0076] The above-mentioned candidate gene RPL4 gene sequence is as shown in SEQ ID.NO.19 or its complementary nucleotide sequence, and its primer sequence can be seen in Table 1.

[0077] The above-mentioned candidate gene UEP gene sequence is as shown in SEQ ID.NO.20 or its complementary nucleotide sequence, and its primer sequence can be seen in Table 1.

[0078] The above-mentioned candidate gene PEPC gene sequence is as shown in SEQ ID.NO.21 or its complementary nucleotide sequence, and its primer sequence can be seen in Table 1.

[0079] The above-mentioned candidate gene MDHI gene sequence is as shown in SEQ ID.NO.22 or its complementary nucleotide sequence, and its primer sequence can be seen in Table 1.

[0080] The above-mentioned candidate gene elF2α gene sequence is as shown in SEQ ID.NO.23 or its complementary nucleotide sequence, and its primer sequence can be seen in Table 1.

[0081] The above-mentioned candidate gene ACTIN gene sequence is as shown in SEQ ID.NO.24 or its complementary nucleotide sequence, and its primer sequence can be seen in Table 1.

[0082] S4. Fluorescent quantitative PCR amplification of candidate reference genes

[0083] Fluorescent quantitative PCR was carried out according to the instructions of the BIO-RAD CFX Connect TM Optics Module analyzer and the instructions of the Premix DimerEraser(PerfectReal Time) kit for operation to perform Real-Time PCR amplification of stable reference gene candidates. Pre-denaturation at 90°C for 3 min; denaturation at 95°C for 30 sec; extension at 72°C for 1 min; 35 cycles; different annealing temperatures were set according to different primers. After the cycle was completed, a melting curve was drawn: 55°C - 95°C, and fluorescence signals were collected every 0.5°C.

[0084] S5. Establishment of a standard curve for primer amplification of candidate reference genes

[0085] The specificity of each primer was judged by whether the melting curve was a single peak. In addition, the cDNA template was diluted in a 10-fold gradient to establish a standard curve for primer amplification of the reference gene, and the amplification efficiency (E) of each pair of primers was determined according to the slope of the standard curve. E = 10 (-1 / 斜率) -1, and whether the amplification efficiency was between 80% and 110% was used as a standard to judge whether the primer was applicable. The fluorescent quantitative reaction system and cycling program of the standard curve were the same as described above. The SYBR fluorescent reagent was purchased from Takara (Dalian) Co., Ltd.

[0086] S6. Data processing and analysis

[0087] The qRT-PCR data of different tissue parts of sea buckthorn under different degrees of salt stress treatment (sample cycle threshold, Ct value) were sorted and summarized by Excel 2003 software. The expression stability of different reference genes in various tissue parts of sea buckthorn was evaluated using geNorm, BestKeeper, NormFinder, and RefFinder reference gene stability analysis software. The evaluation basis is that the geNorm software calculates the average expression stability measurement value M according to the pairwise variation between two reference genes, and the smaller the M value, the stronger the stability; NormFinder calculates the stability value S (StabilityValue) for reference genes based on analysis of variance, and the smaller the stability value, the higher the stability. BestKeeper evaluates the expression stability of candidate genes by calculating the standard deviation (SD) and percentage covariance (CV) of the geometric mean of three replicates of all candidate genes in each sample. The lower the SD value and CV value, the more stable the expression of the candidate gene. RefFinder performs a comprehensive ranking. By integrating 3 general analysis programs (geNorm, Normfinder, and BestKeeper) and the ct method, a comprehensive evaluation of candidate reference genes is obtained, which helps to select more stable reference genes.

[0088] S7. Result Analysis

[0089] (1) Analysis of the cycle threshold (Ct) values of 20 candidate reference genes in all samples

[0090] Based on the results of fluorescence quantitative PCR detection, the Ct averages of 20 candidate reference genes in the roots, stems, and leaves of sea buckthorn under different concentrations of salt stress treatment were statistically summarized, and the results are shown in Tables 2 to 4:

[0091] Table 2: Ct Means of 20 Candidate Reference Genes in Sea Buckthorn Roots

[0092] Gene Name CK 200 400 600 800 1000 ACTIN 19.15 20.37 21.14 20.02 21.26 22.43 18S 23.14 23.85 23.71 23.22 24.02 24.11 HIS3 18.44 18.60 19.02 19.26 18.70 19.82 UBI-lp 16.86 17.55 17.21 16.74 16.68 16.22 TUB 21.23 22.42 23.38 22.47 23.05 22.30 CAP1 21.52 21.90 22.66 21.70 23.12 23.45 elF2α 24.13 25.72 25.93 24.87 25.79 26.12 UEP 21.96 22.36 22.73 21.97 23.64 23.82 PEPC 22.28 22.08 22.50 22.04 22.42 22.95 MDHI 23.55 24.19 24.25 23.97 23.92 24.32 RPL4 24.99 25.26 26.11 24.36 24.96 24.81 CAC 25.36 25.97 25.95 25.52 26.11 26.29 RPL13 22.59 22.96 23.17 22.30 23.20 23.21 TATA 25.92 26.26 26.75 26.37 26.89 27.27 DnaJ-pl 21.92 22.39 23.06 22.17 23.12 23.73 CYP2 24.59 24.69 25.17 24.93 24.93 25.04 TUBβC 23.55 23.96 24.94 24.74 25.97 29.00 28S 25.72 26.27 25.44 26.30 25.53 26.29 UBQ10 19.67 20.68 20.28 19.80 19.21 19.22 TUA 24.27 22.33 22.67 22.69 23.65 23.40

[0093] Table 3: Ct Means of 20 Candidate Reference Genes in Sea Buckthorn Stems

[0094]

[0095]

[0096] Table 4: Ct Means of 20 Candidate Reference Genes in Sea Buckthorn Leaves

[0097] Gene Name CK 200 400 600 800 1000 ACTIN 20.62 21.68 22.43 22.68 22.06 21.34 18S 23.6 23.8 24.1 23.77 24.22 24.42 HIS3 18.92 19.52 20.04 19.84 20.24 20.16 UBI-lp 16.82 17.53 16.98 16.88 16.39 18.44 TUB 23.42 24.02 23.99 23.83 24.25 24.9 CAP1 23.61 24.49 24.26 23.81 23.47 23.12 elF2α 23.02 24.11 24.37 23.35 24.35 24.99 UEP 23.03 23.47 24.08 23.89 23.23 23.46 PEPC 21.4 21.23 21.15 21.44 22.33 23.02 MDHI 23.5 23.59 23.78 24.13 24.23 25.04 RPL4 26.49 26.79 26.92 26.89 29.89 26.85 CAC 25.77 26.22 26.59 27.11 26.26 26.75 RPL13 23.9 23.68 24.54 24.55 25.28 25.44 TATA 26.19 26.43 27.27 27.55 28.2 27.98 DnaJ-pl 22.64 22.71 23.28 23.36 24.89 24.81 CYP2 23.37 23.44 23.06 23.44 23.23 23.09 TUBβC 24.52 25.7 25.4 25.81 26.82 26.31 28S 25.22 25.33 25.81 26.44 26.06 26.49 UBQ10 18.78 19.55 19.11 18.84 18.01 18.18 TUA 23.77 24.95 24.47 23.92 23.85 24.36

[0098] In this example, 20 genes were selected as candidate reference genes, and the root, stem, and leaf tissue samples of seabuckthorn treated with different salt stress concentrations were used as templates to amplify different candidate reference genes respectively. The specificity of the primers was evaluated through the melting curves of real-time fluorescence quantitative PCR amplification. The results showed that all target reference genes had specific single peaks, that is, all primers had good specificity and could be used for the analysis of qRT-PCR gene expression levels. The analysis results of the expression levels of 20 candidate reference genes found that their Ct values were different in all samples (Table 2-4). In all samples, the Ct values varied between 15.65 and 29.89, with an average value of 23.20. The expression ranges of MDHI and 18S genes were the smallest (23.44 - 25.04; 23.14 - 24.77), and the ranges of TUBβC and RPL4 were the largest (22.64 - 29.00, 24.36 - 29.89).

[0099] (2) Evaluation of the stability of 20 candidate reference genes

[0100] The analysis software Bestkeeper, GeNorm, NormFinder, and RefFinder were used to analyze the expression stability of 20 candidate reference genes of seabuckthorn respectively.

[0101] The results of the stability analysis of 20 candidate reference genes of seabuckthorn by Bestkeeper are shown in Table 5 below:

[0102] Table 5: Stability analysis of 20 candidate reference genes based on BestKeeper software

[0103]

[0104] The BestKeeper software calculates the coefficient of variation (CV) and standard deviation (SD). Lower SD and CV values represent high stability, and the gene stability is negatively correlated with SD ± CV. The BestKeeper analysis results show that the gene stabilities are different in different seabuckthorn tissues under salt stress. CYP2 has the highest stability in root samples, and TUBβC is the most unstable reference gene. CAC is the most stable in stem samples, while TUBβC is the most unstable reference gene. The most stable gene in leaves is CYP2, and the most unstable gene is DnaJ-pl. The most stable gene in all tissues is MDHI, and the most unstable gene is TUBβC.

[0105] The results of the stability analysis of 20 candidate reference genes of seabuckthorn by NormFinder software are shown in Table 6 below:

[0106] Table 6: Stability analysis of 20 candidate reference genes based on NormFinder software

[0107]

[0108]

[0109] The smaller the stability value of the gene obtained by NormFinder software, the better its stability; the larger the value, the worse its stability. The results show that in the roots of Hippophae rhamnoides, the M values of 20 candidate reference genes are between 0.203 - 0.866, in the stems between 0.222 - 0.928, and in the leaves between 0.197 - 0.708. Among them, the most stable gene in the roots is TATA, while TUBβC is the most unstable reference gene; in the stems, the most stable gene is RPL13, and the most unstable reference gene is UBQ10; in the leaves, the most stable gene is 18S, and RPL4 is the most unstable gene; the most stable gene in all tissues is 18S, and the most unstable gene is UBQ10.

[0110] The results of the stability analysis of 20 candidate reference genes of Hippophae rhamnoides by GeNorm software are shown in Table 7 below:

[0111] Table 7: Stability analysis of 20 candidate reference genes based on GeNorm software

[0112]

[0113] GeNorm software evaluates the stability of candidate reference genes according to the stability value M. The threshold of the M value is 1.5. If the M value of the obtained gene is less than 1.5, it indicates that the candidate gene is suitable as a reference, and the smaller the M value, the more stable the expression of the reference gene. The results show that under salt stress, the most suitable reference genes in the roots, stems, and leaves of Hippophae rhamnoides are different. Among them, PEPC (M = 0.37) is the gene with the most stable expression in the roots, while TUBβC (M = 0.87) is the most unstable gene. HIS3 (M = 0.29) is the gene with the most stable expression in the stems, and UBQ10 (M = 0.92) is the most unstable gene. MDHI and 28S (M = 0.35) are the genes with the most stable expression in the leaves, while RPL4 (M = 0.88) is the most unstable gene. TATA and DnaJ - pl (M = 0.44) are the genes with the most stable expression in all tissues, while RPL4 (M = 1.00) is the most unstable gene.

[0114] (3) Comprehensive ranking analysis

[0115] Through the analysis of the above three software, it is found that there are inconsistent phenomena in the stable reference genes obtained by different software, and it is impossible to accurately obtain the stable reference genes in different parts of Hippophae rhamnoides under different stress conditions based on the above analysis results. Therefore, in this experiment, RefFinder was used for further analysis. The analysis results of RefFinder are shown in Table 8.

[0116] Table 8: Comprehensive ranking of the stability of 20 candidate reference genes

[0117]

[0118] The comprehensive analysis results of the stability of 20 candidate reference genes in seabuckthorn showed that the stability of each candidate reference gene in the roots, stems and leaves of seabuckthorn under salt stress was inconsistent. Among them, CAC and 18S were the most stable reference genes expressed in the roots, while TUBβC and TUA were the most unstable reference genes expressed. CAC and RPL13 were the most stable reference genes expressed in the stems, while UBQ10 and TUBβC were the most unstable reference genes expressed. Among them, 18S and TUB were the most stable reference genes expressed in the leaves. 18S and MDHI were expressed in all tissue samples, while the most unstable genes expressed in the leaves were RPL4 and UBQ10, and the expression of TUBβC and UBQ10 was the most unstable in all tissue samples. CAC was the most stable expression in the roots and stems of seabuckthorn, and 18S was the most stable expression in the leaf samples.

[0119] From the above, in this example, the genes with the most stable expression in different tissue samples under different concentrations of salt stress were screened. Therefore, when studying the functions of stress-resistant genes in seabuckthorn under salt stress, if different stable reference genes are selected for different tissue parts, the CAC gene is used for the roots, the CAC gene is used for the stems, and the 18S gene is used for the leaves. Therefore, using the CAC gene and the 18S gene as reference genes simultaneously can lay a certain application foundation for the research on the gene expression stability and reliability of seabuckthorn under salt stress, as well as the screening and cultivation of salt-tolerant seabuckthorn germplasm resources.

[0120] The above describes the present invention and its implementation manners. This description is not restrictive. What is shown in the embodiments is only one of the implementation manners of the present invention, and the actual structure is not limited thereto. Generally speaking, if those of ordinary skill in the art are inspired by it and design similar structural manners and embodiments without creative efforts without departing from the purpose of the present invention, they shall fall within the protection scope of the present invention.

Claims

1. A reference gene for fluorescence quantitative PCR under seabuckthorn salt stress, characterized in that: The internal reference gene is 18S gene and / or CAC gene; the sequence of the 18S gene is as shown in SEQ ID.NO.1 or a complementary nucleotide sequence; the sequence of the CAC gene is as shown in SEQ ID.NO.2 or a complementary nucleotide sequence.

2. The internal reference gene for fluorescence quantitative PCR under seabuckthorn salt stress according to claim 1, characterized in that: The internal reference genes are 18S gene and CAC gene.

3. A method for screening internal reference genes for fluorescence quantitative PCR under seabuckthorn salt stress, characterized in that: It includes the following steps: S1, salt stress treatment of different tissues of seabuckthorn: seabuckthorn seedlings were randomly divided into groups and subjected to salt stress treatment of different concentrations. The salt stress control group (CK) was watered normally, and the roots, stems and leaves of seabuckthorn in the control treatment and salt stress treatment were collected respectively; S2, RNA extraction from different tissues of seabuckthorn and preparation of cDNA templates: seabuckthorn roots, stems and leaves were ground into fine powder in liquid nitrogen, and total RNA from the roots, stems and leaves of seabuckthorn was extracted using a plant RNA rapid extraction kit and stored in a -80°C refrigerator for later use; use II 1 st The total RNA of seabuckthorn was reverse transcribed using the Strand cDNA Synthesis Kit, and the obtained reverse transcription product was stored in a refrigerator at -20°C; S3, select candidate internal reference genes and synthesize primers: According to the sequencing results of the seabuckthorn transcriptome obtained in step S2, the candidate internal reference gene sequences of the fluorescent quantitative PCR are searched, the fluorescent quantitative PCR amplification primers are designed and synthesized, and the amplification annealing temperature of the candidate internal reference gene is obtained by PCR amplification; S4, performing real-time fluorescence quantitative PCR analysis on the cDNA products of seabuckthorn roots, stems and leaves obtained in step 2; S5, statistical analysis of the Ct values ​​obtained by real-time fluorescence quantitative PCR in various tissue parts under different salt stresses; S6, geNorm, BestKeeper, and NormFinder internal reference gene stability analysis software were used to evaluate the expression stability of different internal reference genes in various tissues of seabuckthorn under different salt stress levels; S7, RefFinder was used for comprehensive analysis to statistically rank the stability rankings of the candidate reference genes in different tissue locations, and the most stably expressed reference genes in different tissue locations were determined by combining the results of geNorm, BestKeeper, and NormFinder reference gene stability analysis software.

4. The special primers for internal reference genes of seabuckthorn fluorescence quantitative PCR under salt stress according to claim 1 or 2, characterized in that: The forward primer sequence of the 18S gene is shown in SEQ ID.NO.3, and the reverse primer sequence is shown in SEQ ID.NO.4, as shown below: F:5 ， -CAAATGTACGCATACCAAGGACA SEQ ID.NO.3; R:5 ， -GGGTTTCTAATGACACGCAAAAG SEQ ID.NO.4; The forward primer sequence of the CAC gene is shown in SEQ ID.NO.5, and the reverse primer sequence is shown in SEQ ID.NO.6, as shown below: F:5 ， -CAAATGTACGCATACCAAGGACA SEQ ID.NO.5; R:5 ， -GGGTTTCTAATGACACGCAAAAG SEQ ID.NO.6。 5. Use of the internal reference gene for fluorescence quantitative PCR under seabuckthorn salt stress according to claim 1 or 2 in seabuckthorn fluorescence quantitative PCR.

6. Use of the dedicated primers for internal reference genes of seabuckthorn fluorescence quantitative PCR under salt stress as claimed in claim 4 in seabuckthorn fluorescence quantitative PCR.

7. Use of the internal reference gene for fluorescence quantitative PCR under seabuckthorn salt stress according to claim 1 or 2 in screening or researching seabuckthorn salt-tolerant genes.

8. Use of the dedicated primers for internal reference genes of seabuckthorn quantitative fluorescence PCR under salt stress as claimed in claim 4 in screening or research of seabuckthorn salt-tolerant genes.

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