Bok choy high temperature stress reference gene, screening method and application thereof

Abstract

The application belongs to the field of agricultural biotechnology, discloses Brassica parachinensis high-temperature stress internal reference genes, a screening method and application thereof, and specifically discloses Brassica parachinensis high-temperature stress internal reference genes. According to high-temperature stress transcriptome data of Brassica parachinensis, 16 genes stably expressed in the gene expression profile of Brassica parachinensis under high-temperature stress are screened as the high-temperature stress internal reference genes of Brassica parachinensis for the first time, which provides effective stable internal reference genes for the research on the heat-resistant related genes of Brassica parachinensis and the functions thereof in the future, is beneficial to improving the stability and reliability of the gene expression analysis research of Brassica parachinensis under stress, enriches the types of internal reference genes used in the gene expression research related to high-temperature stress of Brassica parachinensis, lays a good foundation for the accurate analysis of gene expression detection, and has good application value.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biotechnology, specifically relating to the internal reference gene for high temperature stress in Chinese cabbage, its screening method, and its application. Background Technology

[0002] Chinese cabbage (Brassica campestris L.ssp.chinensis var. utilis Tsen et Lee), also known as flowering cabbage, is a variety of the Brassica subspecies in the Brassicaceae family. Chinese cabbage prefers cool temperatures; high-temperature stress easily leads to small, poor-quality flower stalks, reduced individual plant weight, and lower marketable yield. Therefore, screening and cultivating heat-resistant Chinese cabbage germplasm is an important goal in Chinese cabbage breeding. In gene expression studies, real-time quantitative PCR (qRT-PCR) technology is frequently used to verify transcriptome results and analyze expression characteristics. Selecting suitable internal reference genes is a prerequisite for ensuring the accuracy of qRT-PCR analysis results. Ideally, the expression of internal reference genes is unaffected by growth and development stages and experimental conditions. However, in practical applications, internal reference genes are usually significantly affected by species type, tissue / organ, and environmental conditions. Therefore, it is necessary to screen suitable internal reference genes according to specific experimental conditions.

[0003] Currently, commonly used internal reference genes in cruciferous vegetables include actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-tubulin (TUB), and transcription elongation factor (EF1). With the development of transcriptome sequencing technology (RNA-seq), it has become possible to discover new internal reference genes. Summary of the Invention

[0004] The first objective of this invention is to provide a reference gene for high-temperature stress in Chinese cabbage.

[0005] A second aspect of the present invention is to provide primers for amplifying the internal reference gene of the first aspect of the present invention.

[0006] A third aspect of the present invention is to provide a reagent kit.

[0007] The fourth aspect of this invention aims to provide the application of the internal reference gene of the first aspect of this invention, the primers of the second aspect of this invention, and / or the kit of the third aspect of this invention in the screening of heat-resistant genes in Chinese cabbage and / or the preparation of products for screening heat-resistant genes in Chinese cabbage.

[0008] The fifth aspect of this invention aims to provide the application of the internal reference gene of the first aspect of this invention, the primers of the second aspect of this invention, and / or the kit of the third aspect of this invention in the analysis of heat resistance gene expression in Chinese cabbage and / or the preparation of products for the analysis of heat resistance gene expression in Chinese cabbage.

[0009] The sixth aspect of this invention aims to provide the internal reference gene of the first aspect of this invention, the primers of the second aspect of this invention, and / or the kit of the third aspect of this invention for detecting changes in the expression level of the heat resistance gene of Chinese cabbage in different tissues, different growth and development stages, or different stress conditions, and / or for preparing products for detecting changes in the expression level of the heat resistance gene of Chinese cabbage in different tissues, different growth and development stages, or different stress conditions.

[0010] The seventh aspect of this invention aims to provide a method for screening internal reference genes for high-temperature stress in Chinese cabbage, as described in the first aspect of this invention.

[0011] The objective of the eighth aspect of this invention is to provide a real-time quantitative PCR detection method for heat resistance genes in Chinese cabbage.

[0012] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0013] In previous studies using qRT-PCR to investigate gene expression levels in heat-tolerant DH lines CX1-7 and heat-sensitive DH lines CX7-3 of Chinese cabbage before and after high-temperature stress, the inventors found significant errors in validating RNA-seq results using different internal control genes. Therefore, this study used an RNA-Seq database of samples from heat-tolerant DH lines CX1-7 and heat-sensitive DH lines CX7-3 before and after high-temperature stress to identify stably expressed genes as candidate internal controls. Four software programs—GeNorm, NormFinder, BestKeeper, and ReFinder—were used to analyze the expression stability of these candidate internal control genes under different experimental conditions, ultimately identifying stably expressed genes to provide a reference for subsequent research on gene expression related to high-temperature stress in Chinese cabbage.

[0014] In a first aspect, the present invention provides an internal reference gene for high-temperature stress in Chinese cabbage, said internal reference gene comprising at least one of the following: cyclic cytokinin CYP gene, transcription elongation factor EF1 gene, actin Actin gene, ribosomal protein 28sRPS27 gene, β-tubulin TUB gene, ribosomal protein 40sRPS17 gene, casein kinase CKⅠ gene, thioredoxin TRX gene, ribosomal protein 60sRPL18 gene, ribosomal protein 50sRPL15 gene, ubiquitin ligase UBI gene, respiratory burst oxidase NADPH gene, 2,4-dienoyl-CoA reductase DECR1 gene, F-box gene, peroxisome protein PMP gene, and c30129.graph_c0 gene.

[0015] Preferably, the nucleotide sequence of the cyclic cytoplasmic protein CYP gene is shown in SEQ ID NO:1; the nucleotide sequence of the transcription elongation factor EF1 gene is shown in SEQ ID NO:2; the nucleotide sequence of the actin gene is shown in SEQ ID NO:3; the nucleotide sequence of the ribosomal protein 28sRPS27 gene is shown in SEQ ID NO:4; the nucleotide sequence of the β-tubulin TUB gene is shown in SEQ ID NO:5; the nucleotide sequence of the ribosomal protein 40sRPS17 gene is shown in SEQ ID NO:6; the nucleotide sequence of the casein kinase CKⅠ gene is shown in SEQ ID NO:7; the nucleotide sequence of the thioredoxin TRX gene is shown in SEQ ID NO:8; the nucleotide sequence of the ribosomal protein 60sRPL18 gene is shown in SEQ ID NO:9; the nucleotide sequence of the ribosomal protein 50sRPL15 gene is shown in SEQ ID NO:10; the nucleotide sequence of the ubiquitin ligase UBI gene is shown in SEQ ID NO:11; and the nucleotide sequence of the respiratory burst oxidase NADPH gene is shown in SEQ ID NO:11. The nucleotide sequence of the 2,4-dienoyl-CoA reductase DECR1 gene is shown in SEQ ID NO:12; the nucleotide sequence of the F-box gene is shown in SEQ ID NO:14; the nucleotide sequence of the peroxisome protein PMP gene is shown in SEQ ID NO:15; and the nucleotide sequence of the c30129.graph_c0 gene is shown in SEQ ID NO:16.

[0016] In a second aspect, primers are provided for amplifying the internal reference gene of the first aspect of the invention.

[0017] Preferably, the primer sequences for amplifying the cyclophilic protein CYP gene are shown in SEQ ID NO:17 and SEQ ID NO:18; the primer sequences for amplifying the transcription elongation factor EF1 gene are shown in SEQ ID NO:19 and SEQ ID NO:20; the primer sequences for amplifying the actin gene are shown in SEQ ID NO:21 and SEQ ID NO:22; the primer sequences for amplifying the ribosomal protein 28sRPS27 gene are shown in SEQ ID NO:23 and SEQ ID NO:24; the primer sequences for amplifying the β-tubulin TUB gene are shown in SEQ ID NO:25 and SEQ ID NO:26; the primer sequences for amplifying the ribosomal protein 40sRPS17 gene are shown in SEQ ID NO:27 and SEQ ID NO:28; the primer sequences for amplifying the casein kinase CKⅠ gene are shown in SEQ ID NO:29 and SEQ ID NO:30; and the primer sequences for amplifying the thioredoxin TRX gene are shown in SEQ ID NO:31 and SEQ ID NO:28. The primer sequences for amplifying the 60sRPL18 ribosomal protein gene are shown in SEQ ID NO:32; the primer sequences for amplifying the 50sRPL15 ribosomal protein gene are shown in SEQ ID NO:35 and SEQ ID NO:36; the primer sequences for amplifying the ubiquitin ligase UBI gene are shown in SEQ ID NO:37 and SEQ ID NO:38; the primer sequences for amplifying the respiratory burst oxidase NADPH gene are shown in SEQ ID NO:39 and SEQ ID NO:40; the primer sequences for amplifying the 2,4-dienoyl-CoA reductase DECR1 gene are shown in SEQ ID NO:41 and SEQ ID NO:42; the primer sequences for amplifying the F-box gene are shown in SEQ ID NO:43 and SEQ ID NO:44; and the primer sequences for amplifying the peroxisome protein PMP gene are shown in SEQ ID NO:45 and SEQ ID NO:36. As shown in NO:46; the primer sequences for amplifying the c30129.graph_c0 gene are shown in SEQ ID NO:47 and SEQ ID NO:48.

[0018] A third aspect of the present invention provides a kit comprising primers from the second aspect of the present invention.

[0019] A fourth aspect of the present invention provides the application of the internal reference gene of the first aspect of the present invention, the primers of the second aspect of the present invention, and / or the kit of the third aspect of the present invention in the screening of heat resistance genes in Chinese cabbage and / or the preparation of products for screening heat resistance genes in Chinese cabbage.

[0020] Preferably, the Chinese cabbage includes one or more of the following: heat-resistant DH series CX1-7, CX3-2, CX5-1, 008 Chinese cabbage, pointed-leaf 50-day Chinese cabbage, and heat-sensitive DH series or varieties CX7-3, CX4-1, CX6-5, CX5-4, and Hangyu 49-1 Chinese cabbage.

[0021] A fifth aspect of the present invention provides the application of the internal reference gene of the first aspect of the present invention, the primers of the second aspect of the present invention, and / or the kit of the third aspect of the present invention in the analysis of heat resistance gene expression in Chinese cabbage and / or the preparation of products for the analysis of heat resistance gene expression in Chinese cabbage.

[0022] Preferably, the Chinese cabbage includes one or more of the following: heat-resistant DH series CX1-7, CX3-2, CX5-1, 008 Chinese cabbage, pointed-leaf 50-day Chinese cabbage, and heat-sensitive DH series or varieties CX7-3, CX4-1, CX6-5, CX5-4, and Hangyu 49-1 Chinese cabbage.

[0023] A sixth aspect of the present invention provides the application of the internal reference gene of the first aspect of the present invention, the primers of the second aspect of the present invention, and / or the kit of the third aspect of the present invention in detecting changes in the expression level of the heat resistance gene of Chinese cabbage in different tissues, different growth and development stages, or different stress conditions, and / or in preparing products for detecting changes in the expression level of the heat resistance gene of Chinese cabbage in different tissues, different growth and development stages, or different stress conditions.

[0024] Preferably, the different tissues include roots, leaves, petioles, stems, and flowers.

[0025] Preferably, the stress condition is treatment at 35-40°C for 0.25-48 hours.

[0026] Preferably, the Chinese cabbage includes one or more of the following: heat-resistant DH series CX1-7, CX3-2, CX5-1, 008 Chinese cabbage, pointed-leaf 50-day Chinese cabbage, and heat-sensitive DH series or varieties CX7-3, CX4-1, CX6-5, CX5-4, and Hangyu 49-1 Chinese cabbage.

[0027] A seventh aspect of the present invention provides a method for screening internal reference genes for high-temperature stress in Chinese cabbage, as described in the first aspect of the present invention, comprising the following steps:

[0028] S1. Extract the genome of Chinese cabbage under different high-temperature stress treatments and screen candidate internal reference genes based on nucleotide sequences;

[0029] S2. Design primers based on the nucleotide sequence of the candidate reference gene to obtain primers that specifically amplify the candidate internal reference gene;

[0030] S3. Stability analysis of candidate internal reference genes was performed using real-time quantitative PCR.

[0031] S4. The expression stability of candidate internal reference genes was evaluated using GeNorm, NormFinder, Bestkeeper, and ReFinder software.

[0032] Preferably, the temperature of the high-temperature stress treatment in step S1 is 30-40°C; more preferably, it is 37°C.

[0033] Preferably, in step S4, the expression stability M value, coefficient of variation (CV value), and standard deviation (SD value) of each candidate internal reference gene under different treatments are calculated using GeNorm, NormFinder, and Bestkeeper software, respectively. The expression stability of the candidate internal reference genes is then sorted from smallest to largest according to the M value, CV value, and SD value. The smaller the M value, CV value, and SD value, the more stable the corresponding internal reference gene.

[0034] The stability rankings obtained from each software were scored using ReFinder software, and the geometric mean was calculated to obtain a comprehensive index ranking. The smaller the mean, the higher the ranking, indicating that the expression of the internal reference gene is more stable.

[0035] An eighth aspect of the present invention provides a real-time quantitative PCR detection method for a heat resistance gene in Chinese cabbage, wherein the method uses the internal reference gene of the first aspect of the present invention as an internal reference gene.

[0036] The beneficial effects of this invention are:

[0037] This invention, for the first time, screened 16 genes stably expressed in the gene expression profile of Chinese cabbage under high-temperature stress based on transcriptome data of Chinese cabbage under high-temperature stress, as internal reference genes for high-temperature stress in Chinese cabbage. This provides effective and stable internal reference genes for future research on heat tolerance-related genes and their functions in Chinese cabbage, which is conducive to improving the stability and reliability of gene expression analysis research under high-temperature stress in Chinese cabbage, enriching the types of internal reference genes used in gene expression research related to high-temperature stress in Chinese cabbage, laying a good foundation for accurate gene expression detection analysis, and has good application value.

[0038] Further investigation into the expression of candidate internal reference genes in samples from different DH lines or varieties under high-temperature stress at different times was conducted to identify stable and reliable internal reference genes required for gene expression level analysis in Chinese cabbage under high-temperature stress. This aims to provide better internal reference genes for screening heat-resistant genes and gene expression studies in Chinese cabbage. The results showed that BrCKⅠ and BrCYP exhibited the best expression stability under different high-temperature stress times, while BrCKⅠ and BrUBI showed the most stable expression levels in different tissues. Among different DH lines or varieties of Chinese cabbage, Br28sRPS27 and BrCYP showed the best expression stability. Among all tested samples, BrCKⅠ, BrCYP, and Br28sRPS27 demonstrated the best expression stability.

[0039] The detection primer pairs proposed in this invention are specific, optimize the PCR amplification procedure, greatly improve detection efficiency, shorten detection time, enhance the reliability of detection results, and improve the stability, reliability, and reproducibility of the analysis of gene expression under high temperature stress in Chinese cabbage.

[0040] The present invention discloses a method for screening internal reference genes for high-temperature stress in Chinese cabbage. Stability analysis of different candidate internal reference genes was performed using Genorm, NormFinder, and BestKeeper software. The stability rankings obtained from each software were assigned scores using ReFinder software, and the geometric mean was calculated to obtain a comprehensive index ranking. A comprehensive analysis of the results from each software successfully screened out suitable stable internal reference genes. Detailed Implementation

[0041] The present invention will be further described in detail below through specific embodiments.

[0042] It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0043] Unless otherwise specified, the materials and reagents used in this embodiment are commercially available.

[0044] Example 1: Screening of internal reference genes for high temperature stress in Chinese cabbage and design of corresponding qRT-PCR primers.

[0045] Using the heat-resistant DH line CX1-7 and the heat-sensitive DH line CX7-3 of Chinese cabbage (CX1-7 and CX7-3 have been disclosed in patent publication document CN113348992A) that were both identified as heat-resistant during the seedling stage and in the field, the heat stress treatment temperature was 37℃ (the specific treatment was carried out in “Pang Qiangqiang et al. Analysis of heat resistance during germination and seedling stages of different Chinese cabbage varieties and screening of identification indicators [J]. Northwest Agricultural Journal, 2020, 29(2):295-305.”). An RNA-seq database was established based on four transcriptome samples (CX1-7CK, CX1-7T6, CX7-3CK, and CX7-3T6) from heat-tolerant and heat-sensitive DH seedlings of Chinese cabbage under high-temperature stress treatment for 0 h (normal growth temperature, CK) and 6 h (T6). A total of 46,204 unigene RPKM values ​​were obtained, from which 16 stable unigenes were identified as candidate internal reference genes: the cyclic protein CYP gene (c29858.graph_c0, BrCYP), transcription elongation factor EF1, and others. Genes (c28644.graph_c0, BrEF1), actin gene (c28257.graph_c0, BrActin), ribosomal protein 28sRPS27 gene (c23582.graph_c1, Br28sRPS27), β-tubulin TUB gene (c33247.graph_c2, BrTUB), ribosomal protein 40sRPS17 gene (c25116.graph_c0, Br40sRPS17), casein kinase CKⅠ gene ( c36193.graph_c0, BrCKⅠ), thioredoxin TRX gene (c26646.graph_c0, BrTRX), ribosomal protein 60sRPL18 gene (c37397.graph_c0, Br60sRPL18), ribosomal protein 50sRPL15 gene (c27286.graph_c0, Br50sRPL15), ubiquitin ligase UBI gene (c31909.graph_c0, BrUBI), respiratory burst oxidase NADPH gene (c 31115.graph_c0, BrNADPH), 2,4-dienoyl-CoA reductase DECR1 gene (c34752.graph_c0, BrDECR1), F-box gene (c35581.graph_c0, BrF-box), peroxisome protein PMP gene (c33960.graph_c0, BrPMP), unknown protein gene or c30129.graph_c0 gene (c30129.graph_c0, BrUCP) (see Table 1 for details).The nucleotide sequences of the cyclic cytoplasmic protein CYP gene are shown in SEQ ID NO:1; the nucleotide sequences of the transcription elongation factor EF1 gene are shown in SEQ ID NO:2; the nucleotide sequences of the actin gene are shown in SEQ ID NO:3; the nucleotide sequences of the ribosomal protein 28sRPS27 gene are shown in SEQ ID NO:4; the nucleotide sequences of the β-tubulin TUB gene are shown in SEQ ID NO:5; the nucleotide sequences of the ribosomal protein 40sRPS17 gene are shown in SEQ ID NO:6; the nucleotide sequences of the casein kinase CKⅠ gene are shown in SEQ ID NO:7; the nucleotide sequences of the thioredoxin TRX gene are shown in SEQ ID NO:8; the nucleotide sequences of the ribosomal protein 60sRPL18 gene are shown in SEQ ID NO:9; the nucleotide sequences of the ribosomal protein 50sRPL15 gene are shown in SEQ ID NO:10; the nucleotide sequences of the ubiquitin ligase UBI gene are shown in SEQ ID NO:11; and the nucleotide sequences of the respiratory burst oxidase NADPH gene are shown in SEQ ID NO:11. The nucleotide sequence of the 2,4-dienoyl-CoA reductase DECR1 gene is shown in SEQ ID NO:12; the nucleotide sequence of the F-box gene is shown in SEQ ID NO:14; the nucleotide sequence of the peroxisome protein PMP gene is shown in SEQ ID NO:15; and the nucleotide sequence of the c30129.graph_c0 gene is shown in SEQ ID NO:16. Further qRT-PCR primers were designed for the 16 candidate internal reference genes screened above, and their sequence listing information is shown in Table 2.

[0046] Table 1. Unigenes corresponding to candidate internal reference genes and their FPKM values ​​in RNA-Seq expression profiles.

[0047]

[0048]

[0049] Table 2. Sequence lengths and primer sequences of candidate internal reference genes.

[0050]

[0051] Example 2: Stability analysis of candidate internal reference gene expression in heat-tolerant and heat-sensitive DH series Chinese cabbage leaves under different periods of high-temperature stress.

[0052] The heat-resistant DH line CX1-7 and the heat-sensitive DH line CX7-3 of Chinese cabbage were used as experimental materials. Plump, uniformly sized Chinese cabbage seeds were sown in seedling pots, with one seedling per pot. When the seedlings had 4 or 5 true leaves, they were pre-cultured in an artificial climate chamber at 25℃ / 18℃ (day / night), 12h / 12h (day / night), 80% light, and 70% humidity, followed by a high-temperature treatment at 37℃. The third true leaf of the Chinese cabbage seedlings was collected after 0h (CK, i.e., plants growing normally under pre-culture conditions), 0.25h, 0.5h, 1h, 3h, 6h, 12h, 24h, and 48h of the high-temperature treatment, and used for further processing. Ten seedlings were randomly selected from each treatment, with three biological replicates. Total RNA was extracted from the third true leaf of Chinese cabbage from each treatment according to the Trizol reagent instructions (Beijing TransGen Biotech Co., Ltd.). Genomic DNA was removed using a cDNA synthesis kit (Beijing TransGen Biotech Co., Ltd.), and cDNA was synthesized by reverse transcription. The cDNA product was diluted 3-fold and used as a template for qRT-PCR amplification. The reagents used for qRT-PCR amplification were as follows: Premix Ex Taq™ kit. Prepare a 20 μL qRT-PCR amplification reaction system as follows: 5 μL 2×SYBR Premix Ex Taq™, 0.5 μL forward primer (10 μmol / L), 0.5 μL reverse primer (10 μmol / L), 4 μL cDNA template, and 10 μL sterile distilled water. qRT-PCR reaction program: 95℃ pre-denaturation for 30 s; 95℃ denaturation for 5 s, 60℃ de-denaturation for 20 s, 72℃ extension for 20 s, 40 cycles. Each reaction was repeated three times. After the qRT-PCR reaction, the amplification status of each gene was automatically analyzed, and the corresponding Ct values ​​were exported (Table 3). The stability of each candidate internal control gene was analyzed using GeNorm, Bestkeeper, and Normfinder software based on the Ct values ​​obtained after the qRT-PCR reaction. GeNorm software calculated the expression stability M value of each candidate internal control gene; a higher M value indicates poorer stability. NormFinder software calculates the stability value of each candidate internal reference gene by combining within-group and between-group variances; the smaller the value, the more stable the internal reference gene. Bestkeeper allows direct input of Ct values ​​and compares the stability of each internal reference gene by calculating the standard deviation (SD) and coefficient of variation (CV); the smaller the standard deviation and coefficient of variation, the better the stability.

[0053] As shown in Table 4, the candidate reference genes with the best expression stability under different time periods of high temperature stress in Chinese cabbage, as analyzed by GenNorm, NormFinder, and BestKeeper, are BrCKⅠ, Br28sRPS27, and BrCKⅠ, respectively.

[0054] Table 3. Average Ct values ​​of candidate internal reference genes in heat-tolerant and heat-sensitive Chinese cabbage leaves at different times of high temperature stress.

[0055]

[0056] Table 4. Expression stability of candidate internal reference genes in heat-tolerant and heat-sensitive Chinese cabbage leaves at different times of high temperature stress.

[0057]

[0058]

[0059] Example 3: Stability analysis of candidate internal reference genes in different tissues of Chinese cabbage under high temperature stress

[0060] The heat-resistant DH line CX1-7 and the heat-sensitive DH line CX7-3 of Chinese cabbage were used as experimental materials. Plump, uniformly sized seeds were selected and sown in nutrient pots. They were cultivated using conventional methods until bolting and flowering, then transferred to an artificial climate chamber at 25℃ / 18℃ (day / night), 12h / 12h (day / night), 80% light, and 70% humidity for 3 days of pre-cultivation. Afterward, high-temperature stress treatment was applied at 37℃ / 27℃ (day / night), with all environmental conditions except temperature remaining consistent with the pre-cultivation conditions. Roots, leaves, petioles, bolting stems, and flowers from 0h and 6h of high-temperature treatment were collected, frozen in liquid nitrogen, and stored at -80℃ for later use. RNA extraction, cDNA reverse transcription, and qRT-PCR analysis were performed on the obtained samples (specific steps and methods are the same as in Example 2). The Ct values ​​of the candidate internal reference gene in different tissues of high-temperature stressed Chinese cabbage are shown in Table 5. The Ct values ​​obtained after qRT-PCR were analyzed using GeNorm, Bestkeeper, and Normfinder software to obtain the stability differences of each candidate internal reference gene in different tissues of heat-resistant and heat-sensitive Chinese cabbage.

[0061] The results are shown in Table 6. Among the different tissues of heat-resistant and heat-sensitive Chinese cabbage under high temperature stress, BrCKⅠ showed the best expression stability among the 16 candidate internal reference genes, followed by BrUBI.

[0062] Table 5. Average Ct values ​​of candidate reference genes for high temperature stress in different tissues of heat-resistant and heat-sensitive Chinese cabbage.

[0063]

[0064] Table 6. Expression stability of candidate reference genes for high temperature stress in different tissues of heat-resistant and heat-sensitive Chinese cabbage.

[0065]

[0066] Example 4: Stability analysis of candidate internal reference genes in leaves of 10 Chinese cabbage DH lines or varieties under high temperature stress

[0067] The heat-resistant DH series or varieties CX1-7, CX3-2, CX5-1, 008 Chinese cabbage, and 50-day pointed-leaf Chinese cabbage, and the heat-sensitive DH series or varieties CX7-3, CX4-1, CX6-5, CX5-4, and Hangyu 49-1 Chinese cabbage were used as materials (published in "Pang Qiangqiang et al. Analysis of heat resistance during germination and seedling stages of different Chinese cabbage varieties and screening of identification indicators. Northwest Agricultural Journal, 2020, 29(02):295-305." and "Pang Qiangqiang et al. Evaluation of heat resistance of Chinese cabbage and response of enzymatic antioxidant system to high temperature stress. Zhejiang Agricultural Journal, 2020, 32(01):72-79."). Plump and uniformly sized Chinese cabbage seeds were sown in nutrient pots. When the seedlings had 4 or 5 true leaves, they were transplanted into an artificial climate chamber at 25℃ / 18℃ (day / night), 12h / 12h (day / night), 80% light, and 70% humidity for 3 days of pre-culture, followed by high-temperature stress treatment at 37℃. The third true leaves from the 0h (CK) and 6h high-temperature treatments were taken, frozen in liquid nitrogen, and stored at -80℃ for later use. RNA extraction, cDNA reverse transcription, and qRT-PCR analysis were performed on the obtained samples (specific steps and methods are the same as in Example 2). The Ct values ​​of the candidate internal reference genes in the leaves of 10 Chinese cabbage DH lines or varieties under high-temperature stress were obtained (Table 7). The Ct values ​​obtained after qRT-PCR reaction were analyzed using GeNorm, Bestkeeper, and Normfinder software to obtain the stability differences of each candidate internal reference gene in different Chinese cabbage DH lines or varieties under high-temperature stress.

[0068] The results are shown in Table 8. Under high temperature stress, among different DH lines or varieties of Chinese cabbage, the BrCYP gene showed the best stability in both GenNorm and BestKeeper analyses, while the Br28sRPS27 gene showed the best stability in NormFinde analysis.

[0069] Table 7. Average Ct values ​​of the internal reference gene for high temperature stress in leaves of 10 Chinese cabbage varieties.

[0070]

[0071]

[0072] Table 8. Expression stability of internal reference genes under high temperature stress in leaves of 10 Chinese cabbage DH lines or varieties.

[0073]

[0074] Example 5: Comprehensive Analysis of the Expression Stability of Candidate Internal Reference Genes under Different Experimental Conditions (Different Durations of High Temperature Stress, Different Tissues, Different DH Lines or Varieties) in Heat-Tolerant and Heat-Sensitive Chinese Peppers

[0075] This embodiment is used for a comprehensive analysis of the expression stability of candidate internal reference genes under different experimental conditions (different durations of high-temperature stress, different tissues, different DH lines or varieties) in heat-resistant and heat-sensitive Chinese cabbage. The treatment methods and data processing for different durations of high-temperature stress are the same as in Example 2; the treatment methods and data processing for different tissues of Chinese cabbage under high-temperature stress are the same as in Example 3; and the treatment methods and data processing for different DH lines or varieties are the same as in Example 4.

[0076] Further analysis of the stability rankings obtained using the GeNorm, NormFinder, and Bestkeeper methods was performed using ReFinder software to calculate the geometric mean and obtain a comprehensive index ranking. The smaller the comprehensive index, the more stable the expression of the internal reference gene.

[0077] Table 9 shows the overall stability ranking of the 16 candidate internal control genes under different experimental conditions. As can be seen from Table 9, the top three internal control genes with the highest stability under different time periods of high-temperature stress are BrCKⅠ, BrCYP, and BrPMP; the top three internal control genes with the highest stability under different tissues under high-temperature stress are BrCKⅠ, BrUBI, and Br40sRPS17; and the top three internal control genes with the highest stability under different DH lines or varieties under high-temperature stress are Br28sRPS27, BrCYP, and Br60sRPL18. Therefore, the optimal internal control gene for gene expression analysis of all samples under different experimental conditions (different time periods of high-temperature stress, different tissues, and different DH lines or varieties) is BrCKⅠ, followed by BrCYP and Br28sRPS27.

[0078] Table 9. Comprehensive analysis results of ReFinder software stability under different experimental conditions for candidate internal reference genes.

[0079]

[0080] The embodiments of the present invention have been described in detail above. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, unless otherwise specified, the embodiments of the present invention and the features thereof can be combined with each other.

Claims

1. Application of primers used to amplify the internal reference gene for high temperature stress in Chinese cabbage in any one of a1~a2; a1. Screening for heat-resistant genes in Chinese cabbage; a2. Prepare products for screening heat-resistant genes in Chinese cabbage; The internal reference gene for high temperature stress in Chinese cabbage is the casein kinase CKⅠ gene, and its nucleotide sequence is shown in SEQ ID NO:

7.

2. The use of the kit containing the primers described in claim 1 in any one of a1 to a2; a1. Screening for heat-resistant genes in Chinese cabbage; a2. Prepare products for screening heat-resistant genes in Chinese cabbage.

3. Application of primers used to amplify the internal reference gene for high temperature stress in Chinese cabbage in any one of b1~b2; b1. Analysis of gene expression for heat tolerance in Chinese cabbage; b2. Application in the preparation of products for analyzing the expression of heat resistance genes in Chinese cabbage; The internal reference gene for high temperature stress in Chinese cabbage is the casein kinase CKⅠ gene, and its nucleotide sequence is shown in SEQ ID NO:

7.

4. The use of a kit containing the primers described in claim 3 in any one of b1 to b2; b1. Analysis of gene expression for heat tolerance in Chinese cabbage; b2. Application in the preparation of products for analyzing the expression of heat resistance genes in Chinese cabbage.

5. Application of primers used to amplify the internal reference gene for high temperature stress in Chinese cabbage in any one of c1~c2; c1. To detect the changes in the expression level of heat resistance genes in Chinese cabbage under different tissues, different growth and development stages, or high temperature stress conditions; c2. Application in the preparation of products for detecting changes in the expression level of heat resistance genes in different tissues, different growth and development stages, or under high temperature stress; The internal reference gene for high temperature stress in Chinese cabbage is the casein kinase CKⅠ gene, and its nucleotide sequence is shown in SEQ ID NO:

7.

6. The application according to claim 1, 3, or 5, characterized in that, The nucleotide sequences of the primers are shown in SEQ ID NO:29 and SEQ ID NO:

30.

7. The use of a kit containing the primers described in claim 5 in any one of c1 to c2; c1. To detect the changes in the expression level of heat resistance genes in Chinese cabbage under different tissues, different growth and development stages, or high temperature stress conditions; c2. Application in the preparation of products for detecting changes in the expression level of heat resistance genes in different tissues, different growth and development stages, or under high temperature stress.

8. A real-time quantitative PCR method for detecting heat resistance genes in Chinese cabbage, characterized in that, The method uses the high-temperature stress internal reference gene of Chinese cabbage as described in claim 1 as an internal reference gene.

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