An internal reference gene for detecting and analyzing genes related to camellia fragrance formation and its application
By using the PP2A gene as the internal reference gene in camellia, the stability problem in detecting and analyzing the related genes of camellia flower fragrance in the prior art is solved, and more efficient and reliable detection results are achieved.
Patent Information
- Application Number
- CN202410981426.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-07-22
- Publication Date
- 2025-04-18
- Estimated Expiration
- 2044-07-22
AI Technical Summary
The prior art is difficult to effectively detect and analyze the genes related to the formation of camellia flower fragrance, and lacks stable internal reference genes, which affects the accuracy and reliability of the detection results.
Camellia PP2A gene was used as the internal reference gene, and corresponding real-time fluorescence quantitative PCR primers were designed to be used for expression analysis of genes related to Camellia scent formation to ensure the stability and reproducibility of the detection results.
By using the PP2A gene as the internal reference gene, the efficiency and credibility of camellia floral gene detection are improved, and the stability and reliability of the test results are ensured.
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Figure CN118834985B_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of gene detection technology, and more specifically to an internal reference gene for detecting and analyzing genes related to the formation of camellia fragrance and its application. Background Art
[0002] Camellia (Camellia japonica L) is a plant of the genus Camellia in the family Theaceae and is one of the top ten traditional famous flowers in my country. Camellia has a wide variety of resources, rich colors, large and colorful flowers, and has high ornamental and economic value. It is widely used in courtyard cultivation, potted flower production, park greening, etc., and has a high market share in the New Year flower market. Among the tens of thousands of camellia varieties in the world, there are only more than 10 varieties with fragrance, which is the so-called "good flowers are not fragrant". Aromatic camellia varieties are rare varieties, and cultivating new varieties that combine color, flower shape and fragrance has become one of the main directions of camellia breeding. Compared with other species of the genus Camellia, such as Camellia oleifera, Camellia sinensis, and Camellia sasanqua, research on the fragrance of camellia is relatively lagging, and there are few reports on molecular-level research on the formation of camellia fragrance.
[0003] Transcriptome sequencing technology is often used in the study of floral fragrance biosynthesis pathways and the screening of key differential genes in floral fragrance formation. Real-time fluorescence quantitative PCR technology is widely used in the analysis and verification of gene expression patterns due to its advantages such as high sensitivity, strong specificity and convenient operation. Many factors such as RNA quality, reverse transcription efficiency, primer specificity and PCR amplification efficiency can affect the accuracy of real-time fluorescence quantitative PCR results. Introducing appropriate internal reference genes for standard correction is conducive to improving the accuracy of real-time fluorescence quantitative PCR results.
[0004] The main floral fragrance components of camellia are monoterpenes and benzene ring / phenylpropanoid substances. There are obvious differences in the floral fragrance components and contents among different flowering stages and different varieties of camellia. Currently, research on the function of camellia fragrance genes is relatively rare, and research on the internal reference genes for the formation of camellia fragrance has not been reported.
[0005] Therefore, how to provide stable internal reference genes for the detection and analysis of genes related to the formation of camellia fragrance is a technical problem that technicians in this field urgently need to solve. Summary of the invention
[0006] In view of this, the purpose of the present invention is to provide an internal reference gene for camellia fragrance formation related gene detection and analysis and its application. For the first time, the camellia PP2A gene is used as an internal reference gene for expression analysis of camellia fragrance formation related genes, which can be stably expressed in different tissue parts, different flowering stages and different varieties of camellia. The designed real-time fluorescence quantitative PCR primers can improve the stability, reliability and repeatability of the detection results when used for camellia fragrance gene detection and analysis.
[0007] In order to achieve the above object, the present invention adopts the following technical solution:
[0008] An internal reference gene for detecting and analyzing genes related to camellia fragrance formation, wherein the internal reference gene is the camellia PP2A gene, and the nucleotide sequence is shown in SEQ ID NO.39.
[0009] Preferably, during the detection and analysis, the primer sequence of the internal reference gene is as follows:
[0010] Forward primer: 5'-CTGCATCGAAAGACAGGGTA-3', SEQ ID NO. 5;
[0011] Reverse primer: 5'-CTGACATCAACATCTGGGTC-3', SEQ ID NO.6.
[0012] Preferably, the camellia fragrance formation-related genes are: CaHMGR, CaDXS1, CaDXS2, CaMDS, CaGGPPS, CaLIS / NES1, CaLIS / NES2, CaSAMT and CaPAR.
[0013] Another object of the present invention is to provide: application of the above-mentioned internal reference gene in the analysis of camellia floral fragrance gene expression.
[0014] Another object of the present invention is to provide: a real-time fluorescence quantitative PCR detection and analysis method using the Camellia PP2A gene as an internal reference gene. During the detection and analysis, the primer sequences for the Camellia PP2A gene are designed as shown in SEQ ID NO.5 and SEQ ID NO.6.
[0015] It can be seen from the above technical solution that compared with the prior art, the present invention has the following beneficial effects:
[0016] The present invention screened 10 candidate internal reference genes (α-TUB, CYP, PP2A, ACTIN1, ACTIN2, GAPDH, UBQ, AQP, UBC and HIS) according to the transcriptome data of camellia, respectively, and measured the expression levels in different tissue parts, different flowering stages and flowers of different varieties of camellia, and combined with geNorm, NormFinder and Bestkeeper three analysis software to evaluate the expression stability of the 10 candidate internal reference genes, and finally screened protein phosphatase 2a (PP2A) as the most ideal internal reference gene by the geometric mean method, providing a technical reference for subsequent camellia flower fragrance gene expression analysis research. The provided real-time fluorescence quantitative PCR detection and analysis method using the camellia PP2A gene as an internal reference gene can effectively improve the detection efficiency and credibility of the camellia flower fragrance gene. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.
[0018] Figure 1 The following are the selection of test materials, including A: samples of different tissue parts of 'Xiang Fei', 1-5 are roots, stems, young leaves, mature leaves and flowers in full bloom; B: flower samples of 'Xiang Tai Yang' at different flowering stages, 1-4 are bud stage, early flowering stage, full bloom stage and late flowering stage; C: flower samples of different camellia varieties in full bloom, 1-4 are 'Xiang Tai Yang', 'Xiang Fei', 'Tian Kate' and 'Chi Dan' respectively;
[0019] Figure 2 Agarose gel electrophoresis of 10 candidate internal reference genes;
[0020] FIG3 is a melting curve diagram of 10 candidate internal reference genes; Figure 3-A - Figure 3-J They represent 10 different internal reference genes respectively;
[0021] Figure 4 The following are: Ct value distribution diagrams of 10 candidate internal reference genes;
[0022] Figure 5 For: Stability analysis of 10 candidate reference genes in all samples;
[0023] Figure 6 To: Analysis of the stability of 10 candidate reference genes in different tissues of 'Xiang Fei';
[0024] Figure 7 To: Stability analysis of 10 candidate reference genes at different flowering stages of 'Xiang Taiyang';
[0025] Figure 8 To: Analysis of the stability of 10 candidate reference genes in different camellia varieties;
[0026] Fig. 9 Figure 2: qRT-PCR validation of the relative expression of camellia fragrance-related genes with PP2A as the internal reference gene, where A: expression of structural genes in the precursor pathway of terpenoid synthesis in camellia; B: expression of key enzyme genes in the synthesis of terpenoids; C: expression of key enzyme genes in the synthesis pathway of benzene ring / phenylpropanoid compounds; V1: light fragrance type 'Xiang Taiyang'; V2: strong fragrance type 'Xiang Fei'; V3: no fragrance type 'Chi Dan';
[0027] Fig.10 Figure 2 is a qRT-PCR validation diagram of the relative expression levels of camellia fragrance-related genes using GAPDH as the internal reference gene, where V1: light fragrance type 'Xiang Taiyang'; V2: strong fragrance type 'Xiang Fei'; V3: no fragrance type 'Chi Dan'. DETAILED DESCRIPTION
[0028] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.
[0029] The instruments and reagents used in each embodiment are as follows:
[0030] ABI 7500 fluorescence quantitative PCR instrument (Applied Biosystems, USA);
[0031] RNA extraction kit (Bioteke Corporation, Dalian);
[0032] Qubit 2.0 Fluorometer (Thermo Scientific, USA);
[0033] Agilent 2100 Bioanalyzer (Agilent Technologies, USA);
[0034] HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Novozymes, Nanjing);
[0035] ChamQ Universal SYBR qPCR MasterMix Kit (Novozymes, Nanjing);
[0036] Taq PCR SuperMix (+dye) kit (Quanshijin, Beijing);
[0037] Primer synthesis was completed by Shanghai Shenggong Bioengineering Technology Service Co., Ltd., and the remaining biochemical reagents were all domestically produced analytical grade.
[0038] Example 1
[0039] Screening of candidate internal reference genes and primer design
[0040] According to the full name of the reference gene, the Gene ID number and sequence of the gene were searched in the transcriptome sequencing results of Camellia japonica, and the gene sequence was compared through NCBI (http: / / www.ncbi.nlm.nih.gov / ). Then, the expression FPKM value of the gene in different samples was searched according to the Gene ID number. According to the stability and consistency level of the expression FPKM value between different groups of samples, 10 candidate reference genes such as PP2A were preliminarily screened, and the corresponding primer sequences were designed, as shown in Table 1:
[0041] Table 1 Primers for qRT-PCR analysis of 10 candidate reference genes and 9 floral fragrance-related genes
[0042]
[0043]
[0044] Example 2
[0045] Specificity analysis of 10 candidate reference genes
[0046] (1) Test materials and sensory evaluation
[0047] Four representative camellia varieties with obvious olfactory differences were selected as test materials, namely 'Scented Sun' (C.japonica L.var.'Scented Sun'), 'High Fragrance' (C.japonica L.var.'SweetEmilyKate') and 'Chidan' (C.japonica L.var.'Chidan'); they were uniformly planted in the flower germplasm resource nursery of the Crop Research Institute of the Fujian Academy of Agricultural Sciences, and the cultivation conditions and management methods of all varieties were consistent.
[0048] Flower samples of four camellia cultivars, 'Xiang Taiyang', 'Xiang Fei', 'Tian Kate' and 'Chi Dan', were collected at 8:00-9:00 a.m. on sunny days during the peak flowering period for sensory evaluation. The final floral fragrance types are summarized as shown in Table 2.
[0049] Table 2 Sensory evaluation of the aroma of different camellia varieties
[0050] Variety name Floral scent type Points Fragrance Description 'Fragrant Sun' Light fragrance 6.3b Lily of the Valley 'Fragrant Concubine' Strong aroma 9.0a Rose scent 'Sweet Kate' Sweet fragrance 6.1b Fruity 'Red Dan' No scent 1.2c -
[0051] (2) Total RNA extraction and cDNA synthesis
[0052] Preparation of experimental materials: There are three groups of samples: 1. Select samples from different tissue parts of 'Xiang Fei', including roots, stems, young leaves, mature leaves and flowers in full bloom ( Figure 1 -A). 2. Collect flower samples of 'Xiang Yang' at different flowering stages, including bud stage, early flowering stage, full flowering stage and late flowering stage ( Figure 1 -B). 3. Collect healthy and pest-free camellia varieties, including 'Xiang Taiyang', 'Xiang Fei', 'Tian Kate' and 'Chi Dan', at full flowering stage ( Figure 1 -C). Samples were collected from each group at 8:00-9:00 a.m. on a sunny day, with 3 biological replicates for each group. All samples were immediately wrapped in tin foil and placed in liquid nitrogen, pre-cooled and stored in a -80°C refrigerator for later use.
[0053] Total RNA from each sample was extracted using a universal RNA extraction kit (Bioteke Corporation, Dalian). RNA concentration was measured with high precision using a Qubit 2.0 fluorometer (Thermo Scientific, USA) and RNA quality was tested by 1% agarose gel electrophoresis. RNA integrity was accurately tested using an Agilent 2100 bioanalyzer (Agilent Technologies, USA).
[0054] The extracted sample RNA was used as a template to synthesize the first strand of cDNA by reverse transcription using the HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Novozyme, Nanjing). The synthesized cDNA was diluted 3 times and stored at -20°C for later use.
[0055] (3) Primer design and verification
[0056] The corresponding primers were designed by PrimerPremier 5.0 and DNAMAN 9.0 software, as shown in Table 1. RT-PCR was performed using Taq PCR SuperMix (+dye) kit (Quanshijin, Beijing), and the PCR amplification products were detected by 1.5% agarose gel electrophoresis. Figure 2 shown.
[0057] Reaction system: The total volume of the reaction system is 50 μL, containing 1 μL of cDNA, 1 μL of 10 μM forward and reverse primers, Taq PCR SuperMix (+dye) 25 μL, Nuclease-free water 22 μL.
[0058] Reaction procedure: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 5 s, annealing at 50°C for 30 s, extension at 72°C for 30 s, 35 cycles; final extension at 72°C for 5 min, and storage at 4°C.
[0059] Result analysis: The amplification products of the 10 internal reference genes had single bands, indicating that the primers could amplify specifically and there were no primer dimers ( Figure 2 ), which can be further used for qRT-PCR research.
[0060] (4) qRT-PCR analysis of candidate internal reference genes
[0061] qRT-PCR reactions were performed using the ChamQ Universal SYBR qPCR MasterMix Kit (Nanobio, Nanjing) in an ABI 7500 fluorescent quantitative PCR instrument (Applied Biosystems, USA).
[0062] Reaction system: The total volume of the reaction system is 20 μL, containing 10 μL of 2×ChamQ Universal SYBR qPCR MasterMix, 1 μL of diluted cDNA, 0.4 μL each of 10 μM forward and reverse primers, and 8.2 μL of sterile water.
[0063] The reaction procedure was as follows: pre-denaturation at 95°C for 30 s; denaturation at 95°C for 10 s, and annealing at 60°C for 30 s, for a total of 40 cycles, with 3 technical replicates for each reaction.
[0064] The product specificity was detected by melting curve. The melting curve program was: 95°C, 15 s; 60°C, 1 min; 95°C, 15 s. Each reaction was repeated 3 times.
[0065] Result analysis: The melting curves of the 10 candidate internal reference genes all showed obvious single peaks (Figure 3), and there were no primer dimers, indicating that the internal reference gene primers had good specificity, which was consistent with the RT-PCR results and could be further analyzed.
[0066] Example 3
[0067] Analysis of the expression stability of internal reference genes in Camellia japonica
[0068] According to the analysis results of geNorm, NormFinder and BestKeeper, combined with the geometric mean method, the expression stability of all candidate internal reference genes was comprehensively evaluated, and then the most ideal internal reference genes were screened out. The specific process is as follows:
[0069] (1) Analysis of cycle threshold (Ct) values of 10 candidate reference genes in all samples
[0070] Results: The Ct values of the 10 candidate reference genes were significantly different in all samples ( Figure 4 The Ct value range of PP2A and ACTIN1 genes was the smallest (25.47-27.75; 25.88-29.09), while the Ct value range of GAPDH and ACTIN2 genes was the largest (21.66-29.14; 31.18-38.13).
[0071] (2) Stability analysis of internal reference genes using geNorm software
[0072] The geNorm software determines the stability of candidate reference genes based on the expression stability M value. The smaller the M value, the more stable the reference gene.
[0073] Results: In all samples, the most stable reference genes were PP2A and ACTIN1, followed by α-TUB, and then CYP; in different tissues of 'Xiangfei', the most stable reference genes were PP2A and ACTIN1, followed by UBQ, and then α-TUB; in different flowering stages of 'Xiangtaiyang', the most stable reference genes were PP2A and UBC, followed by ACTIN1, and then α-TUB; in different camellia varieties, the most stable reference genes were PP2A and ACTIN1, followed by α-TUB, and then AQP (Table 3 and Table 4). Figure 5-8 ).
[0074] Table 3 Analysis of the stability of candidate reference genes of Camellia japonica by geNorm software
[0075]
[0076]
[0077] (3) NormFinder software internal reference gene stability analysis
[0078] NormFinder software determines the stability of each candidate reference gene based on its stability value S value, where the lower the S value, the higher the stability.
[0079] Result analysis: The stability values S of the 10 candidate reference genes in all samples were ranked as PP2A < ACTIN1 < α-TUB < AQP < UBC < HIS < CYP < ACTIN2 < UBQ < GAPDH (Table 4). The most stable reference gene in all samples, different tissue parts of 'Xiangfei', and different flowering stages of 'Xiangtaiyang' was PP2A; among different camellia varieties, the most stable reference gene was AQP.
[0080] Table 4 Stability analysis of candidate reference genes of camellia by NormFinder software
[0081]
[0082] (4) Stability analysis of reference genes by BestKeeper software
[0083] The BestKeeper software determines the stability of candidate reference genes based on two variables, the standard deviation (SD) and the coefficient of variation (CV). A stable reference gene should have a smaller SD value. If the SD value is greater than 1, it indicates that the stability of the gene is poor.
[0084] Result analysis: In all samples, the SD values of the 10 candidate reference genes were ranked as PP2A < ACTIN1 < α-TUB < AQP < UBC < CYP < UBQ < GAPDH < ACTIN2 < HIS; in different tissue parts of 'Xiangfei', the most stable reference gene was α-TUB; in different flowering stages of 'Xiangtaiyang' and different camellia varieties, the most stable reference gene was PP2A (Table 5).
[0085] Table 5 Stability analysis of candidate reference genes of camellia by BestKeeper software
[0086]
[0087] (5) Comprehensive ranking analysis
[0088] Due to the different principles of the three statistical algorithms of geNorm, NormFinder, and BestKeeper, there are differences in the results of the most stable reference genes. Therefore, the geometric mean method is used to comprehensively analyze the total ranking values of each reference gene. The smaller the geometric mean value, the higher the stability of the reference gene.
[0089] Results analysis: In all samples, different tissue parts of 'Xiangfei' and different camellia varieties, the most stable reference genes were PP2A and ACTIN1; in different flowering stages of 'Xiang Taiyang', the most stable reference genes were PP2A and UBC (Table 6). The comprehensive evaluation results showed that PP2A had the lowest geometric mean and the best expression stability, so PP2A was the most ideal reference gene for this study, and the nucleotide sequence is shown in SEQ ID NO.39:
[0090]
[0091] Table 6 Comprehensive ranking of 10 candidate reference genes
[0092]
[0093]
[0094] Example 4
[0095] Verification of the PP2A internal reference gene in Camellia japonica
[0096] The expression patterns of nine key camellia fragrance genes, CaHMGR, CaDXS1, CaDXS2, CaMDS, CaGGPPS, CaLIS / NES1, CaLIS / NES2, CaSAMT and CaPAR, in the full flowering period of the light-fragrance 'Xiang Taiyang', the strong-fragrance 'Xiang Fei' and the non-fragrance 'Chi Dan' were used to verify the stability of the optimal reference gene PP2A in camellia. The relative expression of the target gene was calculated using 2 -△△Ct Law.
[0097] Results: The expression levels of the five structural genes in the precursor pathway of terpenoids in the light-fragrance 'Xiang Taiyang' were significantly different from those in the non-fragrance 'Chidan'; the expression levels of CaDXS2, CaMDS, and CaGGPPS in the strong-fragrance 'Xiang Fei' were significantly different from those in the non-fragrance 'Chidan' ( Fig. 9 -A). The expression levels of key enzyme genes CaLIS / NES1 and CaLIS / NES2 in the two aroma varieties were significantly different from those in the non-aroma 'Chidan' ( Fig. 9 -B). The expression levels of key enzyme genes CaSAMT and CaPAR in the synthesis pathway of benzene ring / phenylpropanoid compounds in the strong-flavor 'Xiangfei' were significantly higher than those in the light-flavor 'Xiangtaiyang' and the unflavored 'Chidan' ( Fig. 9 -C). The results of qRT-PCR analysis were consistent with the results of transcriptome gene expression.
[0098] In addition, when the less stable GAPDH was used as the internal reference gene, the expression levels of CaLIS / NES1, CaLIS / NES2, and CaPAR had a large deviation from the results of transcriptome gene expression, and also differed from the expression pattern when the PP2A gene was used as the internal reference gene. Among them, the expression level of the CaPAR gene fluctuated greatly ( Fig.10 ). Therefore, it is more accurate and reliable to use PP2A gene as an internal reference gene for the expression analysis of genes related to the formation of camellia floral fragrance.
[0099] The various embodiments in the specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.
[0100] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.
Claims
1. Application of Camellia PP2A gene as an internal reference gene in the analysis of camellia floral fragrance gene expression, characterized in that: The nucleotide sequence of the internal reference gene is shown in SEQ ID NO.
39.
2. A real-time fluorescence quantitative PCR detection and analysis method using the camellia PP2A gene as an internal reference gene for camellia floral fragrance gene expression analysis, characterized in that: During the detection and analysis, the primer sequences designed for the Camellia PP2A gene were as shown in SEQ ID NO.5 and SEQ ID NO.6; Forward primer: 5'-CTGCATCGAAAGACAGGGTA-3', SEQ ID NO. 5; Reverse primer: 5'-CTGACATCAACATCTGGGTC-3', SEQ ID NO.6.