Application of Actin gene and / or RPL35 gene in preparation of reference gene for quantitative analysis of gene expression in camellia oleifera kernel development stage

Abstract

The invention discloses application of an Actin gene and / or an RPL35 gene in preparation of a reference gene for quantitative analysis of gene expression in a camellia oleifera seed kernel development stage. Candidate genes which are stably expressed in different development stages are screened based on transcriptome data, the expression stability of the candidate genes is evaluated through qRT-PCR in combination with a plurality of stability evaluation methods (geNorm, NormFinder, BestKeeper, Delta-Ct and RefFinder) systems, and finally Actin and RPL35 are determined as optimal reference genes. The invention also provides a specific primer of the reference gene, and the specific primer has the characteristics of high amplification efficiency, strong specificity and the like, is suitable for standardized detection of the expression quantity of the target gene in the camellia oleifera seed kernel development stage, and can effectively improve the accuracy and repeatability of gene expression analysis; and a stable and reliable molecular tool is provided for subsequent grease accumulation and functional gene research.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of molecular biology, and particularly relates to use of Actin gene and / or RPL35 gene in preparation of an internal reference gene for quantitative analysis of gene expression in an oil-tea camellia seed development stage BACKGROUND

[0002] Real-time fluorescent quantitative PCR (qRT-PCR) is a commonly used gene expression analysis technology, and is widely used in plant physiological development, environmental stress response and functional gene research. In order to ensure the accuracy and reliability of the qRT-PCR results, it is crucial to select a suitable internal reference gene for expression standardization. An ideal internal reference gene should be stably expressed in different tissues, different development stages and various treatment conditions.

[0003] Oil-tea camellia (Camellia oleifera) is an important woody oil crop in southern China, and its seed kernel is the main organ of oil accumulation. With the deepening of molecular breeding and oil synthesis mechanism research, the demand for gene expression research in the seed kernel development stage is increasing. However, there is currently a lack of stably verified internal reference genes in the oil-tea camellia seed kernel, and most related researches borrow common housekeeping genes such as GAPDH, UBQ, etc. in other species or tissues. These genes have large expression fluctuations under different experimental conditions, which makes it difficult to meet the demand for accurate expression analysis in the oil-tea camellia seed kernel development stage.

[0004] Therefore, it is urgent to screen and verify the internal reference genes that are stably expressed in different development stages of the oil-tea camellia seed kernel through high-throughput transcriptome data, so as to improve the accuracy and repeatability of qRT-PCR analysis, and provide a reliable molecular basis for oil synthesis, development regulation and other researches. SUMMARY

[0005] The application aims to overcome the deficiencies of the prior art, and provides use of Actin gene and / or RPL35 gene in preparation of an internal reference gene for quantitative analysis of gene expression in an oil-tea camellia seed development stage.

[0006] In order to achieve the above-mentioned purpose, the technical scheme provided by the application is as follows:

[0007] The application provides use of Actin gene and / or RPL35 gene in preparation of an internal reference gene for quantitative analysis of gene expression in an oil-tea camellia seed development stage, wherein the sequence of the Actin gene is as shown in SEQ ID NO. 17, and the sequence of the RPL35 gene is as shown in SEQ ID NO. 18.

[0008] Preferably, the oil-tea camellia seed development stage includes a fruit swelling period, a seed swelling period, an initial oil accumulation period, a rapid oil accumulation period and a seed maturation period.

[0009] The application also provides a primer pair for detecting expression of an Actin gene and an RPL35 gene by qRT-PCR; the forward primer of the Actin gene is 5'-GGAATGGAAGCTGCAGGGAT-3'(SEQ ID NO. 1), and the reverse primer is 5'-TCCTTGCTCATACGGTCTGC-3'(SEQ ID NO. 2);

[0010] the forward primer of the RPL35 gene is 5'-TTGTCCATTGCCCAGGTGTT-3'(SEQ ID NO. 3), and the reverse primer is 5'-GATGCTTGGTGAGGCGTCTA-3'(SEQ ID NO. 4).

[0011] The application also provides a kit for quantitative analysis of gene expression in the development stage of Camellia oleifera seeds, wherein the kit contains the primer pair described above.

[0012] Preferably, the kit contains the Actin gene and the RPL35 gene.

[0013] The application also provides a method for screening a stable reference gene in the development stage of Camellia oleifera seeds, which comprises the following steps:

[0014] (1) collecting Camellia oleifera seed tissues in different development stages or under different treatment conditions, and obtaining transcriptome data thereof;

[0015] (2) screening genes with log2TPM greater than 3 and standard deviation less than 1;

[0016] (3) calculating the expression variation coefficient CV, and selecting candidate reference genes with CV less than 0.2;

[0017] (4) combining qRT-PCR detection and stability evaluation by geNorm, NormFinder, BestKeeper, Delta-Ct and RefFinder software to screen stable reference genes.

[0018] Preferably, the candidate internal reference genes include MGST3, Actin, RPL35, GRP, HMG, SelT, SAP4 and RPS17 genes, and sequences of the MGST3, Actin, RPL35, GRP, HMG, SelT, SAP4 and RPS17 genes are shown in SEQ ID NO. 19, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 22, SEQ ID NO. 21, SEQ ID NO. 23, SEQ ID NO. 24 and SEQ ID NO. 20 respectively.

[0019] The application will be further described below:

[0020] The application aims at the problem that there is no special internal reference gene for seed kernel tissue in the current qRT-PCR expression analysis of Camellia oleifera, and based on transcriptome data, a plurality of development period seed kernel samples are screened, 8 candidate internal reference genes are preliminarily selected, and the stability of the genes is systematically evaluated by qRT-PCR technology combined with five methods of geNorm, NormFinder, BestKeeper, Delta-Ct and RefFinder, and finally Actin and RPL35 are selected as the most stable internal reference genes; and specific primer pairs are provided, which have high amplification efficiency and good specificity. The internal reference gene and the primer thereof can be used for gene expression quantitative analysis in the development process of Camellia oleifera seed kernel, solve the problem that there is no stable internal reference gene in the current Camellia oleifera seed kernel functional gene research, and improve the reliability of qRT-PCR data and the repeatability of research.

[0021] Specifically, the application preliminarily selects candidate genes with high expression and stability by analyzing transcriptome data of Camellia oleifera seed kernel at a plurality of development stages (including fruit swelling period, seed swelling period, oil initial accumulation period, oil rapid accumulation period and seed maturation period). The candidate internal reference genes with stable expression are screened according to the expression amount (log2TPM>3), standard deviation (SD<1) and coefficient of variation (CV<0.2). Further, the expression stability of the candidate internal reference genes is verified by qRT-PCR experiment, and the stability thereof is comprehensively evaluated by five algorithms of geNorm, NormFinder, BestKeeper, Delta-Ct and RefFinder. Finally, the Actin gene and the RPL35 gene are determined as the internal reference genes with the most stable expression in the development stage of Camellia oleifera seed kernel.

[0022] Compared with the prior art, the application has the following beneficial effects:

[0023] The application first systematically screens and verifies the stable reference genes expressed in the development stage of Camellia oleifera seeds based on transcriptome data, effectively overcoming the poor stability of traditional housekeeping genes; the screened Actin and RPL35 reference genes have the characteristics of high consistency of cross-stage expression and small fluctuation of Ct value, and are suitable for the expression research of multiple functional genes such as oil synthesis and endosperm development; the specific primers provided have high amplification efficiency and strong specificity, and can significantly improve the accuracy, stability and repeatability of the qRT-PCR analysis results; the reference genes and the application method thereof can be widely popularized to the fields of Camellia oleifera related breeding, transgenic detection, molecular regulation mechanism research and the like.

[0024] In summary, the application screens the candidate genes with stable expression in different development stages based on transcriptome data, and systematically evaluates the expression stability of the candidate genes through qRT-PCR combined with multiple stability evaluation methods (geNorm, NormFinder, BestKeeper, Delta-Ct and RefFinder), and finally determines that Actin and RPL35 are the most suitable reference genes. The application also provides specific primers of the reference genes, which have the characteristics of high amplification efficiency and strong specificity, are suitable for the standardized detection of the expression amount of target genes in the development stage of Camellia oleifera seeds, and can effectively improve the accuracy and repeatability of gene expression analysis, thereby providing a stable and reliable molecular tool for subsequent oil accumulation and functional gene research. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 It is a flow chart for the screening and functional enrichment of candidate reference genes: A is a wein diagram of stably expressed genes between three sample groups; B to D are respectively KEGG pathway enrichment analysis diagrams of stably expressed genes in three sample groups.

[0026] Figure 2 It is a log2TPM heat map of eight candidate reference genes in different samples.

[0027] Figure 3 It is a qRT-PCR melting curve diagram of each reference gene.

[0028] Figure 4 It is a Ct value distribution diagram of eight candidate reference genes in different development stage samples.

[0029] Figure 5 It is a stability comprehensive ranking result of each reference gene evaluated by five algorithms.

[0030] Figure 6 It is a linear regression relationship diagram between the log2TPM value and the Ct value of each candidate reference gene. DETAILED DESCRIPTION

[0031] The technical solutions provided by the present application are described in detail below in combination with embodiments, but they should not be understood as limiting the scope of protection of the present application.

[0032] In the embodiments of the present application, the nucleotide sequences of the candidate reference genes are shown in Table 1.

[0033] Table 1

[0034]

[0035]

[0036] The specific primers of the candidate reference genes are shown in Table 2.

[0037] Table 2 Specific primers of each candidate reference gene

[0038]

[0039] Note: F represents forward primer, and R represents reverse primer.

[0040] 1 Materials and methods

[0041] 1.1 Sample collection and transcriptome data processing

[0042] Camellia oleifera cultivar 'Huashuo' (hexaploid cultivar) was selected as the research object, and seed samples were collected from five consecutive development stages: T1 (fruit expansion stage), T2 (seed expansion stage), T3 (initial oil accumulation stage), T4 (rapid oil accumulation stage), and T5 (seed maturation stage), with three biological replicates for each stage. To evaluate the stability of the reference genes under hormone treatment, ethylene-treated samples were also selected, and the data were obtained from the NCBI database (PRJNA693152, PRJNA668531, PRJNA947537).

[0043] 1.2 RNA extraction and cDNA synthesis

[0044] All samples were immediately frozen using liquid nitrogen and stored at -80℃ after collection. Total RNA was extracted using a TIANGEN plant RNA extraction kit, and the quality was determined by agarose gel electrophoresis and microplate spectrophotometry. Reverse transcription was performed using a Takara PrimeScript RT kit, which included a gDNA removal step. The obtained cDNA was used for subsequent qRT-PCR reactions.

[0045] 1.3 Transcriptome data processing and candidate gene screening

[0046] The original RNA-Seq data was filtered by fastp software, and the adapter and low-quality sequences were removed. Then, HISAT2 was used to align to the Camellia oleifera hexaploid reference genome, and read counts were generated by FeatureCounts and normalized to TPM values. The primary screening criteria were: log2TPM > 3, and the expression standard deviation (SD) < 1. Further calculation of the expression coefficient of variation CV screened the genes with CV < 0.2 as the expression stable candidate genes.

[0047] 1.4 Functional annotation and enrichment analysis

[0048] Functional annotation was performed using eggNOG-mapper, and pathway enrichment analysis was performed on the screened candidate genes using KEGG database to identify their potential functions in metabolic regulation, energy synthesis, protein degradation, etc.

[0049] 1.5 qRT-PCR primer design and reaction conditions

[0050] Eight candidate internal reference genes (MGST3, Actin, RPL35, GRP, HMG, SelT, SAP4, RPS17) with high expression stability were selected, and qRT-PCR primers were designed using Primer Premier 5.0, and synthesized by a commercial company. ChamQ Universal SYBR qPCR Master Mix (Vazyme) was used for qRT-PCR reaction, and the reaction system was 10 μL, including cDNA template 1 μL, primers each 0.2 μL, reaction solution 5 μL, ddH2O 3.6 μL. The reaction program was: 95℃ pre-denaturation for 30s; 40 cycles: 95℃ for 10s, 60℃ for 30s; finally, melt curve analysis (65-95℃, every 0.5℃ warming). At each development stage, 3 technical repeats were performed.

[0051] 1.6 Amplification efficiency verification and expression stability analysis

[0052] Primer specificity was confirmed by melt curve analysis, and standard curve was established by cDNA gradient dilution series. The amplification efficiency calculation formula was: E = (10^(-1 / slope)-1) x 100, and the correlation coefficient R 2 was used to judge the linear range. The Ct values of the candidate internal reference genes were calculated by four methods of geNorm, NormFinder, BestKeeper, and Delta-Ct to analyze the expression stability, and RefFinder platform was used to comprehensively weight and sort the above results to obtain the final stability ranking.

[0053] 1.7 qRT-PCR and transcriptome data correlation analysis

[0054] Linear fitting analysis was performed on the Ct values ​​of each gene and the corresponding log2TPM values ​​of the transcriptome to assess the consistency between the two expression modes and provide data support for internal reference screening.

[0055] 2 Results

[0056] 2.1 Screening and functional enrichment analysis of candidate internal reference genes

[0057] To identify reference genes that are stably expressed during the development of Camellia oleifera kernels, this study screened 1550 co-expressed candidate genes from three RNA-Seq datasets. Figure 1 The Venn diagram analysis reveals gene overlap: Group 1 and Group 2 share 81 genes, Group 1 and Group 3 share 356 genes, and Group 2 and Group 3 share 326 genes. KEGG pathway enrichment analysis was then performed on these candidate genes. Figure 1 B–1D was found to be involved in core metabolic pathways such as fatty acid synthesis, protein degradation, energy metabolism, and carbohydrate metabolism, and is closely related to the seed kernel development process.

[0058] 2.2 Candidate gene expression heatmap display and log2TPM analysis

[0059] Figure 2 The log2TPM expression heatmaps of eight candidate internal reference genes (MGST3, Actin, RPL35, GRP, HMG, SelT, SAP4, and RPS17) in different samples are shown. These genes all showed log2TPM values ​​greater than 6 in all samples, with a maximum value approaching 13, indicating high transcriptional levels, which is beneficial for qRT-PCR detection. Furthermore, their expression variation was relatively small across different developmental stages, meeting the basic requirements for use as internal reference genes.

[0060] 2.3 Validation of qRT-PCR amplification specificity and evaluation of efficiency

[0061] To ensure the primers could be used for subsequent quantitative expression analysis, melting curve analysis was performed on the qRT-PCR amplification products of the eight candidate internal reference genes. Figure 3 (A–3H). All primers exhibited a single melting peak, without any impurity peaks or primer dimers, indicating good specificity of the amplification products. Further calculation of the amplification efficiency of each primer using a standard curve, as shown in Table 2, reveals that the efficiencies of all primers ranged from 91.4% to 109.2%, with a correlation coefficient R0. 2 All values ​​were between 0.982 and 0.999, indicating that each primer had good linear correlation and amplification performance.

[0062] Table 3. Statistical table of amplification performance indicators of primers for each candidate internal reference gene.

[0063]

[0064] 2.4 Ct value distribution and expression fluctuation analysis

[0065] Figure 4 The Ct value distribution of 8 candidate reference genes in different developmental stage samples was shown. The Ct value of Actin gene was the most concentrated in all samples, with the smallest variation range and a standard deviation of 1.073, showing high stability; RPL35 was second, with a standard deviation of 1.901, also a relatively stable gene. In contrast, SAP4 (4.700), HMG (3.836), SelT (5.607), RPS17 (7.065), and MGST3 (7.213) had larger Ct value differences and were not suitable as reference genes for qRT-PCR experiments of different developmental stages of Camellia oleifera seeds.

[0066] 2.5 Comprehensive evaluation of candidate gene expression stability by multiple algorithms

[0067] To comprehensively evaluate the stability of reference genes, four methods, geNorm, NormFinder, BestKeeper, and Delta-Ct, were used for individual ranking, and RefFinder platform was used to integrate all scoring results. Figure 5 The results showed that in the geNorm analysis, Actin and RPL35 had the best pairing stability with the lowest M value; NormFinder results showed that Actin was the most stable, followed by RPL35; in the BestKeeper analysis, Actin had the smallest standard deviation and coefficient of variation of Ct value; in the Delta-Ct method, Actin and RPL35 had the smallest Ct difference in all sample comparisons.

[0068] Figure 5 For RefFinder comprehensive ranking, the results showed that Actin and RPL35 had the lowest comprehensive score and the strongest stability, and were recommended as combined reference genes. GRP was second, and could be used as an auxiliary reference under specific conditions.

[0069] 2.6 Consistency verification of qRT-PCR and transcriptome expression data

[0070] To further verify the reference value of transcriptome data in the screening process, the log2TPM value of each candidate reference gene was linearly fitted with its qRT-PCR Ct value. Figure 6 The results showed that the two were significantly negatively correlated, with a fitting equation of Ct = –2.258 × log2TPM + 49.816, and a determination coefficient R 2 of 0.46. This correlation further supports the feasibility of preliminary screening by transcriptome data, and verifies the reliability of qRT-PCR experimental results.

[0071] The above merely describes the preferred embodiments of the present application, and it should be pointed out that those skilled in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered as falling within the protection scope of the present application.

Claims

1. The use of the Actin gene and / or RPL35 gene in the preparation of internal reference genes for quantitative analysis of gene expression during the developmental stage of Camellia oleifera seed kernels, wherein the sequence of the Actin gene is shown in SEQ ID NO.17 and the sequence of the RPL35 gene is shown in SEQ ID NO.

18.

2. The use as described in claim 1, characterized in that, The developmental stages of Camellia oleifera seeds include the fruit enlargement stage, the seed enlargement stage, the initial oil accumulation stage, the rapid oil accumulation stage, and the seed maturity stage.

3. Primer pairs for qRT-PCR detection of Actin and RPL35 gene expression, characterized in that, The forward primer for the Actin gene is 5'-GGAATGGAAGCTGCAGGGAT-3', and the reverse primer is 5'-TCCTTGCTCATACGGTCTGC-3'. The forward primer for the RPL35 gene is 5'-TTGTCCATTGCCCAGGTGTT-3', and the reverse primer is 5'-GATGCTTGGTGAGGCGTCTA-3'.

4. A kit for quantitative analysis of gene expression during the developmental stage of Camellia oleifera seed kernels, characterized in that, The kit contains the primer pair as described in claim 3.

5. The kit for quantitative analysis of gene expression during the developmental stage of Camellia oleifera seed kernels as described in claim 4, characterized in that, The kit contains the Actin gene and the RPL35 gene.

6. A method for screening internal reference genes stably expressed during the developmental stage of Camellia oleifera seed kernels, characterized in that, The method for screening Camellia oleifera seed kernels that stably express internal reference genes during the developmental stage includes the following steps: (1) Collect Camellia oleifera seed kernel tissues at different developmental stages or under different treatment conditions, and obtain their transcriptome data; (2) Screen for genes with log2TPM greater than 3 and standard deviation less than 1; (3) Calculate the expression coefficient of variation (CV) and select candidate internal reference genes with a CV less than 0.2; (4) Combine qRT-PCR detection and the stability evaluation of geNorm, NormFinder, BestKeeper, Delta-Ct and RefFinder software to screen out stable internal reference genes.

7. The method for screening stable expression of internal reference genes at the developmental stage of Camellia oleifera seed kernels as described in claim 6, characterized in that, The candidate internal reference genes include MGST3, Actin, RPL35, GRP, HMG, SelT, SAP4, and RPS17 genes; the sequences of the MGST3, Actin, RPL35, GRP, HMG, SelT, SAP4, and RPS17 genes are shown in SEQ ID NO.19, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.22, SEQ ID NO.21, SEQ ID NO.23, SEQ ID NO.24, and SEQ ID NO.20, respectively.

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