Reference gene emtbp1 in fruit development of callitris intratropica and primer and application thereof

Abstract

The present application relates to a rough-leafed plantain fruit development internal reference gene EmTBP1 , primer and application thereof, and belongs to the technical field of biology. The present application provides a rough-leafed plantain fruit internal reference gene EmTBP1 , wherein the nucleotide sequence of the internal reference gene EmTBP1 is shown in SEQ ID NO. 17. The internal reference gene has extremely high expression stability, and can be applied to quantitative detection, screening and expression analysis of rough-leafed plantain fruit genes, especially functional genes in the development process. The correction effect of the internal reference gene EmTBP1 significantly improves the specificity, stability and accuracy of gene expression analysis data, and provides more possibilities for the research of rough-leafed plantain fruit development functional genes.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and particularly relates to an internal reference gene for fruit development in *Prunus cerasifera*. EmTBP1 Its primers and applications. Background Technology

[0002] Coarse chaff tree ( Ehretia macrophylla Wall . It belongs to the genus *Thick Shell* of the family Boraginaceae. Ehretia (Alternative name for wild loquat) The fruit of the wild loquat is rich in pectin (over 10%), and the degree of methylation of the pectin is over 70%, making it a fast-setting pectin with high economic value. Every 100g of wild loquat peel contains 6909.17 ± 176.85 GAE and 3066.96 ± 169.06 CAE, respectively, and the peel extract has strong antioxidant activity.

[0003] In-depth research into the molecular regulatory mechanisms of fruit development in *Prunus cerasifera* is beneficial for the development and utilization of *Prunus cerasifera*. Analyzing gene expression patterns using quantitative real-time PCR (qRT-PCR) is an important method for uncovering gene function; however, qRT-PCR experiments require relatively stable internal reference genes to avoid differences in mRNA concentration between samples.

[0004] Currently, there is a lack of stable and effective internal reference genes in the expression pattern analysis of genes related to fruit development in *Symplocos buergeriana*, which restricts the research progress on the molecular regulatory mechanisms during fruit development. Therefore, providing a stable and effective internal reference gene related to fruit development in *Symplocos buergeriana* is of great significance. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide an internal reference gene for the fruit of the *Prunus cerasifera*. EmTBP1 The primers and applications of *Symplocos buergeriana* were studied to improve the specificity, stability, and accuracy of gene detection in *Symplocos buergeriana* fruits.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a reference gene for the fruit of the Chinese privet tree. EmTBP1 The internal reference gene EmTBP1 The nucleotide sequence is shown in SEQ ID NO.17.

[0007] This invention provides a set of reference genes for the fruit of the Chinese privet tree. EmTBP1 The amplification primers, wherein the amplification primers are based on the internal reference gene. EmTBP1 Based on the gene sequence, and following the principles of real-time quantitative PCR primer design, an internal reference gene was designed. EmTBP1 Amplification primers.

[0008] Preferably, the nucleotide sequences of the amplification primers are shown in SEQ ID NO.52 and SEQ ID NO.53.

[0009] This invention provides the amplification primers described above for preparing internal reference genes from *Prunus cerasifera* fruit. EmTBP1 Applications in testing reagents.

[0010] This invention provides a method for detecting internal reference genes in the fruit of *Prunus cerasifera*. EmTBP1 The reagents include the amplification primers.

[0011] This invention provides the aforementioned amplification primers or reagents for the internal reference gene in the fruit of *Prunus cerasifera*. EmTBP1 Applications in quantitative detection.

[0012] This invention provides the aforementioned internal reference gene. EmTBP1 The application of the amplification primers or reagents described herein in the quantitative detection of genes in the fruit of *Prunus thunbergii*.

[0013] Preferably, the quantitative detection includes real-time quantitative PCR detection.

[0014] This invention provides the aforementioned internal reference gene. EmTBP1 The application of the amplification primers or reagents described herein in the screening or expression analysis of functional genes related to fruit development of *Prunus cerasifera*.

[0015] Preferred internal reference gene EmTBP1 Used as a correction gene.

[0016] The beneficial effects of this invention are: This invention provides an internal reference gene for the fruit of the *Pterocarya stenoptera*. EmTBP1 Its nucleotide sequence is shown in SEQ ID NO.17. This internal reference gene is stably expressed at different developmental stages of *Symplocos lucida* fruit, unaffected by changes in fruit development, exhibiting extremely high expression stability. It can be used for the quantitative detection, screening, and expression analysis of genes in *Symplocos lucida* fruit, especially functional genes related to development. Through the internal reference gene... EmTBP1 The correction effect significantly improves the specificity, stability and accuracy of gene expression analysis data of *Symplocos buergeriana* fruit, providing strong support for the functional gene expression analysis of *Symplocos buergeriana* fruit. Attached Figure Description

[0017] Figure 1 The melting curve of the candidate internal reference gene is shown in the RT-qPCR melting curve.

[0018] Figure 2 This invention presents the results of expression stability analysis of 19 established candidate internal reference genes based on geNorm analysis.

[0019] Figure 3 This invention presents the results of Norm Finder analysis on the expression stability of 19 established candidate internal reference genes.

[0020] Figure 4 This invention presents the results of expression stability analysis of 19 established candidate internal reference genes based on Best Keeper analysis.

[0021] Figure 5 This invention presents the results of Delta CT analysis on the expression stability of 19 established candidate internal reference genes.

[0022] Figure 6 This invention presents the results of expression stability analysis of 19 established candidate internal reference genes based on RefFinder comprehensive analysis.

[0023] Figure 7 This is the result of verifying the stability of the candidate internal reference gene in Example 3 of the present invention. Detailed Implementation

[0024] This invention provides a reference gene for the fruit of the Chinese privet tree. EmTBP1 The internal reference gene EmTBP1 The nucleotide sequence is shown in SEQ ID NO.17.

[0025] The reference gene for the fruit of the *Pterocarya stenoptera* provided by this invention EmTBP1 This gene was screened from fruit samples taken 10–100 days after flowering of *Symplocos rubrum*, exhibiting extremely high expression stability, with its expression level almost unaffected by changes in developmental progress. Its expression stability is significantly superior to other candidate internal reference genes and homologs of commonly used internal reference genes.

[0026] The internal reference gene provided by this invention EmTBP1 This method can be applied to the quantitative detection, screening, and expression analysis of genes in *Paeonia lactiflora* fruits, especially functional genes related to fruit development. Because *Paeonia lactiflora* fruits are rich in pectin, flavonoids, and polyphenols, these genes are stable expressions during fruit development. EmTBP1 This gene can be used as an internal reference gene for the quantitative detection, screening, and expression analysis of functional genes related to pectin, flavonoid, or polyphenol synthesis in the fruit of *Prunus cerasifera*. EmTBP1 The correction effect significantly improves the specificity, stability and accuracy of gene expression analysis data of *Symplocos buergeriana* fruit, providing strong support for the functional gene expression analysis of *Symplocos buergeriana* fruit.

[0027] This invention also provides a set of reference genes for the fruit of the *Pterocarya stenoptera*. EmTBP1 The amplification primers, wherein the amplification primers are based on the internal reference gene. EmTBP1 Based on the gene sequence, and following the principles of real-time quantitative PCR primer design, an internal reference gene was designed. EmTBP1 The amplification primers are described. The preferred nucleotide sequences of the amplification primers are shown in SEQ ID NO. 52 and SEQ ID NO. 53. The amplification primers of this invention are based on an internal reference gene. EmTBP1 The design is highly specific.

[0028] This invention provides the amplification primers described above for preparing internal reference genes from *Prunus cerasifera* fruit. EmTBP1 Applications in testing reagents.

[0029] This invention also provides a method for detecting internal reference genes in the fruit of *Prunus cerasifera*. EmTBP1 The reagents include the amplification primers.

[0030] In this invention, the reagent is preferably a kit, which, in addition to the amplification primers, preferably also includes other reagents required for the qRT-PCR reaction system, such as SYBR Green real-time PCR premix and sterile enzyme-free water.

[0031] This invention also provides the aforementioned amplification primers or reagents for the internal reference gene in the fruit of *Prunus cerasifera*. EmTBP1 Applications in quantitative detection, wherein the quantitative detection preferably includes real-time quantitative PCR detection.

[0032] The present invention also provides the aforementioned internal reference gene. EmTBP1 The application of the amplification primers or reagents described above in the quantitative detection of genes in *Prunus cerasifera* fruit. Preferably, the *Prunus cerasifera* fruit genes include functional genes related to the development of *Prunus cerasifera* fruit, and these functional genes preferably include those related to pectin, flavonoid, or polyphenol synthesis. The quantitative detection preferably includes real-time quantitative PCR. In the detection, the internal reference gene... EmTBP1 It is preferred as a correction gene to correct quantitative errors caused by experimental factors such as the amount of RNA template input, reverse transcription efficiency and sample loading operation between fruit samples, thereby ensuring the accuracy and comparability of qRT-PCR detection results.

[0033] The present invention also provides the aforementioned internal reference gene. EmTBP1 The application of the aforementioned amplification primers or reagents in the screening or expression analysis of functional genes related to fruit development in *Prunus cerasifera*. As one embodiment, the screening method and expression analysis method preferably include real-time quantitative PCR detection.

[0034] The preferred functional genes related to fruit development of *Pterocarya stenoptera* include those related to pectin, flavonoid, or polyphenol synthesis. The internal reference gene is used in the screening or expression analysis of relevant functional genes. EmTBP1 The preferred gene serves as a correction gene, acting as an internal standard. It can effectively correct for differences in template concentration between samples caused by RNA initiation amount, reverse transcription efficiency, and sample loading operations, ensuring the accuracy of the relative quantification results of the target gene. Subsequently, by comparing the differential expression of the target gene in different fruit development stages and tissue parts (such as pulp and peel), candidate genes that are significantly associated with developmental process or quality traits can be screened out, and the relationship between their expression and phenotypic parameters can be analyzed, providing theoretical support for gene function analysis and molecular breeding.

[0035] The present invention does not impose any special limitations on the specific steps of the real-time fluorescence quantitative PCR detection method; conventional steps in the field can be used.

[0036] In this invention, the fruits of the *Symplocos lucida* preferably include fruits from 0 to 100 days after flowering, specifically fruits from 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 days after flowering.

[0037] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0038] Unless otherwise specified, the following embodiments are all conventional methods.

[0039] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0040] Example 1 Screening of candidate internal reference genes in fruits of *Symplocos rubrum* at different developmental stages 1. Experimental materials and transcriptome sequencing This invention uses *Phyllostachys edulis* (Chinese privet) from the Pingdingshan University planting base as material. Samples of *Phyllostachys edulis* fruit were collected 10 to 100 days after flowering. The sampling times were 10, 30, 60, 80, 90, and 100 days. Three biological replicates were set up for each resource for each sampling period. After collection, the samples were quick-frozen in liquid nitrogen and then sent for transcriptome sequencing.

[0041] 2. Screening of candidate internal reference genes Identify commonly used internal reference genes in the model plant Arabidopsis thaliana. TIP41-L ( AT4G34270 ), TUB (AT5G12250) , TBP (At1g55520) , SAND (AT2G28390) , PP2A (AT3G21650) , GAPDH (At1g42970) , eIF4α (AT3G13920) , EFa (AT5G60390) and ACT2 (At5g09810) Homology alignment of homologous genes in rice bran yielded nine traditional candidate internal reference genes (SEQ ID NO. 1-9 in Table 1). Simultaneously, based on the transcriptome data obtained from sequencing, the mean (Mean) and coefficient of variation (CV) of each gene transcript were calculated. Following a screening process based on Mean > 50 and CV < 0.2, ten new candidate internal reference genes (SEQ ID NO. 10-19 in Table 1) were obtained. The candidate internal reference genes and their nucleotide sequences are shown in Table 1 below.

[0042] Table 1. Information on candidate internal reference genes

[0043] SEQ ID NO. 17 Example 2 Determination of internal reference genes in fruits of *Symplocos rubra* at different developmental stages 1. Total RNA extraction and cDNA synthesis (1) The sample preserved in step 1 of Example 1 was ground and pulverized in liquid nitrogen, and total RNA was extracted using the Polymer M5 HiPerTotal RNA Extraction Reagent (TRIgent) kit. The operation steps were performed according to the kit steps. After the Caulis Scabiosifolia RNA sample passed the agarose gel electrophoresis test, cDNA was synthesized using the Polymer M5 Superplus qPCR RT kit with gDNA remover kit. The operation steps were performed according to the kit instructions.

[0044] (2) The cDNA synthesized in different groups in step (1) (10d-1, 10d-2, 10d-3, 30d-1, 30d-2, 30d-3, 60d-1, 60d-2, 60d-3, 80d-1, 80d-2, 80d-3, 90d-1, 90d-2, 90d-3, 100d-1, 100d-2 and 100d-3 (corresponding to the cDNA obtained from the three biological replicates with sampling times of 10, 30, 60, 80, 90 and 100d respectively) were stored in a -20℃ refrigerator for later use.

[0045] 2. Primer design Based on the 19 internal reference genes obtained in Example 1 above, specific primers corresponding to the 19 internal reference genes were designed using Primer3Plus software. The designed primers were scored with a melting temperature (Tm) of 60±2℃, a primer length of 18~22bp, and an amplification length of 80~250bp as the standard. Primers with high scores were selected for synthesis. The primer sequences are shown in Table 2 below.

[0046] Table 2 Specific primer information

[0047] 3. qRT-PCR reaction After mixing the cDNA sample obtained from reverse transcription in step 1, dilute it sequentially to 5 ppm. 0 5 -1 5 -2 5 -3 5 -4Five concentration gradients were used to obtain dilutions. Using these dilutions as templates and the primers listed in Table 2, a real-time quantitative PCR (qRT-PCR) kit (2×SYBR Green qPCR Premix, Krypton) was used. The qRT-PCR reaction system was prepared on ice (10 μL): 1 μL sample solution, 0.2 μL forward primer, 0.2 μL reverse primer, 5 μL 2×SYBR Green qPCR Premix, and 3.6 μL sterile enzyme-free water. After mixing and centrifugation, the mixture was placed on a real-time quantitative PCR instrument for reaction. Three biological replicates were set for each sample. The qRT-PCR program was: 95℃ pre-denaturation for 30 s, 95℃ denaturation for 15 s, 60℃ extension for 30 s, for 40 cycles, with fluorescence signal acquisition at 60℃. The melting curve program was: 95℃, 15 s; 65℃, 1 min; 95℃, 20 s, with a 0.2℃ step every 20 s; 30℃, 1 min. Gene expression data (Cq values) and melting curves were obtained at 97℃. The reaction was performed on an ABI ViiATM 7 real-time quantitative PCR system (Applied Biosystems).

[0048] 4. Standard curve construction and primer amplification efficiency analysis The linear relationship between cDNA template mass concentration (logarithmic value) and CT value was analyzed using Excel software to obtain the correlation coefficient (R²). 2 ) and slope (S), using the formula E = (5 -1 / S The amplification efficiency (E value) of the primers was calculated by multiplying the result by 1) × 100%, and the results are shown in Table 3 below. Primers with good specificity should have an E value between 90% and 110%, and a correlation coefficient R0. 2 >0.98. As shown in the table, all 19 pairs of amplification primers have good specificity.

[0049] Table 3. Analysis of Amplification Efficiency of Candidate Internal Reference Genes by Quantitative Real-Time PCR

[0050] Simultaneously, the RT-qPCR melting curves of 19 candidate internal reference genes were obtained as follows: Figure 1 As shown, by Figure 1 It can be seen that the melting curves of the 19 candidate internal reference genes all showed single peaks, overlap, and no impurity peaks, and there were no primer dimers. The curves showed no non-specific amplification and good repeatability, indicating that the above primers were reasonably designed, had good specificity, and could be used for RT-qPCR detection of *Symplocos buergeriana* fruit.

[0051] 5. Analysis of the stability of internal reference gene expression cDNA from fruits at different developmental stages of *Prunus cerasifera* was diluted 20-fold and amplified by quantitative real-time PCR. The reaction system preparation and amplification procedure were the same as in step 3. The amplification results were analyzed using the online software RefFinder (http: / / blooge.cn / RefFinder / ). The stability of candidate internal reference genes in fruits at different developmental stages of *Prunus cerasifera* was determined by overall sorting based on the geometric mean of the sorting values ​​from geNorm, NormFinder, Best Keeper, and Delta CT software. Figures 2-5 As shown in the figure. The geNorm software evaluates the expression stability of candidate genes based on the average expression stability index (M). A smaller M value indicates better stability, and an M value less than 1.5 is considered a relatively stable threshold. NomFinder uses the expression stability value (SV) to represent the stability of candidate genes; a smaller SV value indicates a more stable candidate gene. Best Keeper software judges the stability of internal reference genes by comparing the standard deviation (SD) of Ct values; a smaller SD value indicates a more stable candidate gene, and an SD value less than 1 is considered stable. Delta CT analysis ranks candidate genes based on their average standard deviation; a smaller average standard deviation indicates a more stable internal reference gene.

[0052] The geometric mean of the stability rankings obtained from the above analyses was calculated using the RefFinder URL. A smaller geometric mean indicates more stable expression; conversely, a larger geometric mean indicates less stable expression. The results show ( Figure 6 The stability of the candidate internal reference gene is: EmTBP1 > EmANT > EmEIF2S3 > EmEIF4A3A > EmAPX1 > EmTIP > EmTUBb > EmTBP > EmUBC28 > EmUBC17 > EmEFa > EmeIF4A > EmGAPDH > EmUP > EmCLC > EmMON1 > EmPP2A > EmMP > EmACT7 The number one gene is EmTBP1 The geometric mean is 2.78, making it the most stable gene; the gene ranked last is... EmACT7 The geometric mean is 19, making it the most unstable gene.

[0053] Example 3 Validation of the stability of internal reference gene expression Select the most stably expressed internal reference gene EmTBP1 and the most unstable internal reference gene EmACT7 Stability verification was performed, and the gene being verified was... EmSBT1.3 The nucleotide sequence of this gene is shown in SEQ ID NO.58. This gene was designed. EmSBT1.3 The detection primer sequences are shown below: EmSBT1.3 -F (SEQ ID NO.59): 5'-TGCTATCGTGAAGGTCGAGC-3' EmSBT1.3 -R (SEQ ID NO.60): 5'-GGCTTCTTACCCTGTGCACT-3' SBT1.3 is an apoplastically localized serine protease with dual functions of proteolytic and catalytic activity regulation, and is widely expressed in various tissues and developmental stages. Transcriptome sequencing results revealed that this... EmSBT1.3 The expression level of *Prunus cerasifera* fruit at different developmental stages showed a trend of first decreasing, then increasing, and then decreasing again. The RT-qPCR data corrected with internal reference genes were compared with the transcriptome data, and the results are as follows: Figure 7 As shown, Figure 7 FPKM represents transcriptome-based expression levels, while EmACT7 represents transcriptome-based expression levels. EmACT7 Expression levels of the internal reference gene after validation; EmTBP1 is based on EmTBP1 Expression levels of the internal reference gene were validated, and the results showed that... EmTBP1 After the internal reference gene is calibrated, EmSBT1.3 The expression trend is consistent with the expression pattern of the transcriptome, but using EmACT7 After the internal reference gene is calibrated, EmSBT1.3 The gene expression was downregulated 60 to 80 days after flowering, which differed from the transcriptome sequencing results, indicating that the accuracy of gene expression levels was not high.

[0054] In conclusion, it can be determined that EmTBP1 The gene can be used as an internal reference gene for quantitative expression analysis of fruit development in *Symplocos buergeriana*. It can be applied to the fluorescence quantitative and expression analysis of fruit-related genes in *Symplocos buergeriana*, which can significantly improve the accuracy of expression analysis of functional genes related to fruit development. It provides strong support for the expression analysis of genes related to fruit development, pectin synthesis, and flavonoid metabolism in *Symplocos buergeriana*, and has potential application significance.

[0055] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A reference gene for the fruit of the Chinese privet tree. EmTBP1 Its characteristics are, The internal reference gene EmTBP1 The nucleotide sequence is shown in SEQ ID NO.

17.

2. A set of reference genes in the fruit of *Prunus cerasifera* EmTBP1 The amplification primers are characterized by, The amplification primers are based on the internal reference gene described in claim 1. EmTBP1 Based on the gene sequence, and following the principles of real-time quantitative PCR primer design, an internal reference gene was designed. EmTBP1 Amplification primers.

3. The amplification primer according to claim 2, characterized in that, The nucleotide sequences of the amplification primers are shown in SEQ ID NO. 52 and SEQ ID NO.

53.

4. The amplification primers according to any one of claims 2 to 3 in the preparation of the internal reference gene of *Prunus cerasifera* fruit. EmTBP1 Applications in testing reagents.

5. A method for detecting internal reference genes in the fruit of *Prunus cerasifera*. EmTBP1 The reagent is characterized by, The reagent includes the amplification primers as described in any one of claims 2 to 3.

6. The amplification primers according to any one of claims 2-3 or the reagent according to claim 5 in the internal reference gene of *Prunus cerasifera* fruit. EmTBP1 Applications in quantitative detection.

7. The internal reference gene according to claim 1 EmTBP1 The application of the amplification primers according to any one of claims 2 to 3 or the reagents according to claim 5 in the quantitative detection of genes in the fruit of *Symplocos buergeriana*.

8. The application according to any one of claims 6 or 7, characterized in that, The quantitative detection includes real-time quantitative PCR detection.

9. The internal reference gene according to claim 1 EmTBP1 The application of the amplification primers according to any one of claims 2 to 3 or the reagents according to claim 5 in the screening or expression analysis of functional genes related to fruit development of *Symplocos buergeriana*.

10. The application according to claim 7 or 9, characterized in that, Internal reference gene EmTBP1 Used as a correction gene.

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