CN117210611B - Internal reference gene of balsam pear with different tissues and fruits with different maturity stages and application thereof - Google Patents
Internal reference gene of balsam pear with different tissues and fruits with different maturity stages and application thereof Download PDFInfo
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Abstract
The invention discloses an internal reference gene of fruits with different tissues and different maturity stages of balsam pears, a detection primer and application thereof, wherein the internal reference gene is McHMG1/2, and the nucleotide sequence of the McHMG1/2 gene is shown as SEQ ID NO. 1. According to a large amount of data analysis and experience accumulation, the inventor screens out the reference gene McHMG1/2 with the most stable expression in different tissues and fruits at different maturity stages of the balsam pear from a plurality of candidate new reference genes for the first time through a strict reference screening program, can be suitable for the expression analysis of the related genes or other functional genes of the ripe fruits of the balsam pear at different tissues and maturity stages, obviously improves the stability, reliability and accuracy of the obtained data, and provides powerful support for the accurate quantification of the expression of the functional genes.
Description
Technical Field
The invention belongs to the technical field of fluorescent quantitative PCR detection, and in particular relates to an internal reference gene, a detection primer and application of the internal reference gene for fruits of different organs and different maturity stages of balsam pears.
Background
Balsam pear (Momordica charantia l.) belongs to annual climbing herbs of cucurbitaceae and is widely planted in tropical and subtropical areas of the world. The extracts of various organs of balsam pear, especially stems, leaves and fruits, have remarkable pharmacological actions, including anti-diabetes, insect expelling, anti-tumor, anti-inflammatory and the like. The bitter gourd fruits in the green ripe period have unique flavor and health benefits, are bitter in nature and cold in taste and rich in nutrition, contain active ingredients such as flavone, polysaccharide, biological peptide, momordica glycoside, momordicin and the like, and are taken as vegetables with medicated diet in China. However, the fruit of Momordica charantia, which is a typical respiratory-modified fruit, rapidly yellow ripens and softens soon after harvest, resulting in a drastic decrease in its edible and commercial value. Along with the deep research of genetic improvement of balsam pear, the research of the accurate expression mode of the balsam pear maturation related gene by utilizing the molecular biological technology becomes important research content.
Real-time fluorescent quantitative PCR (qRT-PCR) techniques are commonly used to analyze the expression pattern of a gene of interest. The qRT-PCR technology is used for accurately detecting the expression mode of the balsam pear maturation related genes, and reference genes which can be stably expressed in different balsam pear tissues, especially in fruits at different maturation stages, need to be screened out as references.
Currently, most of the molecular biology studies of Momordica charantia are also using conventional internal genes such as actin-7 (ACT 7), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 18S ribosomal RNA (18 SrRNA), peptidyl-prolyl cis-trans isomerase (CYP), and elongation factor 1-alpha (EF 1 alpha), etc. However, there are ongoing studies showing that most conventional reference genes are not stably expressed in different plant species, different tissues and organs, experimental conditions and developmental stages. Whether these conventional reference genes can be used to precisely quantify the expression levels of the genes associated with the maturation of balsam pear fruits is not known. A recent study of the reference gene of Momordica charantia has also used only the leaf tissue of seedlings, but not all around.
Until now, the use of systematically validated reference genes in different balsam pear tissues, in particular in fruits at different maturity stages, has not been reported.
Disclosure of Invention
Based on the above, the invention aims to provide reference genes of fruits with different tissues and different maturity stages of balsam pears, detection primers and application thereof, wherein the reference genes can be stably expressed in the fruits with different tissues and different maturity stages of balsam pears.
The technical scheme for realizing the aim of the invention comprises the following steps.
In a first aspect of the invention, an internal reference gene of fruits with different tissues and different maturity stages of balsam pears is provided, which is characterized in that the internal reference gene is McHMG1/2, and the nucleotide sequence of the McHMG1/2 gene is shown as SEQ ID NO. 1.
In a second aspect of the invention, a specific primer for detecting internal reference genes of different tissues and fruits in different maturity stages of balsam pears is provided, wherein the specific primer comprises a forward primer shown as SEQ ID NO.2 and a reverse primer shown as SEQ ID NO. 3.
In a third aspect, the invention provides an application of the reference gene or the specific primer in analysis of relative expression amounts of different tissues of balsam pear and related genes of fruits in different maturity stages.
In a fourth aspect of the present invention, there is provided a detection reagent or kit comprising the above specific primer
In a fifth aspect of the invention, a real-time fluorescence quantitative PCR detection method for fruit related genes of different tissues and different maturity stages of balsam pear is provided, wherein McHMG1/2 genes are used as internal reference genes in the real-time fluorescence quantitative PCR detection method, and the nucleotide sequence of the McHMG1/2 genes is shown as SEQ ID NO. 1.
According to a large amount of data analysis and experience accumulation, the inventor screens out the reference gene McHMG1/2 with the most stable expression in different tissues and fruits at different maturity stages of the balsam pear from a plurality of candidate new reference genes for the first time through a strict reference screening program, can be suitable for the expression analysis of the related genes or other functional genes of the ripe fruits of the balsam pear at different tissues and maturity stages, obviously improves the stability, reliability and accuracy of the obtained data, and provides powerful support for the accurate quantification of the expression of the functional genes.
Drawings
FIG. 1 is a schematic diagram of Momordica charantia for different tissues or organs and different maturation stages of RNA-Seq in example 1 of the present invention; wherein A is root; b is leaf; c is male flowers; d is female flowers; e is stem; f is green ripe bitter gourd fruit; g is bitter gourd fruit in the color breaking period; h is semi-yellow bitter gourd fruit; i is bitter gourd fruit in full yellow period; the scale in B-E is 1 cm; A. the scale bar in F-I is 5 cm.
FIG. 2 shows the specificity analysis of qRT-PCR amplification primers in example 2 of the present invention.
FIG. 3 is a heat map of expression levels (A) and a range of expression levels (B) of 5 reference genes (ERG) and 11 candidate Novel Reference Genes (NRG) established in example 2 of the present invention; wherein red represents high transcript levels and blue represents low transcript levels; r, root; s, stems; fm, male flowers; ff, female flowers; l, leaves; pg, green ripe pulp; pb, broken pulp; pt, semi-yellow flesh; py, full yellow stage pulp.
FIG. 4 is an analysis of expression stability of 5 reference genes and 11 candidate novel reference genes established in example 2 of the present invention; wherein A-E are based on geNorm, bestKeeper and DeltaC respectively t Stability values for 16 candidate reference genes for the methods, normFinder and RefFinder; f is the paired variation (V) analysis by the gemm software.
FIG. 5 is a graph showing the stability verification of the reference gene McHMG1/2 in example 3 of the present invention; wherein, R, root; s, stems; fm, male flowers; ff, female flowers; l, leaves; pg, green ripe pulp; pb, broken pulp; pt, semi-yellow flesh; py, full yellow stage pulp.
Detailed Description
The present invention will be described more fully hereinafter in order to facilitate an understanding of the present invention. This invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.
The experimental procedures, which do not address the specific conditions in the examples below, are generally followed by conventional conditions, such as those described in Green and Sambrook et al, molecular cloning, an experimental guideline (Molecular Cloning: A Laboratory Manual, 2013), or by the manufacturer's recommendations. The various chemicals commonly used in the examples are commercially available.
In the present invention, the inventors first calculated the Coefficient of Variation (CV), the tissue-specific coefficient (τ) and the transcripts per million mapping reads per kilobase of Transcripts (TPM) per CV for each gene transcript based on the transcriptome sequencing (RNA-Seq) data of fruits of different tissues and different maturity stages of Momordica charantia<0.5、τ<0.5 and average log 2 (TPM)>5, screening 11 candidate new reference genes according to the standard; then, with the correlation coefficient (R 2 ) The amplification efficiency is close to 1, the amplification efficiency is close to 100%, a single melting curve peak is used as a standard, and specific amplification primers are designed for 11 obtained candidate new reference genes; then, cDNA from different tissues and fruits at different maturity stages of balsam pear is used as a template, and fluorescent quantitative PCR reaction is carried out on a CFX96 real-time fluorescent quantitative PCR instrument to obtain expression data C of each reference gene q Value according to the obtained C q The value is determined by gemum, normFinder, ΔC t And BestKeeper software is used for evaluating the stability of candidate new reference genes, and RefFinder software is used for comprehensively analyzing four stability evaluation results, and the gene McHMG1/2 with the strongest comprehensive stability is selected as the new reference gene of fruits of different tissues and different maturity stages of the balsam pear; finally, the novel reference gene McHMG1/2 is used for surfacing the fruit maturation related genesThe arrival calibration analysis verifies its reliability. According to a large amount of data analysis and experience accumulation, the invention gets rid of the selection restriction of the traditional reference genes through a strict reference screening program, and firstly screens the reference genes with the most stable expression in different tissues of balsam pear and fruits in different maturity stages from a plurality of candidate new reference genes to be used as new reference genes with fluorescent quantification. The internal reference gene McHMG1/2 obtained by screening is suitable for expression analysis of fruit ripening related genes or other functional genes in balsam pear fruits in different ripening stages, has common application, can obviously improve the stability, reliability and accuracy of the obtained data, and provides powerful support for accurate quantification of functional gene expression.
In some embodiments of the invention, reference genes of fruits with different tissues and different maturity stages of balsam pears are disclosed, wherein the reference genes are McHMG1/2, and the nucleotide sequence of the McHMG1/2 genes is shown as SEQ ID NO. 1.
Nucleotide sequence of McHMG1/2 Gene (SEQ ID NO. 1)
TTTCAATTTGGCCTCTCGGACTTAATATCTCTCTTCTTTTTACTATCTTTCG
GTTCCCCTTTCCTCACACGCAGGTCCGCTCCCTCCACCCTCTTCAAACCCT
AAACCTAACCCAGCCTCGCTCCGGAGCCCCCCACCGAGGTTCGTTTCATC
GATCTCCTTCCAATTCCGACCTAATTTCACGTCTTTTCATTGTTTTCCTTCG
AGATTGTTGCTGATCCGATCTTATAACTGCAGGATTCGACATGAAAGGTG
GAAAATCGAAGTCCGAGTCGAAGAAAGCAGACGCGAAGTGAGTTTTTTTT
TTTTTTTTCTCGCCCTATTTCTCCGTCGGATGTGTTTAATTTATAGCTCTGA
TCGGCCCTAGATTTAAGAAGCTTCGTTGCTTAAATTACCTTTGTAAAAGCT
ATGCGTTGACCTTTTCTTTTTTGATTGATCGGTTTTGGCGATCTTTTTTGTA
GGCTTTCTGTGAAGAAAGGTGCAGCCAAAGCGGGCGCAACACGGGGTAA
GAAAGCAGGGAAGGATCCTAACAAACCTAAGCGGCCGGCCAGTGCCTTC
TTCGTTTTTATGTAATTATTTCATTTTTCAATTTACTTTTAGATGTCTGAAT
TACGCTGCTTGACTGCAAATTATTCATTCGGATGGTTGTAGAGTTGGAAA
AATTGCAAACAAGGAAGAAAAGAACTATGAAGTTGTTTTCGGTGATTTTT
GTTTAAGGATGTTAAATAAGCCGGCTTTCAACAAAACGATCTCTGTGTTA
TATGTGTATTTAGCTAGTGGACCCGTTCTTTTGTTTTATCTGTAAGTCGATT
ACGTATCTAAAATCTGTGTTGTCATGTTGTTGCACGTTTTGTAATTAAAGG
GAGGAGTTCAGGAAGAAGTTTAATGAAGAGAATCCGAATAACAAAGCAG
TATCCGCTGTAAGCTATTTACCTTTGTCTAATTGTCAATTCTTTTTTCTTGT
CCCCATTTCATATTATATAAATTCTTTTATGGAAATGAGCAGTTATAATTG
ACCTACAAATGATACCATCAGGTTGGTAAAGCTGCTGGACAGAAATGGA
AATCATTGTCAGATGCTGTACGTGATACCTCTCGTAGTATTTTCTAAATTT
CTCCTGGTATTATAAACTCTGTCTGAATTACTGTTTATAATATCAGGAAAA
AGCACCTTACATTGCTAAGGCTGACAAGAGGAAGGTTGAATATGAGAAA
AATATGAAGGCCTATAACAAGAAACAGGTACTGAAAACTTTCCGAATCTT
ATATTTTGCAGCTGTATTTTATTTTGTAAATTCATTATCTCTCCCTGCTCCA
TTTGAGTTCAGGCCAGTGGAGCCAATGCTGCTGAAGAAGATGAATCTGAG
AAGTCCATGTCTGAGGTGAATGATGATGAGGATGGAGACGAGGGCAGCG
AAGAGGTTAGTGGGTTGAAGAATTTTTTCCAGTCATTATTGTGCTTTGTAC
CCTTTCACTTATTTCATTTCTCTCTTGTGGTTGCAGGAGGAAGATGACGAG
TAATGAAGATGTAGATGGTAGTAAAGTCATTTAGGCTGTCAGTGAACTTG
ATCGTTCTATATTAATGGTCTGATCATTTCCTGTTAATTGTTTTTTATATCA
TGCAAATATATTAGTATCTCCCTGTTATTTTGTTTGTGCATTCAGAAACGA
ATGGTTGTAATGGGAGATCATCTGTCTTGTACCTTACAATGTTAGTTATTC
CAATCCAACTATGTTTTTTTTTTTCAACTGAAATGGCAACAACTTTCCACT
CCACATAGAGGAGATGAAGATGCA
In other embodiments of the present invention, a specific primer for detecting reference genes of different tissues and fruits of different maturity of Momordica charantia is disclosed, the specific primer comprising a forward primer as shown in SEQ ID NO.2 and a reverse primer as shown in SEQ ID NO. 3.
Forward primer McHMG1/2-F (SEQ ID NO. 2): GCACCTTACATTGCTAAGGC
The reverse primer McHMG1/2-R (SEQ ID NO. 3): CTCAGACATGGACTTCTCAG
In other embodiments of the invention, a detection reagent or kit comprising the above specific primers is disclosed.
In other embodiments of the invention, the use of the reference gene, the specific primer or a detection reagent or kit containing the specific primer in the analysis of the relative expression levels of the genes related to different tissues and fruits of balsam pear in different maturity stages is disclosed.
In some of these embodiments, the method of gene relative expression analysis is a real-time fluorescent quantitative PCR method.
In other embodiments of the invention, a real-time fluorescence quantitative PCR detection method for fruit related genes of different tissues and different maturity stages of balsam pear is disclosed, wherein McHMG1/2 gene is used as an internal reference gene, and the nucleotide sequence of the McHMG1/2 gene is shown as SEQ ID NO. 1.
In some embodiments, the reaction system of the real-time fluorescent quantitative PCR detection method is: 0.05-0.15 mu g cDNA, 8-12 mu L2 XSYBR mixture, 0.3-0.5 mu L10 mu M forward primer, 0.3-0.5 mu L10 mu M reverse primer, ddH 2 O was added to 20. Mu.L.
In some of these embodiments, the forward primer and the reverse primer are mixed in an equimolar ratio.
In some of these embodiments, the reaction procedure of the real-time fluorescent quantitative PCR detection method is: pre-denaturation at 94 ℃ for 30 seconds; 94℃for 5 seconds and 60℃for 45 seconds, 40 cycles.
The balsam pear variety K13 used in the following examples was derived from the second institute of vegetable research, the national academy of agricultural sciences, guangdong.
The invention is described in detail below with reference to the drawings and the specific embodiments.
EXAMPLE 1 screening of candidate novel reference genes for fruits of Momordica Charantia in different tissues and maturity
The embodiment screens candidate reference genes with the most stable expression in fruits of different tissues and fruit maturity stages of balsam pears based on RNA-Seq data, and specifically comprises the following steps:
1. sampling of Experimental Material and RNA-Seq
The balsam pear K13 planted in the white cloud planting base of Guangdong province agricultural academy of sciences is an experimental material. The roots, leaves, male flowers, female flowers and stems of the balsam pear, and the balsam pear fruits in green ripe stage, broken color stage, semi-yellow stage and full-yellow stage were sampled respectively (fig. 1). After the sample was isolated, it was snap frozen using liquid nitrogen and sent to commercial company for RNA-Seq, and the backup sample was stored in-80℃refrigerator.
2. RNA-Seq data analysis
Based on the transcriptome data, CV values, τ values, and TPM values were calculated for each gene transcript. The calculation formulas are respectively as follows:
sigma and mu are Standard Deviation (SD) and mean, respectively, of gene expression FPKM values in all tissues.
x i The expression level FPKM of the gene in the tissue i is given, and n is the number of tissues.
TPM i And FPKM i Is the TPM value and FPKM value of the genes in the organization i, sigma j FPKM j Is the sum of the FPKM values of all genes in the tissue i.
3. Screening of candidate novel reference genes
According to CV <0.5, τ <0.5 and average log2 (TPM) >5, genes with the expression level of front 11, namely DNAJ, HSCP2, ARF1, UP, HMG1/2, TRXH-1, PHOS32, GAPDH2, RPL35-2, UBC36 and RPS8, are selected as candidate new reference genes. As controls, 5 established reference genes of balsam pear were selected from McCYP, mcEF1 a, mcTIP41, mcACT7 and McGAPDH.
EXAMPLE 2 determination of novel reference genes for different tissues and mature fruits of Momordica Charantia
The most suitable reference gene was selected from the 11 candidate novel reference genes of example 1 using a total of 5 established balsam pear reference genes of McCYP, mcEF1 alpha, mcTIP41, mcACT7 and McGAPDH as controls. The method comprises the following steps:
1. total RNA extraction and cDNA Synthesis
(1) Extraction of total RNA
The samples stored in a-80℃refrigerator of example 1 were used, frozen with liquid nitrogen and ground into powder in a mortar. Total RNA was extracted from each sample as per the instructions of the extraction kit from TransZol Up Plus RNA. The mass and concentration of total RNA were measured using a NanoDrop 2000 spectrophotometer. The 260/280 value is between 1.8 and 2.1, the purity is higher, and the method is suitable for subsequent experiments.
(2) cDNA Synthesis
Oligo (dT) as described in TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix Kit kit 18 As a reverse transcription primer, total RNA (. About.2. Mu.g) was reverse transcribed into 20. Mu.L of cDNA.
2. qRT-PCR primer design and verification
(1) qRT-PCR primer design
Based on the obtained 11 candidate new reference genes and 5 established balsam pear reference gene sequences, the design principle of qRT-PCR primers is followed: GC content is 50% -60%, melting temperature (Tm) is 58-60 ℃, primer length is 20-22 bp, amplification length is 90-150 bp, and specific quantitative primer of the gene is designed. The sequences are shown in Table 1, and were synthesized by commercial companies, synthesized using PAGE purification, and diluted to working fluid for use. The information of the 5 established reference genes and the 11 candidate new reference genes is specifically shown in Table 1.
TABLE 1
(2) qRT-PCR reaction
PCR reactions were performed on a CFX96 real-time fluorescent quantitative PCR instrument. The reaction system was placed on ice as shown in table 2.
TABLE 2
| Reaction components | Volume of each component |
| 2×SYBR Green Mix | 10μL |
| cDNA(～2μg/μL) | 1μL |
| Forward primer F (10. Mu.M) | 0.4μL |
| Reverse primer R (10. Mu.M) | 0.4μL |
| ddH 2 O | 8.2μL |
qRT-PCR run program:
determination of melting Curve
Each reaction was repeated 3 times. Obtaining expression data C of each reference gene q Values.
(3) qRT-PCR primer verification
Generating a standard curve by using a series of 5-fold serial diluted cDNA as a template, and calculating the amplification efficiency and R of the primer by the standard curve 2 . The results are shown in Table 3.
TABLE 3 Table 3
Only the amplification efficiency is 90 to 110 percent and R 2 >A primer of 0.98 was considered as a pass primer. As can be seen from the results of Table 3, the amplification efficiency and R of the primers used 2 And all are qualified.
The results of the specificity analysis on qRT-PCR amplification primers are shown in FIG. 2, and it is clear from FIG. 2 that the melting curves of 5 conventional reference genes and 11 novel reference genes all show single, overlapping and no impurity peak. It shows that no primer dimer exists, no nonspecific amplification exists, and all primers can be used for balsam pear qRT-PCR experiments.
3. Stability analysis of the 11 reference genes screened
The method comprises the following steps:
(1) Gene expression profiling
A heat map was constructed from transcriptome data of all reference genes in different tissues/organs (heat map using log 2 (FPKM) values were generated) and expression data of 16 genes was displayed using the Cq values. The results are shown in FIG. 3.
The heat map results show that: the expression fluctuations of the candidate new reference gene in different tissues/organs are smaller than those of the established reference gene (a in fig. 3). Suitable reference genes should have moderate transcript levels (Cq values 15 to 30) to provide the most accurate normalization.
Expression data for 16 genes are shown using the Cq values, 75% of which are in the "box" range and 25% in the "vertical line" range. Whiskers represent 95% confidence intervals. The straight line in the box represents the median of the Cq value and the dot represents the outlier. C (C) q The results of the value analysis show that all reference genes meet the basic requirements of standardized use except for McGAPDH (Cq value of 20.46 to 32.63). According to C q The value-plotted box-plot shows that the expression range of most candidate new reference genes is more stable than the established reference genes and is more suitable as a reference gene (B in fig. 3).
(2) Evaluation of Gene expression stability
Expression C detected in each tissue sample for each gene q The values are input into analysis software by gemum, normFinder, ΔC t And BestKeeper four software on 16 genes stability evaluation (figure 4A ~ D). The stability evaluation results of the four kinds of software were comprehensively analyzed using refFinder software (E in FIG. 4). The paired mutation (V) analysis by gemm software (a threshold of 0.15 (V value) was typically used to determine if additional reference genes were required to be added, and the result showed that the minimum number of reference genes normalized to different tissues and fruit ripening stages was 2 (F in fig. 4).
The specific analytical data are shown in Table 4.
TABLE 4 Table 4
The smaller the value, the more stable the gene expression. The results in Table 4 show that McHMG1/2 is the gene with the highest comprehensive stability, so that it is used as a novel reference gene for fruits of different tissues and different maturity stages of balsam pears.
EXAMPLE 3 verification of expression stability of reference McHMG1/2
Stability verification (data correction) was performed using the two reference genes that are most stably expressed (McHMG 1/2 and McPHOS 32) and the reference gene that is least stably expressed (McHSCP 2), alone or in combination. The two genes are verified to be fruit maturation related genes McACO1 and McACO2, and the corrected qRT-PCR data and the FPKM data of the original RNA-Seq are subjected to expression pattern comparison. The results are shown in fig. 5, where the data are the mean of ± SE (n=3) of three independent biological replicates. The different letters represent the significance differences (P < 0.05) determined by the Duncan multiple comparison method.
A and C in FIG. 5 show the transcript levels of the two fruit ripening related genes McACO1 and McACO2 in fruits of different tissues and at different ripening stages. B and D in FIG. 5 are qRT-PCR analyses of two fruit ripening related genes McACO1 and McACO2 in fruits of different tissues and different ripening stages. The results show that: the expression pattern of McHMG1/2 alone was similar to that of McHMG1/2 and McPHOS32 in combination and very similar to that represented by FPKM data, whereas McHSCP2 alone showed abnormalities.
In conclusion, mcHMG1/2 is taken as an internal reference gene and is most suitable for quantitative expression analysis of fruits of different organs and different maturity stages of balsam pears.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (6)
- The application of the McHMG1/2 gene as an internal reference gene of fruits of different tissues and different maturity stages of balsam pears is characterized in that the nucleotide sequence of the McHMG1/2 gene is shown as SEQ ID NO. 1.
- The application of the McHMG1/2 gene in analysis of relative expression amounts of fruit related genes of different tissues and different maturity stages of balsam pears is characterized in that the nucleotide sequence of the McHMG1/2 gene is shown as SEQ ID NO. 1.
- 3. The application of the specific primer for detecting the McHMG1/2 gene in the analysis of the relative expression amounts of fruit related genes of different tissues and different maturity stages of balsam pear is characterized in that the nucleotide sequence of the McHMG1/2 gene is shown as SEQ ID NO. 1; the specific primer comprises a forward primer shown as SEQ ID NO.2 and a reverse primer shown as SEQ ID NO. 3.
- 4. The application of the reagent for detecting the McHMG1/2 gene in analysis of relative expression amounts of fruit related genes of different tissues and different maturity stages of balsam pear is characterized in that the nucleotide sequence of the McHMG1/2 gene is shown as SEQ ID NO. 1.
- 5. The application of the kit for detecting the McHMG1/2 gene in analysis of relative expression amounts of fruit related genes of different tissues and different maturity stages of balsam pear is characterized in that the nucleotide sequence of the McHMG1/2 gene is shown as SEQ ID NO. 1.
- 6. The use according to any one of claims 2 to 5, wherein the method of analysis of the relative expression level of genes is a real-time fluorescent quantitative PCR method.
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| Identification and Analysis of Reference and Tissue-Specific Genes in Bitter Gourd Based on Transcriptome Data;Yangyi Zheng 等;Horticulturae;第9卷;1-15 * |
| LOC111012664 HMG1/2-like protein [ Momordica charantia (bitter melon) ].GenBank.2022,全文. * |
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