CN116751884A - Fluorescent quantitative internal reference gene for stems and tendrils of different parts of Chinese yam, and primers and application thereof - Google Patents
Fluorescent quantitative internal reference gene for stems and tendrils of different parts of Chinese yam, and primers and application thereof Download PDFInfo
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Abstract
The invention relates to a fluorescent quantitative internal reference gene of different parts of Chinese yam and a primer and application thereof, belonging to the technical field of molecular biology of Chinese yam. The reference geneMYB7The nucleotide sequence of (2) is shown as SEQ ID NO. 1. Reference geneMYB7Nucleotide sequence of the PCR amplification primer: the upstream primer is shown as SEQ ID NO. 2; the downstream primer is shown as SEQ ID NO. 3. The invention screens out internal reference genes with stable expression of the stems and the tendrils at different positions of the Chinese yam, and provides reference basis for research works such as gene function verification and the like of the growth and development of the Chinese yam stems and tendrils and metabolic mechanism in later period development.
Description
Technical Field
The invention belongs to the technical field of yam molecular biology, and particularly relates to an internal reference gene for fluorescent quantification of stems and tendrils at different parts of yam, and primers and application thereof.
Background
The real-time fluorescent quantitative PCR (qRT-PCR) technology is one of the most conventional and effective means for researching gene expression in molecular biology, and the product change in the whole PCR reaction process can be monitored in real time by adding a specific fluorescent group into a PCR reaction system, so that the qualitative and quantitative expression analysis of a target gene is achieved. qRT-PCR has the advantages of strong specificity, high sensitivity, good repeatability, simplicity, high efficiency and the like, but is influenced by factors such as RNA quality, template cDNA quality, primer specificity, PCR amplification rate and the like, and proper internal reference genes need to be selected for correction and standardization in the qRT-PCR process. Genes that constitute the cytoskeleton or are involved in the basic metabolic activity of cells are often used as reference genes, which are relatively stable in expression. Currently, there are many housekeeping genes commonly used, including genes encoding actin (actin), ribosomal RNA (ribosomalRNA, rRNA), glyceraldehyde-3-phosphate dehydrogenase (GADPH), tubulin (TUB), and the like.
The Chinese yam stem and tendril is an important organ of Chinese yam, has the functions of supporting leaves, storing organic substances, transporting nutrient substances and the like, and simultaneously, the stem and tendril also transport photosynthetic products manufactured by the leaves to roots through phloem to supply root growth and development and normal physiological activities. Thus, the growth conditions of the stem and vine affect the growth of the whole plant. However, there are few reports about the reference gene of yam. The expression studies of the yam anthocyanin related genes DaF3H and flavonol synthase DaFLS1 genes used action 2, action 1 as reference genes, but these documents have not analyzed the stability of these reference genes. The patent (patent number ZL 202011469612.7) discloses the application of the F-box gene of the Chinese yam in the fluorescent quantitative expression of different tissues of the Chinese yam, and the other patent (patent number ZL 202011469619.9) discloses the application of the CKI-2 gene of the Chinese yam in the fluorescent quantitative expression of different growth stages of leaves of the Chinese yam, but the two patents do not relate to the stability of the expression of the F-box gene of the Chinese yam and the CKI-2 gene in other tissues such as stems and tendrils. The stem and tendrils of the Chinese yam are important components of Chinese yam plants, and have important functions of supporting growth of leaves and the like. In order to research the expression of important genes of Chinese yam in the stem and tendrils in future, stable genes need to be found to be used as reference genes. Therefore, how to overcome the defects of the prior art is a problem which needs to be solved in the technical field of yam molecular biology at present.
Disclosure of Invention
The invention aims to solve the defects of the prior art and provides an internal reference gene for fluorescent quantification of stems and tendrils at different parts of Chinese yam, and a primer and application thereof. The invention screens out internal reference genes with stable expression of the stems and the tendrils at different positions of the Chinese yam, and provides reference basis for research works such as gene function verification of the growth development and metabolism of the Chinese yam stems and tendrils in later period.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
fluorescent quantitative reference genes of different parts of the Chinese yam stem and tendrils, wherein the nucleotide sequence of the reference gene MYB7 is shown as SEQ ID NO. 1.
Further, it is preferable that the PCR amplification primer nucleotide sequence of the reference gene MYB 7: the upstream primer is shown as SEQ ID NO. 2; the downstream primer is shown as SEQ ID NO. 3.
The invention also provides a screening method of the fluorescent quantitative reference genes of the stems and the tendrils at different positions of the Chinese yam, which comprises the following steps:
s1, selecting 8 gene sequences with E values close to zero or zero from candidate reference genes;
s2, designing primers for 8 genes screened in the step S1, and performing PCR amplification by using the primers to verify the specificity of the primers;
s3, extracting total RNA of the stems and the tendrils at different positions, performing reverse transcription to synthesize first-chain cDNA, performing real-time fluorescent quantitative PCR analysis by adopting primers designed in the S2, calculating Ct values, and analyzing the expression stability of 8 genes screened in the step S1;
s4, comprehensively determining fluorescent quantitative internal reference genes of different parts of the yam with the most stable expression.
Further, it is preferable that 8 genes are Act-1, alpha-TUB, EF-1alpha, F-box, GADPH, HIS, MYB7, TRX-2 in step S1.
Further, it is preferable that in step S2, the PCR reaction system is 20 μl: comprises 17.0 mu LGreenmix,1.0 mu L cDNA,1.0 mu L10 mu mol/L upstream primer, 1.0 mu L10 mu mol/L downstream primer;
reaction conditions: pre-denaturation at 95℃for 1min; then denaturation at 95℃for 10s, annealing at 60℃for 30s, pre-extension at 72℃for 10s, and cycling for 30 times.
Further, it is preferable that in step S3, the real-time fluorescent quantitative PCR reaction system is 20 μl: comprises 10.0 mu.L of qPCRMasterMixSYBRGreen I, 1.0 mu.L of cDNA,0.8 mu.L of 10 mu mol/L upstream primer, 0.8 mu.L of 10 mu mol/L downstream primer, ROX0.4 mu.L, ddH 2 O7.0μL；
Two-step PCR reaction conditions: pre-denaturation at 95℃for 1min; then denaturation at 95℃for 10s, annealing at 60℃for 30s, extension at 72℃for 10s, and circulation 40 times.
Further, in step S4, it is preferable that the expression stability of each candidate reference gene is analyzed by DeltaCT, geNorm, normfinder, bestKeeper, comparative Δct method.
The invention also provides application of the fluorescent quantitative internal reference genes of different parts of the Chinese yam in Chinese yam gene expression analysis.
The invention further provides application of the fluorescent quantitative internal reference genes of different parts of the Chinese yam in improvement of Chinese yam germplasm resources.
The patent (patent number ZL 202011469612.7) discloses the application of the F-box gene of the Chinese yam in the fluorescent quantitative expression of different tissues of the Chinese yam, the other patent (patent number ZL 202011469619.9) discloses the application of the CKI-2 gene of the Chinese yam in the fluorescent quantitative expression of different growth stages of leaves of the Chinese yam, but the two patents do not relate to the research on the expression stability of the F-box gene of the Chinese yam and the CKI-2 gene in other tissues such as the stem and the vine, and the research process of the invention finds that the expression stability of the two disclosed genes in different parts of the stem and the vine of the Chinese yam is not high.
Compared with the prior art, the invention has the beneficial effects that:
first, the present invention adds a new candidate gene for screening reference genes, and finds a more stable gene. According to the invention, 8 genes, namely Actin gene 1 (Actin 1, ACT-1), MYB7 (MYB family tra nscription facto 7), F-box (tubby-like F-box protein), histidine family protein (Histone superfamily, HIS), thioredoxin2 (TRX-2), alpha-tubulin beta (alpha-TUB), encoding eukaryotic translation elongation factor (elongation factor alpha, EF-1 alpha), glyceraldehyde-3-phosphate dehydrogenase (glycoaldehyde-3-phosphate dehydrogenase, GADPH), are selected as candidate genes of different part stems, and the MYB7 gene is found to be more stable than the published CKI-2 gene (the gene is an internal gene of different growth-stage leaves, the patent number is 202011469619.9) and the F-box gene (the gene is an internal gene of different tissues, and the patent number is ZL 202011469612.7).
Secondly, the invention adopts new plant materials (different parts of the Chinese yam stem and tendrils) to screen the reference genes, and finds the reference genes more suitable for the new materials (different parts of the Chinese yam stem and tendrils). The yam stem is not only an important component of the yam plant, but also performs an important function. The invention screens the stem and vine internal reference gene MYB7 of different parts of the Chinese yam, provides an application technology, lays a foundation for researching the stem and vine growth and development related genes of the Chinese yam later, and is irreplaceable in the prior art.
Thirdly, the invention uses a low ROXqRT-PCR two-step method and combines a plurality of reference gene screening software (geNorm, normfinder and DeltaCT) to evaluate the gene stability respectively, finally, the stability of the former several software evaluations is comprehensively evaluated through a finer website, and the reference genes which are extremely stable in expression in different parts of the Chinese yam stems are screened out, so that the error of the gene expression evaluation can be reduced to a large extent.
In a word, the invention provides a reference basis for research works such as gene function verification of the growth and the metabolism of the Chinese yam stem vines and the like in the later period.
Drawings
FIG. 1 is an RNA extraction band; in the figure, 5s represents 5s ribosomal RNA,18s represents 18s ribosomal RNA, and 28s represents 28s ribosomal RNA; as can be seen from FIG. 1, the extracted RNA electrophoresis band is complete and clear, which indicates that RNA is not degraded and has high purity concentration;
FIG. 2 is a gel electrophoresis chart of PCR amplification of 8 candidate genes; m in the figure represents DNAMake, which is used for comparing the molecular weight of PCR amplified bands of 11 candidate genes;
FIG. 3 shows the Plot peaks of MYB7 gene on different parts of the Chinese yam, wherein 1 is the upper end and the lower end of the Chinese yam vine part 1, 2 is the upper end and the lower end of the Chinese yam vine part 2, 3 is the upper end and the lower end of the Chinese yam vine part 3, 4 is the upper end and the lower end of the Chinese yam vine part 4, and 5 is the upper end and the lower end of the Chinese yam vine part 5;
FIG. 4 shows the relative expression levels of genes of the vine of the Chinese yam auxin transport vector at different positions; in the figure, (a) is the gene expression level of the auxin transport vector at the upper end of the stem at different positions of the Chinese yam; (b) The gene expression level of the auxin transport carrier is used for the lower ends of different parts of the stems of the Chinese yam.
Detailed Description
The present invention will be described in further detail with reference to examples.
It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The specific techniques or conditions are not identified in the examples and are performed according to techniques or conditions described in the literature in this field or according to the product specifications. The materials or equipment used are conventional products available from commercial sources, not identified to the manufacturer.
1 implementation materials
Taking the oxtail yam as an implementation material, taking 10 parts of upper end tissues and lower end tissues of 5 parts on the stem and tendril as internal reference gene screening materials. The 10 parts of materials are as follows: the terminal bud is 0.5cm lower than the stem tendril (the upper end of the 1 st part); the first leaf below the terminal bud is upwards 0.5cm of the vine (the lower end of the 1 st part), the vine is light green in color and has a diameter of 0.5-1 mm; a stem tendril (the upper end of the 2 nd part) with 0.5cm of the 3 rd leaf with the terminal bud downwards; the stem with 0.5cm upward top bud and 4 th leaf (the lower end of the 2 nd part) is emerald green and has a diameter of 1.2-1.7 mm; a stem tendril (the upper end of the 3 rd part) with 0.5cm of the 7 th leaf with the terminal bud downwards; the stem with 0.5cm upward top bud and 8 th leaf (the lower end of 3 rd part) has green and purple color and diameter of 1.7-2.0 mm; a stem vine (the upper end of the 4 th part) with 0.5cm from the 9 th leaf with the top bud down and a stem vine (the lower end of the 4 th part) with 0.5cm from the 10 th leaf with the top bud down, wherein the stem vine is semi-green and semi-purple and has a diameter of 2.0-2.5 mm; the stem with 0.5cm of top bud downward 11 th leaf (upper end of 5 th position) and 0.5cm of top bud downward 12 th leaf upward (lower end of 5 th position), the color of this stem is basically purple stem diameter about 3mm. A total of 10 different sites of stem and vine tissue were sampled. The materials of each part are sampled repeatedly for 3 times, weighed, split charged with liquid nitrogen and quick frozen, and stored in a refrigerator at the temperature of minus 80 ℃ for standby.
2 internal reference gene primer design
Based on yam transcriptome CDS data, 8 genes were selected as candidate internal genes, including 8 genes of Actin gene 1 (action 1, ACT-1), MYB7 (MYB family transcription facto), F-box (TUB-like F-box protein), histidine family protein (Histone superfamily, HIS), thioredoxin2 (TRX-2), α -tubulin gene (α -tubulin beta, α -TUB), encoding eukaryotic translation elongation factor (elongation factor alpha, ef-1 α), glyceraldehyde-3-phosphate dehydrogenase (Glyceraldehyde-3-phosphate dehydrogenase, GADPH), and the obtained transcriptome sequences were checked by Blast-N in NCBI, and sequences with E values near zero or zero were selected for primer design. The primer sequences were designed on-line by PrimerBlast according to qRT-PCR primer design principles (Table 1) and were delegated to the synthesis of the corresponding primers by Kunming, biotechnology Co. Wherein, the gene accession numbers of Act-1, alpha-TUB, EF-1 alpha, F-box, GADPH, HIS, MYB and TRX-2 are XM039280107.1, XM039274609.1, XM039289033.1, XM039258576.1, XM39277937.1, XM039270868.1, XM039269262.1 and XM039279110.1 respectively.
TABLE 1 qRT-PCR primer sequences of candidate internal reference genes of yam
3 extraction of Total RNA and Synthesis of cDNA Strand 1
Reference Eastep TM The Super total RNA extraction kit (marine product) uses instructions to extract total RNA from different parts of the stems and tendrils of Chinese yam. RNA integrity and purity were checked by mass concentration 1.2% agarose gel electrophoresis, and RNA concentration was checked using a DenoviXDS-11 instrument. Synthesis of cDNA strand 1 reference TSI NGKE company reverse transcription kit Goldenstar TM RT6cDNASynthesis kit instructions, obtained cDNA stored in-20 ℃ refrigerator.
4 primer specific PCR verification
The Chinese yam stem and tendril cDNA is used as a template, and a PCR reaction system is 20 mu L: includes 17.0. Mu. LGreenMix, 1.0. Mu. LcDNA, 1.0. Mu.L upstream primer (10. Mu. Mol/L), 1.0. Mu.L downstream primer (10. Mu. Mol/L). Reaction conditions: pre-denaturation at 95℃for 1min; then denaturation at 95℃for 10s, annealing at 60℃for 30s, pre-extension at 72℃for 10s, and cycling for 30 times.
5 real-time fluorescent quantitative PCR amplification
The assay was performed using the abiquantsudio real-time fluorescent quantitative PCR system. The qRT-PCR reaction system was 20. Mu.L: comprises 10.0 mu L of PCRMastermix (SYBRGreen I), 1.0 mu L of cDNA,0.8 mu L of upstream primer (10 mu mol/L), 0.8 mu L of downstream primer (10 mu mol/L), ROX0.4 mu L and ddH 2 O7.0. Mu.L. The reaction conditions of the two-step method are adopted: pre-denaturation at 95℃for 1min; then denaturation at 95℃for 10s, annealing at 60℃for 30s, pre-extension at 72℃for 10s, and circulation 40 times.
6 data processing
The RT-qPCR data of the stems and the tendrils at different positions of the Chinese yam are arranged and summarized through Excel2016 software; the stability of the expression of the different reference genes was assessed using the reference gene stability analysis website (http:// www.ciidirsinaloa.com.mx/refFinder-master /) which includes the DeltaCT, geNorm, normfinder and Comparative ΔCt methods 4 assessment methods, specific analysis steps and parameter settings reference Wu Jianyang et al (Wu Jianyang et al. Method for reference gene stability analysis using gemm, normFinder and BestKeeper software [ J ]. Modern agriculture technology, 2017 (5): 4 ]) data processing analysis methods.
Results 7 results
7.1RNA purity and concentration analysis
Extracting total RNA of rhizoma Dioscoreae, wherein the concentration of total RNA is 410.635 + -65.23 ng/ul, A 260 /A 230 ＝1.987～2.264，A 260 /A 280 The requirements of 1.900-2.188 are satisfied, which means that the sample RNA has good integrity and high concentration, and can be used for subsequent implementation (as shown in fig. 1).
7.2 PCR-specific analysis of reference genes
The cDNA of the stem and the tendril at different positions is used as a template, and 8 candidate gene primers are subjected to PCR to obtain clear and single strips (shown in figure 2), so that the specificity of the primers is good, and the primers can be used for qRT-PCR implementation. The on-machine inspection of the internal reference gene primers of the stems and the tendrils at different positions of the Chinese yam only has a single signal peak, and has no impurity peak and non-specific amplification phenomenon, which shows that the accuracy of the primers used for qRT-PCR is high.
7.3 analysis of expression abundance of reference genes
qRT-PCR amplification is carried out by using the verified primer, and the expression abundance of the gene in the stems and tendrils at different positions of the Chinese yam is estimated by analyzing the Ct value of the candidate gene. The Ct value of the candidate internal reference genes of the stems and the tendrils of different parts of the Chinese yam is between 15.45 and 32.05, and the difference value of the maximum value and the minimum value of the Ct is sequenced from low to high: HIS is more than TRX-2 and EF-1 alpha and MYB7 and GADPH is more than alpha-TUB and F-box is more than ACT-1, and the expression abundance of HIS, TRX-2 and MYB7 is high. Since the Ct value is inversely related to the expression abundance, that is, the smaller the Ct difference, the greater the expression abundance.
7.4 stability analysis of reference Gene expression
Stability of the genes was evaluated on-line based on Refinder, and the stability values and sequencing results of each software are shown in table 2. The first two of the stem and vine internal reference genes MYB7 of different positions of the Chinese yam are arranged in DeltaCT, geNorm, normfinder and BestKeeper 4 evaluation values, and finally, the first of the complete delta Ct rows shows that the stem and vine internal reference genes MYB7 of different positions of the Chinese yam are the most stable housekeeping genes.
Table 2 evaluation results of Ct values of reference genes of different parts of Dioscorea opposita
7.5 stability verification of reference Gene expression
Performing real-time fluorescent quantitative PCR reaction on MYB7 genes in different parts of stem and tendril tissues of the Chinese yam, and verifying the stability of the reference genes through peak heights. The dissolution curve temperature of MYB7 is 80-83 ℃, and the difference of the Plot peak expression heights at different positions is small (figure 3), which shows that the stability of MYB7 gene is reliable.
7.6 application of reference Gene
And (3) taking the MYB7 gene as a fluorescent quantitative reference gene, and carrying out real-time fluorescent quantitative PCR analysis on the 7 auxin transport carrier genes of the Chinese yam obtained by homologous cloning of Yunnan agricultural university. The 7 genes are respectively named as DoAUX1, doAUX2, doAUX3, doPIN1, doPIN3, doPIN6 and DoPIN7, and the GeneBank accession numbers are OQ706632, OQ722352, OQ722353, OQ740044, OQ740045, OQ740046 and OQ740047. The system for performing qRT-PCR reaction was 20. Mu.L: includes 10.0. Mu.L of qPCR Master mix (SYBRGreen), 1.0. Mu.L of cDNA, 0.8. Mu.L of upstream primer (10. Mu. Mol/L), 0.8. Mu.L of downstream primer (10. Mu. Mol/L), ROX 0.4. Mu.L, ddH2O 7.0. Mu.L. Two-step reaction conditions: pre-denaturation at 95℃for 1min; then denaturation at 95℃for 10s, annealing at 60℃for 30s, pre-extension at 72℃for 10s, and circulation 40 times. The CT value of the gene was measured, and then the relative expression amount of the target gene of the sample was calculated using the following formula:
Δct (test sample) =ct (test sample, gene of interest) -Ct (mean value of reference gene in test sample);
Δct (control sample) =ct (control sample, gene of interest) -Ct (mean value of reference gene in control sample);
ΔΔct= Δct (test sample) - Δct (control sample mean);
relative expression level of test sample=2 －△△Ct 。
The relative expression of each gene in the stem and tendrils at different positions is calculated by using the formula.
As a result, the relative expression amount of the target gene has a remarkable change trend (shown in figure 4), and the expression amounts of the genes of the yam auxin transport carrier genes in different positions of the stems and vines accord with the growth trend of the yams and the stems and vines. The result of the example is reliable, and the MYB7 reference gene obtained by screening is suitable for real-time fluorescence quantitative analysis of stem and vine functional genes at different parts of Chinese yam.
The foregoing has shown and described the basic principles, principal features and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and that the above embodiments and descriptions are merely illustrative of the principles of the present invention, and various changes and modifications may be made without departing from the spirit and scope of the invention, which is defined in the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.
Sequence listing
Claims (9)
1. Fluorescent quantitative reference gene of different parts of Chinese yam stem and tendril, characterized in that the reference geneMYB7The nucleotide sequence of (2) is shown as SEQ ID NO. 1.
2. The fluorescent quantitative reference gene for different parts of the stem and tendrils of Chinese yam according to claim 1, wherein the reference geneMYB7Nucleotide sequence of the PCR amplification primer: the upstream primer is shown as SEQ ID NO. 2; the downstream primer is shown as SEQ ID NO. 3.
3. The screening method of fluorescent quantitative reference genes of different parts of the Chinese yam, which is characterized by comprising the following steps:
s1, selecting 8 gene sequences with E values close to zero or zero from candidate reference genes;
s2, designing primers for 8 genes screened in the step S1, and performing PCR amplification by using the primers to verify the specificity of the primers;
s3, extracting total RNA of the stems and the tendrils at different positions, performing reverse transcription to synthesize first-chain cDNA, performing real-time fluorescent quantitative PCR analysis by adopting primers designed in the S2, calculating Ct values, and analyzing the expression stability of 8 genes screened in the step S1;
s4, comprehensively determining fluorescent quantitative internal reference genes of different parts of the yam with the most stable expression.
4. The method for screening fluorescent quantitative reference genes of different parts of Chinese yam according to claim 3, wherein in the step S1, 8 genes areAct-1、α-TUB、EF-1α、F-box、GADPH、HIS、MYB7、TRX-2。
5. The method for screening fluorescent quantitative reference genes of different parts of Chinese yam according to claim 3, wherein in the step S2, the PCR reaction system is 20. Mu.L: comprises 17.0 mu L Green Mix,1.0 mu L cDNA,1.0 mu L10 mu mol/L upstream primer, 1.0 mu L10 mu mol/L downstream primer;
reaction conditions: pre-denaturation at 95℃for 1min; then denaturation at 95℃for 10s, annealing at 60℃for 30s, pre-extension at 72℃for 10s, and cycling for 30 times.
6. The method for screening fluorescent quantitative reference genes of different parts of Chinese yam according to claim 3, wherein in the step S3, the real-time fluorescent quantitative PCR reaction system is 20. Mu.L: comprises 10.0 mu L qPCR Master Mix SYBR Green I, 1.0 mu L cDNA,0.8 mu L10 mu mol/L upstream primer, 0.8 mu L10 mu mol/L downstream primer, ROX0.4 mu L, ddH 2 O 7.0μL；
PCR reaction conditions: pre-denaturation at 95℃for 1min; then denaturation at 95℃for 10s, annealing at 60℃for 30s, extension at 72℃for 10s, and circulation 40 times.
7. The method for screening quantitative fluorescent reference genes of different parts of Chinese yam according to claim 3, wherein in the step S4, the expression stability of each candidate reference gene is analyzed by using Delta CT and geNorm, normfinder, bestKeeper, comparative Delta Ct methods.
8. The use of the fluorescent quantitative reference genes of different parts of the Chinese yam vines in the analysis of Chinese yam gene expression.
9. The use of the fluorescent quantitative reference genes of different parts of the Chinese yam vines in the improvement of Chinese yam germplasm resources.
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