CN115852014A - Screening method, primers and application of internal reference genes related to growth and flowering of schima superba - Google Patents
Screening method, primers and application of internal reference genes related to growth and flowering of schima superba Download PDFInfo
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Abstract
The invention discloses a screening method, primers and application of internal reference genes related to the growth and flowering of schima superba. The screening method comprises the following steps: (1) Respectively taking M mature leaves of the single plant of the schima superba with obvious volume difference and N different tissues of any 1 single plant of the schima superba, then extracting total RNA from all samples, and carrying out reverse transcription on cDNA; (2) Respectively designing primers according to the nucleotide sequences of the 18 candidate reference genes to obtain PCR primers; (3) evaluation of stability: respectively carrying out qRT-PCR amplification by using the cDNA as a template and PCR primers to obtain a CT value, and further calculating to obtain an average standard deviation; and then evaluating the stability of the candidate reference genes by using different methods, and screening to obtain the reference genes or the combination of the reference genes of the schima superba. The selected reference gene has high stability, pertinence and wide applicability, and can provide important reference for the research of the gene function of the physiological process related to the schima superba.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a screening method, primers and application of internal reference genes related to the growth and flowering of schima superba.
Background
The schima superba is an important material and forest fire prevention tree species widely distributed in southern areas of China, and is also an important ornamental tree species for forest flowers. At present, researchers in China develop long-time breeding research on the tree species by using conventional means, and select and breed a series of improved tree species. However, with the spread of molecular biotechnology in forest breeding, the tree species has primarily entered the molecular breeding line. Obtaining important forest traits needed by people by modifying the expression of specific genes is the most effective genetic improvement means in the biological field at present, and obtaining functional genes with the regulation and control target traits is the premise of realizing the technology.
The expression verification of candidate genes by using a fluorescent quantitative PCR (qRT-PCR) method is a common means, and the method needs normalization analysis of target genes on reference genes with stable expression in different materials. Therefore, screening of suitable reference genes for different materials is the basis for developing candidate gene expression studies.
In the prior research, the screening of Reference Genes of different tissues such as the secondary xylem of the woody plant, the secondary phloem, the mature leaf, the bud, the annual fruit and the root of the tissue culture seedling subculture is reported (Zhongyi Yang, rui Zhang, zhochun Zhou. Identification and variation of Reference Genes for Gene Expression in Schima superba.2021,12,732. Doi. However, these reference genes have a limited range of use, and are not suitable for individuals with significantly different volume traits, nor for different tissues of schima superba with floral organs as the main material.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides a method for screening a reference gene related to the growth and flowering of schima superba.
The invention also aims to provide a reference gene related to the growth and flowering of the schima superba, which is suitable for research on the growth, development and flowering of the schima superba.
The invention further aims to provide application of the internal reference gene related to the growth and flowering of the schima superba.
The purpose of the invention is realized by the following technical scheme:
a screening method of a reference gene related to the growth and flowering of schima superba, comprises the following steps:
(1) Selecting materials:
respectively taking M mature leaves of the single plant of the schima superba with obvious volume difference to obtain M experimental materials with different volume sources; respectively taking N different tissues of any other 1 schima superba single plants (except the obtained M schima superba single plants with obvious volume difference) to obtain N experimental materials with different tissue sources; then, combining the experimental materials with different volume sources and the experimental materials with different tissue sources into a whole sample material, namely obtaining (M + N) samples; finally, extracting total RNA from (M + N) samples respectively, and carrying out reverse transcription on cDNA; wherein M is more than or equal to 10, N is more than or equal to 12;
(2) Designing a primer:
respectively designing primers according to the nucleotide sequences of the candidate reference genes to obtain PCR primers for specifically amplifying the candidate reference genes; wherein the candidate reference genes comprise EF 1-alpha 1, EF 1-alpha 2, GAPDH1, EF2, NADP-GPD, CYS, EF-Ts, ubiquitin1, ubiquitin2, ABC transporter1, ABC transporter2, IF2, NADH1, EF-1 gamma, eIF2, IF-IIA, ACTIN1 and ACTIN2 genes;
(3) Evaluation of stability:
respectively carrying out qRT-PCR amplification by using cDNA as a template and PCR primers of the specific amplification candidate reference genes to obtain a CT value, and further calculating to obtain an average standard deviation (mSD); then, aiming at experimental materials with different volume sources, experimental materials with different tissue sources and all sample materials, at least three methods of delta Ct, geonorm, normFinder, bestKeeper and RefFinder are used for evaluating the stability of the candidate reference genes; and sequencing the candidate reference genes from strong to weak according to the stability evaluation result, and screening to obtain the reference genes or reference gene combinations of the schima superba.
The significant difference in the step (1) is obtained by calculating the family filial generation with the significant difference in volume through analysis of variance (ANOVA), wherein the volume has a fixed calculation formula as follows: HT 1.01545 ×DBH 1.81296 ×6.29692×10 -5 Wherein HT is the tree height and DBH is the diameter at breast height.
The different tissues in the step (1) comprise mature leaves, petioles, buds, petals, stamens, pistils, immature fruits, mature fruits, newly grown flower organs and branches at different development stages.
The newly-formed flower organ is the whole flower organ which is just formed but not unfolded.
The branches in different development stages are continuously taken from the tail end of the branch to the old branch from the tender branch.
The value ranges of M and N in the step (1) can be carried out according to actual needs, and the total sample volume (M + N) is more than or equal to 22.
Designing the PCR primers of the specific amplification candidate reference genes in the step (2) according to the nucleotide sequence of the reference genes to ensure that the size of the PCR product fragment is between 80 and 300bp (preferably between 101 and 250 bp), then screening according to the gel electrophoresis band and the qRT-PCR dissolution curve, and selecting the primers with single electrophoresis band and corresponding to the dissolution curve which is a single peak as the PCR primers of the specific amplification candidate reference genes; preferably shown as SEQ ID NO. 1-36 respectively; more preferably, it is represented by SEQ ID NO.1 to 4, SEQ ID NO.25 to 26, and SEQ ID NO.29 to 30.
Nucleotide sequences of the EF 1-alpha 1, EF 1-alpha 2, GAPDH1, EF2, NADP-GPD, CYS, EF-Ts, ubiquitin1, ubiquitin2, ABC transporter1, ABC transporter2, IF2, NADH1, EF-1 gamma, eIF2, IF-IIA, ACTIN1 and ACTIN2 genes in the step (2) are respectively shown as SEQ ID NO. 37-54.
The reference genes in the step (2) are preferably EF 1-alpha 1, EF 1-alpha 2, NADH1 and eIF2, and the gene sequences are respectively shown as SEQ ID NO.37, SEQ ID NO.38, SEQ ID NO.49 and SEQ ID NO. 51.
The screening in step (3) may select the candidate genes comprehensively ranked first and/or second as an internal reference gene or combination of internal reference genes for schima superba.
In the step (3), for experimental materials with different volume sources, the reference gene is preferably at least one of NADH1 gene and eIF2 gene.
In the step (3), the reference gene is preferably at least one of EF 1-alpha 1 and EF 1-alpha 2 genes of the experimental materials of different tissue sources.
In the step (3), the internal reference gene of all the sample materials is preferably at least one of EF 1-alpha 1 and eIF2 genes.
An internal reference gene related to the growth and flowering of schima superba, which comprises the following specific parts:
(i) For experimental material from single plants of schima superba of different volumes, the reference gene is at least one of NADH1 and eIF2 genes;
(ii) For experimental materials from different tissues of the same single plant of the schima superba, the reference gene is at least one of EF 1-alpha 1 and EF 1-alpha 2 genes;
(iii) The reference gene for experimental material from different volumes of wood lotus and from different tissues of wood lotus (i.e. for all sample material) is at least one of the EF1- α 1 and eIF2 genes.
The experimental material in the step (i) is preferably a single plant of the schima superba with a remarkable volume difference; more preferably mature leaves of individual plants of schima superba with significant volume differences.
In the step (i), the primer sequence for amplifying the NADH1 gene is shown as SEQ ID NO. 25-26, and the primer sequence for amplifying the eIF2 gene is shown as SEQ ID NO. 29-30.
The different tissues described in step (ii) include mature leaves, petioles, buds, petals, stamens, pistils, immature fruits, mature fruits, new flower organs and shoots at different developmental stages.
The branches in different development stages are continuously taken from the tail end of the branch to the old branch from the tender branch.
In the step (ii), the primer sequence for amplifying the EF 1-alpha 1 gene is shown as SEQ ID No. 1-2, and the primer sequence for amplifying the EF 1-alpha 2 gene is shown as SEQ ID No. 3-4.
In the step (iii), the primer sequence for amplifying the EF 1-alpha 1 gene is shown as SEQ ID NO. 1-2, and the primer sequence for amplifying the eIF2 gene is shown as SEQ ID NO. 29-30.
The application of the internal reference gene related to the growth and flowering of the schima superba in real-time fluorescent quantitative PCR (qRT-PCR) is disclosed.
The application of the internal reference gene related to the growth and flowering of the schima superba in the expression analysis of the related genes of the growth, flowering and/or seed (seed formation) of the schima superba.
The application of the reference gene related to the growth and flowering of the schima superba in screening the genes related to the growth, flowering and/or seed formation of the schima superba.
The application of the reference gene related to the growth and flowering of the schima superba in the breeding aspect of the schima superba.
Compared with the prior art, the invention has the following advantages and effects:
(1) The invention screens the stable expression reference genes aiming at the economic character gene function research requirements of materials, ornamental flowers, seed formation and the like, screens candidate reference genes by adopting transcriptome primary selection, qRT-PCR verification and multiple methods for comparative analysis, has simple and efficient technical process, is suitable for the screening requirements of the reference genes of most forest related characters, has higher stability and pertinence and wide applicability by adopting the multiple methods for selecting the reference genes, and provides important reference for the gene function research of the physiological process related to the wood lotus.
(2) The invention solves the problem of lack of internal reference genes in the quantitative research process of target gene expression quantity by utilizing a qRT-PCR method for arborous tree species, mainly relates to individuals with significantly different volume traits and different tissues mainly comprising flower organs, evaluates the stability of 18 candidate internal reference genes by utilizing different algorithms such as delta Ct, geNorm, normFinder, bestKeeper, refFinder and the like, and comprehensively analyzes to show that NADH1 and eIF2 are suitable for different volume arborous single plants, EF 1-alpha 1 and EF 1-alpha 2 are suitable for different tissues (including flower organs and the like), and EF 1-alpha 1 and eIF2 are suitable for all samples.
(3) The method of the invention has wide material sources: aiming at the most important economic character of the wood lotus, the technology selects test materials with remarkable phenotype difference, and is beneficial to breeding of high-yield excellent wood lotus strains and gene function research; in addition, the flower organs are closely related to seed formation, and related tissue materials are selected, so that the gene function research of physiological processes related to flowering and seed formation of the schima superba is facilitated. The above is the most concerned research hotspot of the schima superba, and the screening of the reference gene aiming at the related tissue materials is beneficial to the gene function research.
(4) The reference genes in the method of the invention have rich types: compared with the prior art, the candidate reference genes related by the technology have obvious differences, and mainly relate to 18 genes such as EF 1-alpha 1, EF 1-alpha 2, GAPDH1, EF2, NADP-GPD, CYS, EF-Ts, ubiquitin1, ubiquitin2, ABC transporter1, ABC transporter2, IF2, NADH1, EF-1 gamma, eIF2, IF-IIA, ACTIN1, ACTIN2 and the like, wherein EF 1-alpha, EF2, NADP-GPD, CYS, EF-Ts, ubiquitin, ABC transporter, IF, NADH, EF-1 gamma, eIF and IF-IIA are unreported gene types; wherein EF 1-alpha 1, EF 1-alpha 2, CYS, NADH1 and eIF2 are candidate reference genes with the most stable expression, and provide important reference for future gene research.
Drawings
FIG. 1 is a flow chart of the present invention technique.
FIG. 2 is an agarose gel electrophoresis of 18 candidate reference gene amplification products (DNA Marker: 100-5000 bp).
FIG. 3 is a graph showing melting curves of 18 candidate reference genes.
FIG. 4 is a graph showing the distribution of CT values of candidate reference genes in all samples (boxes indicate 25 th and 75 th percentiles; squares indicate medians; and X indicates the maximum and minimum values).
FIG. 5 is a graph showing the results of analysis of expression stability of candidate reference genes and the number of optimal reference genes based on the geNorm analysis; wherein a and d are analysis results of different volume wood lotus; b and e are analysis results of different tissues; c and f are the results of the analysis of all samples.
FIG. 6 is a graph showing the results of expression stability tests of candidate reference genes based on RefFinder analysis; wherein a is the test result of different volume wood lotus; b is the test results of different tissues; c is the test result of all samples.
FIG. 7 is a graph showing the results of comparative analyses of the expression amounts of the objective genes SsCSL1 and SsCSL2 using 6 reference genes; wherein a is a target gene SsCSL1; b is a target gene SsCSL2.
Detailed Description
The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated. The test methods in the following examples, in which specific experimental conditions are not specified, are generally performed according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. Unless otherwise specified, reagents and starting materials for use in the present invention are commercially available.
Example 1
(1) Plant material selection and transcriptome sequencing (technical flow sheet as shown in figure 1):
randomly selecting 10 individual plants of Schima superba (from Guangdong province forestry science research institute) with significant differences in volume (significant differences are 10 family filial generations with significant differences in volume calculated by analysis of variance (ANOVA). The fixed formula of the volume is HT 1.01545 ×DBH 1.81296 ×6.29692×10 -5 Wherein HT is tree height and DBH is diameter at breast height. Selecting 1 plant from each family for 10 individual plants), and collecting mature leaves; meanwhile, any other individual plant of the schima superba (not from the 10 schima superba plants) is selected, collecting mature leaves, petioles, buds, petals, stamens,Taking 12 tissues such as pistils, immature fruits, mature fruits, newly-grown flower organs (namely the whole flower organ which is just formed but not developed) and branches in different development stages (starting from the tail end of the branch, and continuously taking the branches in 3 different development stages from twigs to old branches), respectively extracting total RNA (ribonucleic acid), carrying out reverse transcription cDNA (complementary deoxyribonucleic acid), and then entrusting Beijing Nuo Gongyuan scientific and technology GmbH to carry out transcriptome sequencing.
(2) Candidate reference gene screening, primer design and feature analysis
The FPKM values of all genes obtained by the sequence determination of the schima superba transcriptome are combined with the gene annotation function, and the gene with the most stable expression is preliminarily selected as a candidate internal reference gene. Specifically, FPKM was used to calculate the standard deviation and the variation coefficient of each gene in 22 samples (22 samples are respectively the mature leaf samples of the above 10 Lophania arborea plants (respectively named as: SS1-L, SS-L … … SS 10-L) and 12 tissues of 1 Lophania arborea (respectively named as: mature leaf (L), petiole (Pe), bud (Pi), petal (YF), stamen (S), pistil (Bu), immature fruit (F0), mature fruit (Fr), new flower organ (P) and shoots (B1, B2, B3) at different developmental stages), wherein the single plant is not derived from the above 10 Lophania arborea plants but any other Lophania arborea plants with different NADH, and 18 or more samples (evenly divided into 3 copies, namely 3 copies) are taken from each tissue, and the common internal reference candidate genes reported in the literature are combined to determine 18 travertins (Table 1) including the sequences of EF 1-alpha 1, GAEF 1- α 1, GATTIN 1- α 1, GAEI 2, ACTEF 1-2-GAII, GAF 2-P, and ACTEF 2-NO, and ACTEF 1-2.
Then, appropriate primers are designed by using Primer 3, the length of a PCR product is usually between 80 and 300bp, the length of the product designed by the technology is between 101 and 250bp, the length of each gene amplification product is verified by using PCR, and the detailed information of the primers of 18 candidate internal reference genes for qRT-PCR analysis is shown in Table 1. Primer specificity was evaluated by gel electrophoresis bands (FIG. 2) and qRT-PCR dissolution curves (FIG. 3), with a single electrophoresis band and a single peak dissolution curve for the appropriate primer. The distribution range of the amplification efficiency of each primer is 77.9-118.1%, and the correlation coefficient is 0.995-0.999.
TABLE 1 primer information and PCR amplification efficiency for 18 candidate genes
(3) Evaluation of expression stability of candidate reference Gene
Obtaining CT values of 18 candidate reference genes in the 22 samples through qRT-PCR, namely, carrying out qPCR amplification by using the cDNA as a template and adopting primers in the table 1 to obtain the CT values, and repeating for three times; among them, ACTIN1 had the lowest average CT value (21.45), NADP-GPD had the highest average CT value (26.79), and there were significant differences in the transcription levels between the respective reference genes (FIG. 4).
By log 2 Fold Change The method calculates the transcription level of 18 genes in 22 samples, and the result shows that the expression level of the candidate genes in different tissue types has irregular difference change. Therefore, other reference gene analysis software needs to be continuously utilized to candidate the expression stability of the reference gene.
The Mean standard deviation (mSD) of the CT value of the reference gene in each sample can be used to evaluate the stability of the reference gene, the lower mSD is, the better the stability is, and the stability of the candidate reference gene in different tissues in the present study is ranked in table 2.
TABLE 2 stability evaluation of Delta Ct against 18 candidate genes
| Ranking | Different volume of material | mSD | Different tissues | mSD | All are provided with | |
| 1 | NADH1 | 0.28 | EF1-α1 | 0.54 | EF1-α1 | 0.55 |
| 2 | eIF2 | 0.28 | EF1-α2 | 0.55 | eIF2 | 0.57 |
| 3 | EF2 | 0.29 | EF2 | 0.57 | EF2 | 0.59 |
| 4 | EF1-α1 | 0.3 | CYS | 0.58 | ABCtransporter2 | 0.59 |
| 5 | IF2 | 0.31 | ABCtransporter2 | 0.59 | IF2 | 0.59 |
| 6 | Ubiquitin1 | 0.31 | Ubiquitin1 | 0.6 | NADH1 | 0.59 |
| 7 | ACTIN1 | 0.32 | eIF2 | 0.6 | IF-IIA | 0.64 |
| 8 | EF1-α2 | 0.32 | IF-IIA | 0.62 | Ubiquitin1 | 0.64 |
| 9 | Ubiquitin2 | 0.33 | IF2 | 0.62 | Ubiquitin2 | 0.64 |
| 10 | EF-1γ | 0.34 | NADH1 | 0.63 | CYS | 0.65 |
| 11 | ABCtransporter1 | 0.38 | ACTIN1 | 0.67 | EF1-α2 | 0.66 |
| 12 | ABCtransporter2 | 0.38 | Ubiquitin2 | 0.68 | ABCtransporter1 | 0.69 |
| 13 | IF-IIA | 0.39 | ABCtransporter1 | 0.7 | EF-Ts | 0.69 |
| 14 | GAPDH1 | 0.42 | EF-Ts | 0.71 | EF-1γ | 0.7 |
| 15 | CYS | 0.43 | EF-1γ | 0.79 | ACTIN1 | 0.74 |
| 16 | EF-Ts | 0.45 | GAPDH1 | 1.18 | GAPDH1 | 1.18 |
| 17 | NADP-GPD | 0.57 | NADP-GPD | 1.45 | NADP-GPD | 1.23 |
| 18 | ACTIN2 | 0.64 | ACTIN2 | 1.54 | ACTIN2 | 1.73 |
The expression stability of the gene was evaluated by calculating the expression stability M value of the reference gene using the geonorm software, with smaller M values indicating higher expression stability. The results show that eIF2| NADH1 (0.127) is the most suitable reference gene combination for different wood products of Schima superba (Table 3; FIG. 5 a), EF1- α 1 EF1- α 2 (0.158) is the most suitable reference gene combination for different tissues of Schima superba (Table 3; FIG. 5 b), and eIF2| IF2 (0.228) is the most suitable reference gene combination for all samples (Table 3; FIG. 5 c). Furthermore, further analysis by geNorm revealed that V2/V3 was less than 0.15 in different tissue types, indicating that the number of genes most suitable for normalization was 2 (fig. 5d, 5e, 5 f).
TABLE 3 stability of 18 candidate reference genes based on the analysis of geNorm
The NormFinder software is used for screening the internal reference gene by calculating the Stability Value (SV) of the expression of the internal reference gene, and the smaller the SV, the better the stability is. The stability of 18 candidate genes was analyzed using the NormFinder software (table 4) and the results showed that NADH1 ranked first (SV = 0.08) and ACTIN2 ranked last (SV = 0.594) in different volume wood grains; EF1- α 1 was the best choice among different tissues (SV = 0.2), ACTIN2 was the least stable (SV = 1.458); EF1- α 1 was most stable in expression in all samples (SV = 0.233) and ACTIN2 was least stable (SV = 1.668).
TABLE 4 stability of 18 candidate reference genes based on NormFinder analysis
| Ranking | Different volume of material | SV | Different tissues | SV | All are | |
| 1 | NADH1 | 0.08 | EF1-α1 | 0.2 | EF1-α1 | 0.233 |
| 2 | eIF2 | 0.114 | EF1-α2 | 0.218 | eIF2 | 0.283 |
| 3 | EF2 | 0.126 | CYS | 0.233 | Ubiquitin1 | 0.292 |
| 4 | EF1-α1 | 0.148 | EF2 | 0.256 | ABCtransporter2 | 0.294 |
| 5 | IF2 | 0.161 | Ubiquitin1 | 0.272 | NADH1 | 0.327 |
| 6 | Ubiquitin1 | 0.175 | ABCtransporter2 | 0.287 | EF2 | 0.336 |
| 7 | ACTIN1 | 0.187 | ACTIN1 | 0.348 | IF2 | 0.336 |
| 8 | Ubiquitin2 | 0.188 | eIF2 | 0.381 | ABCtransporter1 | 0.379 |
| 9 | EF1-α2 | 0.199 | IF-IIA | 0.386 | EF-Ts | 0.385 |
| 10 | EF-1γ | 0.211 | NADH1 | 0.393 | EF-1γ | 0.408 |
| 11 | ABCtransporter1 | 0.268 | IF2 | 0.395 | CYS | 0.412 |
| 12 | IF-IIA | 0.27 | EF-Ts | 0.42 | IF-IIA | 0.412 |
| 13 | ABCtransporter2 | 0.282 | ABCtransporter1 | 0.442 | Ubiquitin2 | 0.414 |
| 14 | GAPDH1 | 0.332 | Ubiquitin2 | 0.463 | ACTIN1 | 0.433 |
| 15 | CYS | 0.339 | EF-1γ | 0.548 | EF1-α2 | 0.467 |
| 16 | EF-Ts | 0.368 | GAPDH1 | 1.03 | GAPDH1 | 1.031 |
| 17 | NADP-GPD | 0.515 | NADP-GPD | 1.372 | NADP-GPD | 1.123 |
| 18 | ACTIN2 | 0.594 | ACTIN2 | 1.458 | ACTIN2 | 1.668 |
The stability of the genes was evaluated by calculating the Standard Deviation (SD) and the Coefficient of Variation (CV) using bestkieper software. In general, the stability of a gene is determined by the SD value, the lower the SD value, the better the stability. The results are shown in table 5, and the results of the stability analysis of 18 genes by Bestkeeper show that EF1- α 2 is suitable for different volume lotus, CYS has the highest stability in different tissues and total samples, and ACTIN2 has the least stable expression in all three sample combinations.
TABLE 5 stability of 18 candidate reference genes based on BestKeeper analysis
Finally, the stability of 18 candidate reference genes was evaluated using RefFinder software. The results show that NADH1 and eIF2 are relatively stably expressed in different Schima superba (FIG. 6 a), EF 1-alpha 1 and EF 1-alpha 2 are the most suitable choices in different tissues (FIG. 6 b), and EF 1-alpha 1 and eIF2 are more stable than other genes in the total sample (FIG. 6 c).
(4) Target gene verification
And (3) selecting stable-expression reference genes such as CYS, NADH1, eIF2, EF 1-alpha 1, EF 1-alpha 2 and the like and the expression-most-unstable ACTIN2 according to analysis results of each software, carrying out expression comparison analysis on two target genes SsCSL1 (SEQ ID NO. 55) and SsCSL2 (SEQ ID NO. 56) in the 22 samples (calculating the average value and standard deviation of the genes in each sample by using Excel, displaying the average value and the standard deviation by using a bar chart), and repeating for three times. Wherein, the primer sequence for amplifying the SsCSL1 and SsCSL2 genes is shown as SEQ ID NO. 57-60.
The results are shown in FIG. 7: the result shows that when the objective genes SsCSL1 and SsCSL2 are analyzed by taking ACTIN2 as an internal reference gene, the expression patterns of the objective genes SsCSL1 and SsCSL2 are obviously different from those of other internal reference genes, and the result shows that the internal reference genes such as CYS, NADPH1, eIF2, EF 1-alpha 1, EF 1-alpha 2 and the like have obvious advantages.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. A screening method of an internal reference gene related to the growth and flowering of schima superba is characterized by comprising the following steps:
(1) Selecting materials:
respectively taking M mature leaves of the single plant of the schima superba with obvious volume difference to obtain M experimental materials with different volume sources; respectively taking N different tissues of any other 1 schima superba single plant to obtain N experimental materials with different tissue sources; then, combining the experimental materials with different volume sources and the experimental materials with different tissue sources into a whole sample material, namely obtaining M + N samples; finally, extracting total RNA from the M + N samples respectively, and carrying out reverse transcription on cDNA; wherein M is more than or equal to 10, N is more than or equal to 12;
(2) Designing a primer:
respectively designing primers according to the nucleotide sequences of the candidate reference genes to obtain PCR primers for specifically amplifying the candidate reference genes; wherein the candidate reference genes comprise EF 1-alpha 1, EF 1-alpha 2, GAPDH1, EF2, NADP-GPD, CYS, EF-Ts, ubiquitin1, ubiquitin2, ABC transporter1, ABC transporter2, IF2, NADH1, EF-1 gamma, eIF2, IF-IIA, ACTIN1 and ACTIN2 genes;
(3) Evaluation of stability:
respectively carrying out qRT-PCR amplification by using cDNA as a template and PCR primers of the candidate internal reference genes for specific amplification to obtain a CT value, and further calculating to obtain an average standard deviation; then, aiming at experimental materials with different volume sources, experimental materials with different tissue sources and all sample materials, at least three methods of delta Ct, geonorm, normFinder, bestKeeper and RefFinder are used for evaluating the stability of the candidate reference genes; and sequencing the candidate reference genes from strong to weak according to the stability evaluation result, and screening to obtain the reference genes or reference gene combinations of the schima superba.
2. The method for screening the reference gene related to the growth and flowering of schima superba according to claim 1, wherein the reference gene is selected from the group consisting of:
the different tissues in the step (1) comprise mature leaves, petioles, buds, petals, stamens, pistils, immature fruits, mature fruits, newly-grown flower organs and branches at different development stages;
the branches in different development stages are continuously taken from the tail end of the branch to the old branch from the tender branch;
the PCR primers for specifically amplifying the candidate reference genes in the step (2) are respectively shown as SEQ ID NO. 1-36.
3. The method for screening the reference gene related to the growth and flowering of schima superba according to claim 1, wherein the reference gene is selected from the group consisting of:
nucleotide sequences of the EF 1-alpha 1, EF 1-alpha 2, GAPDH1, EF2, NADP-GPD, CYS, EF-Ts, ubiquitin1, ubiquitin2, ABC transporter1, ABC transporter2, IF2, NADH1, EF-1 gamma, eIF2, IF-IIA, ACTIN1 and ACTIN2 genes in the step (2) are respectively shown as SEQ ID NO. 37-54.
4. The method for screening the reference gene related to the growth and flowering of schima superba according to claim 1, wherein the reference gene is selected from the group consisting of:
the internal reference genes in the step (2) are EF 1-alpha 1, EF 1-alpha 2, NADH1 and eIF2, and the gene sequences are respectively shown as SEQ ID NO.37, SEQ ID NO.38, SEQ ID NO.49 and SEQ ID NO. 51.
5. The method for screening the reference gene related to the growth and flowering of schima superba according to claim 1, wherein the reference gene is selected from the group consisting of:
selecting the candidate genes comprehensively ranked in the step (3) as the reference genes or the reference gene combination of the schima superba;
in the step (3), for experimental materials with different volume sources, the reference gene is at least one of NADH1 gene and eIF2 gene;
in the step (3), for experimental materials of different tissue sources, the reference gene is at least one of EF 1-alpha 1 and EF 1-alpha 2 genes;
in the step (3), the reference gene is at least one of EF 1-alpha 1 and eIF2 genes of all sample materials.
6. An internal reference gene related to the growth and flowering of schima superba, which is characterized by comprising the following specific components:
(i) For experimental material from single plants of schima superba of different volumes, the reference gene is at least one of NADH1 and eIF2 genes;
(ii) For experimental materials from different tissues of the same single plant of the schima superba, the reference gene is at least one of EF 1-alpha 1 and EF 1-alpha 2 genes;
(iii) The reference gene is at least one of EF 1-alpha 1 and eIF2 genes for experimental material from different volume wood lotus and different tissues of the wood lotus.
7. The reference gene related to the growth and flowering of schima superba according to claim 6, wherein:
(ii) the experimental material in step (i) is a mature leaf of a single plant of schima superba with a significant volume difference;
in the step (i), the primer sequence for amplifying the NADH1 gene is shown as SEQ ID NO. 25-26, and the primer sequence for amplifying the eIF2 gene is shown as SEQ ID NO. 29-30;
(iii) the different tissues of step (ii) comprise mature leaves, petioles, buds, petals, stamens, pistils, immature fruits, mature fruits, new flower organs and shoots at different developmental stages;
the branches in different development stages are continuously taken from the tail end of the branch to the old branch from the tender branch;
in the step (ii), the primer sequence for amplifying the EF 1-alpha 1 gene is shown as SEQ ID NO. 1-2, and the primer sequence for amplifying the EF 1-alpha 2 gene is shown as SEQ ID NO. 3-4;
in the step (iii), the primer sequence for amplifying the EF 1-alpha 1 gene is shown as SEQ ID NO. 1-2, and the primer sequence for amplifying the eIF2 gene is shown as SEQ ID NO. 29-30.
8. Use of the reference gene related to the growth and flowering of schima superba as claimed in claim 6 or 7 in real-time fluorescent quantitative PCR or schima superba breeding.
9. Use of the internal reference gene related to the growth and flowering of schima superba as defined in claim 6 or 7 in the expression analysis of schima superba growth, flowering and/or seed-related genes.
10. Use of the reference gene related to the growth and flowering of schima superba as claimed in claim 6 or 7 for screening genes related to the growth, flowering and/or seed formation of schima superba.
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