CN115852014B - Screening method, primer and application of reference genes related to growth and flowering of xylocarpus - Google Patents
Screening method, primer and application of reference genes related to growth and flowering of xylocarpus Download PDFInfo
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Abstract
The invention discloses a screening method, primers and application of internal reference genes related to growth and flowering of xylocarpa. The screening method comprises the following steps: (1) Mature leaves of M wood lotus single plants with obvious volume difference and N different tissues of any 1 wood lotus single plant are respectively taken, then total RNA is extracted from all samples, and cDNA is reversely transcribed; (2) Respectively designing primers according to nucleotide sequences of 18 candidate internal reference genes to obtain PCR primers; (3) stability evaluation: respectively carrying out qRT-PCR amplification by using the PCR primer by taking cDNA as a template 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 of the wood lotus or the combination thereof. The reference genes selected by the invention have higher stability, pertinence and wide applicability, and can provide important reference for the functional research of the genes of the related physiological processes of the wood and the lotus.
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 growth and flowering of xylocarpa.
Background
The wood lotus is an important wood used in the south of China and a forest fire prevention tree species, and is also an important ornamental tree species for the trees and flowers. At present, domestic researchers have developed long-time breeding researches on tree species by using conventional means, and a series of improved tree species are bred. However, with the popularity of molecular biology techniques in tree breeding, the tree species have been initially introduced into molecular breeding lines. The expression of the specific gene is modified to obtain the important forest character required by people, which is the most effective genetic improvement means in the current biological field, and the obtaining of the functional gene with the regulatory target character is the precondition for realizing the technology.
The verification of candidate gene expression by using a fluorescent quantitative PCR (qRT-PCR) method is a common means, and the method needs to perform normalized analysis of target genes in reference genes with stable expression of different materials. Therefore, screening of suitable reference genes for different materials is the basis for developing candidate gene expression studies.
The prior study reports (Zhongyi Yang, rui Zhang, zhichun Zhou identification and Validation of Reference Genes for Gene Expression Analysis in Schima superba.2021,12,732.Doi: org/10.3390/genes 12050732) that the internal genes of different tissues such as wood-load secondary xylem, secondary phloem, mature leaves, buds, annual fruits and tissue culture seedling subcultured roots were screened, and SsuACT, ssuRIB was finally selected as a suitable combination of internal genes. However, these internal genes have a limited range of application, and are not suitable for individuals with significant differences in volume characteristics, nor for different tissues of the wood load using flower organs as the main material.
Disclosure of Invention
The primary aim of the invention is to overcome the defects and shortcomings of the prior art and provide a screening method of reference genes related to growth and flowering of the wood lotus.
The invention also aims to provide an internal reference gene related to growth and flowering of the xylocarpus, which is suitable for research on growth and development of the xylocarpus and flowering.
It is still another object of the present invention to provide the use of the reference gene related to the growth and flowering of said xylem.
The aim of the invention is achieved by the following technical scheme:
a screening method of reference genes related to growth and flowering of xylocarpus comprises the following steps:
(1) And (3) material selection:
respectively taking mature leaves of M individual wood lotus plants with obvious volume differences to obtain M experimental materials with different volume sources; respectively taking N different tissues of other arbitrary 1 wood lotus single plants (other arbitrary single plants except the M wood lotus single plants with obvious volume difference) to obtain experimental materials with N different tissue sources; then, experimental materials with different material volumes and experimental materials with different tissue sources are combined into all sample materials, namely (M+N) samples are obtained; finally, extracting total RNA from the (M+N) samples respectively, and reversely transcribing cDNA; wherein M is more than or equal to 10, and N is more than or equal to 12;
(2) Primer design:
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 internal 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) Stability evaluation:
respectively carrying out qRT-PCR amplification by using cDNA as a template and using PCR primers of the specific amplification candidate reference genes to obtain CT values, and further calculating to obtain average standard deviation (mSD); then, evaluating the stability of the candidate reference genes by using at least three of delta Ct, geNorm, normFinder, bestKeeper and refFinder methods according to experimental materials from different volumes, experimental materials from different tissues and all sample materials; and sequencing the candidate reference genes according to the stability from strong to weak according to the stability evaluation result, and screening to obtain reference genes or reference gene combinations of the wood lotus.
The significant difference in the step (1) is family offspring with significant volume difference obtained by calculation of analysis of variance (ANOVA), wherein the volume has a fixed calculation formula: HT (HT) 1.01545 ×DBH 1.81296 ×6.29692×10 -5 Wherein HT is tree height and DBH is chest diameter.
The different tissues in step (1) comprise mature leaves, petioles, buds, petals, stamens, pistils, immature fruits, mature fruits, new flower organs and branches at different developmental stages.
The newly formed flower organ is the whole flower organ which is just formed but not yet developed.
The branches in different development stages are branches from the tail ends of the branches, and more than 3 branches in different development stages are continuously taken from the tender branches to the old branches.
The value ranges of M and N in the step (1) can be carried out according to actual needs, and the total sample size (M+N) is more than or equal to 22.
Designing PCR primers of the specific amplification candidate reference genes in the step (2) according to the nucleotide sequence of the reference genes, enabling the sizes of PCR product fragments to be 80-300 bp (preferably 101-250 bp), screening according to gel electrophoresis bands and qRT-PCR dissolution curves, and selecting primers corresponding to single electrophoresis bands and single dissolution curves as the PCR primers of the specific amplification candidate reference genes; preferably as shown in SEQ ID NO. 1-36 respectively; further preferable are those shown as SEQ ID NO. 1-4, SEQ ID NO. 25-26 and SEQ ID NO. 29-30.
The 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 in 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 shown in SEQ ID NO.37, SEQ ID NO.38, SEQ ID NO.49 and SEQ ID NO.51 respectively.
The screening in step (3) may select the candidate genes of the first and/or second comprehensive ordering as reference genes or reference gene combinations of the wood lotus.
In the step (3), the reference gene is preferably at least one of NADH1 and eIF2 genes for the experimental materials from different volumes.
In the step (3), for the experimental materials of different tissue sources, the reference gene is preferably at least one of EF 1-alpha 1 and EF 1-alpha 2 genes.
In step (3), the reference gene is preferably at least one of EF 1-. Alpha.1 and eIF2 genes for all sample materials.
An internal reference gene related to the growth and flowering of the lotus, which is specifically as follows:
(i) For experimental materials of individual wood lotus plants from different volumes, the internal reference gene is at least one of NADH1 and eIF2 genes;
(ii) For experimental materials of different tissues from the same single plant of the same lotus, the internal reference gene is at least one of EF 1-alpha 1 and EF 1-alpha 2 genes;
(iii) For experimental materials from different wood charges of different volumes and different tissues of wood charges (i.e., for all sample materials), the internal reference is at least one of the EF1- α1 and eIF2 genes.
The experimental material in the step (i) is preferably a single wood lotus plant with remarkable volume difference; more preferably mature leaves of individual plants of Mulotus with significant differences in volume.
In the step (i), the primer sequences for amplifying the NADH1 gene are shown in SEQ ID NO. 25-26, and the primer sequences for amplifying the eIF2 gene are shown in 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 floral organs and shoots at different stages of development.
The branches in different development stages are from the tail ends of the branches, and more than 3 branches in different development stages are continuously taken from the tender branches to the old branches.
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 xylocarpa in real-time fluorescence quantitative PCR (qRT-PCR).
The application of the internal reference genes related to the growth and the flowering of the wood lotus in the expression analysis of the genes related to the growth, the flowering and/or the seed (seed formation) of the wood lotus.
The application of the internal reference gene related to the growth and the flowering of the xylocarpus in screening genes related to the growth, the flowering and/or the seed formation of the xylocarpus.
The application of the internal reference gene related to the growth and flowering of the nucifera in the aspect of nucifera breeding.
Compared with the prior art, the invention has the following advantages and effects:
(1) The invention screens the reference genes with stable expression aiming at the functional research demands of economic trait genes such as wood, ornamental flowers, seed formation and the like, adopts transcriptome primary selection, qRT-PCR verification and multiple methods for comparison analysis to screen candidate reference genes, has simple and efficient technical process, is suitable for the screening demands of reference genes with most forest related traits, has higher stability and pertinence and wide applicability, and provides important references for the functional research of genes with related physiological processes of wood and lotus.
(2) The invention solves the problem of reference gene deficiency in the quantitative research process of target gene expression quantity of the nux vomica seeds by using a qRT-PCR method, mainly relates to individuals with remarkable differences in volume characters and different tissues mainly including flower organs, and evaluates the stability of 18 candidate reference genes by using different algorithms such as delta Ct, geNorm, normFinder, bestKeeper, refFinder, and comprehensive analysis shows that NADH1 and eIF2 are suitable for different material building block 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 has the advantages of wide sources of the materials: aiming at the most important economic character of the wood lotus, the technology selects test materials with obvious difference in phenotype, thereby being beneficial to the breeding of high-yield excellent wood lotus strain and the research of gene functions; in addition, the flower organs are closely related to seed formation, and the selection of related tissue materials is helpful for researching gene function research of physiological processes related to flowering and seed formation of the Mulotus. The research hot spot which is the most focused on the nux vomica is described above, and screening of internal reference genes for related tissue materials is helpful for gene function research.
(4) The reference genes in the method are rich in variety: the candidate internal reference genes related to the technology are obviously different from the prior art, 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, compared with the prior art, 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 references for future gene research.
Drawings
Fig. 1 is a flow chart of the technique of the present invention.
FIG. 2 is an agarose gel electrophoresis chart (DNA Marker: 100-5000 bp) of amplified products of 18 candidate reference genes.
FIG. 3 is a melting graph of 18 candidate reference genes.
FIG. 4 is a graph of CT distribution of candidate internal genes over all samples (box plot represents 25 th and 75 th percentiles; square represents median; x represents maximum and minimum).
FIG. 5 is a graph showing the results of analysis of the expression stability of candidate reference genes and the number of optimal reference genes based on the gemum analysis; wherein a and d are analysis results of different volumes of wood lotus; b and e are analysis results of different tissues; c and f are the analysis results of all samples.
FIG. 6 is a graph of results of expression stability tests for candidate reference genes based on RefFinder analysis; wherein a is the test result of different timber volumes of the wood lotus; b is a test result of different organizations; c is the test result of all samples.
FIG. 7 is a graph showing the results of comparative analysis of the expression levels of the target genes SsCSL1 and SsCSL2 using 6 reference genes; wherein a is a target gene SsCSL1; b is the target gene SsCSL2.
Detailed Description
The present invention will be described in further detail with reference to examples, but embodiments of the present invention are not limited thereto. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art. The test methods for specific experimental conditions are not noted in the examples below, and are generally performed under conventional experimental conditions or under experimental conditions recommended by the manufacturer. The reagents and starting materials used in the present invention are commercially available unless otherwise specified.
Example 1
(1) Plant material selection and transcriptome sequencing (technical flow diagram is shown in fig. 1):
randomly selecting 10 individual wood plants (from the forestry science institute of Guangdong province) with remarkable volume difference (the remarkable difference is family offspring with remarkable volume difference obtained by calculation of analysis of variance (ANOVA) of calculation, wherein the volume has a fixed calculation formula of HT 1.01545 ×DBH 1.81296 ×6.29692×10 -5 Where HT is tree height and DBH is thoracodiameter. 10 individual plants are 1 for each family), and mature leaves are collected; meanwhile, any other wood lotus single plant (not from the 10 wood lotus single plants) is selected, 12 tissues such as mature leaves, petioles, buds, petals, stamens, pistils, immature fruits, mature fruits, new flower organs (i.e. the whole flower organs which are just formed but not yet developed) and branches at different development stages (branches at 3 different development stages are continuously taken from the tail end of the branch from the tender branch to the old branch) are collected as materials, total RNA is respectively extracted, cDNA is reversely transcribed, and then the Beijing Nodeum source technology Co Ltd is entrusted for transcriptome sequencing.
(2) Candidate reference gene screening, primer design and feature analysis
And (3) preliminarily selecting the gene with the most stable expression as a candidate reference gene by utilizing all gene FPKM values obtained by sequencing of the wood lotus transcriptome and combining the gene annotation function. Specifically, FPKM was used to calculate standard deviations and variation coefficients of each gene in 22 samples (22 samples are respectively named as SS1-L, SS2-L … … SS 10-L) and 12 tissues (respectively named as mature leaf (L), petiole (Pe), bud (Pi), petal (YF), stamen (S), pistil (Bu), immature fruit (F0), mature fruit (Fr), new-born flower organ (P) and branches (B1, B2, B3) of different development stages of the 10 wood-lotus individuals, wherein the individual is not derived from the 10 wood-lotus individuals, is any other wood-lotus individuals, takes more than 18 samples (each is equally named as 3 copies, namely 3 copies)), and preliminarily determines 18 candidate internal reference genes (Table 1) including EF 1-alpha 1, EF 1-alpha 2, GAeF 1, EF 35-84, ueif-84, uportF 1, ABC 2, ACID 2, ABC 1-F2, and the sequence of which are shown in SEQ ID 1-alpha 1, ABC 2, ABC 1-F2, and the like.
Then, a proper Primer is designed by utilizing Primer 3, the length of a PCR product is generally 80-300 bp, the length of the product designed by the technology is 101-250 bp, the length of amplified products of each gene is verified by utilizing PCR, and detailed information of the primers of 18 candidate internal reference genes for qRT-PCR analysis is shown in table 1. Primer specificity was assessed by gel electrophoresis bands (FIG. 2) and qRT-PCR dissolution curves (FIG. 3), with electrophoresis bands single and dissolution curves as single peaks being suitable primers. 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 of 18 candidate genes and PCR amplification efficiency
(3) Evaluation of expression stability of candidate reference Gene
Obtaining CT values of 18 candidate internal reference genes in the 22 samples through qRT-PCR, namely carrying out qPCR amplification by using the cDNA as a template and adopting the primers in the table 1 to obtain 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 was a significant difference in transcription level between the reference genes (FIG. 4).
Using log 2 Fold Change The method calculates the transcription levels of 18 genes in 22 samples, and the result shows that the expression levels of candidate genes in different tissue types have irregular difference changes. Therefore, it is necessary to continue to analyze the stability of the expression of the candidate reference gene by using other reference genes.
The mean standard deviation (Mean standard deviation, mSD) of the CT values of the reference genes in each sample can be used to evaluate the stability of the reference genes, the lower mSD the better the stability of the reference genes, the ranking of the candidate reference genes in different tissues in this study is shown in Table 2.
TABLE 2 evaluation of the stability of DeltaCt on 18 candidate genes
| Ranking | Different volumes of material | mSD | Different textures | mSD | All of which | mSD |
| 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 gemm software, and a smaller M value indicates a higher expression stability. The results showed that eIF2|NADH1 (0.127) was the optimal reference gene combination for different wood volumes (Table 3; FIG. 5 a), EF1- α1|EF1- α2 (0.158) was the optimal reference gene combination for different wood textures (Table 3; FIG. 5 b), and that for eIF2|IF2 (0.228), all samples were the optimal reference gene combinations (Table 3; FIG. 5 c). Furthermore, further analysis by gemm showed that V2/V3 was less than 0.15 in different tissue types, indicating that the most suitable normalized gene numbers were 2 (FIG. 5d, FIG. 5e, FIG. 5 f).
TABLE 3 stability of 18 candidate reference genes based on geNorm analysis
The NormFinder software was used to screen the reference gene by calculating the Stability Value (SV) of the expression of the reference gene, with smaller SV indicating better stability. Stability of 18 candidate genes was analyzed using normfilter software (table 4), showing that NADH1 ranks first (sv=0.08) and ACTIN2 ranks last (sv=0.594) in different wood boluses; EF1- α1 is the best choice in different tissues (sv=0.2), ACTIN2 is the least stable (sv=1.458); EF1- α1 had the highest expression stability in all samples (sv=0.233) and ACTIN2 had the lowest stability (sv=1.668).
TABLE 4 NormFinder-based analysis of stability of 18 candidate reference genes
| Ranking | Different volumes of material | SV | Different textures | SV | All of which | SV |
| 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 Standard Deviation (SD) and Coefficient of Variation (CV) using bestdeeper software. In general, the stability of a gene is determined by the SD value, and the lower the SD value, the better the stability. As shown in Table 5, the results of the stability analysis of Bestkeeper on 18 genes show that EF 1-alpha 2 is suitable for different wood boluses, CYS has the highest stability in different tissues and total samples, and ACTIN2 is the least stable in all three sample combinations.
TABLE 5 stability of 18 candidate reference genes based on BestKeeper analysis
Finally, stability of 18 candidate reference genes was assessed using refFinder software. The results show that NADH1 and eIF2 are relatively stable in expression in different wood volumes (FIG. 6 a), EF1- α1 and EF1- α2 are the most suitable choices in different tissues (FIG. 6 b), and EF1- α1 and eIF2 are better stable in the total sample than other genes (FIG. 6 c).
(4) Verification of target Gene
And selecting internal reference genes with stable expression such as CYS, NADH1, eIF2, EF 1-alpha 1, EF 1-alpha 2 and the like and ACTIN2 with the least stable expression according to analysis results of all software, carrying out expression comparison analysis of two target genes SsCSL1 (SEQ ID No. 55) and SsCSL2 (SEQ ID No. 56) in the 22 samples (calculating average value and standard deviation of the genes in all samples by using Excel and displaying by using a bar graph), and repeating the steps. Wherein, the primer sequences for amplifying SsCSL1 and SsCSL2 genes are shown in SEQ ID NO. 57-60.
The results are shown in FIG. 7: the result shows that the expression patterns of the target genes SsCSL1 and SsCSL2 are obviously different from other reference genes when the ACTIN2 is used as the reference genes for analysis, and the expression patterns of the target genes SsCSL1 and SsCSL2 show that the reference genes such as CYS, NADPH1, eIF2, EF 1-alpha 1, EF 1-alpha 2 and the like have obvious advantages.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (5)
1. The screening method of the reference genes related to the growth and flowering of the xylocarpa is characterized by comprising the following steps:
(1) And (3) material selection:
respectively taking mature leaves of M individual wood lotus plants with obvious volume differences to obtain M experimental materials with different volume sources; respectively taking N different tissues of other arbitrary 1 wood lotus single plants to obtain N experimental materials with different tissue sources; then, experimental materials with different material volumes and experimental materials with different tissue sources are combined into all sample materials, namely M+N samples are obtained; finally extracting total RNA from M+N samples respectively, and reversely transcribing cDNA; wherein M is more than or equal to 10, and N is more than or equal to 12;
(2) Primer design:
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, and the nucleotide sequences of the candidate reference genes are respectively shown in SEQ ID NO. 37-54;
(3) Stability evaluation:
respectively carrying out qRT-PCR amplification by using PCR primers of specific amplification candidate reference genes by taking cDNA as a template to obtain CT values, and further calculating to obtain average standard deviation; then, evaluating the stability of candidate reference genes by at least three of father Ct, geNorm, normFinder, bestKeeper and RefFinder methods according to experimental materials from different volumes, experimental materials from different tissues and all sample materials; sequencing the candidate reference genes according to the stability from strong to weak according to the stability evaluation result, and screening to obtain reference genes or reference gene combinations of the wood lotus;
in the step (1), the experimental materials of different tissue sources comprise mature leaves, petioles, buds, petals, stamens, pistils, immature fruits, mature fruits, new-born organs and branches of different development stages;
the branches in different development stages are branches in different development stages from the tail ends of the branches, and more than 3 branches are continuously taken from the tender branches to the old branches;
in the step (3), for the experimental materials with different material volumes, the internal reference gene is at least one of NADH1 and eIF2 genes;
in the step (3), for the experimental materials with different tissue sources, the internal reference gene is at least one of EF 1-alpha 1 and EF 1-alpha 2 genes;
in step (3), the internal reference is at least one of EF 1-alpha 1 and eIF2 genes for all sample materials.
2. The screening method of internal reference genes related to growth and flowering of xylem according to claim 1, wherein the screening method comprises the following steps:
in the step (2), PCR primers of the specific amplification candidate reference genes are respectively shown in SEQ ID NO. 1-36.
Application of EF1-alpha 1 gene, EF 1-alpha 2 gene, NADH1 gene and eIF2 gene as reference genes in real-time fluorescence quantitative PCR analysis of wood load, which is characterized in that:
(i) For experimental materials of single wood lotus plants from different volumes, at least one of NADH1 and eIF2 genes is used as an internal reference gene;
(ii) For experimental materials of different tissues from the same single plant of the same lotus, at least one of EF 1-alpha 1 and EF 1-alpha 2 genes is used as an internal reference gene;
(iii) For experimental materials of different tissues from different wood single plants and from the same wood single plant, at least one of EF 1-alpha 1 and eIF2 genes is used as an internal reference gene;
the nucleotide sequence of the EF 1-alpha 1 gene is shown as SEQ ID NO. 37;
the nucleotide sequence of the EF 1-alpha 2 gene is shown as SEQ ID NO. 38;
the nucleotide sequence of the NADH1 gene is shown as SEQ ID NO. 49;
the nucleotide sequence of the eIF2 gene is shown as SEQ ID NO. 51;
in (i), the experimental material is mature leaves of single wood lotus plants with remarkable volume difference;
in (ii), the test material comprises mature leaves, petioles, buds, petals, stamens, pistils, immature fruits, mature fruits, new floral organs, and shoots at different stages of development;
the branches in different development stages are branches from the tail ends of the branches, and more than 3 branches in different development stages are continuously taken from the tender branches to the old branches.
4. A use according to claim 3, characterized in that: the primer sequence for amplifying the NADH1 gene is shown as SEQ ID NO. 25-26, the primer sequence for amplifying the eIF2 gene is shown as SEQ ID NO. 29-30, 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.
5. Use according to claim 3 or 4, characterized in that: the application is applied to the expression analysis of the xylogen SsCSL1 or SsCSL2.
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