CN112342310A

CN112342310A - Internal reference gene PP2A of glehnia littoralis as well as screening method and application thereof

Info

Publication number: CN112342310A
Application number: CN202011358754.6A
Authority: CN
Inventors: 李莉; 周义峰; 李乃伟
Original assignee: Institute of Botany of CAS
Current assignee: Institute of Botany of CAS
Priority date: 2019-07-22
Filing date: 2019-07-22
Publication date: 2021-02-09
Anticipated expiration: 2039-07-22
Also published as: CN112322637B; CN112322773B; CN112342310B; CN112322773A; CN110331228B; CN112322637A; CN110331228A

Abstract

The invention discloses an internal reference gene of glehnia littoralis and a screening method and application thereof, belonging to the technical field of plant genetic engineering. In particular to an internal reference gene of glehnia littoralis and a primer thereof, wherein the internal reference gene is PP 2A; and provides a real-time fluorescent quantitative PCR screening method of the internal reference gene of the glehnia littoralis, and fills the blank that the research field of the glehnia littoralis lacks a proper and universal internal reference gene.

Description

Internal reference gene PP2A of glehnia littoralis as well as screening method and application thereof

The application is a divisional application;

filing date of original application: 2019-07-22;

application No.: CN 201910659220.8;

the invention creates the name: internal reference gene of glehnia littoralis and screening method and application thereof;

Technical Field

The invention belongs to the technical field of plant genetic engineering, and particularly relates to an internal reference gene of glehnia littoralis, a screening method and application thereof.

Background

Glehnia littoralis, an umbelliferae perennial, is a basic source plant of the traditional Chinese medicinal material radix glehniae in China. Radix glehniae, ginseng, radix scrophulariae, salvia miltiorrhiza and codonopsis pilosula are called as five-ginseng, have the effects of nourishing yin, clearing lung, benefiting stomach and promoting fluid, are mainly used for treating diseases such as lung heat, yin deficiency, dry cough, stomach dryness, throat dryness, thirst and the like, are commonly used in clinical and health care, and tender leaves on the overground part of the radix glehniae can be eaten as vegetables, so that the economic value is high.

The glehnia littoralis grows naturally in coastal sandy beach zones, is a genuine medicinal material production area of glehnia littoralis in coastal provinces of China, such as Liaoning, Jiangsu, Shandong, Zhejiang, Fujian, Guangdong and Hainan, and is an important source area of glehnia littoralis commercial medicinal materials. However, for reasons of coastal development and the like, the range of coastal land production areas of phellopterin is gradually reduced or even disappears; in recent years, with the development and utilization of new coastal reclaimed land in China, space and opportunity are provided for the scale recovery of the coastal region production area of the glehnia littoralis, however, the salt tolerance of the existing main planting of the glehnia littoralis is insufficient due to long-term inland cultivation and artificial seed selection, and the difficulty in the recovery planting of the coastal saline-alkali land is great, so that the research on the salt tolerance mechanism of the glehnia littoralis is urgently needed.

In addition, the mechanism of the genuine formation of the glehnia littoralis medicinal material is not clear, and the specific coastal high-salt living environment is probably an important reason influencing the accumulation of the medicinal effective components and the genuine formation of the medicinal material, so that the specific influence mechanism has great research significance. To solve the above two problems, researches on salt tolerance mechanism, breeding, anabolism mechanism of active ingredients, etc. of Eucheuma Gelatinosum are inevitably carried out, and researches in the field of plant molecular biology are required for Eucheuma Gelatinosum.

Gene expression analysis is one of important means in the research of the field of plant molecular biology, and is important in the aspects of searching plant related genes and regulation and control mechanisms, disclosing plant elegance and the like. In the study of gene expression level, reference genes are required to be used as references, such as a real-time fluorescent Quantitative PCR (qRT-PCR) technology, and in order to obtain more accurate and reliable results, the reference genes are required to carry out standardized measurement on the expression level of target genes.

When the relative quantitative expression of genes is analyzed through qRT-PCR, in order to eliminate the influence of the difference of initial templates and the difference of reverse transcription efficiency of different samples, a stable reference gene needs to be introduced to correct the expression result. The internal reference genes are various housekeeping genes, and are stably expressed in the intracellular composition and help to maintain the functions of the cells. The ideal reference gene should satisfy the following conditions: 1) the absence of pseudogenes (pseudogenes) to avoid non-specific amplification of the gene DNA; 2) high or moderate expression, excluding low expression; 3) stably expresses in different types of cells and tissues (such as normal cells and cancer cells), and the expression quantity is approximate without significant difference; 4) the expression level is independent of the cell cycle and whether the cell is activated; 5) the stable expression level is similar to that of the target gene; 6) is not influenced by any endogenous or exogenous factors, such as any experimental treatment measures. As reference genes, there have been conventionally used in plants, for example, 18S rRNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Actin (Actin), etc. However, perfect reference genes almost do not exist, most of the reference genes are not constantly expressed in different environmental conditions, different developmental stages and tissues and organs, and are relatively stable only in a certain test range, and meanwhile, different plants have the most suitable reference genes, and the stability of the reference genes of different tissues of the same plant is also different. Therefore, if a gene is used as an internal reference gene under any test condition, the reliability of the research result is directly affected, and even wrong results are generated, so that the selection of a proper internal reference gene for a specific test condition and a sample type for standardization is very important for obtaining an accurate gene expression result.

Meanwhile, when more and more researchers analyze gene expression, the researchers tend to select a research method for jointly correcting a plurality of internal reference genes, for example, two or more stable internal reference genes are arranged, the obtained result is homogenized, and the target gene is corrected, so that the experimental error can be reduced, and the reliability of the research result is increased. For example, in the study of expression of rice BAHD acyltransferase, researchers evaluated five internal reference genes, and finally determined that the two most stable internal reference genes Ubq5 and Cc55 were used together for the correction of the fluorescent quantitative PCR results (Bartley LE et al. overexpression of a BAHD acyl transferase, OsAt10, water cell wall hydroxyl acid content and dsaccharation. plant physical. 2013,161: 1615-. Similarly, in the expression analysis of HKTS family genes of potassium channel, researchers applied two internal reference genes UBQ5 and 18sRNA for simultaneous correction to increase the stability of the results (Wang R, sting W, Xiao L, Jin Y, Shen L, Zhang W. the rice high-affinity site transporter 1; 1is included in the dissolved and regulated by an MYB-type transport factor. plant physiology.2015, 168(3): 1076-90). Therefore, after screening reference genes according to specific experimental conditions and samples and evaluating stability, evaluating the reference genes with stability in the front has important use value in multi-reference correction.

At present, no research report on the development, screening and stability verification of the internal reference genes of coralline exists, and for the reasons, the development of the stable internal reference genes and primers of coralline has important practical application value in the research of coralline molecular biology and the research of umbelliferae plants.

Disclosure of Invention

1. Problems to be solved

Aiming at the lack of an effective means for carrying out related gene expression analysis on glehnia littoralis in the prior art, the invention provides an internal reference gene of the glehnia littoralis, a screening method and application thereof; through a glehnia littoralis transcriptome sequencing database, internal reference gene development is carried out, four different experimental treatment conditions are selected for stability screening and verification, and the method is suitable for stable internal reference genes and primers thereof under the treatment research of salt stress, drought stress, abscisic acid (ABA) and methyl jasmonate (MeJA) of glehnia littoralis.

2. Technical scheme

In order to solve the problems, the technical scheme adopted by the invention is as follows:

an internal reference gene of glehnia littoralis, wherein the internal reference gene is PP 2A; the nucleotide sequence of the PP2A is SEQ ID NO. 1.

PCR primers for amplifying the above-mentioned reference genes.

Preferably, the size of the amplified fragment of the primer is 100-250 bp.

Preferably, the sequence of the primer pair for amplifying PP2A is SEQ ID NO.15/SEQ ID NO. 16.

The real-time fluorescent quantitative PCR screening method of the internal reference gene of the glehnia littoralis comprises the following steps:

(1) selecting root tissue samples of 4 template glehnia littoralis treated by salt stress, drought stress, abscisic acid and methyl jasmonate respectively;

(2) screening out internal reference genes of the glehnia littoralis by using transcriptome sequencing data of the glehnia littoralis;

(3) designing a reference gene primer of real-time fluorescent quantitative PCR by taking the selected reference gene sequence as a template;

(4) carrying out real-time fluorescence quantitative PCR; and performing statistical analysis on the obtained real-time fluorescence quantitative PCR data through four statistical software of delta Ct, GeNorm, NormFinder and BestKeeper, respectively screening out the optimal reference gene and reference gene combination, and then performing comprehensive ranking analysis through comprehensive analysis software RankAggreg R. It should be noted that, the step (1) may be located after the step (2) or after the step (3); however, step (1) must be preceded by step (4).

Preferably, before the real-time fluorescent quantitative PCR in the step (4), the specificity of the internal reference gene primer is identified by general PCR (using DNA polymerase supplied by Nanjing Novozam Biotech Co., Ltd.: Green Taq Mix; identification conditions: 95 ℃ for 3min, 95 ℃ for 15s, 56 ℃ for 15s, 72 ℃ for 60s/kb, 30 cycles; 72 ℃ for 5 min).

Preferably, in the step (4), the real-time fluorescent quantitative PCR amplification procedure is: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 15s, annealing at 60 ℃ for 15s, and extension at 72 ℃ for 25s, 40 cycles were run.

Preferably, in the step (4), the obtained real-time fluorescence quantitative PCR data is subjected to statistical analysis through four statistical software of delta Ct, GeNorm, NormFinder and BestKeeper, the optimal reference genes and reference gene combinations are respectively screened out, and then comprehensive ranking analysis is performed through comprehensive analysis software RankAggreg R.

Preferably, the real-time fluorescent quantitative PCR screening method of the internal reference gene of the glehnia littoralis comprises the following specific steps:

1) the material and the processing method are as follows: selecting sandculture glehnia littoralis with consistent growth vigor, and respectively carrying out salt stress, drought stress, abscisic acid and methyl jasmonate treatment on different glehnia littoralis by utilizing a NaCl solution with the concentration of 200mM, a PEG (polyethylene glycol) 6000 solution with the mass concentration of 20%, an ABA (abscisic acid) solution with the mass concentration of 100 mu M and a methyl jasmonate solution with the mass concentration of 100 mu M to obtain template glehnia littoralis; sampling the root of each template glehnia littoralis at three time points of which the processing time is 0h, 6h and 24h, and performing three times of repeated sampling on the glehnia littoralis at each time point;

2) RNA extraction and detection: washing all root tissue samples of the glehnia littoralis by using deionized water, drying by using absorbent paper, and quickly freezing by using liquid nitrogen; extracting RNA of the root tissue sample of the glehnia littoralis by using a Trizol Reagent (Takara) kit (the extraction mode is consistent with the content in the kit specification); detecting the integrity of the extracted RNA by electrophoresis, carrying out electrophoresis for 10min by using 1% agarose gel and TAE buffer solution under the voltage of 170V, and observing and analyzing by using a gel imaging system; performing purity detection (OD 260/280 of pure RNA is between 1.8 and 2.2) and concentration quantification on the extracted RNA by using NanoDrop ND-2000;

3) synthesizing cDNA by RNA reverse transcription: taking 0.5-1 μ g RNA sample of Eucheuma Gelatinosum, and using reverse transcription kit PrimeScript^TMThe RT reagent Kit carries out reverse transcription reaction on RNA with g DNA Eraser (Perfect Real Time);

4) screening candidate reference genes: screening candidate internal reference genes of the glehnia littoralis for stability analysis by using glehnia littoralis transcriptome sequencing data and referring to gene homology of a model plant arabidopsis thaliana;

5) designing and detecting a specific primer: designing an internal reference gene Primer for real-time fluorescence quantitative PCR by using the screened candidate internal reference gene sequence as a template and using a site Primer 3web (http:// Primer3.ut. ee /);

the specificity of the primer is preliminarily identified by common PCR by taking cDNA obtained by reverse transcription of a template glehnia littoralis as a template.

6) Establishing a reference gene primer standard curve: establishing a standard curve of each internal reference gene primer: diluting cDNA obtained by reverse transcription into 6 concentration gradients (1, 1/5, 1/25, 1/125, 1/625 and 1/3125) as templates for establishing a standard curve; carrying out real-time fluorescent quantitative PCR by taking the primer as a guide;

7) fluorescent quantitative PCR amplification: carrying out real-time fluorescence quantitative PCR amplification on the internal reference gene by taking cDNA obtained by reverse transcription as a template to obtain a corresponding Ct value; the amplification procedure was: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 15s, annealing at 60 ℃ for 15s, extension at 72 ℃ for 25s, and running for 40 cycles; drawing a standard curve by using the obtained fluorescent quantitative PCR result to obtain the amplification efficiency and slope of each candidate gene, wherein the primer amplification efficiency of the candidate reference gene is 88-108 percent;

8) analyzing the stability of the reference gene by using four statistical analysis methods of delta Ct, GeNorm, NormFinder and BestKeeper;

9) carrying out comprehensive statistical ranking on the candidate internal reference gene stability ranking obtained by the four statistical analysis methods by using a RankAggreg R program package to obtain a comprehensive result;

10) by use of 2^-ΔΔCtThe method is used for analyzing and verifying the expression quantity of target genes by using reference genes, wherein delta Ct is Ct (target gene) -Ct (reference gene), delta Ct is delta Ct (treatment) -delta Ct (control), 2^-ΔΔCtRelative expression amount.

The application of the internal reference gene of the glehnia littoralis in researching the expression level of the target gene of the glehnia littoralis.

3. Advantageous effects

Compared with the prior art, the invention has the beneficial effects that:

(1) in the face of the current situation that the salt tolerance mechanism, breeding, anabolism mechanism of active ingredients and other aspects of the glehnia littoralis are urgently needed to be researched in the existing glehnia littoralis industry, and the gene expression of different parts, different tissues and different conditions of the glehnia littoralis is analyzed by utilizing PCR (polymerase chain reaction), the inventor screens out a plurality of candidate internal reference genes in glehnia littoralis species, discloses an internal reference gene sequence and further screens out an internal reference gene with better stability; fills the blank that a proper and universal reference gene is lacked in the research field of the glehnia littoralis;

(2) according to the invention, a plurality of internal reference genes are screened from coral vegetable species, and real-time fluorescent quantitative PCR primers are designed according to the screening, so that the primer amplification efficiency is high through verification, the specificity is strong (see table 1 and figure 1 for details), the blank that a proper and universal internal reference gene is lacked in the coral vegetable research field is filled, the blank that a proper fluorescent quantitative PCR primer is lacked in the coral vegetable research field is also filled, and an effective internal reference gene correction tool is provided for the coral vegetable gene expression analysis, screening and verification work in the future.

(3) Aiming at the research blank of the coral vegetable species stable reference gene, the invention screens the reference gene on the basis of transcriptome data, analyzes and comprehensively compares the stability of the reference gene by using various statistical methods; different treatment conditions are set, such as salt stress, drought stress and hormone stress, so that the requirements of most physiological researches can be met; the materials are medicinal active parts, so the research is more targeted; under the conditions of salt stress, drought stress and abscisic acid treatment, the PP2A stability is comprehensively ranked fourth (top 30%) in thirteen candidate genes, and the stability is ranked sixth (top 50%) in methyl jasmonate treatment, and comprehensively belongs to a reference gene with better stability under the stress treatment (shown in figure 4), so that the requirement of real-time fluorescence quantitative detection of the expression level of the glehnia littoralis gene is met, the glehnia littoralis gene has better correction capability, researchers can independently use the reference gene in the stress treatment research, can also combine with other reference genes with better stability to jointly correct the expression level of a target gene, more selections are provided for the researchers according to the research content of different requirements to obtain stable and reliable results, the work efficiency of the research and the cost are improved, and the stability of the research results is further ensured, Reliability and repeatability.

(4) The reference gene provided by the invention can also provide reference value for the research of other plants in the Umbelliferae.

Drawings

FIG. 1is a sequence electrophoresis diagram obtained by using Eucheuma Gelatinosum cDNA as template for amplification of multiple primers provided by the present invention; m: the DNA Marker has the strip size sequence of 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp from top to bottom; the genes represented by the numbers from 1 to 13 are respectively PP2A, UBQ10, ACT, EF 1-alpha, GAPDH, alpha-TUB, beta-TUB, PTBP1, EXP1, EXP2, TIP41-like, SAND family and CYP 2;

FIG. 2 is a Ct value distribution diagram of fluorescent quantitative PCR of a plurality of reference genes;

FIG. 3 is a graph of the ranking of the expression stability values (M) of a plurality of candidate reference genes using GeNorm software, the lower the M value, the more stable the gene; wherein FIG. 3A shows NaCl treatment conditions; FIG. 3B shows PEG treatment conditions; FIG. 3C depicts ABA treatment conditions; FIG. 3D shows MeJA processing conditions;

FIG. 4 is a graph of the rankings obtained by the four statistical analysis methods using the RankAggreg R package to obtain a composite ranking; FIG. 4A shows NaCl treatment conditions; FIG. 4B shows PEG treatment conditions; FIG. 4C shows ABA treatment conditions; FIG. 4D shows MeJA processing conditions;

FIG. 5 shows the effect of the candidate reference gene of the present invention in application, which is verified by analyzing the change in the expression level of the target PYL gene of Eucheuma Gelatinosum under four stress treatments; FIG. 5A shows NaCl treatment conditions; FIG. 5B shows PEG treatment conditions; FIG. 5C depicts ABA treatment conditions; fig. 5D shows MeJA processing conditions.

Detailed Description

Herein, the following: the coral vegetable transcriptome sequencing data are as follows: reference is made to Li L, Li M, Qi X, Tang X, Zhou Y. De novo transcription sequencing and analysis of genes related to salt stress in Glehnialite transformation. PeerJ.2018,6: e 5681;

Δ Ct references described herein: silver N, Best S, Jiang J, the in SL.selection of housekeeping genes for gene expression students in human reticulocytes using real-time PCR. BMC Mol biol.2006; 7: 33;

GeNorm reference: vandesoplex J, De Preter K, Pattyn F, Poppe B, Van Roy N, Paepe A, et al, accurate normalization of real-time quantitative RT-PCR data by geometrical averaging of multiple internal control genes, Genome biol.2002; 3: 7;

NormFinder reference: andersen CL, Jensen JL,

TF.Normalization of real-time quantitative reverse transcription-PCR data:a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets.Cancer Res.2004；64:5245–5250；

bestkoeper reference: pfaffl MW, TiChopad A, Prgomet C, Neuvians TP. determination of stable housekeeping genes, differentiated regulated target genes and sample integration BestKeeper-Excel-based tool using pair-wise corerrelations Biotechnol Lett.2004; 26: 509-;

the RankAggreg R package is referred to: pihur V, DattaS, Datta s.rank aggregate, an R package for weighted rank aggregate, BMC Bioinformatics 2009,10: 6;

biological replicates described herein: each individual coral vegetable is one repetition;

the invention is further described with reference to specific examples.

Example 1

This example shows the internal reference gene of Eucheuma Gelatinosum of the present invention. Provides a plurality of internal reference genes of the glehnia littoralis: PP2A (protein phosphatase 2), UBQ10 (ubiquitin 10), ACT (actin), EF1- α (elongation factor), GAPDH (glyceraldehyde-3-phosphate dehydrogenase), α -TUB (α -tubulin), β -TUB (β -tubulin), PTBP1 (polypyrimidine sequence binding protein), EXP1 (expansin 1), EXP2 (expansin 2), TIP41-like (TIP-like 41 protein), SAND family protein, CYP2 (cyclophilin); the nucleotide sequence of the PP2A is SEQ ID NO. 1; the nucleotide sequence of UBQ10 is SEQ ID NO. 2; the nucleotide sequence of the ACT is SEQ ID NO. 3; the nucleotide sequence of the EF 1-alpha is SEQ ID NO. 4; the nucleotide sequence of the GAPDH is SEQ ID NO. 5; the nucleotide sequence of the alpha-TUB is SEQ ID NO. 6; the nucleotide sequence of the beta-TUB is SEQ ID NO. 7; the nucleotide sequence of the PTBP 1is SEQ ID NO. 8; the nucleotide sequence of the EXP 1is SEQ ID NO. 9; the nucleotide sequence of the EXP2 is SEQ ID NO. 10; the nucleotide sequence of the TIP41-like is SEQ ID NO. 11; the nucleotide sequence of the SAND family is SEQ ID NO. 12; the nucleotide sequence of the CYP2 is SEQ ID NO. 13.

Example 2

In this example, fluorescent quantitative PCR primers for amplifying the reference gene of example 1 were provided,

the size of the amplified fragment of the primer is 100-250 bp.

The sequences of the primer pairs for amplifying the PP2A are SEQ ID NO.15/SEQ ID NO. 16; the sequences of a primer pair for amplifying UBQ10 are SEQ ID NO.17/SEQ ID NO. 18; the sequence of a primer pair for amplifying the ACT is SEQ ID NO.19/SEQ ID NO. 20; the sequence of a primer pair for amplifying EF 1-alpha is SEQ ID NO.21/SEQ ID NO. 22; the sequences of the primer pairs for amplifying GAPDH are SEQ ID NO.23/SEQ ID NO. 24; the sequence of a primer pair for amplifying alpha-TUB is SEQ ID NO.25/SEQ ID NO. 26; the sequence of a primer pair for amplifying beta-TUB is SEQ ID NO.27/SEQ ID NO. 28; the sequence of a primer pair for amplifying the PTBP 1is SEQ ID NO.29/SEQ ID NO. 30; the sequences of the primer pair for amplifying the EXP1 are SEQ ID NO.31/SEQ ID NO. 32; the sequences of the primer pair for amplifying the EXP2 are SEQ ID NO.33/SEQ ID NO. 34; the sequences of a primer pair for amplifying TIP41-like are SEQ ID NO.35/SEQ ID NO. 36; the sequence of a primer pair for amplifying the SAND family is SEQ ID NO.37/SEQ ID NO. 38; the sequences of the primer pair for amplifying the CYP2 are SEQ ID NO.39/SEQ ID NO. 40.

Example 3

The embodiment provides a real-time fluorescent quantitative PCR method for screening the internal reference gene of the glehnia littoralis, which comprises the following steps: (1) selecting root tissue samples of the glehnia littoralis which are respectively treated by salt stress, drought stress, abscisic acid and methyl jasmonate;

(2) screening out candidate internal reference genes of the glehnia littoralis by using transcriptome sequencing data of the glehnia littoralis;

(3) the selected candidate internal reference genes are taken as templates (namely PP2A, UBQ10, ACT, EF 1-alpha, GAPDH, alpha-TUB, beta-TUB, PTBP1, EXP1, EXP2, TIP41-like, SAND family and CYP 2; the specific sequences can be seen in sequence tables SEQ ID NO. 1-SEQ ID NO.13), and primers for real-time fluorescence quantitative PCR of each internal reference gene are designed and obtained (see the embodiment 2 for details);

(4) and (3) performing real-time fluorescence quantitative PCR, performing statistical analysis on the obtained real-time fluorescence quantitative PCR data (Ct value), and screening out the optimal reference gene.

Example 4

This example differs from example 3 only in that: the step (1) is positioned after the step (2).

Example 5

This example differs from example 3 only in that: the step (1) is positioned after the step (3).

Example 6

This example differs from example 3 only in that: the step (1) is a material and processing method: selecting sand-culture glehnia littoralis with consistent growth vigor, and respectively treating different glehnia littoralis with a NaCl solution with the concentration of 200mM, a PEG 6000 solution with the mass concentration of 20%, an ABA solution with the mass concentration of 100 mu M and a methyl jasmonate solution with the mass concentration of 100 mu M to obtain a template glehnia littoralis; the root of each template glehnia littoralis was sampled at three time points at treatment times of 0h, 6h, and 24h, and three biological replicates were sampled at each time point.

Example 7

The real-time fluorescent quantitative PCR screening method of the internal reference gene of the glehnia littoralis provided in the embodiment comprises the following steps: (1) selecting root tissue samples of the glehnia littoralis which are respectively treated by salt stress, drought stress, abscisic acid and methyl jasmonate;

(3) the primers of real-time fluorescence quantitative PCR of each reference gene are designed and obtained by taking the selected reference gene as a template (namely PP2A, UBQ10, ACT, EF 1-alpha, GAPDH, alpha-TUB, beta-TUB, PTBP1, EXP1, EXP2, TIP41-like, SAND family and CYP 2; the specific sequence can be seen in SEQ ID NO. 1-SEQ ID number 13 of a sequence table) (see the embodiment 2 for details);

(4) preliminarily identifying the specificity of the reference gene primer by common PCR;

using a DNA polymerase provided by tokyo kezan biotechnology limited: green Taq Mix; identification conditions: 95 ℃ for 3min, 95 ℃ for 15s, 56 ℃ for 15s, 72 ℃ for 60s/kb for 30 cycles; 5min at 72 ℃;

(5) and (3) performing real-time fluorescence quantitative PCR, performing statistical analysis on the obtained real-time fluorescence quantitative PCR data (Ct value), and screening out the optimal reference gene.

Example 8

This example differs from example 1 in that: the step (4) is as follows: carrying out real-time fluorescence quantitative PCR; carrying out quantitative analysis on the obtained real-time fluorescence quantitative PCR data through a standard curve, and screening out an optimal reference gene and an optimal reference gene combination;

wherein, the real-time fluorescent quantitative PCR amplification procedure comprises the following steps: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 15s, annealing at 60 ℃ for 15s, and extension at 72 ℃ for 25s, 40 cycles were run.

Example 9

The embodiment provides a method for screening the stability of an internal reference gene of glehnia littoralis, which comprises the following specific steps:

1) the material and the processing method are as follows: selecting sand-culture glehnia littoralis with consistent growth vigor, and respectively treating different glehnia littoralis with a NaCl solution with the concentration of 200mM, a PEG 6000 solution with the mass concentration of 20%, an ABA solution with the mass concentration of 100 mu M and a methyl jasmonate solution to obtain a template glehnia littoralis;

the roots of each template glehnia littoralis were sampled at three time points at treatment times of 0h, 6h, and 24h, and three biological replicates (one replicate per individual) were sampled for each time point.

2) RNA extraction and detection: washing the extracted root (medicinal part) tissue sample of the glehnia littoralis with deionized water, drying the tissue sample with absorbent paper, and quickly freezing the tissue sample with liquid nitrogen; extracting RNA of the root tissue sample of the glehnia littoralis by using a Trizol Reagent (Takara) kit (the extraction mode is consistent with the content in the kit specification);

detecting the integrity of the extracted RNA by electrophoresis, carrying out electrophoresis for 10min by using 1% agarose gel and TAE buffer solution under the voltage of 170V, observing by using a gel imaging system, and analyzing; performing purity detection (OD 260/280 of pure RNA is between 1.8 and 2.2) and concentration quantification on the extracted RNA by using NanoDrop ND-2000;

3) synthesizing cDNA by RNA reverse transcription: taking 0.5-1 μ g RNA sample and using reverse transcription kit PrimeScript^TMThe RT reagent Kit carries out reverse transcription on RNA with g DNA Eraser (Perfect Real Time) to obtain cDNA.

5) designing and detecting a specific primer: designing an internal reference gene Primer for real-time fluorescence quantitative PCR by using the screened internal reference gene as a template and using a site Primer 3(http:// Primer3.ut. ee /);

the specificity of the primers was identified by ordinary PCR using cDNA obtained by reverse transcription as a template, using DNA polymerase provided by limited Biotech, Nanjing Novozam: green Taq Mix; identification conditions: 95 ℃ for 3min, 95 ℃ for 15s, 56 ℃ for 15s, 72 ℃ for 60s/kb for 30 cycles; 5min at 72 ℃.

6) Establishing a reference gene primer standard curve: establishing a standard curve of each internal reference gene primer: diluting cDNA obtained by reverse transcription into 6 concentration gradients (1, 1/5, 1/25, 1/125, 1/625 and 1/3125) as templates for establishing a standard curve; performing real-time fluorescent quantitative PCR by taking the primer as a guide, and drawing a standard curve;

7) and (3) PCR amplification: carrying out real-time fluorescence quantitative PCR amplification on the internal reference gene by taking cDNA obtained by reverse transcription as a template to obtain a corresponding Ct value;

the amplification procedure was: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 15s, annealing at 60 ℃ for 15s, extension at 72 ℃ for 25s, and running for 40 cycles; and (3) obtaining the amplification efficiency and the slope of each candidate gene by using the obtained data result, wherein the amplification efficiency of the primers is between 88 and 108 percent.

Example 10

This example shows the development and screening process of the candidate internal reference genes of glehnia littoralis and the stability evaluation of the candidate internal reference genes under different stress treatments. The germplasm resources of the template glehnia littoralis in the embodiment are from Fujian plain pond coastal areas and are cultivated in a germplasm resource garden of the plant research institute of Chinese academy of sciences of Jiangsu province, namely Nanjing Zhongshan;

(1) the material and the processing method are as follows:

pre-treatment: collecting seedling (the underground part is about 3-6cm in length) of Eucheuma Gelatinosum in nursery, transplanting into flowerpot cleaned with river sand, irrigating Hoagland nutrient solution, culturing in illumination incubator at 26 deg.C and 22 deg.C, 14h/10h day and night circulation, and relative humidity of 75%, and culturing for three months;

sampling: selecting sand-cultured glehnia littoralis with consistent growth vigor, and respectively treating different glehnia littoralis with a NaCl solution with the concentration of 200mM, a PEG 6000 solution with the mass concentration of 20%, an ABA solution with the mass concentration of 100 mu M and a methyl jasmonate solution with the mass concentration of 100 mu M to obtain a template glehnia littoralis;

sampling the root of each template glehnia littoralis at three time points of 0h, 6h and 24h of processing time, performing three times of biological repeated sampling (one is repeated for each single plant) on the glehnia littoralis at each time point, washing the collected root tissue sample of the template glehnia littoralis by using deionized water, drying the sample by using absorbent paper, quickly freezing the sample by using liquid nitrogen, and placing the sample in a refrigerator at the temperature of-80 ℃ for extracting RNA;

(2) RNA extraction and cDNA synthesis: extracting RNA of root tissue samples of 4 different templates of glehnia littoralis by using a Trizol Reagent (Takara) kit (the extraction mode is consistent with the content in the kit specification);

(3) mu.g of RNA sample was sampled using reverse transcription kit PrimeScript^TMThe RT reagent Kit with gDNA Eraser (Perfect Real Time) performs reverse transcription to obtain cDNA (the reverse transcription mode is consistent with the content in the Kit instruction).

(4) Internal reference gene screening and specific primer design: according to the glehnia littoralis transcriptome sequencing database information obtained by the research team, the gene homology of a model plant Arabidopsis thaliana is referred, and homologous genes in the glehnia littoralis are screened out to be used as candidate internal reference genes (shown in a sequence table SEQ ID NO. 1-SEQ ID NO. 13); using the screened candidate internal reference gene as a template, designing a Primer by using Primer 3web (http:// Primer3.ut. ee /) according to a gene sequence, and amplifying the fragment with the size of 100-250bp (see Table 1 for details);

using cDNA obtained by reverse transcription of a template glehnia littoralis as a template, identifying the specificity of a primer through common PCR, observing a band of a PCR product under a gel imaging system after electrophoresis, selecting the primer with correct size of the band, single band and no primer dimer as shown in figure 1, and recovering and sequencing the PCR product to further determine whether the target candidate gene amplification product is correct.

(6) Establishing a reference gene primer standard curve: and (3) preparing respective standard curves for the primers of each pair of reference genes, and calculating the amplification efficiency of the corresponding primers. Diluting cDNA reverse transcription of the template glehnia littoralis into 6 gradients (1, 1/5, 1/25, 1/125, 1/625 and 1/3125) by taking 5 times as a template for establishing a standard curve; qRT-PCR is carried out by using a qTOWER2.2 Real-Time PCR System instrument, the amplification efficiency and the slope of each candidate gene are obtained by using the obtained data result, and the amplification efficiency of the primer is between 88 and 108 percent (detailed in Table 1);

TABLE 1 candidate internal reference genes of Gynura corallina and corresponding primers

(7) qRT-PCR: carrying out real-time fluorescence quantitative PCR amplification on the internal reference gene by taking cDNA obtained by reverse transcription as a template to obtain a corresponding Ct value; SYBR Premix ExTaqTM II (TliRNaseH Plus) (Takara) fluorescent quantitation kit, amplification program: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 15s, annealing at 60 ℃ for 15s, extension at 72 ℃ for 25s, and running for 40 cycles; and collecting a melting curve signal, wherein the generated melting curve is a single peak, which indicates that the specificity of the primer is good, the Ct value data is automatically read by a real-time fluorescence quantitative PCR instrument, and the distribution range of the Ct value is shown in figure 2. The Ct value is inversely proportional to the expression quantity of the gene, the larger the Ct value is, the lower the expression quantity of the gene is, and conversely, the smaller the Ct value is, the higher the expression quantity of the gene is represented;

(8) analysis of stability of internal reference gene: analyzing the stability of the reference gene by using four statistical analysis methods of delta Ct, GeNorm, NormFinder and BestKeeper;

(9) the RankAggreg R program package is used for carrying out the comprehensive analysis of the stability of the internal reference genes. Δ Ct is ranked according to the degree of dispersion of the Ct value data, the smaller the mSD (mean StdDev), the higher the gene stability. Table 2 is a case of ranking the stability of multiple candidate reference genes under four different stress treatments using the Δ Ct method. FIG. 3 shows that GeNorm measures the stability of a gene according to the average variation M, and the smaller the M value, the better the stability. Table 3 shows that NormFinder is based on analysis of variance, and the stability of the internal reference gene is directly evaluated. Table 4 shows that BestKeeper judges the stability of the reference gene by the standard mutation coefficient (SD), and the smaller the SD, the more stable the gene. As shown in fig. 4, finally, according to the stability ranking obtained by the four methods, a RankAggreg R program package is used for making a comprehensive ranking, and under four treatment conditions of coral vegetable salt stress, drought stress, ABA and MeJA, the CYP2 gene comprehensive stability is ranked first and has the best stability;

(10) method for calculating relative expression amount of gene and application 2^-ΔΔCtThe method comprises the following specific steps: Δ Ct (target gene) -Ct (reference gene), Δ Δ Ct ═ Δ Ct (treatment) - Δ Ct (control), 2^-ΔΔCtRelative expression amount.

TABLE 2 stability analysis of multiple candidate reference genes by Delta Ct method

TABLE 3 stability analysis of multiple candidate reference genes under four stresses by the NormFinder method

TABLE 4 stability analysis of multiple candidate reference genes under four stresses by BestKeeper method

Example 11

This example is to verify the effect of the application of the candidate reference gene provided by the present invention. PYL (abscisic acid receptor analogous protein) is selected as a target gene, and is a key component in an ABA signal transduction pathway, and the expression level of the PYL is generally increased after the PYL is stressed. According to the research of NaCl differential transcriptome sequencing of glehnia littoralis, the expression level of the glehnia littoralis PYL gene is definitely up-regulated under the salt treatment. Therefore, in this embodiment, the reference genes with better and poorer stability provided by the present invention (fig. 4) are respectively selected, and the qRT-PCR method is used to verify and compare the expression levels of the glehnia littoralis PYL gene under salt treatment, drought treatment, ABA treatment and MeJA treatment. The method comprises the following specific steps:

(1) experimental materials and treatment methods: the germplasm resource of the template glehnia littoralis comes from Fujian Tan coast, and is cultivated in a germplasm resource garden of the plant institute of Chinese academy of sciences of Jiangsu province and Nanjing Zhongshan;

the stress treatment method comprises the following steps: collecting seedling (underground part length about 3-6cm) of Eucheuma Gelatinosum in nursery garden, transplanting into flowerpot cleaned with river sand, irrigating Hoagland nutrient solution, culturing in illumination incubator at 26 deg.C and 22 deg.C, 14h/10h day and night circulation, relative humidity 75%, culturing for three months, selecting healthy plants with similar growth vigor, soaking flowerpot of Eucheuma Gelatinosum in 200mM NaCl (salt stress), 20% PEG 6000 (drought stress), and 100 μ M ABA, taking root (medicinal part) tissue samples of glehnia littoralis in 100 mu MMeJA solution in 0, 6 and 24 hours respectively, washing the tissue samples with deionized water, sucking the samples with absorbent paper, quickly freezing the samples with liquid nitrogen, placing the samples in a refrigerator at the temperature of-80 ℃ for RNA extraction, wherein each sampling has three biological repetitions (one repetition per single plant);

detecting the integrity of the extracted RNA by electrophoresis, carrying out electrophoresis for 10min by using 1% agarose gel and TAE buffer solution under the voltage of 170, observing by using a gel imaging system, and analyzing; performing purity detection (OD 260/280 of pure RNA is between 1.8 and 2.2) and concentration quantification on the extracted RNA by using NanoDrop ND-2000;

(3) mu.g of RNA sample was sampled using reverse transcription kit PrimeScript^TMCarrying out reverse transcription by an RT reagent Kit with gDNA Eraser (Perfect Real Time) to obtain cDNA (the reverse transcription mode is consistent with the content in the Kit instruction);

(4) and (3) a target gene PYL sequence for verifying the application effect of the reference gene and a specific primer design: obtaining a PYL nucleic acid sequence (shown as a sequence table SEQ ID NO.14) by referring to a PYL gene AT5G05440 of a model plant Arabidopsis thaliana according to information of a phellopterin transcriptome sequencing database obtained by a research team, designing a Primer by using a Primer 3web (http:// Primer3.ut. ee /) according to the gene sequence, wherein a PYL fluorescence quantitative upstream Primer sequence is shown as a sequence table SEQ ID NO.41, a downstream Primer sequence is shown as a sequence table SEQ ID NO.42, and a target fragment is 137 bp;

(5) establishing a standard curve of a target gene PYL primer: diluting cDNA reverse transcription of the template glehnia littoralis into 6 gradients (1, 1/5, 1/25, 1/125, 1/625 and 1/3125) by taking 5 times as a template for establishing a standard curve;

qRT-PCR is carried out by using a qTOWER2.2 Real-Time PCR System instrument, the amplification efficiency and the slope of the gene are calculated by using the obtained data result, the amplification efficiency of the PYL primer is 92.22 percent, and the slope is 0.9974;

(6) qRT-PCR: taking cDNA obtained by reverse transcription as a template, and simultaneously carrying out real-time fluorescence quantitative PCR amplification on the selected and verified reference gene and the target gene to obtain a corresponding Ct value;

SYBR Premix ExTaqTM II (TliRNaseH Plus) (Takara) fluorescent quantitation kit, amplification program: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 15s, annealing at 60 ℃ for 15s, extension at 72 ℃ for 25s, and running for 40 cycles; and collecting a melting curve signal, wherein the generated melting curve is a single peak, which indicates that the specificity of the primer is good, and the Ct value data is automatically read by a real-time fluorescence quantitative PCR instrument. The Ct value is inversely proportional to the expression quantity of the gene, the larger the Ct value is, the lower the expression quantity of the gene is, and conversely, the smaller the Ct value is, the higher the expression quantity of the representative gene is; method for calculating relative expression amount of gene and application 2^-ΔΔCtThe method comprises the following specific steps: Δ Ct (target gene) -Ct (reference gene), Δ Δ Ct ═ Δ Ct (treatment) - Δ Ct (control), 2^-ΔΔCtRelative expression amount.

(7) Different reference genes are selected to carry out qRT-PCR verification to verify the application effect of the reference genes provided by the invention:

as shown in fig. 4, the results of the comprehensive stability evaluation of the reference genes, under the NaCl treatment condition, four reference genes were applied, wherein CYP2 and EXP1 were reference genes with relatively good proven stability, and GAPDH and UBQ10 were reference genes with relatively poor stability, respectively; the qRT-PCR result shows that the expression level of PYL is changed more greatly when the PYL is treated for 24h by NaCl through correction by GAPDH and UBQ10 compared with the control, and the result is relatively stable by using CYP2 and EXP1 with good stability, so that the possibility of false positive result can be reduced to a certain extent by using the reference gene with good stability (see figure 5A).

Under the PEG treatment condition, four reference genes are respectively applied, wherein CYP2 and ACT are the reference genes with relatively good stability, and beta-TUB and UBQ10 have poor stability; as can be seen from the results of qRT-PCR, the PYL expression levels varied greatly at 24h in PEG treatment using the relatively unstable β -TUB and UBQ10 genes, and the use of UBQ10 showed a trend opposite to that of the use of other internal reference genes, while the use of CYP2 and ACT, which are highly stable, showed a consistent trend and good reproducibility (see FIG. 5B).

Under the ABA treatment condition, four reference genes are respectively applied, wherein CYP2 and ACT are reference genes with relatively good stability, and beta-TUB and UBQ10 are poor in stability; as seen in the qRT-PCR result, the PYL expression amount after ABA treatment is inconsistent and is in an opposite trend at 24h by using unstable beta-TUB and UBQ10 genes, while the PYL expression trend is consistent and the result is more reliable by using CYP2 and ACT with better stability (see FIG. 5C).

Under MeJA treatment conditions, four internal reference genes are respectively applied, wherein CYP2 and EXP2 are internal reference genes with better evaluation relative stability, and beta-TUB and EF 1-alpha have poorer relative stability; the qRT-PCR result shows that PYL expression trends obtained by applying the four genes are almost consistent, which shows that the repeatability of each internal reference gene is good and the result is reliable under MeJA treatment (see figure 5D).

According to the embodiment, the high-stability reference gene developed by the invention has a good effect on target gene expression analysis, clearly shows the expression change trend of the target gene PYL after various stresses are treated for 6h and 24h, and if CYP2 is used alone, the test operation is simple and convenient, the cost is reduced, and meanwhile, the reliability is realized, and the high-stability reference gene can also be combined with other reference genes for correction.

Sequence listing

<110> institute of plant of Chinese academy of sciences of Jiangsu province

<120> internal reference gene of glehnia littoralis and screening method and application thereof

<160> 42

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1764

<212> DNA

<213> PP2A

<400> 1

atgtcgaatg ttgaagagcc attgtaccca atagctgtgt tgattgatga gctgaagaat 60

gacgatattc agttgcgact taattctatt agaaggcttt cgactattgc tcgtgctctc 120

ggggaggaac ggacaagaag ggaattgatt ccgtttttga gcgagaacaa tgatgatgac 180

gatgaggttc ttcttgcaat ggccgaagag ttgggagtat ttattcctta tgttggaggt 240

gtggaacacg ctcatgtttt gctccccact cttgaaaatc tttgcactgt tgaagaaact 300

tgtgtcaggg acaaagccgt ggagtcgctt tgtagaattg gatctcagat gagggagagt 360

gatttaaatg actattttgt tcctctagtg aagaggttgg cagcaggtga atggtttact 420

gctcgagttt ctgcttgtgg attgtttcac atcgcatatc ccagtgcacc agagacgttg 480

aaaaccgaat tgcgatcgat atacagtcag ttgtgtcagg atgacatgcc tatggttagg 540

agatctgctg ctacaaattt ggggaagttt gctgcaacca ttgaacctgc tcatctcaag 600

actgatatta tgtcaatatt tgaggatctt acccaggatg atcaagattc tgttcggttg 660

ctagctgttg aaggatgtgc tgctcttgga aagttgttgg aacctcaaga ttgtgtagca 720

catatacttc ccgttattgt caacttttcc caggataagt cgtggcgtgt tcgttacatg 780

gttgccaatc agctctatga attatgtgaa gctgtgggac ctgaacctac caggacggaa 840

ttagttcctg catatgtgcg acttcttcga gataatgaag ccgaagtacg tatagctgct 900

gctggcaaag tcaccaagtt ctgcagaatt cttaatcctg agctagcaat tcagcacatc 960

cttccatgtg tgaaggagtt atcatctgat tcttctcagc atgttagatc tgctctggcc 1020

tcggttataa tgggcatggc tcctgtatta ggaaaggaag caacaatcga gcagcttctt 1080

ccaatatttc tttcccttct gaaggatgaa tttcctgatg tgcgactcaa cattattagc 1140

aagctagatc aagttaatca ggttattgga atagatcttt tgtcccagtc tctgctacca 1200

gccattgttg agcttgcagg ggataggcat tggagagttc gactagcaat tattgagtat 1260

attccgctat tagctagcca attaggagta ggcttcttcg atgataagct tggtactctc 1320

tgtatggagt ggctaaagga taaggtttgc tcgattcgag atgctgctgc tgataacttg 1380

aagcgccttg cagaagaatt tggcccagag tgggcgatgc agcatatcat tccacaggtg 1440

ttggacatga ttaataaccc tcattacctg tatcggatga caatcctgcg tgctatatct 1500

ctacttgctc ctgtcatggg cccagaaatc acatgttcta aactgctacc tgttctagtt 1560

actgcatcaa aggacagagt ggcgaacatc aaattcaatg tggcaaaggt gctgcaatcc 1620

cttattactg tagtcgatca gtctgtggtg gagtcaacaa ttcgcccttg tctggtggaa 1680

cttagcgagg acccggatgt cgatgttcgt ttctttgcca atgaagcact tcatgctatt 1740

gatcatgaca tgatgtcaag ctag 1764

<210> 2

<211> 612

<212> DNA

<213> UBQ10

<400> 2

atgcaaattt ttgtcaagac cctcactggg aagacgatta ccttggaagt agagagctcg 60

gatacaattg acaatgtgaa ggcgaaaatt caagacaagg aaggaatccc tcctgatcag 120

caaaggttga tatttgctgg caagcagtta gaggatggaa ggactttggc cgattacaat 180

attcagaagg aatcaaccct tcatttggtg ctgaggctga ggggtggaat gcagattttt 240

gtgaagactt tgacggggaa aaccatcacc ctggaggtgg agagctcgga caccattgat 300

aatgtcaaag caaaaataca ggacaaagaa ggtatcccac cagaccagca gaggctgatt 360

tttgctggca agcagcttga ggatggtcgt acacttgcag actacaacat ccaaaaggaa 420

tctacccttc acttggtgct acgtctcaga ggagggatgc agatctttgt caagactttg 480

actggtaaga ccattactct ggaggttgaa agctcggata ccattgataa tgtgaaggca 540

aagatccaag acaaggaggg aatcccacca gatcagcaga ggttgatttt tgctggaaag 600

cagttggaag at 612

<210> 3

<211> 1134

<212> DNA

<213> ACT

<400> 3

atggccgatg ctgaggatat ccagccccta gtttgtgaca atggaactgg aatggtgaag 60

gctggttttg ctggtgatga tgctcccaga gcagtattcc ccagtattgt tggtaggccc 120

agacatactg gtgttatggt cgggatgggg cagaaggatg cctatgttgg tgatgaagcc 180

caatcgaaga gaggtattct taccttgaaa tatccgattg agcacggtat tgtgagtaat 240

tgggatgaca tggagaaaat ttggcatcat accttttaca atgagcttcg agttgctcct 300

gaggagcacc cagttctttt gactgaagcg cctctcaatc ccaaggccaa cagggagaaa 360

atgactcaga ttatgtttga gacgtttaat gttcctgcta tgtatgttgc catccaggct 420

gttctttctc tgtatgcaag tggtcgtact actggtattg tgctggattc tggtgatggt 480

gtgagccata ctgtaccaat ttacgaagga tatgcccttc cccatgccat tctccgtctc 540

gaccttgctg gtcgtgatct cactgattct ctcatgaaga tcttaacgga gagaggttac 600

atgttcacca ccactgctga gcgggaaatt gttcgtgaca tgaaggagaa acttgcctat 660

gttgctcttg actacgagca agagcttgaa acctcaaaga gtagctcttc tgtggaaaag 720

aactatgaat tgcctgacgg acaagttatt acaattggag ctgagagatt ccgttgccca 780

gaagtcctgt tccagccgtc tctgatcggg atggaagctg ctggaatcca tgaaaccact 840

tacaactcca tcatgaagtg tgatgtcgat atcagaaagg atctctatgg aaacatagtg 900

ctcagtggtg gttcaacaat gttccccggt attgcagatc gtatgagcaa ggaaattact 960

gcccttgcac ccagcagcat gaagatcaaa gttgttgcac cacccgagag aaaatacagt 1020

gtctggattg gaggatccat tcttgcatct ctcagcacct tccaacagat gtggatatcc 1080

aagggcgaat atgacgagtc tggcccatca atcgtgcaca ggaagtgttt ttaa 1134

<210> 4

<211> 1341

<212> DNA

<213> EF1α

<400> 4

atgggtaagg aaaagattca tatcagtatt gtggtcattg gccatgtcga ctctggaaag 60

tctaccacaa ctggtcatct tatctacaag ctaggtggta tcgacaagcg tgtgattgaa 120

aggttcgaga aggaagctgc tgagatgaac aaacgttcat tcaagtacgc atgggttctt 180

gacaagctta aggctgagcg tgaacgtggt attaccattg atattgctct ttggaagttt 240

gagactacca agtactactg cacagttatt gatgctccag ggcatcgtga tttcattaag 300

aacatgatta ctggaacttc tcaggctgat tgtgctgtcc tgatcattga ctccaccact 360

ggaggttttg aagctggtat ctctaaggat gggcaaactc gtgagcatgc tttgcttgca 420

tttacacttg gtgtcaagca gatgatctgt tgctgcaaca agatggatgc tacaaccccc 480

aagtactcca agtctagatt cgaagaaatt gtgaaggagg tttcttctta tttgaagaag 540

gttgggtaca accccgacaa aattgctttc attcccatct ctggatttga gggtgacaac 600

atgattgata ggtctaccaa ccttgactgg tacaagggac caactcttct tgaagctctt 660

gaccagatct ctgagcccaa gagaccctca gacaagcccc ttcgtctccc acttcaggat 720

gtttacaaga ttggaggtat tggaactgtg ccagtgggac gtgttgaaac tggtgtgatc 780

aagcctggta tggttgtgac ttttggtcct tcagggttga ccactgaagt caagtctgtt 840

gagatgcatc atgaggctct ccaggaggct cttcctggtg acaatgttgg attcaatgtt 900

aagaatgttg ctgttaagga tctcaagcgt ggatatgttg cctccaactc caaggatgat 960

cccgccaaag aggctgccaa tttcactgct caagttatca tcatgaacca ccctggtcag 1020

atctcaaatg gttatgctcc agtgcttgat tgccatacct gtcacattgc tgttaagttt 1080

gctgaaatcc aaaccaagat tgatcgtcga tctggtaagg agatcgagaa ggagcccaag 1140

tttttgaaga atggtgatgc tggatttgtt aagatgattc caaccaagcc catggtggtc 1200

gagaccttta tgacctaccc tcctcttgga aggtttgctg taagggacat gaggcagact 1260

gttgctgtgg gagtcatcaa gagtgtggag aagaaggaac ctaccggagc caaggtcaca 1320

aaggcggcaa tcaagaagaa a 1341

<210> 5

<211> 1010

<212> DNA

<213> GAPDH

<400> 5

atggcaccaa tcaagatcgg aatcaacggt ttcggaagaa ttggacgatt ggttgctaga 60

gttgttctgc aaagagatga tgttgagctt gttgctgtta acgatccatt tatctcaact 120

gattacatga catacatgtt caagtatgac agtgttcacg gtgcatggaa gcatcatgaa 180

ctcaaggtta aggatgagaa gactcttctc ttcggtgcga agcctgttgc tgtctttggt 240

tgcaggaacc cagaggagat cccatgggct agcactggtg cagagtatat tgttgaatcc 300

actggtgtct tcactgacaa ggaaaaggct gctgcacatt tgaagggagg tgcaaagaag 360

gtcatcatat ctgccccaag caaagatgct ccaatgtttg tcgttggtgt caatgagaag 420

gaatacaagt ctgacctcca cattgtttcc aatgctagtt gcacaacaaa ttgccttgct 480

cccctagcta aggtgatcaa tgataggttt ggcattgttg aggggcttat gacaactgtt 540

cattcaatca ctgccacaca aaaaactgtt gacggacctt ctgcgaagga ctggagaggt 600

ggaagagctg cttcattcaa catcattcct agcagcactg gagctgccaa ggctgttgga 660

aaagtgctac cttctctgaa tgggaagttg accggaatgt cattccgagt tcctactgtg 720

gatgtctcag ttgttgatct cactgtcagg ctggaaaaga aggctactta tgaacaaatt 780

aaagctgcca ttaaggagga gtctgaggga aagcttaagg gaatcttggg ttacactgaa 840

gatgatgtgg tttccacaga ctttgtgggt gacagcaggt caagcatctt tgatgccaaa 900

gctggaattg ctctaaatga caactttgtc aagcttgttt cgtggtatga caacgaatgg 960

ggatacagca cccgagtggt tgacttgatc gttcatatgg catctgttca 1010

<210> 6

<211> 1356

<212> DNA

<213> α-TUB

<400> 6

atgagggagt gcatttcagt tcacatcggt caggccggta ttcagatcgg taacgcttgc 60

tgggaacttt actgcctcga gcacggcatt cagcctgatg gccaaatgcc aagtgacaaa 120

actgtcggtg gaggtgatga tgctttcaac actttcttta gtgaaactgg tgctggaaag 180

catgtgcctc gagcaatctt tgtggatctt gagcccactg tcattgatga agtgaggact 240

ggaacatatc gtcagctctt tcatcctgaa cagctgatta gcggaaaaga agatgcagct 300

aacaactttg ctcgtggaca ctataccatt ggaaaggaga ttgttgatct ttgcctggat 360

cgtatcagga agcttgctga caattgcact ggtctccagg gtttccttgt ttttaatgct 420

gttggaggag gcactggttc tggtttgggt tcccttcttc tggaacgtct ctccgtggac 480

tatggcaaaa agtcaaaact tggattcact gtttatcctt caccacagat ctctacctct 540

gttgttgagc cttacaacag tgtgctttcg acccactcac ttttggagca caccgatgtt 600

tctgtgctgc tggataatga ggctatatat gatatttgca agcgttccct tgacattgag 660

cgacccacct ataccaacct taatcgattg gtttctcagg tcatttcctc tttgaccgct 720

tccttgaggt ttgatggagc cttgaatgtt gatgtgactg agttccagac taatctggtg 780

ccatacccaa ggatccactt catgctttct tcttatgccc ctgttatctc cgctgagaag 840

gcctaccatg aacagctatc tgttgcagag atcaccaaca gtgcatttga gccctcttct 900

atgatggcca agtgtgatcc tcgccatgga aagtacatgg cttgctgtct gatgtaccga 960

ggtgatgtgg tgcccaaaga tgtgaatgca gctgttggta ccattaagac caagcgcacc 1020

atccagtttg ttgattggtg cccaactggt tttaagtgcg gtatcaacta tcaggcccca 1080

actgttgttc caggtggtga tcttgccaaa gtgcagagag ctgtatgcat gatctcaaat 1140

tcgaccagtg ttgcagaggt tttctcacgc atagacacta aatttgacct aatgtactca 1200

aagagggctt tcgttcactg gtatgttggc gagggtatgg aagaaggtga attctctgaa 1260

gcacgtgagg atcttgctgc ccttgagaag gactatgagg aggttggtgc agagtctgct 1320

gagggggatg atgaggacga gggagaagat tactga 1356

<210> 7

<211> 1344

<212> DNA

<213> β-TUB

<400> 7

atgagagaaa ttcttcacat tcagggcggt caatgtggaa accagatcgg agcaaagttc 60

tgggaagtga tctgcgccga gcacgggatc gattcgacag ggcgttacca gggagacact 120

gaaattcaat tggagcgaat caatgtgtat tacaatgaag ccagttctca gaggtatgtt 180

cccagggctg tgcttatgga tctggagcct ggtactatgg atagtctccg atctggaccc 240

tacggtcaga tcttcaggcc tgataacttt gtgtttggtc aatctggtgc tggtaataat 300

tgggccaaag gtcactatac tgaaggtgct gagttaatcg actcggtgct tgatgttgtg 360

aggaaggaag ctgagaattg tgactgtctt caagggtttc aggtgtgtca ttcactcggt 420

ggtggaaccg gatctggaat gggtacactt ctgatttcaa aaatcagaga ggagtatcct 480

gaccgtatga tgcttacttt ctcagttttc ccatcaccca aggtgtctga tactgtggtt 540

gagccttata atgccactct ttctgttcat caacttgttg aaaatgctga tgagtgcatg 600

gttttggaca atgaggctct ttatgacatt tgcttccgca ccttgaagct taccacacct 660

agctttggtg atctaaacca cttgatttcg gccactatgt ctggtgttac atgctgcttg 720

cgtttccctg gtcagttgaa ctccgatctc aggaagttgg ctgtaaatct cattcccttc 780

cccaggttgc acttctttat ggttggattt gcacctctta cctcccgtgg ttcccagcaa 840

taccgtgcat tgagtgtacc tgagcttacc cagcagatgt gggattcaaa gaacatgatg 900

tgcgcagctg atccccgcca tggtagatac ttgacagctt ctgctgtgtt cagaggaaag 960

atgagcacta aagaggttga tgagcagatg atcaacgtcc agaacaagaa ctcttcctac 1020

tttgttgaat ggatcccaaa caatgtgaag tcaactgttt gtgacatccc accaactggt 1080

ctgaagatgg cttcaacctt cattgggaat tcaacttcaa ttcaagagat gtttaggcgt 1140

gtgagtgagc agttcacagc tatgttcagg aggaaagctt tcttgcattg gtataccggt 1200

gagggcatgg acgagatgga gttcactgag gctgagagca acatgaatga tcttgtttcg 1260

gagtaccagc agtaccagga tgccactgct gacgaggagg gtgactattt cgaagaagaa 1320

gaagaggatg gccaagacat gtaa 1344

<210> 8

<211> 1163

<212> DNA

<213> PTBP1

<400> 8

atgtcgaatt caaatcagcc tcaatttcga tacacacaga ctccttctaa agtgcttcac 60

ttgcgtaact tgccttggga gtgtattgaa gaagagctcg tcgagctttg caggcctttt 120

ggtaagatcg ttaacaccaa gtgcaatgtc ggcgctaatc gcaatcaagc cttcgttgaa 180

tttgtggatc ttaatcaggc cattaatatg gtttcatatt atgcttcatc atcagaacct 240

gcatctgttc ggggtaaaca tgtttatata cagtattcaa acagacatga aattgtcaac 300

aacaagggtc caggtgatgt tccgggaaat gtcttgctgg taaccattga gggtgtagaa 360

gccggtgatg taagcattga tgtgattcac ttggtcttct cggcttttgg atttgtgcac 420

aagattgcta cttttgagaa ggcagcaggt tttcaggcac taatccagtt tactgatgct 480

gagactgctc tttcagcaag ggaagcttta gatggcagaa gtattccaag gtacttgctt 540

ccagaacatg ttggttcttg caatctgcgc atctcatatt cagctcacac agatctaaac 600

atcaagttcc aatcacaccg tagccgggac tatacaaatc catatcttcc tgttaatgca 660

actgcaattg agggatttgt ccagcctgtt gtaggtcctg acggaaagaa aaaagaaccg 720

gagagtaatg tacttcttgc ctcaattgaa aataggatct atgatgtcac tgtagatgtt 780

cttaacacgg tattctctgc atttggcacg gttcagaaaa ttgctatatt cgagaagaac 840

gcgacaactc aggctctaat tcagtatcct gatgtcaaca ctgccgccgt agctaaagat 900

gctctagagg gacactgcat atatgatggt ggctactgta agcttcatat atcatactct 960

cgtcatactg atctcaatgt aaaggccttc agcgataaaa gtagggatta tacagtacca 1020

gagtccggtt ttgctgctgg tctgcctgct ggagcaacag tctggcagaa tcctcatgct 1080

gctgctccgg tctttattgg gagcgaattt gctagtatca attatgggca gcctcaaggc 1140

tctcccggtc aaggacctcc tgg 1163

<210> 9

<211> 1179

<212> DNA

<213> EXP1

<400> 9

atgtcgaaaa ctgaagacga agaggagcgc cggagaaagt acgaggaagc tctcgaagtc 60

aaatctctcc gccgtatcat cagcgcctat ctcaattacc cagaggctgc agaggaggac 120

ttgaaaagat atgaaagatc ttatagaagg cttccaccaa cccataaggg tctcctgtct 180

caccttcctg taaaatatcg aagactgcga aggtgtatat ctaagaattc atattttata 240

tttgaaatgc taaaggcatt tgaacctccc cttgatctga gccaagacct tgacatatgt 300

gaacaagatc cgcagaatat cttagacgat accaaagaaa ccaattattt ttcttgtggg 360

tctgcatcaa ccagtaaaac aggatgtcat ccagggtgca atgaagctgt cagtggagag 420

gaggggagcg tgttattagg atctcccaag gaggagaaac ttgggctttt tattgattcg 480

gacaccggga gccgtcatat tttggaatgt gatgccacag cagataaagc tggtaacaac 540

ggtgttaaga ttaaaaaaac ttcacactct aatgcagact ccaataataa tgagaaactt 600

gggcttttca ttgagtctga cactgggaac cgtcatgttt tggaatgtga taccaaagca 660

gatgaggctg gtaacaacgg tgttaggata caggaaactt cgtactctaa tgcagactcc 720

aattataatg tgtcttcatc tcctgattgg ttggatccat cactgcagtc gcatgttcct 780

ctagttgatg tagataaggt tcgatgtatt ataagaaata ttgtaagaga ttgggctgca 840

gagggacaac aagaacgtga tcagtgctat acgcctattc ttgaagagct taaatcacaa 900

tttcctaatc gaagtaaagg gagccctcct gcatgtttag ttccgggtgc tggacttggt 960

agactggctt tggaaatttc atgtcttggt tttgcaagcc aaggaaatga attttcatac 1020

tatatgatga tctgctcgag ttttattctt aaccaagcgg aaagggctaa tgaatggact 1080

atccatcctt ggattcatag caattgcaat tcactttctg acagtgacca gcttcgtcct 1140

atttcaatac cagatattca tcctgccagg aataactga 1179

<210> 10

<211> 981

<212> DNA

<213> EXP2

<400> 10

atggcaagag gagagtgggg gtactataat ggaagaacga aatggtgttc ttatagaaga 60

accactttga ttatttgttc aattaacatt ggtgttgctc tttatgttct tcacactctt 120

tataactctc tttacaccta cccttttaat gatcctcaaa aagctgctag gtacactcct 180

gatcagatta ggaaaatgga agaatcaaat gatattagaa aagcctcaca acccactgaa 240

cttattaaat tggtgaatga aataaggaag gattttttac aagaagagaa gagggttgat 300

ttgccatcaa atttgaaaca ccaggtaatt gatgagattg tggaattatt gaggagcttg 360

aagtcctcca atgcgactgt tcaaaatgaa gcagttgaaa gatggcgcaa gcaaaaaata 420

agagaagcta gaggggtggc tcggggagat attctgaatc caaacattct gccaaaggaa 480

gcaaaaattc ttgcaagaac gttgaagtct cgctgggatg agtttagaga agaaatcggt 540

ctctggatac ctgttgcaat cgttaacaag gaacatgatg acaagcctga gggtgaagaa 600

gagtttgaca gcgaaatatt agccggcaga cagcttcctc ccgagtgcca tactgaactt 660

catacagatt atggtggggc agctgttcgc tggggcctta cccaccataa agagagcgct 720

tatgattgtt gtcaagcttg tctggatcaa gccaaaaatg caagagaagg cgaaaagcgc 780

tgcaatatat gggtgtactg cccttcagag ggtggatgtt actcaccaga tatatatgaa 840

cacaaacagc aagaatgctg gctgaaatat gacgagaaac cccaagtaag ctttaaggac 900

aaatactccg aatcattcag aaactcgcat ccaaatgttc cactggttgt tccatgggta 960

gctgggattg taagtgtata a 981

<210> 11

<211> 684

<212> DNA

<213> TIP41-like

<400> 11

atggtttttg gggaaagttc attggttctc aagcacttga agagcgatgt aaagattcat 60

ttcaacgcat ttgattctct agttggttgg aagcaggaaa aattaccacc agttgaggtc 120

cctgcagcag caaaatggaa atttagaagc aaacctttcc agcaggtgat attagattat 180

gactacacat ttacaacacc atattgtgga agtgaaactg ttgagaaaaa ctcagagagg 240

gatacaatct ctgatgaagg cagttgcaag cttcgttggg aggactgcga ggaacgaatt 300

aatttgactg cacttgcatc aaaagagcct attctcttct atgatgaggt gatcttctat 360

gaagatgaat tggctgatag tggagtgtcg cttttaacag taaaagtgag agtgatgcca 420

agctgttggt ttcttctctt gcgtttttgg cttagagttg atggtgtgct tatgcgttta 480

agggacacac gcatccattg catttttggt gagggtaaaa caccagttat tctgagagaa 540

tgttgctgga gagaggccac atttcaagca ctagcttcta aaggatatcc ttctgattgt 600

gctgcgtata ttgatccaag cagcatcggc caaagacttc ctatcatttt gcataagacc 660

caaaagctta taattcctga ttaa 684

<210> 12

<211> 2007

<212> DNA

<213> SAND family

<400> 12

atgttaccag aagatgatgc caactcctca tcagaaaccg actcaattga ccaaaaccct 60

aaccctacca cttcaattga ccaatctctc gacgctattg aaggtcaatt aacctctatt 120

tcactcaatc accaccactc aaaaccccca tttcaccctc ctcttcccca aaatatcgat 180

acattgcctt cccattcgca ttcgcaactc caacaaccac cagcaccagc tgaaaatctc 240

caaaatatcg atacattacc ttcccattca cattcgaaac tccaacaagt agcagtagct 300

gaaaatatcg gttcattacc ttcggattca cattcgcggg ccggacaagt agttgaaaat 360

tctggacaag tggatatatt aggttcggat tcctatacga aagtagagaa ggaagtagtt 420

ggaaattcga gaggcgaagg agtgttgtgg aggaataatt cggatgtgga agttgaggtg 480

gaagggcaag ggagtccgag tagtagtgga tatgctggag gaaaggggac tagtagtagt 540

ggtagtagtg gtataagtgg ttcaggtatc gaggagatta gtggcggcga tgacgaggtg 600

gttaacagga gtggttcttt tggtggtagt gtggattccg agtgggtccc tgggaaacgg 660

catgttaatg aagatgatgc ttctgtttca tggaggaaaa ggaagaagca tttttttatc 720

ttaagccatt ccggaaaacc aatatattca agatatggag atgaacacag actagcagga 780

ttttcagcaa ctttgcaagc catcatttcc ttcgtggaga atgggggaga tcgcgtgaag 840

ttggttaggg cgggaaaaca ccaggtggtt tttcttgtta aaggaccaat atatctagtt 900

tgcataagct gtacagaaga gcctcatgaa tccctcagtg aacaactgga acttctttat 960

ggccagatga tacttattct gacaaagtct ataaatagat gctttgagaa gaatccgaaa 1020

tttgatatga cacctttgct tgggggaaca gatgctgtgt tctcttctct catccactcg 1080

tttagttgga accctgccac ttttcttcat gcctactctt gtcttcccct tgcttatcca 1140

acaaggcaag ccgccggtgc catattgcag gatgttgctg agtcaggtgt cctcttcgcg 1200

atattaatgt gtaaacacaa ggtcatcagt ctggttggtg cacaaaaagc gtctcttcat 1260

cccgatgata tgctcttgct tgccaacttt gtgatgtcat ctgaatcatt caggacatct 1320

gaatctttct ctccaatctg tcttccgaga tacaatccaa tggcattttt atatacttat 1380

gtgtattatc ttgatgctga tacttatttg atgttgctta ctgctaatcc tgatgcattt 1440

catcgtctaa aagattggag gatccgtatc gaaatggtcc ttctgaagtc aaatgttctt 1500

aatgaagctc aaaggtcgat gttggatggt ggcatgcgtg tcgaagatgt gcctgttaat 1560

ccatctcctc gctcgggatc tttgtcatct catttaggtc agcctagacc tccaccggac 1620

tctgcagatg ggtgtaaggc actgttaggt ggtcctgctg ggctttggca cttcgtttac 1680

cgcagtatat atctagatca atatgtatct tctgagttct catcaccgat caacacccct 1740

aaacaacaga aaagattata tagagcatat cagaagctgt atacttctat gcatgatata 1800

gaacttggtc ctcacaaaac ccagtttaga agggacgaga actatgttct actctgctgg 1860

gttactcagg attttgaact ttatgcagca tttgatcctc tagcagacaa ggcactggct 1920

ataaagacat gcaaccgagt atgccaatgg gttaaagatg tggaaaatga agtttttttg 1980

ttgggagcaa gccccttttc atggtga 2007

<210> 13

<211> 1491

<212> DNA

<213> CYP2

<400> 13

atgtcatcta tctacgtatc ggagcctccg accaaaggca aagtttcgct caagacaaca 60

tacggtccat tggacataga gctatggccg aaagaggctc ctaaagctgt gcgcaacttc 120

gttcagctct gtctcgaagg ttattatgat gacacaattt ttcatcgtat aattaagtca 180

tttatggtcc aaggtggtga tcctactggc actggcaaag gtggtgaaag tatatatgga 240

ggtacatttt ctgatgagtt ccattcccgc cttaggttca accacagggg cttggttgca 300

tgtgcgaatg ctggatcacc aaattcaaat gggagtcagt tttttataac cttggatcgt 360

tgtgattggc ttgatcgtaa acataccatt ttcggaaagg taactggaga ttcactatac 420

aatctcttaa acttttccga ggttgaaact gataaggatg atcgaccagt agaatctccc 480

cctaaattga tttcagttga ggtgatatgg aacccttttg atgatattgt tccaagggca 540

gcccctgcta aagctttggt ctcctcaaat gatagtggca acagagatac aaaaaggaaa 600

gcgtcaaaaa agctaaactt gctttcattt ggagaagaag ctgaagaaga ggaaaaagaa 660

ttggcagctg tgaagatgaa aattagaagt agtcacgatg tattagatga tcctcgtttg 720

ctgaaggaag acggttcaac cagcaaaccg agtgaatcag aagccaaagc tatgaaagat 780

atgcagttaa gtgttagaga agctctaagt tcaaagaagg atgaatcatg gaaagagacg 840

cacagtaaat tttcagagac ccttcctgat agcgatgacg atgaggccaa ctttgataac 900

aggatgcgat tacaaatact taagaaaaga aaggagcttg gagatcattc aactaagcaa 960

aagtcacaca atgcgagttc aagtccaaga aaccgtgaac gctcctattc tcctcccagg 1020

tcaaatgcca aaaattccga tgatcaacca aaagtggaga agttggcttt gaagaaggga 1080

ataggatcag aagccagggc cgagcgtttg gccaatgcgg atgtggactt gcaactgttg 1140

ggagaagctg aacgagaaag gcagttacaa aagcagaaga agcgccgatg tcatgggcac 1200

gaagaggatg tgctagcaaa gcttgagaag ttcaaggcca ccatgtcctc caaatctgtt 1260

ggagctgatg gtgaatctgg aggacacaag gaagaggact tgtctgactg gacaaaagtt 1320

aagctgaagt ttgaacctca atccgggaag gataatatga ctcgcaccga gaatgtgaat 1380

gactatgtat ttcatgatcc tcttctggag aagggaaaag agaagttcaa caaaatgcaa 1440

gcaaagcaaa agcgacgaga acgagaatgg gctggaaagt cacttacata a 1491

<210> 14

<211> 603

<212> DNA

<213> PYL

<400> 14

atgccttcat ctcttcgacg tcaaagaatc tacaacaccg attacaacta ccaaaaaaaa 60

tcacataata atgtgcatac aattattcca ccacctctag ggcttccgga aaatatcaaa 120

caccaccaca cacacatgat tagctataac caaagcagct ccgccgtggt ccaaaccatc 180

tccgcaccta tatccaccgt gtggtccatt attcgaaact tcgaaaagcc acaaatttac 240

aagcacttta tcaaaagctg ccatgtcatc cacggggatg gatccgtagg tagtctccgg 300

gaagtccacg tcatctctgg cctgcccgcg gtgtcgagca tcgagaggct agatattctt 360

gatgaagagt gtcacattat tagttttagt gtagtaggag gtgatcatcg gttgaacaat 420

tatcggtcag tgacgacgct acacaagacg gagaccggaa atggtacggt ggtggtggag 480

tcatatgtcg tggatgtgcc ggaggggaat actaaagagg agacttgtgg atttgcaaat 540

acaattgtga catgtaattt acattctttg gcaaagattg ctgaaaactt gagtaataag 600

taa 603

<210> 15

<211> 20

<212> DNA

<213> Artificial sequence

<400> 15

gcaaccattg aacctgctca 20

<210> 16

<211> 20

<212> DNA

<213> Artificial sequence

<400> 16

gaacacgcca cgacttatcc 20

<210> 17

<211> 20

<212> DNA

<213> Artificial sequence

<400> 17

tgaggggtgg aatgcagatt 20

<210> 18

<211> 20

<212> DNA

<213> Artificial sequence

<400> 18

tgcaagtgta cgaccatcct 20

<210> 19

<211> 20

<212> DNA

<213> Artificial sequence

<400> 19

accatcacca gaatccagca 20

<210> 20

<211> 20

<212> DNA

<213> Artificial sequence

<400> 20

cttcgagttg ctcctgagga 20

<210> 21

<211> 20

<212> DNA

<213> Artificial sequence

<400> 21

aaggatgggc aaactcgtga 20

<210> 22

<211> 20

<212> DNA

<213> Artificial sequence

<400> 22

agcaattttg tcggggttgt 20

<210> 23

<211> 20

<212> DNA

<213> Artificial sequence

<400> 23

accttctttg cacctccctt 20

<210> 24

<211> 20

<212> DNA

<213> Artificial sequence

<400> 24

gctgtctttg gttgcaggaa 20

<210> 25

<211> 20

<212> DNA

<213> Artificial sequence

<400> 25

acaactttgc tcgtggacac 20

<210> 26

<211> 20

<212> DNA

<213> Artificial sequence

<400> 26

tgcctcctcc aacagcatta 20

<210> 27

<211> 20

<212> DNA

<213> Artificial sequence

<400> 27

caggtacact caatgcacgg 20

<210> 28

<211> 20

<212> DNA

<213> Artificial sequence

<400> 28

cgcaccttga agcttaccac 20

<210> 29

<211> 20

<212> DNA

<213> Artificial sequence

<400> 29

ggctacggtg tgattggaac 20

<210> 30

<211> 20

<212> DNA

<213> Artificial sequence

<400> 30

tcggcttttg gatttgtgca 20

<210> 31

<211> 20

<212> DNA

<213> Artificial sequence

<400> 31

agccctcctg catgtttagt 20

<210> 32

<211> 20

<212> DNA

<213> Artificial sequence

<400> 32

acgaagctgg tcactgtcag 20

<210> 33

<211> 20

<212> DNA

<213> Artificial sequence

<400> 33

acacccatat attgcagcgc 20

<210> 34

<211> 20

<212> DNA

<213> Artificial sequence

<400> 34

catgatgaca agcctgaggg 20

<210> 35

<211> 20

<212> DNA

<213> Artificial sequence

<400> 35

agagtgatgc caagctgttg 20

<210> 36

<211> 20

<212> DNA

<213> Artificial sequence

<400> 36

gcctctctcc agcaacattc 20

<210> 37

<211> 21

<212> DNA

<213> Artificial sequence

<400> 37

ggaccatttc gatacggatc c 21

<210> 38

<211> 20

<212> DNA

<213> Artificial sequence

<400> 38

aagcgtctct tcatcccgat 20

<210> 39

<211> 22

<212> DNA

<213> Artificial sequence

<400> 39

gccactatca tttgaggaga cc 22

<210> 40

<211> 20

<212> DNA

<213> Artificial sequence

<400> 40

cgttgtgatt ggcttgatcg 20

<210> 41

<211> 20

<212> DNA

<213> Artificial sequence

<400> 41

tgccttcatc tcttcgacgt 20

<210> 42

<211> 20

<212> DNA

<213> Artificial sequence

<400> 42

catgtgtgtg tggtggtgtt 20

Claims

1. The internal reference gene of the glehnia littoralis is characterized in that: the reference gene is PP 2A; the nucleotide sequence of the PP2A is SEQ ID NO. 1.

2. PCR primers for amplifying the reference gene of claim 1.

3. The PCR primer for amplifying reference gene according to claim 2, characterized in that: the size of the amplified fragment of the primer is 100-250 bp.

4. The PCR primer for amplifying reference gene according to claim 3, wherein: the sequence of the primer pair for amplifying the PP2A is SEQ ID NO.15/SEQ ID NO. 16.

5. The real-time fluorescent quantitative PCR screening method of the internal reference gene of the glehnia littoralis is characterized by comprising the following steps: the method comprises the following steps:

(2) screening candidate internal reference genes of the glehnia littoralis by using transcriptome sequencing data of the glehnia littoralis;

(3) designing a reference gene primer of real-time fluorescent quantitative PCR by taking the selected candidate reference gene sequence as a template;

(4) carrying out real-time fluorescence quantitative PCR; and performing statistical analysis on the obtained real-time fluorescence quantitative PCR data, and respectively screening out the optimal reference gene and the optimal reference gene combination.

6. The real-time fluorescent quantitative PCR screening method of internal reference genes of glehnia littoralis as claimed in claim 5, which is characterized in that: before the real-time fluorescent quantitative PCR in the step (4), the specificity of the reference gene primer is identified through common PCR.

7. The real-time fluorescent quantitative PCR screening method of internal reference genes of glehnia littoralis as claimed in claim 5, which is characterized in that: in the step (4), the real-time fluorescent quantitative PCR amplification procedure is as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 15s, annealing at 60 ℃ for 15s, and extension at 72 ℃ for 25s, 40 cycles were run.

8. The real-time fluorescent quantitative PCR screening method of internal reference genes of glehnia littoralis as claimed in claim 5, which is characterized in that: in the step (4), the obtained real-time fluorescence quantitative PCR data is subjected to statistical analysis through four statistical software of delta Ct, GeNorm, NormFinder and BestKeeper, the optimal reference genes and reference gene combinations are respectively screened out, and then comprehensive ranking analysis is performed through comprehensive analysis software RankAggreg R.

9. The real-time fluorescent quantitative PCR screening method of internal reference genes of glehnia littoralis as claimed in claim 5, which is characterized in that: the method comprises the following specific steps:

1) the material and the processing method are as follows: selecting sand-cultured glehnia littoralis with consistent growth vigor, and respectively treating different glehnia littoralis with a NaCl solution with the concentration of 200mM, a PEG 6000 solution with the mass concentration of 20%, an ABA solution with the mass concentration of 100 mu M and a methyl jasmonate solution with the mass concentration of 100 mu M to obtain a template glehnia littoralis;

sampling the root of each template glehnia littoralis at three time points of which the processing time is 0h, 6h and 24h, and performing three times of biological repeated sampling on the glehnia littoralis at each time point;

2) RNA extraction and detection: washing root tissue samples of all the template glehnia littoralis by using deionized water, drying by using absorbent paper, and quickly freezing by using liquid nitrogen; extracting RNA of a coral vegetable root tissue sample;

detecting the integrity of the extracted RNA by electrophoresis;

performing purity detection and concentration quantification on the extracted RNA by using NanoDrop ND-2000;

3) synthesizing cDNA by RNA reverse transcription: carrying out reverse transcription reaction on RNA of the template glehnia littoralis to obtain cDNA;

4) screening candidate reference genes: utilizing glehnia littoralis transcriptome sequencing data and gene homology of a reference model plant arabidopsis thaliana to screen out candidate internal reference genes of the glehnia littoralis for stability analysis;

5) designing and detecting a specific primer: designing an internal reference gene primer for real-time fluorescent quantitative PCR by taking the screened candidate internal reference gene sequence as a template;

taking cDNA obtained by reverse transcription as a template, and preliminarily identifying the specificity of the primer by common PCR;

6) establishing a reference gene primer standard curve: establishing a standard curve of each internal reference gene primer: diluting cDNA obtained by reverse transcription into 6 concentration gradients to be used as a template for establishing a standard curve; carrying out real-time fluorescent quantitative PCR by taking the primer as a guide;

7) fluorescent quantitative PCR amplification: carrying out real-time fluorescence quantitative PCR amplification on the internal reference gene by taking cDNA obtained by reverse transcription as a template to obtain a corresponding Ct value;

the amplification procedure was: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 15s, annealing at 60 ℃ for 15s, extension at 72 ℃ for 25s, and running for 40 cycles; drawing a standard curve by using the obtained fluorescent quantitative PCR result to obtain the amplification efficiency and the slope of each candidate gene;

10) by use of 2^-ΔΔCtThe method is used for analyzing and verifying the expression quantity of the target gene by using the reference gene.

10. The use of the internal reference gene of Eucheuma Gelatinosum as claimed in claim 1 in the study of expression level of target gene of Eucheuma Gelatinosum.