CN110331228A - The reference gene and its screening technique of glehnia littoralis and application - Google Patents

Abstract

The invention discloses the reference gene of glehnia littoralis and its screening technique and applications, belong to field of plant genetic project technology.More particularly to the reference gene and its primer of glehnia littoralis, the reference gene is PP2A, UBQ10, ACT, EF1- α, GAPDH, α-TUB, β-TUB, PTBP1, EXP1, EXP2, TIP41-like, SAND family, CYP2；And a kind of real-time fluorescence quantitative PCR screening technique of glehnia littoralis reference gene is provided, fill up the blank for lacking suitable, general reference gene in glehnia littoralis research field.

Description

The reference gene and its screening technique of glehnia littoralis and application

Technical field

The invention belongs to field of plant genetic project technology, reference gene and its sieve more specifically to glehnia littoralis Choosing method and application.

Background technique

Glehnia littoralis is Umbelliferae herbaceous plant, is used as medicine with root, name Radix Glehniae is China's traditional Chinese medicine, in " legendary god of farming's sheet Grass warp " in be listed in top grade, property slightly sweet flavor is slightly cold, there is nourishiing yin to clear away the lung-heat, and reinforcing stomach reg fluid effect cures mainly lung-heat yin-deficiency cough caused by dryness, stomach The diseases such as dry dry throat and mouth, clinical to commonly use in health care, at home and abroad market annual requirement is up to 8000 tons or more.

Mainly there are two distributed areas for glehnia littoralis wild resource: one is East Asia glehnia littoralis distributed area, secondly being North America coral Coral dish distributed area, China are one of the main growth regions in East Asia glehnia littoralis distributed area.In China, Liaoning, Jiangsu, Shandong, Zhejiang The coastal seashore in river, Fujian, Guangdong and Hainan is the genunie medicinal materials producing region of glehnia littoralis, is Radix Glehniae commodity in history The important sources of medicinal material.Glehnia littoralis is grown in the sand ground at original seabeach, and well developed root system expands naturally during the growth process Therefore and the good economic kind fixed the sand of seashore rhizomes is formed,.Two are faced in the industrial research of glehnia littoralis at present mainly Problem, problem first is that need carry out the research of glehnia littoralis Mechanisms of Salt Resistance, early period due to coastal development etc., above-mentioned glehnia littoralis Coastal Genuine producing area range is gradually reduced, or even disappears；So the major production areas of glehnia littoralis is gradually upcountry shifted, such as in The hinterland such as illiteracy, Hebei, the main artificial growth for being increasingly becoming glehnia littoralis plant producing region, still, plant in inland artificial growth In producing region, glehnia littoralis growing way is limited, and quality is irregular.Although in recent years, as China coast newly encloses tideland for cultivation the exploitation in soil It utilizes, provides space and opportunity for the recovery of the coastal Genuine producing area scale of glehnia littoralis, however the main plantation of existing glehnia littoralis Due to by inland cultivation and artificial selecting, causing salt tolerance insufficient for a long time, it is very big to restore plantation difficulty in coastal salt-soda soil.Problem Second is that the mechanism that is formed of glehnia littoralis medicinal material genuineness is not yet clear, and its specific coastal living environment with high salt is perhaps shadow The major reason of the accumulation of its effective component and the formation of medicinal material genuineness is rung, specific influencing mechanism has very big research significance.It solves Certainly above-mentioned two problems, it is inevitably each to the anabolism mechanism etc. of the Mechanisms of Salt Resistance of glehnia littoralis, breeding, active constituent A aspect is studied, and is both needed to carry out glehnia littoralis the research of field of plant molecular biology.

Gene expression analysis is one of important means in field of plant molecular biology research, deeply to seeking plant Related gene and Regulation Mechanism, announcement plant profoundness etc. are most important.When studying gene expression dose, need to use interior Join gene as reference, such as real-time fluorescence quantitative PCR (Quantitative real time PCR, qRT-PCR) technology, to obtain More accurately believable measurement must be standardized to the expression of target gene as a result, requiring reference gene.

When analyzing the expression of gene relative quantification by qRT-PCR, in order to eliminate different sample original template differences, reversion The influence for recording efficiency variance, needs to introduce stable reference gene and is corrected to expression of results.Reference gene is usually various Housekeeping gene forms stability expression in the cell, helps to maintain the function of cell.Ideal reference gene should meet with Lower condition: 1) pseudogene (Pseudogene) is not present, to avoid gene DNA non-specific amplification；2) height or moderate expression, Exclude low expression；3) stablize and be expressed in different types of cell and tissue (such as normal cell and cancer cell), and its expression quantity It is approximate, no difference；4) whether expression activates unrelated with cell cycle and cell；5) its stable expression It is horizontal similar to target gene；6) it is not influenced by any endogenous or extrinsic factor, if not by any experiment process measure Influence.Through common such as 18S rRNA, glyceraldehyde 3-phosphate dehydro-genase (GAPDH), actin (Actin) in plant As reference gene.However perfect reference gene there's almost no, most reference gene is in different environmental condition, different Expression is not invariable in stage of development and histoorgan, can only be relatively stable in certain trial stretch, meanwhile, Different plants have most suitable internal reference crt gene and between same plant different tissues reference gene stability there is also Difference.So directly affecting result of study if the reference gene under blindness is using a kind of gene as any experimental condition The degree of reliability, in addition generate mistake as a result, therefore selecting suitable internal reference base for specific experimental condition and sample type It is most important for obtaining accurate gene expression results because being standardized.

And currently, about the exploitation of glehnia littoralis reference gene, screening and stability verifying aspect research report, add Above-mentioned reason, to the stabilization reference gene of glehnia littoralis and its exploitation of primer, in glehnia littoralis molecular biology research and umbrella All there is important practical application value in the research of shape section plant.

Summary of the invention

1. to solve the problems, such as

For the effective means for carrying out related gene expression analysis to glehnia littoralis is lacked in the prior art, the present invention provides coral The reference gene and its screening technique of coral dish and application；By glehnia littoralis transcript profile sequencing data storehouse, carries out reference gene and open Hair chooses four kinds of different experiments treatment conditions and carries out stability screenings and verifying, be suitable for glehnia littoralis salt stress, drought stress, Stabilization reference gene and its primer under abscisic acid (ABA), methyl jasmonate (MeJA) treatment research.

2. technical solution

To solve the above-mentioned problems, the technical solution adopted in the present invention is as follows:

The reference gene of glehnia littoralis, the reference gene are PP2A, UBQ10, ACT, EF1- α, GAPDH, α-TUB, β- One of TUB, PTBP1, EXP1, EXP2, TIP41-like, SAND family, CYP2 or more than one；The PP2A's Nucleotides sequence is classified as SEQ ID NO.1；The nucleotides sequence of the UBQ10 is classified as SEQ ID NO.2；The nucleotides sequence of the ACT It is classified as SEQ ID NO.3；The nucleotides sequence of the EF1- α is classified as SEQ ID NO.4；The nucleotides sequence of the GAPDH is classified as SEQ ID NO.5；The nucleotides sequence of the α-TUB is classified as SEQ ID NO.6；The nucleotides sequence of the β-TUB is classified as SEQ ID NO.7；The nucleotides sequence of the PTBP1 is classified as SEQ ID NO.8；The nucleotides sequence of the EXP1 is classified as SEQ ID NO.9；Institute The nucleotides sequence for stating EXP2 is classified as SEQ ID NO.10；The nucleotides sequence of the TIP41-like is classified as SEQ ID NO.11；Institute The nucleotides sequence for stating SAND family is classified as SEQ ID NO.12；The nucleotides sequence of the CYP2 is classified as SEQ ID NO.13.

For expanding the PCR primer of any of the above-described kind of reference gene.

Preferably, the amplified fragments size of the primer is 100~250bp.

Preferably, the primer pair sequence for expanding PP2A is SEQ ID NO.15/SEQ ID NO.16；For expanding The primer pair sequence for increasing UBQ10 is SEQ ID NO.17/SEQ ID NO.18；Primer pair sequence for expanding ACT is SEQ ID NO.19/SEQ ID NO.20；Primer pair sequence for expanding EF1- α is SEQ ID NO.21/SEQ ID NO.22；With In amplification GAPDH primer pair sequence be SEQ ID NO.23/SEQ ID NO.24；For expanding the primer pair sequence of α-TUB For SEQ ID NO.25/SEQ ID NO.26；Primer pair sequence for expanding β-TUB is SEQ ID NO.27/SEQ ID NO.28；Primer pair sequence for expanding PTBP1 is SEQ ID NO.29/SEQ ID NO.30；For expanding the primer of EXP1 It is SEQ ID NO.31/SEQ ID NO.32 to sequence；Primer pair sequence for expanding EXP2 is SEQ ID NO.33/SEQ ID NO.34；Primer pair sequence for expanding TIP41-like is SEQ ID NO.35/SEQ ID NO.36；For expanding The primer pair sequence of SAND family is SEQ ID NO.37/SEQ ID NO.38；Primer pair sequence for expanding CYP2 is SEQ ID NO.39/SEQ ID NO.40。

The real-time fluorescence quantitative PCR screening technique of the reference gene of above-mentioned glehnia littoralis, comprising the following steps:

(1) the 4 kinds of template corals crossed respectively through salt stress, drought stress, abscisic acid and methyl jasmonate treatment are chosen The root tissue sample of dish；

(2) glehnia littoralis transcript profile sequencing data is utilized, the reference gene of glehnia littoralis is filtered out；

(3) using the reference gene sequence picked out as template, the reference gene primer of real-time fluorescence quantitative PCR is designed；

(4) real-time fluorescence quantitative PCR is carried out；It is counted by tetra- kinds of Δ Ct, GeNorm, NormFinder, BestKeeper Software, it is for statistical analysis to obtained real-time fluorescence quantitative PCR data, optimal reference gene and reference gene are filtered out respectively Combination, then Comprehensive Analysis Software RankAggreg R carries out overall ranking analysis.It should be noted that above-mentioned steps (1) can After being located at step (2), it can also be located at after step (3)；But step (1) must be positioned at before step (4).

Preferably, before carrying out the real-time fluorescence quantitative PCR in the step (4), regular-PCR identification reference gene is first passed through The specificity of primer (uses the archaeal dna polymerase provided by Nanjing Vazyme Biotechnology Co., Ltd.: Green Taq Mix；Mirror Fixed condition: 95 DEG C of 3min, 95 DEG C of 15s, 56 DEG C of 15s, 72 DEG C of 60s/kb, 30 circulations；72℃ 5min).

Preferably, in the step (4), real-time fluorescence quantitative PCR amplification program are as follows: 95 DEG C of initial denaturation 5min；95 DEG C of changes 15s, 60 DEG C of annealing 15s, 72 DEG C of extension 25s of property, run 40 circulations.

Preferably, soft by tetra- kinds of statistics of Δ Ct, GeNorm, NormFinder, BestKeeper in the step (4) Part, it is for statistical analysis to obtained real-time fluorescence quantitative PCR data, optimal reference gene and reference gene group are filtered out respectively It closes, then Comprehensive Analysis Software RankAggreg R carries out overall ranking analysis.

Preferably, the real-time fluorescence quantitative PCR screening technique of the reference gene of the glehnia littoralis, the specific steps are as follows:

1) material and processing method: choosing the consistent sand culture glehnia littoralis of growing way, and it is molten to be utilized respectively the NaCl that concentration is 200mM Liquid, 6000 solution of PEG (polyethylene glycol), 100 μM of ABA (abscisic acid) solution and the 100 μM of MeJA that mass concentration is 20% (methyl jasmonate) solution respectively carries out at salt stress, drought stress, abscisic acid and methyl jasmonate different glehnia littoralises Reason obtains template glehnia littoralis；Handle the time be 0h, 6h, for 24 hours when three time points to the root of every plant of template glehnia littoralis into Row sampling, each time point carry out the duplicate sampling of biology three times to glehnia littoralis；

2) RNA is extracted and is detected: the root tissue sample for all glehnia littoralises taken being rinsed using deionized water, is inhaled Water paper blots, liquid nitrogen flash freezer；The RNA of glehnia littoralis root tissue sample is extracted using Trizol Reagent (TakaRa) kit (extracting mode is consistent with the content in kit specification)；Utilize the integrality for the RNA that electrophoresis detection extracts, 1% agar Sugared gel, TAE buffer, electrophoresis 10min under 170V voltage are observed and are analyzed using gel imaging system；Use NanoDrop ND-2000 is to RNA progress purity detecting (OD260/280 of pure RNA is between 1.8~2.2) of extraction and determining for concentration Amount；

3) RNA reverse transcription synthesizes cDNA: the RNA sample of 0.5-1 μ g glehnia littoralis being taken to use reverse transcription reagent box PrimeScript^TMRT reagent Kit with g DNA Eraser (Perfect Real Time) inverts RNA Record reaction；

4) glehnia littoralis transcript profile sequencing data, the gene of reference model plant Arabidopsis thaliana candidate reference gene screening: are utilized It is homologous to filter out the candidate reference gene for carrying out the glehnia littoralis of stability analysis；

5) design and detection of specific primer: using the candidate reference gene sequence filtered out as template, website is utilized Primer 3web (http://primer3.ut.ee/) carries out the design of the reference gene primer of real-time fluorescence quantitative PCR；

The cDNA obtained using template glehnia littoralis reverse transcription first passes through regular-PCR Preliminary Identification primer specificity as template.

6) it the foundation of reference gene primer standard curve: establishes the standard curve of each reference gene primer: will invert It records obtained cDNA and is diluted to 6 concentration gradients (1,1/5,1/25,1/125,1/625,1/3125), as establishing standard curve Template；It is that guidance carries out real-time fluorescence quantitative PCR with primer；

7) fluorescent quantitative PCR: the cDNA obtained using reverse transcription carries out real time fluorescent quantitative as template, to reference gene PCR amplification obtains corresponding Ct value；Amplification program are as follows: 95 DEG C of initial denaturation 5min；95 DEG C of denaturation 15s, 60 DEG C of annealing 15s, 72 DEG C Extend 25s, runs 40 circulations；Using obtained fluorescent quantitative PCR result, standard curve is drawn, each candidate gene is acquired Amplification efficiency and slope, the primer amplification efficiency of candidate reference gene is between 88%-108%；

8) tetra- kinds of statistical analysis techniques of Δ Ct, GeNorm, NormFinder, BestKeeper are utilized, reference gene is analyzed Stability；

9) the candidate reference gene stability that four kinds of statistical analysis techniques obtain is arranged using RankAggreg R program bag Name carries out comprehensive statistics ranking, obtains synthesis result；

10) 2 are utilized^-ΔΔCtMethod carries out the verifying of target gene expression analysis, Δ Ct=Ct (target to application reference gene Gene)-Ct (reference gene), Δ Δ Ct=Δ Ct (processing)-Δ Ct (control), 2^-ΔΔCt=relative expression quantity.

Application of the reference gene of above-mentioned glehnia littoralis in research glehnia littoralis target gene expression level.

3. beneficial effect

Compared with the prior art, the invention has the benefit that

(1) be badly in need of in the existing glehnia littoralis industry to the Mechanisms of Salt Resistance of glehnia littoralis, breeding, active constituent anabolism The various aspects such as mechanism are studied, using PCR to glehnia littoralis different parts, different tissues, the base when facing different situations Because of the status that expression is analyzed, inventor filters out 13 candidate reference genes in glehnia littoralis species, and discloses internal reference Gene order；The blank for lacking suitable, general reference gene in glehnia littoralis research field is filled up；

(2) present invention filters out 13 reference genes in glehnia littoralis species, and devises real-time fluorescence quantitative PCR with this Primer, verified primer amplification is high-efficient, high specificity (see Table 1 for details and Fig. 1), has not only filled up in glehnia littoralis research field and has lacked The blank of few suitable, general reference gene has also been filled up in glehnia littoralis research field and has lacked suitable quantitative fluorescent PCR and draw The blank of object provides effective reference gene aligning tool for glehnia littoralis gene expression analysis from now on, screening, verifying work.

(3) present invention stablizes the research blank of reference gene for glehnia littoralis species, based on transcript profile data, to upper It states reference gene to be screened, be analyzed using statistical methods and reference gene stability is comprehensively compared；Provided with difference Treatment conditions, such as salt stress, drought stress, hormone stress can satisfy the needs of most of physiological Studies；Materials are medicines With active site, study more targeted；Wherein, in salt stress, drought stress, abscisic acid and four kinds of methyl jasmonate processing Under, CYP2 stability overall ranking equal first (being detailed in attached drawing 4) not only meets real time fluorescent quantitative detection glehnia littoralis gene expression Horizontal requirement, and there is better calibration capability, researcher is carrying out in the research of above-mentioned Stress treatment, and use is same Reference gene CYP2 can be obtained by reliable and stable as a result, while improving research work efficiency, reducing cost, moreover it is possible to protect Demonstrate,prove stability, the reproducibility and reliability of result of study.

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

Detailed description of the invention

The sequence electrophoretogram that Fig. 1 13 primers provided by the invention are expanded using glehnia littoralis cDNA as template；M:DNA Marker, stripe size sequence are followed successively by 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp from top to bottom；Number from Gene representated by 1-13 be respectively PP2A, UBQ10, ACT, EF1- α, GAPDH, α-TUB, β-TUB, PTBP1, EXP1, EXP2, TIP41-like,SAND family,CYP2；

Fig. 2 is the Ct Distribution value figure of the quantitative fluorescent PCR of 13 reference genes；

Fig. 3 is using GeNorm software to 13 candidate reference gene expression stationary value (M) ordering charts, and M value is lower, gene It is more stable；Wherein Fig. 3 A is NaCl treatment conditions；Fig. 3 B is PEG treatment conditions；Fig. 3 C is ABA treatment conditions；Fig. 3 D is MeJA Treatment conditions；

Fig. 4 application RankAggreg R program bag carries out comprehensive analysis to the ranking that four kinds of statistical analysis techniques obtain, and obtains To comprehensive ranking；Fig. 4 A is NaCl treatment conditions；Fig. 4 B is PEG treatment conditions；Fig. 4 C is ABA treatment conditions；Fig. 4 D is MeJA treatment conditions；

Fig. 5 is using candidate reference gene of the invention, to glehnia littoralis target PYL gene expression amount under four kinds of Stress treatments Variation is analyzed, and the effect of candidate reference gene application provided by the invention is verified；Fig. 5 A is NaCl treatment conditions；Fig. 5 B is PEG treatment conditions；Fig. 5 C is ABA treatment conditions；Fig. 5 D is MeJA treatment conditions.

Specific embodiment

It is herein: the glehnia littoralis transcript profile sequencing data: to refer to Li L, Li M, Qi X, Tang X, Zhou Y.De novotranscriptome sequencing and analysis of genes related to salt stress response in Glehnialittoralis.PeerJ.2018,6:e5681；

Δ Ct reference described herein: Silver N, Best S, Jiang J, Thein SL.Selection of housekeeping genes for gene expression studies inhuman reticulocytes using real-time PCR.BMC Mol Biol.2006；7:33；

GeNorm reference: Vandesompele 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 geometric 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；

BestKeeper reference: Pfaffl MW, Tichopad A, Prgomet C, Neuvians TP.Determination of stable housekeeping genes,differentially regulated target genes and sample integrity:BestKeeper-Excel-based tool using pair-wise correlations.Biotechnol Lett.2004；26:509–515；

RankAggreg R program bag is referring to Pihur V, DattaS, Datta S.RankAggreg, an R package for weighted rank aggregation,BMC Bioinformatics 2009,10:6；

Biology as described herein repeats: every single plant glehnia littoralis is a repetition；

The present invention is further described below combined with specific embodiments below.

Embodiment 1

The present embodiment is the reference gene of glehnia littoralis of the present invention.Provide the internal reference base of following 13 glehnia littoralises Cause: PP2A (phosphoprotein phosphatase 2), UBQ10 (ubiquitin 10), ACT (actin), EF1- α (elongation factors), GAPDH (glycerol Aldehyde -3- phosphate dehydrogenase), α-TUB (alpha-tubulin), β-TUB ('beta '-tubulin), PTBP1 (poly pyrimidine sequence combination egg It is white), EXP1 (extension albumen 1), EXP2 (extension albumen 2), TIP41-like (class TIP41 albumen), SAND family (husky family Race's albumen), CYP2 (cyclophilin)；The nucleotides sequence of the PP2A is classified as SEQ ID NO.1；The nucleotides sequence of the UBQ10 It is classified as SEQ ID NO.2；The nucleotides sequence of the ACT is classified as SEQ ID NO.3；The nucleotides sequence of the EF1- α is classified as SEQ ID NO.4；The nucleotides sequence of the GAPDH is classified as SEQ ID NO.5；The nucleotides sequence of the α-TUB is classified as SEQ ID NO.6；The nucleotides sequence of the β-TUB is classified as SEQ ID NO.7；The nucleotides sequence of the PTBP1 is classified as SEQ ID NO.8；Institute The nucleotides sequence for stating EXP1 is classified as SEQ ID NO.9；The nucleotides sequence of the EXP2 is classified as SEQ ID NO.10；The TIP41- The nucleotides sequence of like is classified as SEQ ID NO.11；The nucleotides sequence of the SAND family is classified as SEQ ID NO.12；It is described The nucleotides sequence of CYP2 is classified as SEQ ID NO.13.

Embodiment 2

In the present embodiment, the fluorescence quantification PCR primer for expanding reference gene in embodiment 1 is provided,

The amplified fragments size of primer is 100~250bp.

Primer pair sequence for expanding PP2A is SEQ ID NO.15/SEQ ID NO.16；For expanding drawing for UBQ10 Object is SEQ ID NO.17/SEQ ID NO.18 to sequence；Primer pair sequence for expanding ACT is SEQ ID NO.19/SEQ ID NO.20；Primer pair sequence for expanding EF1- α is SEQ ID NO.21/SEQ ID NO.22；For expanding GAPDH's Primer pair sequence is SEQ ID NO.23/SEQ ID NO.24；Primer pair sequence for expanding α-TUB is SEQ ID NO.25/SEQ ID NO.26；Primer pair sequence for expanding β-TUB is SEQ ID NO.27/SEQ ID NO.28；For The primer pair sequence for expanding PTBP1 is SEQ ID NO.29/SEQ ID NO.30；Primer pair sequence for expanding EXP1 is SEQ ID NO.31/SEQ ID NO.32；Primer pair sequence for expanding EXP2 is SEQ ID NO.33/SEQ ID NO.34；Primer pair sequence for expanding TIP41-like is SEQ ID NO.35/SEQ ID NO.36；For expanding SAND The primer pair sequence of family is SEQ ID NO.37/SEQ ID NO.38；Primer pair sequence for expanding CYP2 is SEQ ID NO.39/SEQ ID NO.40。

Embodiment 3

Present embodiments provide the real time fluorescence quantifying PCR method for screening the reference gene of above-mentioned glehnia littoralis comprising with Lower step: (1) the root group of the glehnia littoralis respectively through salt stress, drought stress, abscisic acid and methyl jasmonate treatment is chosen Tissue samples；

(2) glehnia littoralis transcript profile sequencing data is utilized, glehnia littoralis candidate's reference gene is filtered out；

(3) using the candidate reference gene picked out as template (i.e. PP2A, UBQ10, ACT, EF1- α, GAPDH, α-TUB, β- TUB,PTBP1,EXP1,EXP2,TIP41-like,SAND family,CYP2；Particular sequence may refer to sequence table SEQ ID NO.1~SEQ ID NO.13), the primer for designing and having obtained the real-time fluorescence quantitative PCR of each reference gene (is detailed in implementation Example 2)；

(4) real-time fluorescence quantitative PCR is carried out, it is for statistical analysis to obtained real-time fluorescence quantitative PCR data (Ct value), Filter out optimal reference gene.

Embodiment 4

The present embodiment and the difference of embodiment 3 are only that: step (1) is located at after step (2).

Embodiment 5

The present embodiment and the difference of embodiment 3 are only that: step (1) is located at after step (3).

Embodiment 6

The present embodiment and the difference of embodiment 3 are only that: step (1) is material and processing method: it is consistent to choose growing way Sand culture glehnia littoralis is utilized respectively 6000 solution of PEG, 100 μM that NaCl solution, mass concentration that concentration is 200mM are 20% ABA solution and 100 μM of MeJA solution carry out salt stress, drought stress, abscisic acid and jasmine to different glehnia littoralises respectively Sour methyl esters handles to obtain template glehnia littoralis；Handle the time be 0h, 6h, for 24 hours when three time points to every plant of template glehnia littoralis Root be sampled, each time point carries out the duplicate sampling of biology three times to glehnia littoralis.

Embodiment 7

What is provided in the present embodiment states the real-time fluorescence quantitative PCR screening technique of the reference gene of glehnia littoralis comprising with Lower step: (1) root for the glehnia littoralis crossed respectively through salt stress, drought stress, abscisic acid and methyl jasmonate treatment is chosen Tissue sample；

(2) glehnia littoralis transcript profile sequencing data is utilized, the reference gene of glehnia littoralis is filtered out；

(3) using the reference gene picked out as template (i.e. PP2A, UBQ10, ACT, EF1- α, GAPDH, α-TUB, β-TUB, PTBP1,EXP1,EXP2,TIP41-like,SAND family,CYP2；Particular sequence may refer to sequence table SEQ ID NO.1 ~SEQ ID NO.13), design and obtained the primer (detailed in Example 2) of the real-time fluorescence quantitative PCR of each reference gene；

(4) pass through the specificity of regular-PCR Preliminary Identification reference gene primer；

The archaeal dna polymerase provided by Nanjing Vazyme Biotechnology Co., Ltd.: Green Taq Mix is provided；Identify item Part: 95 DEG C of 3min, 95 DEG C of 15s, 56 DEG C of 15s, 72 DEG C of 60s/kb, 30 circulations；72℃ 5min；

(5) real-time fluorescence quantitative PCR is carried out, it is for statistical analysis to obtained real-time fluorescence quantitative PCR data (Ct value), Filter out optimal reference gene.

Embodiment 8

The present embodiment the difference from embodiment 1 is that: step (4) are as follows: carry out real-time fluorescence quantitative PCR；Pass through standard song Line carries out quantitative analysis to obtained real-time fluorescence quantitative PCR data, filters out optimal reference gene and reference gene combination；

Wherein, real-time fluorescence quantitative PCR amplification program are as follows: 95 DEG C of initial denaturation 5min；95 DEG C of denaturation 15s, 60 DEG C of annealing 15s, 72 DEG C of extension 25s, runs 40 circulations.

Embodiment 9

The stability screening technique of the reference gene of glehnia littoralis is provided in the present embodiment, the specific steps are as follows:

1) material and processing method: choosing the consistent sand culture glehnia littoralis of growing way, and it is molten to be utilized respectively the NaCl that concentration is 200mM Liquid, 6000 solution of PEG, 100 μM of ABA solution and the 100 μM of MeJA solution that mass concentration is 20% are respectively to different corals Coral dish carries out salt stress, drought stress, abscisic acid and methyl jasmonate treatment and obtains template glehnia littoralis；

Handle the time be 0h, 6h, for 24 hours when three time points the root of every plant of template glehnia littoralis is sampled, often A time point carries out the duplicate sampling of biology three times to glehnia littoralis (every single plant is a repetition).

2) RNA is extracted and is detected: root (medicinal part) tissue sample of the glehnia littoralis of extraction is rushed using deionized water It washes, blotting paper blots, liquid nitrogen flash freezer；Glehnia littoralis root tissue sample is extracted using Trizol Reagent (TakaRa) kit RNA (extracting mode is consistent with the content in kit specification)；

Using the integrality for the RNA that electrophoresis detection extracts, 1% Ago-Gel, TAE buffer, electricity under 170V voltage Swim 10min, is observed using gel imaging system, and analyze；Purity inspection is carried out using RNA of the NanoDrop ND-2000 to extraction Survey quantifying for (OD260/280 of pure RNA is between 1.8~2.2) and concentration；

3) RNA reverse transcription synthesizes cDNA: 0.5-1 μ g RNA sample being taken to use reverse transcription reagent box PrimeScript^TM RT Reagent Kit with g DNA Eraser (Perfect Real Time) carries out reverse transcription to RNA, obtains cDNA.

4) glehnia littoralis transcript profile sequencing data, the gene of reference model plant Arabidopsis thaliana candidate reference gene screening: are utilized It is homologous, filter out the candidate reference gene for carrying out the glehnia littoralis of stability analysis；

5) design and detection of specific primer: using the reference gene filtered out as template, website Primer 3 is utilized (http://primer3.ut.ee/) carries out the design of the reference gene primer of real-time fluorescence quantitative PCR；

The cDNA obtained using reverse transcription identifies primer specificity as template, by regular-PCR, only praises life using by Nanjing promise The archaeal dna polymerase that object Science and Technology Ltd. provides: Green Taq Mix；Identification condition: 95 DEG C of 3min, 95 DEG C of 15s, 56 DEG C 15s, 72 DEG C of 60s/kb, 30 circulations；72℃5min.

6) it the foundation of reference gene primer standard curve: establishes the standard curve of each reference gene primer: will invert It records obtained cDNA and is diluted to 6 concentration gradients (1,1/5,1/25,1/125,1/625,1/3125), as establishing standard curve Template；It is that guidance carries out real-time fluorescence quantitative PCR with primer, draws standard curve；

7) PCR amplification: the cDNA obtained using reverse transcription carries out real-time fluorescence quantitative PCR amplification as template, to reference gene, Obtain corresponding Ct value；

Amplification program are as follows: 95 DEG C of initial denaturation 5min；95 DEG C of denaturation 15s, 60 DEG C of annealing 15s, 72 DEG C of extension 25s, operation 40 A circulation；The amplification efficiency and slope of each candidate gene are acquired using obtained data result, primer amplification efficiency is in 88%- Between 108%.

8) tetra- kinds of statistical analysis technique analysis reference genes of Δ Ct, GeNorm, NormFinder, BestKeeper are utilized Stability；

9) the candidate reference gene stability that four kinds of statistical analysis techniques obtain is arranged using RankAggreg R program bag Name carries out comprehensive statistics ranking, obtains synthesis result；

10) 2 are utilized^-ΔΔCtMethod carries out the verifying of target gene expression analysis, Δ Ct=Ct (target to application reference gene Gene)-Ct (reference gene), Δ Δ Ct=Δ Ct (processing)-Δ Ct (control), 2^-ΔΔCt=relative expression quantity.

Embodiment 10

The present embodiment is the exploitation of glehnia littoralis candidate reference gene of the present invention, screening process, and candidate internal reference Stability assessment of the gene under Different stress processing.The germ plasm resource of template glehnia littoralis in the present embodiment is flat from Fujian Pool is coastal, cultivates in Institute of Botany and Nanjing Botanical Garden Mem. Sun Yat-Sen Germplasm Resources；

(1) material and processing method:

Pre-processing: glehnia littoralis seedling (underground part length about 3-6cm) transplanting is in the flowerpot for cleaning river sand in acquisition nursery In, pour Huo Gelan Solution culture method in the light incubator, diurnal temperature is respectively 26 DEG C and 22 DEG C, and 14h/10h is followed round the clock Ring, relative humidity 75% are cultivated three months；

Sampling: choosing the consistent sand culture glehnia littoralis of growing way, is utilized respectively NaCl solution, mass concentration that concentration is 200mM The salt side of body is carried out to different glehnia littoralises respectively for 20% PEG6000 solution, 100 μM of ABA solution and 100 μM of MeJA solution Compel, drought stress, abscisic acid and methyl jasmonate treatment obtain template glehnia littoralis；

Handle the time be 0h, 6h, for 24 hours when three time points the root of every plant of template glehnia littoralis is sampled, often A time point carries out the duplicate sampling of biology three times (every single plant is a repetition) to glehnia littoralis, will be adopted using deionized water The root tissue sample wash of the template glehnia littoralis of collection is clean, and blotting paper blots, liquid nitrogen flash freezer, puts -80 DEG C of refrigerators for RNA's It extracts；

(2) RNA is extracted synthesizes from cDNA: extracting 4 kinds of different moulds using Trizol Reagent (TakaRa) kit The RNA of the root tissue sample of plate glehnia littoralis (extracting mode is consistent with the content in kit specification)；

Using the integrality for the RNA that electrophoresis detection extracts, 1% Ago-Gel, TAE buffer, electricity under 170V voltage Swim 10min, is observed using gel imaging system, and analyze；Purity inspection is carried out using RNA of the NanoDrop ND-2000 to extraction Survey quantifying for (OD260/280 of pure RNA is between 1.8~2.2) and concentration；

(3) 1 μ g RNA sample is taken to use reverse transcription reagent box PrimeScript^TM RT reagent Kit with gDNA Eraser (Perfect Real Time) carries out reverse transcription and obtains the cDNA (content in reverse transcription mode and kit specification Unanimously).

(4) reference gene screening and specific primer design: the glehnia littoralis transcript profile obtained according to this research team Sequencing data storehouse information, the DNA homolog of reference model plant Arabidopsis thaliana filter out gene homologous in glehnia littoralis as candidate Reference gene (see sequence table SEQ ID NO.1~SEQ ID NO.13)；Using the candidate reference gene filtered out as template, according to Gene order Primer 3web (http://primer3.ut.ee/) design primer, amplified fragments size is in 100-250bp Between (see Table 1 for details)；

The cDNA obtained using the reverse transcription of template glehnia littoralis identifies primer specificity as template, by regular-PCR, after electrophoresis The band that PCR product is observed under gel imaging system, selects stripe size correct, band is single, without primer as shown in Figure 1 The primer of dimer, and its PCR product is recycled and is sequenced, to further determine that whether target candidate gene amplification product is correct.

(6) it is bent that respective standard the foundation of reference gene primer standard curve: is made to the primer of each pair of reference gene Line calculates the amplification efficiency of corresponding primer.The cDNA of template glehnia littoralis reverse transcription is diluted to 6 gradients (1,1/ with 5 for multiple 5,1/25,1/125,1/625,1/3125), as the template for establishing standard curve；With qTOWER2.2Real-Time PCR System instrument carries out qRT-PCR, and the amplification efficiency and slope of each candidate gene are acquired using obtained data result, and primer expands Increasing Efficiency (see Table 1 for details) between 88%-108%；

1 glehnia littoralis candidate reference gene of table and its corresponding primer

(7) qRT-PCR: the cDNA obtained using reverse transcription is template, carries out real-time fluorescence quantitative PCR expansion to reference gene Increase, obtains corresponding Ct value；Using SYBR Premix ExTaqTM II (TliRNaseH Plus) (Takara) fluorescent quantitation Kit, amplification program: 95 DEG C of initial denaturation 5min；95 DEG C of denaturation 15s, 60 DEG C of annealing 15s, 72 DEG C of extension 25s, operation 40 follow Ring；Melting curve signal is acquired, the melting curve of generation is simple spike, illustrates that primer specificity is good, Ct Value Data is by real-time Fluorescence quantitative PCR instrument is read automatically, and the distribution of Ct value is as shown in Figure 2.The expression quantity of Ct value and gene is in inverse ratio, and Ct value is got over Greatly, the expression quantity of gene is lower, conversely, Ct value is smaller, the expression quantity for representing gene is higher；

(8) Δ Ct, GeNorm, NormFinder, tetra- kinds of statisticals of BestKeeper reference gene stability analysis: are utilized Analysis method analyzes the stability of reference gene；

(9) reference gene stability comprehensive analysis is carried out with RankAggreg R program bag.Δ Ct is according to Ct Value Data Degree of scatter carries out ranking, and mSD (Mean StdDev) is smaller, and gene stability is higher.Table 2 is using Δ Ct method to 13 Stability ranking of the candidate reference gene in the case where four kinds of Different stress are handled.Fig. 3 is that GeNorm is according to average variation degree M Value measures the stability of gene, and M value is smaller, and stability is better.Table 3 is that NormFinder is based on variance analysis, to internal reference Gene stability is directly evaluated.Table 4 is that BestKeeper with the standard coefficient of variation (SD) judges reference gene stability, SD It is smaller, show that the gene is more stable.As shown in figure 4, finally according to the resulting stability ranking of four kinds of methods, application RankAggreg R program bag does overall ranking, under tetra- kinds of glehnia littoralis salt stress, drought stress, ABA and MeJA treatment conditions, CYP2 gene comprehensive stability ranks the first, and stability is best；

(10) calculation method of gene relative expression quantity applies 2^-ΔΔCtMethod, the specific steps are as follows: Δ Ct=Ct (target base Cause)-Ct (reference gene), Δ Δ Ct=Δ Ct (processing)-Δ Ct (control), 2^-ΔΔCt=relative expression quantity.

Stability analysis of the 2 Δ Ct method of table to 13 candidate reference genes

Stability analysis of the 3 NormFinder method of table to 13 candidate reference genes under four kinds of stress

Stability analysis of the 3 BestKeeper method of table to 13 candidate reference genes under four kinds of stress

Embodiment 11

The present embodiment is the effect of verifying candidate reference gene application provided by the invention.Select PYL (ABA Receptor class Like albumen) it is target gene, PYL is component crucial in ABA signal transduction pathway, its expression quantity meeting usually after being forced It increased.According to the sequencing of glehnia littoralis NaCl differential transcription group studies have shown that glehnia littoralis PYL gene is under salt treatment, expression quantity Clearly there is up-regulation.Therefore, the present embodiment chooses the preferable and poor reference gene (attached drawing of stability provided by the invention respectively 4), expression quantity of the glehnia littoralis PYL gene under salt treatment, Osmotic treatment, ABA, MeJA processing is carried out using qRT-PCR method It verifies, compare application effect.Specific step is as follows:

(1) experimental material and processing method: the germ plasm resource of template glehnia littoralis is coastal from Nanping Prefecture, in Jiangsu Province Institute of Botany, Chinese Academy of Sciences and the cultivation of Nanjing Botanical Garden Mem. Sun Yat-Sen Germplasm Resources；

Stress treatment method is as follows: glehnia littoralis seedling (underground part length about 3-6cm) transplanting is in clean river in acquisition nursery In husky flowerpot, Huo Gelan Solution culture method is poured in the light incubator, diurnal temperature is respectively 26 DEG C and 22 DEG C, 14h/ 10h day-night cycle, relative humidity 75% are cultivated three months, are chosen the close and healthy plant of growing way and are carried out Stress treatment, processing When by the flowerpot of the template glehnia littoralis of sand culture be separately immersed in 200mM NaCl (salt stress), 20%PEG 6000 (arid coerce Compel), 100 μM of ABA, in 100 μM of MeJA solution, root (medicinal part) tissue sample of glehnia littoralis was taken at 0,6,24 hour respectively Product, deionized water are rinsed well, and blotting paper blots sample, and liquid nitrogen flash freezer is put extraction of -80 DEG C of refrigerators for RNA, sampled every time There are three biology to repeat (every single plant is a repetition)；

(2) RNA is extracted synthesizes from cDNA: extracting 4 kinds of different moulds using Trizol Reagent (TakaRa) kit The RNA of the root tissue sample of plate glehnia littoralis (extracting mode is consistent with the content in kit specification)；

Using the integrality for the RNA that electrophoresis detection extracts, 1% Ago-Gel, TAE buffer, electrophoresis under 170 voltages 10min is observed using gel imaging system, and is analyzed；Purity detecting is carried out using RNA of the NanoDrop ND-2000 to extraction (OD260/280 of pure RNA is between 1.8~2.2) and concentration quantify；

(3) 1 μ g RNA sample is taken to use reverse transcription reagent box PrimeScript^TM RT reagent Kit with gDNA Eraser (Perfect Real Time) carries out reverse transcription and obtains the cDNA (content in reverse transcription mode and kit specification Unanimously)；

(4) the target gene PYL sequence and specific primer design of reference gene application effect are verified: according to this research group The glehnia littoralis transcript profile sequencing data storehouse information that team has obtained, the PYL Gene A T5G05440 of reference model plant Arabidopsis thaliana, Obtain PYL nucleic acid sequence (see sequence table SEQ ID NO.14), according to gene order with Primer 3web (http: // Primer3.ut.ee/) design primer, PYL fluorescent quantitation upstream primer sequence are shown in sequence table SEQ ID NO.41, downstream primer Sequence is shown in sequence table SEQ ID NO.42, target fragment 137bp；

(5) foundation of target gene PYL primer standard curve: the cDNA of template glehnia littoralis reverse transcription is dilute for multiple with 5 It is interpreted into 6 gradients (1,1/5,1/25,1/125,1/625,1/3125), as the template for establishing standard curve；

QRT-PCR is carried out with qTOWER2.2Real-Time PCR System instrument, is calculated using obtained data result The amplification efficiency and slope of gene, PYL primer amplification efficiency are 92.22%, slope 0.9974；

(6) qRT-PCR: the cDNA obtained using reverse transcription is template, same to the reference gene and target gene of selecting verifying The amplification of Shi Jinhang real-time fluorescence quantitative PCR, obtains corresponding Ct value；

Using SYBR Premix ExTaqTM II (TliRNaseH Plus) (Takara) fluorescence quantitative kit, amplification Program: 95 DEG C of initial denaturation 5min；95 DEG C of denaturation 15s, 60 DEG C of annealing 15s, 72 DEG C of extension 25s run 40 circulations；Acquisition melts The melting curve of curve signal, generation is simple spike, illustrates that primer specificity is good, Ct Value Data is by real-time fluorescence quantitative PCR Instrument is read automatically.The expression quantity of Ct value and gene is in inverse ratio, and Ct value is bigger, and the expression quantity of gene is lower, conversely, Ct value is smaller, The expression quantity for representing gene is higher；The calculation method of gene relative expression quantity applies 2^-ΔΔCtMethod, the specific steps are as follows: Δ Ct= Ct (target gene)-Ct (reference gene), Δ Δ Ct=Δ Ct (processing)-Δ Ct (control), 2^-ΔΔCt=relative expression quantity.

(7) different reference genes are chosen and carry out the effect that qRT-PCR verifies reference gene application provided by the invention:

As shown in figure 4, under NaCl treatment conditions, applying four respectively to the comprehensive stability assessment result of reference gene A reference gene, wherein CYP2 and EXP1 is the good reference gene of verified relative stability, and GAPDH and UBQ10 is to stablize The poor reference gene of property；For qRT-PCR result as it can be seen that being corrected using GAPDH and UBQ10, NaCl handles the expression of PYL when for 24 hours Measure larger compared with contrast ratio amplitude of variation, and the good CYP2 and EXP1 of stability in use is corrected, it is as a result relatively stable, therefore use The good reference gene of stability can reduce the possibility of false positive results to a certain extent (see Fig. 5 A).

Under PEG treatment conditions, four reference genes are applied respectively, and wherein CYP2 and ACT is that relative stability is preferable Reference gene, and β-TUB and UBQ10 stability are poor；QRT-PCR result as it can be seen that using relatively unstable β-TUB and UBQ10 gene, PEG processing for 24 hours when PYL expression quantity variation it is larger, using UBQ10 even occur with use it is other The opposite trend of reference gene, and stability in use preferable CYP2 and ACT, trend are consistent and reproducible (see Fig. 5 B).

Under ABA treatment conditions, four reference genes are applied respectively, and wherein CYP2 and ACT is that relative stability is preferable Reference gene, and β-TUB and UBQ10 stability are poor；QRT-PCR result is as it can be seen that use unstable β-TUB and UBQ10 base Cause, the expression quantity variation of PYL is inconsistent after ABA processing, is occurring opposite trend for 24 hours, and stability in use is preferable CYP2 and ACT, PYL expression trend are consistent, as a result more reliable (see Fig. 5 C).

Under MeJA treatment conditions, four reference genes are applied respectively, and wherein CYP2 and EXP2 is assessment relative stability Preferable reference gene, β-TUB and EF1- α relative stability are poor；And qRT-PCR result using this four genes as it can be seen that obtain The PYL expression trend arrived is almost consistent, illustrates under MeJA processing, and the repeatability of each reference gene is all relatively good, as a result may be used By (see Fig. 5 D).

According to the present embodiment, the good reference gene of the stability that the present invention develops has preferably target gene expression analysis Effect, clearly shows target gene PYL in various Stress treatment 6h, for 24 hours after gene expression variation tendency, if individually made With CYP2, make test operation simplicity, reduce cost, while there is reliability, can also combine with other reference genes for correcting.

Sequence table

<110>Institute of Botany

<120>reference gene of glehnia littoralis and its screening technique and application

<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 (10)

1. the reference gene of glehnia littoralis, it is characterised in that: the reference gene is PP2A, UBQ10, ACT, EF1- α, GAPDH, α- One of TUB, β-TUB, PTBP1, EXP1, EXP2, TIP41-like, SAND family and CYP2 or more than one；

The nucleotides sequence of the PP2A is classified as SEQ ID NO.1；

The nucleotides sequence of the UBQ10 is classified as SEQ ID NO.2；

The nucleotides sequence of the ACT is classified as SEQ ID NO.3；

The nucleotides sequence of the EF1- α is classified as SEQ ID NO.4；

The nucleotides sequence of the GAPDH is classified as SEQ ID NO.5；

The nucleotides sequence of the α-TUB is classified as SEQ ID NO.6；

The nucleotides sequence of the β-TUB is classified as SEQ ID NO.7；

The nucleotides sequence of the PTBP1 is classified as SEQ ID NO.8；

The nucleotides sequence of the EXP1 is classified as SEQ ID NO.9；

The nucleotides sequence of the EXP2 is classified as SEQ ID NO.10；

The nucleotides sequence of the TIP41-like is classified as SEQ ID NO.11；

The nucleotides sequence of the SAND family is classified as SEQ ID NO.12；

The nucleotides sequence of the CYP2 is classified as SEQ ID NO.13.

2. the PCR primer for expanding any reference gene described in claim 1.

3. according to claim 2 for expanding the PCR primer of reference gene, it is characterised in that: the amplification of the primer Clip size is 100~250bp.

4. according to claim 3 for expanding the PCR primer of reference gene, it is characterised in that: described for expanding The primer pair sequence of PP2A is SEQ ID NO.15/SEQ ID NO.16；

Primer pair sequence for expanding UBQ10 is SEQ ID NO.17/SEQ ID NO.18；

Primer pair sequence for expanding ACT is SEQ ID NO.19/SEQ ID NO.20；

Primer pair sequence for expanding EF1- α is SEQ ID NO.21/SEQ ID NO.22；

Primer pair sequence for expanding GAPDH is SEQ ID NO.23/SEQ ID NO.24；

Primer pair sequence for expanding α-TUB is SEQ ID NO.25/SEQ ID NO.26；

Primer pair sequence for expanding β-TUB is SEQ ID NO.27/SEQ ID NO.28；

Primer pair sequence for expanding PTBP1 is SEQ ID NO.29/SEQ ID NO.30；

Primer pair sequence for expanding EXP1 is SEQ ID NO.31/SEQ ID NO.32；

Primer pair sequence for expanding EXP2 is SEQ ID NO.33/SEQ ID NO.34；

Primer pair sequence for expanding TIP41-like is SEQ ID NO.35/SEQ ID NO.36；

Primer pair sequence for expanding SAND family is SEQ ID NO.37/SEQ ID NO.38；

Primer pair sequence for expanding CYP2 is SEQ ID NO.39/SEQ ID NO.40.

5. the real-time fluorescence quantitative PCR screening technique of glehnia littoralis reference gene, it is characterised in that: the following steps are included:

(1) the 4 kinds of template glehnia littoralises crossed respectively through salt stress, drought stress, abscisic acid and methyl jasmonate treatment are chosen Root tissue sample；

(2) glehnia littoralis transcript profile sequencing data is utilized, the candidate reference gene of glehnia littoralis is filtered out；

(3) using the candidate reference gene sequence picked out as template, the reference gene primer of real-time fluorescence quantitative PCR is designed；

(4) real-time fluorescence quantitative PCR is carried out；It is for statistical analysis to obtained real-time fluorescence quantitative PCR data, it filters out respectively Optimal reference gene and reference gene combination.

6. the real-time fluorescence quantitative PCR screening technique of glehnia littoralis reference gene according to claim 5, it is characterised in that: Before carrying out the real-time fluorescence quantitative PCR in the step (4), the specificity of regular-PCR identification reference gene primer is first passed through.

7. the real-time fluorescence quantitative PCR screening technique of glehnia littoralis reference gene according to claim 5, it is characterised in that: In the step (4), real-time fluorescence quantitative PCR amplification program are as follows: 95 DEG C of initial denaturation 5min；95 DEG C of denaturation 15s, 60 DEG C of annealing 15s, 72 DEG C of extension 25s, runs 40 circulations.

8. the real-time fluorescence quantitative PCR screening technique of glehnia littoralis reference gene according to claim 5, it is characterised in that: It is real-time to what is obtained by tetra- kinds of statistical softwares of Δ Ct, GeNorm, NormFinder, BestKeeper in the step (4) Quantitative fluorescent PCR data are for statistical analysis, filter out optimal reference gene and reference gene combination respectively, then comprehensive analysis Software RankAggreg R carries out overall ranking analysis.

9. the real-time fluorescence quantitative PCR screening technique of glehnia littoralis reference gene according to claim 5, it is characterised in that: Specific step is as follows:

1) material and processing method: choose the consistent sand culture glehnia littoralis of growing way, be utilized respectively concentration be 200mM NaCl solution, 6000 solution of PEG, 100 μM of ABA solution and the 100 μM of MeJA solution that mass concentration is 20% are respectively to different glehnia littoralises It carries out salt stress, drought stress, abscisic acid and methyl jasmonate treatment and obtains template glehnia littoralis；

Handle the time be 0h, 6h, for 24 hours when three time points the root of every plant of template glehnia littoralis is sampled, Mei Geshi Between select and carry out the duplicate sampling of biology three times to glehnia littoralis；

2) RNA is extracted and is detected: the root tissue sample for all template glehnia littoralises taken being rinsed using deionized water, is inhaled Water paper blots, liquid nitrogen flash freezer；Extract the RNA of glehnia littoralis root tissue sample；

Utilize the integrality for the RNA that electrophoresis detection extracts；

Using NanoDrop ND-2000 to the RNA progress purity detecting of extraction and quantifying for concentration；

3) RNA reverse transcription synthesizes cDNA: carrying out reverse transcription reaction to the RNA of template glehnia littoralis, obtains cDNA；

4) glehnia littoralis transcript profile sequencing data, the DNA homolog of reference model plant Arabidopsis thaliana candidate reference gene screening: are utilized Filter out the candidate reference gene for carrying out the glehnia littoralis of stability analysis；

5) using the candidate reference gene sequence filtered out as template, it is fixed the design and detection of specific primer: to carry out real-time fluorescence Measure the design of the reference gene primer of PCR；

The cDNA obtained using reverse transcription passes through the specificity of regular-PCR Preliminary Identification primer as template；

6) it the foundation of reference gene primer standard curve: establishes the standard curve of each reference gene primer: reverse transcription is obtained To cDNA be diluted to 6 concentration gradients, as the template for establishing standard curve；It is that guidance carries out real time fluorescent quantitative with primer PCR；

7) fluorescent quantitative PCR: the cDNA obtained using reverse transcription carries out real-time fluorescence quantitative PCR as template, to reference gene Amplification, obtains corresponding Ct value；

Amplification program are as follows: 95 DEG C of initial denaturation 5min；95 DEG C of denaturation 15s, 60 DEG C of annealing 15s, 72 DEG C of extension 25s, operation 40 follow Ring；Using obtained fluorescent quantitative PCR result, standard curve is drawn, the amplification efficiency and slope of each candidate gene are acquired；

8) tetra- kinds of statistical analysis techniques of Δ Ct, GeNorm, NormFinder and BestKeeper are utilized, reference gene is analyzed Stability；

9) the candidate reference gene stability ranking that four kinds of statistical analysis techniques are obtained using RankAggreg R program bag into Row comprehensive statistics ranking, obtains synthesis result；

10) 2 are utilized^-ΔΔCtMethod carries out the verifying of target gene expression analysis to application reference gene.

10. application of the reference gene of glehnia littoralis described in claim 1 in research glehnia littoralis target gene expression level.