CN106222255B - It is a kind of detect Strawberry Seedlings viability primer and its application - Google Patents
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Abstract
The invention discloses a kind of primers for detecting Strawberry Seedlings viability, including following primer pair: (1) detect the primer pair of GAPDH1 gene: its nucleotide sequence is as shown in NO:3~4 SEQ ID；(2) detect the primer pair of TIM1 gene: its nucleotide sequence is as shown in NO:5~6 SEQ ID.The invention also discloses the methods using above-mentioned primer detection Strawberry Seedlings viability.The present invention establishes the molecular detecting method of a kind of fast and convenient, high specificity, accurately and reliably Strawberry Seedlings viability.
Description
Technical field
The invention belongs to plant biotechnology fields, and in particular to the primer of detection Strawberry Seedlings viability and its application.
Background technique
Strawberry (Fragaria × ananassa Duch.) is rosaceae (Rosaceae) Fragaria (Fragaria) perennial root
Property perennial evergreen herbaceous plant.The fresh and tender succulence of fruit, beautiful in colour, good smell, sweet mouthfeel are moderate, are that China is traditional
One of outstanding berry fruit.Strawberry fruit is rich in multiple nutritional components such as vitamin C, organic acid, sugar, minerals and pectin.Closely
Ellagitannins (Ellagitannins), anthocyanidin (Anthocyanins) and procyanidine contained by discovery strawberry are studied over year
(Proanthocyanidins) generation of the prevention of biological active matters mass-energy cardiovascular and cerebrovascular and cancer such as, therefore strawberry is by consumption
Person's favor.
Seedling vitality power, which refers to that nursery stock is cultivated, makes under specific (optimum growth) environmental condition it survive and grow
Ability.Seedling vitality power is measured in crop breeding, the sale of nursery stock, the plantation of nursery stock, the cultivation of nursery stock and the transport of nursery stock
It is of great significance in equal work.
Currently, nursery stock used is mainly based on the detoxification bare-root seeding of tissue-culturing rapid propagation in strawberry production.From lifting, transport to kind
Plant generally requires 5-7 days, and still, after lifting to before transplanting, root system saves degree, sunning, packet when nursery stock is by lifting
The influence of the production links many factors such as dress, storage, transport, temporary planting and root system disease corruption, in the process, seedling vitality power has
Great changes will have a direct impact on seedling percent at this time.Nursery stock blade and a large amount of dehydrations of root system, the decline of seedling vitality power.Simultaneously
After nursery stock plantation, since seedling root and blade incur loss, it not can be carried out photosynthesis and absorb nutrient, seedling root is crawled
Crawl stem and blade gradually dehydration, cause nursery stock finally dead.If serious, it will cause that strawberry shoot survival percent is low, and planting effect is poor,
Waste a large amount of man power and material.Currently, with the continuous expansion of strawberry cultivating area, Cultivar replacing speed is also increasingly
Fastly, strawberry is listed in advance, the requirement of seedling-raising technique it is also higher and higher.In southern area, since the strawberry nursery middle and later periods is undergone
The weather of high temperature and rainy, the sub- seedling easy infection anthracnose of strawberry, the decline of less serious case's Strawberry Seedlings quality, sub- seedling slow seedling is slow or dead after field planting
It dies, serious person will appear strawberry in the mortality of numerous seedling field, and grower's seedling is caused to lack, and listing is postponed, under product quality
Drop, the serious development for restricting Strawberry industry.Therefore, in order to analyze seedling viability, need it is a kind of easy and quickly simultaneously
Measuring method suitable for a large amount of single plants.Researcher has also carried out many researchs, but result of study mainly passes through selection quantity
The viability of numerous morphological index and Evaluation of physiological fruit nursery stock, people are it is further recognized that traditional form refers to
Mark only reflects the resemblance of nursery stock, and physical signs is by many external worlds such as crop species, nursery stock type, season, times
The influence of factor cannot sensitively react the viability of plant.Nursery stock is a complicated organism, in addition transplanted seedling tree
Surviving afterwards is influenced by environmental conditions very greatly with upgrowth situation, inherent physical signs lower nursery stock there is recovery to give birth to
A possibility that vigor, and mode of appearance observation is difficult to the viability of accurate instruction nursery stock, therefore only with form and physiology
Index can not carry out comprehensive objective appraisal to seedling quality, and molecular diagnostic techniques can not only concentrated expression plant entirety
Viability, and early prediction effect can be played.For the status for being difficult to accurate quickly analysis Strawberry Seedlings viability at present, hair
A kind of method of the detection Strawberry Seedlings viability of based on PCR technology is illustrated.
Summary of the invention
The technical problem to be solved by the present invention is to provide a kind of primer for detecting Strawberry Seedlings viability, to solve existing skill
The problem of Strawberry Seedlings viability judgement inaccuracy in art.
In order to solve the above technical problems, the present invention adopts the following technical scheme:
A kind of primer detecting Strawberry Seedlings viability, including following primer pair:
(1) detect the primer pair of GAPDH1 gene: its nucleotide sequence is as shown in NO:3~4 SEQ ID；
(2) detect the primer pair of TIM1 gene: its nucleotide sequence is as shown in NO:5~6 SEQ ID；
The nucleotide sequence of GAPDH1 gene is as shown in SEQ ID NO:1, such as sequence table of the nucleotide sequence of TIM1 gene
Shown in middle SEQ ID NO:2
2, a kind of method for detecting Strawberry Seedlings viability, includes the following steps:
(1a) extracts the total serum IgE of strawberry sample to be measured, by total serum IgE reverse transcription at cDNA；
(2a) using the cDNA in step (1a) as template, NO:3~4 SEQ ID, NO:5~6 SEQ ID are primer, are utilized
Quantitative fluorescent PCR expands GAPDH1 gene and TIM1 gene respectively；
(3a) when the expression quantity Ct value of GAPDH1 gene and TIM1 gene is all larger than 30, the viability of Strawberry Seedlings is less than
50%.
In step (1a), the strawberry sample to be measured is derived from root, stem, leaf or the bud of strawberry.
The primer of above-mentioned detection Strawberry Seedlings viability applying in protection model of the invention in detection Strawberry Seedlings viability
Within enclosing.
The DNA extraction method of strawberry sample is as follows:
(1) 0.1g Strawberry Leaves, root, stem, bud are weighed, is added in 2.0mL EP pipe, pulverizes in liquid nitrogen；
(2) light and slow to be mixed by inversion after 2 × CTAB lysate of 65 DEG C of 700 μ L preheatings is added, it is placed in 65 DEG C of water-baths
30min is cracked, it is light and slow reverse primary every 5min；
(3) cool after the water bath is over and isometric chloroform be added to room temperature: isoamyl alcohol (24:1), it is light and slow be mixed by inversion after,
10min is placed, 12000rpm is centrifuged l0min；Isometric 1%CTAB is added in new 2.0mL centrifuge tube in Aspirate supernatant
Solution mixes gently, and observation precipitating generates (solution becomes slightly muddy)), if not observing precipitating.Left at room temperature over night；
(4) room temperature 12000rpm is centrifuged 5min；It abandons supernatant and precipitating is dissolved in again in the NaCl solution of 100 μ L 1M, room
Temperature, 12000rpm are centrifuged 5min；Supernatant is shifted into another centrifuge tube, isometric pre- cold isopropanol, light and slow top is added
It mixes, 12000rpm is centrifuged l0min；
(5) supernatant is abandoned, 70% ethanol washing 2 times of lmL are added；After removing ethyl alcohol, the dissolution of 50 μ L distilled waters is added
DNA, -20 DEG C of refrigerators save；
(6) Ago-Gel or polyacrylamide gel electrophoresis, dyeing, the electrophorogram for obtaining DNA；
(7) quality according to the band feature diagnosis Strawberry Seedlings DNA of above-mentioned map.
GAPDH1 gene and TIM1 gene screen as follows to be obtained:
(1) acquisition of candidate reference gene sequence: strawberry Glyceraldehyde-3-phosphate is chosen
Dehydrogenase (GAPDH1), Elongation factor 1-alpha (EF1a), Tubulin alpha (TUBa),
Tubulin beta (TUBb), Mitochondrial import inner membrane translocase (TIM1),
SMAD/FHA domain-containing protein (FHA1), interspacer RNA (16S -23S) region
(RIB413), Actin (ACTIN) candidate gene of totally 9 reference genes as reference gene.
(2) gene corresponding special primer (amplified production list design of primers: is designed according to the candidate gene sequence of acquisition
One, specially solubility curve peak is single, and 2% Ago-Gel test strip is single).
(3) Strawberry Seedlings extract total serum IgE: extracting strawberry leaves using improvement 3%CTAB (cetyl trimethylammonium bromide) method
Piece total serum IgE；Total serum IgE reverse transcription is cDNA (complementary DNA (cDNA)): being produced with total serum IgE (DNA enzymatic processing) reverse transcription of extraction
Object (cDNA) is quantitative fluorescent PCR template.
(4) quantitative fluorescent PCR reacts: using the cDNA of reversion as template, using SYBR Green I (Toyobo) reagent, using
The corresponding special primer of reference gene is in AppliedStepOnePlusTMReal-Time PCR Systems is real
When quantitative PCR apparatus, obtain the expression data Ct value of each gene.
(5) Delta-Ct method, GeNorm (Version 3.5), Bestkeeper are passed through according to gained Ct value
(Version1.0) and NormFinder (Version 0.953) software calculates the stability of candidate reference gene, filters out table
Up to most unstable reference gene and determine the reference gene for being most suitable for detecting Strawberry Seedlings viability.
The utility model has the advantages that
(1) present invention filters out expression with the reduction also variation of Development pattern of Strawberry Seedlings viability for the first time
Reference gene has saved the time, significantly reduces the workload of seedling quality detection.And stability is good, and repeatability is high.
(2) present invention judges the size of Strawberry Seedlings viability according to the degree of injury of DNA for the first time.Its operation is relatively easy,
Cost is relatively low, high sensitivity, and accuracy is high.
(3) present invention can set up the detection architecture of Strawberry Seedlings viability.This detection method can reflect different tree species,
Each sections upgrowth situation, water regime and the seedling growths such as different nursery stock types, different nursery stock sizes, nursery stock root, stem, leaf, bud
The different phase of development is to influence caused by seedling vigor.That is, no matter various in form and physiologically of nursery stock change
Become, can all be reflected on gene expression information, so that predicts nursery stock survives potentiality, this to the evaluation of seedling quality extremely
It is beneficial.
Detailed description of the invention
Shown in FIG. 1 is the electrophoresis detection result phase figure of the Strawberry Leaves DNA extracted in the present invention；
Shown in Fig. 2 is the electrophoresis detection result phase figure of the Strawberry Leaves total serum IgE extracted in the present invention；
Shown in Fig. 3 is the fluorescent quantitation result figure of 2 reference genes GAPDH1 and TIM1 in the present invention；
Shown in Fig. 4 is the semidefinite spirogram of 2 reference genes GAPDH1 and TIM1 in the present invention；
Shown in fig. 5 is the relationship of the variation of the expression of GAPDH1 and TIM1 and Strawberry Seedlings viability.
Specific embodiment
According to following embodiments, the present invention may be better understood.However, as it will be easily appreciated by one skilled in the art that real
It applies content described in example and is merely to illustrate the present invention, without sheet described in detail in claims should will not be limited
Invention.
Embodiment 1:
1. experimental material samples: ' Feng Xiang ' strawberry (' Toyonoka ') seedling is derived from Jiangsu Province Agriculture Science Institute.It will growth
Strawberry Seedlings in order are placed in greenhouse, respectively 0h, 6h, 12h, 18h, for 24 hours, 20 plants are respectively taken after 30h, 36h, 48h time
Kind takes sample blade and stolon in basin, and liquid nitrogen flash freezer is stored in -80 DEG C of ultra low temperature freezers.It is counted after 10 days each
Handle the survival rate of seedling.
2. Strawberry Seedlings extract DNA: extracting genomic DNA using modified Booth's arithmetic, concrete operations are as follows: by fresh blade
(0.1g) is ground in liquid nitrogen, is moved into 1.5mL centrifuge tube rapidly.CTAB (the 2%CTAB:100mM of 600 μ L preheating is added
Tris-HCI, pH8.0；20mM EDTA, pH8.0；1.4M NaCl；Use preceding addition 0.4% β-mercaptoethanol) Extraction buffer.
In 65 DEG C of heat preservation 30min, during which overturn several times.Chloroform/isoamyl alcohol (v/v=24/1) of 600 μ L is added, gently overturns several
It is secondary.12000r/min is centrifuged 10min.Supernatant (about 500 μ L) is moved into new 1.5mL centrifuge tube, be added isometric chloroform/
Isoamyl alcohol gently overturns several times, and 12000r/min is centrifuged 10min.It takes supernatant (about 300 μ L), the cold nothing of diploid product is added
Water-ethanol is mixed by inversion.Choose precipitating, be put into the 1.5mL centrifuge tube containing 350 μ L TE buffers, when largely precipitating dissolution
Afterwards, 12000r/min is centrifuged 10min.Supernatant (about 300 μ L) is transferred in new 1.5mL centrifuge tube, 37 after mixing gently
DEG C water-bath 1h.It is cooled to room temperature, the 3M NaAc (pH5.2) of 1/10 volume is added, the cold nothing of diploid accumulated ice is added after mixing
Water-ethanol is mixed by inversion, -20 DEG C of standing 30min.12000r/min is centrifuged 10min.Supernatant is abandoned, it is heavy with 70% ethanol washing DNA
2 times (will be centrifuged each time after washing) in shallow lake.DNA is air-dried on superclean bench.50 μ L TE dissolving DNAs are added, in -20 DEG C of items
Part saves.
The detection of 3.DNA mass: taking the DNA of 2 μ L, detected with 1% agarose gel electrophoresis, and voltage is 150V left
The right side is taken out when EB reaches glue surface 1/3, in photographic analysis in ultraviolet imagery system.DNA is in Biophotometer (protein nucleic acid
Analyzer) on detectable concentration, system be 1 μ L RNA be added 49 μ L DEPC processing ddH2O, the ddH handled with DEPC2O zeroing,
DNA concentration, OD260/OD280And OD260/OD230Directly read from instrument.Electrophoresis result is as shown in Figure 1.
4. Strawberry Seedlings extract total serum IgE: extracting Strawberry Leaves total serum IgE using improvement 2%CTAB method.By fresh blade (0.1g)
It grinds in liquid nitrogen, moves into 1.5mL centrifuge tube rapidly.It is added and contains 600 μ L CTAB lysate (2%CTAB:100mM
Tris-HCI, pH8.0；20mM EDTA, pH 8.0；1.4M NaCl；Use it is preceding be added 2% beta -mercaptoethanol) 2mL centrifuge tube
In, during which 65 DEG C of 15~30min of water-bath are overturned several times.Isometric chloroform/isoamyl alcohol (v/v=24/1) is added, is vortexed mixed
It is even；12000r/min is centrifuged 10min, takes supernatant, and the water-saturated phenol (pH=4.5) of 1/2 volume is added, and mixes, places on ice
2min is added 1/2 volume of chloroform/isoamyl alcohol (v/v=24/1), and vortex mixes 4~5min；12 000r/min centrifugation
10min takes supernatant, and the NaAc (3M, pH=5.2) of 1/3 volume is added, and mixes, and after standing 5min on ice, adds isometric
Chloroform: isoamyl alcohol (24:1)；12 000r/min are centrifuged 10min, take supernatant, and the LiCl (10mol/L) of 1/4 volume is added ,-
20 DEG C of standing 1h；12 000r/min are centrifuged 10min, abandon supernatant, are precipitated 2 times with 70% ethanol washing, drying at room temperature is dissolved in
The ddH of 50 μ L DEPC processing2In O, -20 DEG C are saved backup.
The detection of 5.RNA mass: taking the RNA of 2 μ L, detected with 1% agarose gel electrophoresis, and voltage is 150V left
The right side is taken out when EB reaches glue surface 1/3, in photographic analysis in ultraviolet imagery system.DNA is in Biophotometer (protein nucleic acid
Analyzer) on detectable concentration, system be 1 μ L RNA sample be added 49 μ L DEPC processing ddH2O, the ddH handled with DEPC2O tune
Zero, DNA concentration, OD260/OD280And OD260/OD230Directly read from instrument.Electrophoresis result is as shown in Figure 2.
6. total serum IgE reverse transcription is cDNA: being tried referring to SMARTTM PCR cDNA Synthesis kit user Manual
Agent box specification is operated, and the total serum IgE of acquisition is inverted to cDNA, as quantitative fluorescent PCR template.
7. utilizing 7 software of Primer 6.0 and Oligo based on obtaining 10 reference gene sequences, it then follows glimmering in real time
The principle of Fluorescent Quantitative PCR design of primers designs real-time fluorescence quantitative PCR primer, and amplified fragments are l00~200bp, glimmering in real time
Fluorescent Quantitative PCR specific primer sequences are as shown in table 1.(PAGE method of purification) is synthesized by Shanghai Dongguo Biology Co., Ltd..
1 reference gene information summary sheet of table
8. real-time fluorescence quantitative PCR is carried out using two-step method, in Applied
StepOnePlusTMThe operation of Real-Time PCR Systems real-time PCR.It is contaminated based on SYBR Green I (Toyobo)
The qPCR of material detects the Ct value of reference gene, reaction system used and procedure reference Takara companyPremix
Ex TaqTMThe specification of I (Tli RNaseH Plus) kit.PCR reaction system is 20 μ L, wherein 2 × SYBR Premix
Ex Taq II (Tli RNaseH Plus) 10 μ L, upstream and downstream quantify 10 μM of primer each 0.8 μ L, template cDNA2 μ L, 50 × ROX
0.4 μ L of Reference Dye, with sterilizing distilled water polishing to 20 μ L.Fluorescent quantitative PCR uses two-step method, i.e., at 95 DEG C
After initial denaturation 30s, first run 40 circulation 95 DEG C of 5s, 60 DEG C of 31s, 95 DEG C of 15s in solubility curve stage of reruning, 60 DEG C
1min, 95 DEG C of 30s, 60 DEG C 15.It is simple spike by the solubility curve that observation Real-Time PCR instrument generates after PCR reaction
Determine the primer without non-specific amplification: experiment obtains quantitative Ct value (table 2) for the stability analysis of later period reference gene.As a result
As shown in Figure 3 and Figure 4.
9. data processing: according to formula Q=E Δ Ct, calculating 10 using Ct value of the Excel 2010 to each amplification sample
The relative expression quantity of a house-keeping gene.Wherein E be gene magnification efficiency, Δ Ct=Ctmin-Ct sample (Ctmin be sample in most
Low Ct value；Ct sample is the Ct value of each sample).It is calculated in strawberry different times blade according to 10 house-keeping genes
Relative expression quantity, respectively apply Delta-Ct method, GeNorm (Version 3.5), Bestkeeper (Version
1.0) expression stability of 10 candidate house-keeping genes is analyzed with NormFinder (Version 0.953) program, is sieved
Select suitable house-keeping gene.
2 house-keeping gene stability summary sheet of table
10. Δ Ct method calculates standard deviation S D according to the Ct value of house-keeping gene.SD value is smaller, and the stability of gene is better.
GeNorm software determines most stable of house-keeping gene according to averagely expression index of stability M value.M value is bigger, and the stability of gene is got over
It is low, it is on the contrary then higher.BestKeeper program calculates standard deviation S D according to the Ct value EXCEL software of house-keeping gene.SD value
Smaller, the stability of gene is better.NormFinder program calculates the stationary value M of gene with EXCEL software.M value is lower, then should
Gene is more stable.The above analysis result it is found that GAPDH1 and TIM1 gene stability is minimum in the present context, expression most
It is easy to be influenced by Strawberry Seedlings state itself and external environment, it is true that the variation of expression also accurately embodies Strawberry Seedlings
Growth conditions, also just most suitable as diagnosis Strawberry Seedlings viability house-keeping gene.
Embodiment 2:
The good Feng Xiang of upgrowth situation (' Toyonoka ') Strawberry seedlings are placed in greenhouse, without any safeguard measure,
Give free rein to wilt, then be poured respectively in 0h, 6h, 12h, 18h, for 24 hours, respectively take 20 plants of kinds in basin after 30h, 36h, 48h time
Water, then the survival rates of each toeatment period Strawberry seedlings is counted after control environment condition is planted 10 days.
The corresponding each period obtained again to the quantitative fluorescent PCR for passing through GAPDH1 and TIM1 gene in embodiment 1
(0h, 6h, 12h, 18h, for 24 hours, 30h, 36h, 48h) Ct value compare.The results are shown in Table 3.
The Ct value of table 3GAPDH1 and TIM1 gene and the relationship of strawberry viability
| GAPDH1 gene C t value | TIM1 gene C t value | Survival rate |
| 21.60291 | 17.55905 | 100.0% |
| 23.2751 | 18.05166 | 100.0% |
| 24.08488 | 18.65912 | 85.6% |
| 27.87724 | 22.05006 | 65.3% |
| 28.99202 | 23.13817 | 58.1% |
| 29.77107 | 25.90221 | 32.3% |
| 32.83171 | 26.24231 | 14.6% |
| 32.83171 | 26.24231 | 0.0% |
Reference gene of the invention is not limited to strawberry ' Feng Xiang ' kind, it has been investigated that, in other kinds of strawberry
It is also suitable, has obtained similar test result.
Claims (1)
1. a kind of method for detecting Strawberry Seedlings viability, which comprises the steps of:
(1a) extracts the total serum IgE of strawberry sample to be measured, by total serum IgE reverse transcription at cDNA；
(2a) using the cDNA in step (1a) as template, NO:3~4 SEQ ID, NO:5~6 SEQ ID are primer, utilize fluorescence
Quantitative PCR expands GAPDH1 gene and TIM1 gene respectively；
(3a) when the expression quantity Ct value of GAPDH1 gene be greater than 30 and TIM1 gene expression quantity Ct value be greater than 26 when, Strawberry Seedlings
Viability is less than 50%；
The strawberry viability refers to the survival rate of wilting Strawberry seedlings；
The strawberry sample is Strawberry Leaves or stolon.
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| CN103898223A (en) * | 2014-04-08 | 2014-07-02 | 北京林业大学 | Method for studying populus diversifolia gene by using multi-reference-gene combination |
| CN105203672A (en) * | 2015-11-04 | 2015-12-30 | 江苏省农业科学院 | Quality assurance and identification method for high-quality strawberries |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103898223A (en) * | 2014-04-08 | 2014-07-02 | 北京林业大学 | Method for studying populus diversifolia gene by using multi-reference-gene combination |
| CN105203672A (en) * | 2015-11-04 | 2015-12-30 | 江苏省农业科学院 | Quality assurance and identification method for high-quality strawberries |
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| Selection and validation of reference genes for gene expression analysis in switchgrass ( panicum virgatum) using quantitative real-time rt-pcr;Gimeno J et al.;《PLOS One》;20140312;第9卷(第3期);摘要，材料和方法，结果，表1-3，图1-4 |
| 苗木失水对生活力的影响;马阿滨等;《林业科技》;19950925(第5期);第12-15页 |
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Inventor after: Zhu Xudong Inventor after: Fang Jinggui Inventor after: Jia Haifeng Inventor after: Wang Baoju Inventor after: Zheng Ting Inventor before: Zhu Xudong Inventor before: Fang Jinggui Inventor before: Jia Haifeng Inventor before: Wang Baoju |