CN108441544A - The detection of expression method and amplimer of sugar grass Sucrose phosphate synthase gene - Google Patents
The detection of expression method and amplimer of sugar grass Sucrose phosphate synthase gene Download PDFInfo
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Abstract
The present invention is with sugar grass LTR102 (female parent), sorghum 654 (male parent) and the F that they hybridize5The Gao Tang groups in generation are examination material with low sugar group, in seedling stage, jointing stage, heading stage and maturity period measure different times sugar grass LTR102, sorghum 654 and F5The activity of cane sugar content and SPS in Gao Tang groups and low sugar colony leaves, determine sugar grass SPS activity and cane sugar content correlation.Qualitative analysis is carried out to above-mentioned examination material different development stage SPS gene expressions, determines the otherness of different times SPS gene expressions.Difference of the above-mentioned examination material of quantitative analysis in different growth period SPS gene expression amounts, and SPS gene expressions relative concentration and cane sugar content are subjected to correlation analysis, the result shows that SPS gene expressions relative concentration and cane sugar content are significantly correlated, result according to the present invention establishes a kind of sugar grass SPS gene expression rapid detection methods, to achieve the purpose that by quickly detecting determining the best harvest time and the screening high sugar content new material of sugar grass.
Description
Technical field
The invention belongs to biotechnologies, are related to sugar grass Sucrose Phosphate Synthase (SPS) gene, and in particular to sweet tea is high
The detection of expression method and amplimer of fine strain of millet Sucrose phosphate synthase gene.
Background technology
Sucrose is the photosynthetic primary product of higher plant, is the principal mode of carbon transport, and is carbohydrate product
Tired and storage principal mode, and " library metabolism " main matrix, with special position (Farrar in plant glycometabolism
et al.,2000).Especially for sugar grass, the good raw material as sugar industry, production alcohol, fiber and feed mainly depends on
In sugared part that cane is rich in, wherein 85% is sucrose, 9% glucose, 6% fructose (Gnansounou et al., 2005).Profit
It uses the sugar of sugar grass to produce the highest attention that alcohol causes people as alternative energy source in recent years, inquires into the sugar of sugar grass
Metabolism switching mechanism is a pith of bioenergy research.
Sugar grass is " double library types " crop, has seed library and stalk library, sucrose is the main of sugar in sweet sorghum stalk
Existence form, accumulation of the sucrose in stalk are related to sugar content and yield in sweet sorghum stalk, and stalk sucrose accumulation
Process is inseparable with Sucrose Metabolism.Sucrose Metabolism is regulated and controled by a variety of enzymes, and there are mainly three types of the key enzymes for regulating and controlling Sucrose Metabolism：Sugarcane
Sugared phosphate synthase (Sucrose Phosphate Synthase, E.C.2.4.1.14, SPS), sucrose synthase
(Sucrosesynthase, E.C.2.4.1.13, SS) and invertase (Invertase, EC3.2.1.26, Inv).Wherein,
SPS catalysing sucroses synthesize, and Inv catalysing sucroses degradation, SS has the double attribute decomposed with synthesis of sucrose, three's co-catalysis
Sucrose enters various metabolic pathways.Three kinds of enzymes play the role of very important in plant growth and development process, therefore, study this
It is most important with the relevant enzyme of Sucrose Metabolism a bit.
The activity of SPS directly reflects the ability of Sucrose synthesis in plant.It is to verify from analysis SPS gene expression amounts
Stalk Sugar Accumulation explains sugared content difference between different cultivars and establishes the effective way of molecular regulation system.And sweet tea is high
Fine strain of millet SPS gene expression detection technique studies have no comprehensive report.
Invention content
In order to solve the above technical problem, the present invention provides a kind of detection of expression of sugar grass Sucrose phosphate synthase gene
Method and amplimer, with the F of sugar grass LTR102 (female parent), sorghum 654 (male parent) and their hybridization5Dai Zhonggao sugar group
And F5Low sugar group is examination material, using high performance liquid chromatography and fluorescent quantitative PCR technique to cane sugar content and sucrose phosphate
The gene expression of synzyme (SPS) is detected, and establishes a kind of high sugared screening varieties rapid detection method of sorghum.
The purpose of the present invention one：Different times sugar grass is measured using high performance liquid chromatography and spectrophotometry
LTR102 (female parent), sorghum 654 (male parent) and F5The activity of cane sugar content and SPS in Gao Tang groups and low sugar colony leaves,
Determine sugar grass SPS activity and cane sugar content correlation.
The object of the invention two：Using round pcr to male parent, female parent and F5Gao Tang groups and low sugar group, difference development
The gene expression of period SPS carries out qualitative analysis, primarily determines the otherness of different times SPS gene expressions.
The purpose of the present invention three：Using fluorescent quantitative PCR technique, quantitative analysis sugar grass LTR102 (female parent), sorghum 654
(male parent) and F5Gao Tang groups and low sugar group different growth period SPS genes quantitative expression situation, and by SPS bases
Because expression relative concentration and cane sugar content carry out correlation analysis, the results showed that SPS gene expressions relative concentration and cane sugar content
Significantly correlated (R=0.972, P<0.05) reaction system that SPS gene expressions detect rapidly, is established in turn according to experimental result
And condition, a kind of Rapid identification standard of the high sugared screening varieties of sugar grass is established, reaches determining optimum harvest date and screening height contains
The purpose of the sorghum variety of sugar provides technical guarantee to put into practice assistant breeding, shortening breeding cycle.
(1)F5The structure of group：
The present invention is female parent with sugar grass LTR102, and sorghum 654 is male parent, and hybridization obtains F5Generation, wherein 87 plant heights sugar and
33 plants of low sugar.
By F5In generation, is divided into Gao Tang groups and low sugar group, and Gao Tang groups and low sugar group are respectively by F587 plant height sugar strain leaves of generation
Piece and 33 plants of low sugar strain blade compositions, and carry out the extraction of enzyme solution, mRNA.
(2) high effective liquid chromatography for measuring cane sugar content is applied；SPS activity is measured using spectrophotometry, sucrose is contained
Amount carries out correlation analysis with SPS activity.
(3) extracting method for establishing sugar grass RNA, using the RNA that improved Trizol method extracts, the RNA extracted is complete
Whole property is fine, and there is no pollutions by RNA, can be used as template in next step is tested.
(4) Sucrose Phosphate Synthase (SPS) gene primer designs：It is compiled according to known one section of sugar grass SPS3-1 genes
Code area cDNA sequence, primer is designed using Oligo6 softwares and Primer primier softwares.It is named as SPS-1, β-
Actin is house-keeping gene.
(5) round pcr is applied to carry out qualitative analysis to sugar grass Sucrose Phosphate Synthase (SPS) gene expression, as a result table
Bright to expand ideal target fragment by primer of β-Actin and SPS-1, bands of a spectrum are clear and without miscellaneous band, and seedling stage just can detect
Go out SPS genes, with the extension of growth period, SPS gene expression amounts are stepped up, and are that expression quantity reaches maximum to the maturity period.
This is consistent with the changing rule of cane sugar content, and the accumulation of sucrose can be influenced with the expression of preliminary proof SPS genes.In qualitative inspection
Quantitative fluorescent PCR detection is carried out on the basis of survey, more accurately understands the changing rule of SPS genes.
(6) fluorescent quantitative PCR technique is applied to carry out quantitative analysis to SPS gene expressions, the results showed that SPS gene expressions
Amount is followed successively by Gao Tang groups, female parent, low sugar group and male parent from high to low.The changing rule of SPS genes contains with sugar grass sucrose
The changing rule of amount and SPS enzymatic activitys is almost the same, and is all to reach maximum value in the maturity period, is the high sugar products kind of sugar grass
Selection and breeding and determine that the best water content in harvest of sugar grass provides theoretical foundation.
The invention is realized in this way sugar grass Sucrose phosphate synthase gene amplimer is on the one hand provided, including
Primer SPS-1 and primer β-Actin, primer SPS-1 include SPS-1 forward primers and SPS-1 reverse primers, nucleotide sequence point
Not such as SEQ ID NO：1 and SEQ ID NO：Shown in 2；Primer β-Actin include β-Actin forward primers and β-Actin reversed
Primer, nucleotide sequence is respectively such as SEQ ID NO：3 and SEQ ID NO：Shown in 4.
Further, the length of the target fragment of the primer SPS-1 amplifications is 121bp；Primer β-the Actin expand
The length of the target fragment of increasing is 210bp.
On the other hand, the present invention also provides the fast PCR examinations of detection sugar grass Sucrose phosphate synthase gene expression
Agent, the PCR reagent include 10 × Buffer, 2.5 μ l, 25mg/ml MgCl22.5 μ l, 100 μm of 0.5 μ l of ol/l dNTP,
5U/ μ l Taq enzymes 0.5 μ l, 0.2 μm of ol/l Forward Primer 1.0 μ l, 0.2 μm of 1.0 μ of ol/l Reverse Primer
1.0 μ l of l, cDNA template, use ddH2O supplies 25 μ l；
Primer in the PCR reagent is primer SPS-1 described in claim 1 or primer β-Actin.
Further, include following response procedures using the method that above-mentioned fast PCR reagent carries out PCR reactions：Pre-degeneration
94 DEG C of 2min → 32 amplification cycles → 72 DEG C of extension 10min → 4 DEG C preserve, and amplification cycles include the following steps：94 DEG C of denaturation
53 DEG C of 30s → annealing, 72 DEG C of 20s → extension 1min 20s.
In another aspect, the present invention also provides a kind of rapid fluorescence quantitative PCRs of detection Sucrose phosphate synthase gene expression
Reagent, 20 μ l of the quantitative fluorescent PCR reagent volume, including：2 × SYBR Premix ExTaq II 10.0 μ l, ddH2O
6.0 μ l, 0.2 μm of ol/l Forward Primer 0.8 μ l, 0.2 μm of ol/l Reverse Primer 0.8 μ l, ROX
0.4 μ l, cDNA templates of Reference DYE, 2.0 μ l；
Primer in the quantitative fluorescent PCR reagent is primer SPS-1 described in claim 1 or primer β-Actin.
Further, include following response procedures using the method that mentioned reagent carries out rapid fluorescence quantitative PCR：Pre-degeneration
95 DEG C of 15min → 35 amplification cycles → 53-95 DEG C of solubility curve programs；Amplification cycles include the following steps：94 DEG C of denaturation
53 DEG C of 20s → annealing, 72 DEG C of 20s → extension 1min 20s.
Compared with the prior art, the advantages of the present invention are as follows：
The primer and detection of expression method are applied to sorghum sugared content, SPS enzymatic activitys, the quick of SPS gene expressions to divide
Analysis eliminates low sugar variety and genetype in the selection of high sugar content kind, accelerates breeding process, determines best water content in harvest, section
Saving practice ground and cost needed for selection and breeding etc. etc. has larger practical value, and then overcomes those since phenotype is reflected
It is fixed difficult, with the very huge difficulty of usual way assisted Selection workload, provide technical guarantee to shorten breeding cycle.
Fill up the state of sugar grass SPS gene expression assisted Selection researchs, inside and outside blank simultaneously.
Description of the drawings
Fig. 1 is the seedling stage SPS gene expression detection bands of a spectrum (PCR method) of the present invention.
Fig. 2 is the jointing stage SPS gene expression detection bands of a spectrum (PCR method) of the present invention.
Fig. 3 is the heading stage SPS gene expression detection bands of a spectrum (PCR method) of the present invention.
Fig. 4 is the maturity period SPS gene expression detection bands of a spectrum (PCR method) of the present invention.
Specific implementation mode
Referring to Fig.1, Fig. 2, Fig. 3 and Fig. 4, sorghum 654 (male parent) (1,5 bands of a spectrum) as shown in the figure, sugar grass LTR102 are (female
This) (2,6 bands of a spectrum), F5High araa gene pond (3,7 bands of a spectrum), F5Low sugar gene pool (4,8 bands of a spectrum), Marker (M bands of a spectrum).1-4 bands of a spectrum
Using the PCR product that primer pair β-Actin go out as primer amplification, fragment length is 210bp or so；5-8 bands of a spectrum are with primer pair SPS-1
For the PCR product that primer amplification goes out, fragment length 121bp.
1. material and method
1.1 test material
Sugar grass LTR102 (female parent), sorghum 654 (male parent) and the F that they hybridize5Generation individual.
1.2 drugs, instrument and equipment
100mmol/L PH7.8 phosphate buffers 1%PVP, 0.1mmol/LEDTA, 0.1% resorcinol, UDPG,
Six phosphofructoses of 100mmol/L, 100mmol/L fructose, agarose, chloroform, EDTA are that domestic analysis is pure, Trizol reagents
Box, pyrocarbonic acid diethyl ester (DEPC), Tris, isopropanol, QuantiTect Reverse Transcription Kid are treasured
Biological (Dalian) Products.
Quantitative fluorescent PCR, temperature gradient PCR, cryogenic freezing centrifuge, electronic balance, electrophoresis apparatus, refrigerator, ultralow temperature
Refrigerator micro-wave oven, micropipettor, ultra-pure water instrument.
1.3 experiment master-plans
Respectively in seedling stage, jointing stage, heading stage, maturity period take LTR102 (female parent), 654 (male parents) and F5Generation individual
Blade → extraction mRNA (improved Trizol method) → reverse transcription is cDNA → Sucrose Phosphate Synthase (SPS) gene magnification primer
Design → carrying out qualitative analysis to SPS gene expressions using round pcr → applies fluorescent quantitative PCR technique to SPS gene expressions
Carry out method → determination that quantitative analysis → analysis sugar content quickly detects SPS gene expressions with SPS expression correlations → foundation
Identification method → for the high sugar products kind of sugar grass selection and breeding and determine that best water content in harvest provides theoretical foundation.
1.4 sugar grass Sucrose Phosphate Synthase (SPS) determinations of activity
1g samples are weighed, cleans and shreds as in precooling mortar, 2.5ml phosphate buffers, ice bath is added to grind.It is ground into homogenate
After pour into centrifuge tube, then with 2.5ml phosphate buffer rinses, pour into centrifuge tube together later.10000xg is centrifuged,
15min takes supernatant spare.It takes 50 μ l crude enzyme liquids to be added in the test tube of respective number, 50 μ l buffer solutions, 20 μ l is added
MgCl2,20 μ l UDPG, 20 μ l fructose-1, 6-diphosphates, oscillation.30 DEG C, water-bath 30min.200 μ l NaOH, boiling water boiling is added
10min, it is cooling.30% hydrochloric acid of 1.5ml, 0.5ml resorcinols is added.80 DEG C of water-bath 10min coolings.The colorimetric at 480nm.
The extraction of 1.5 sugar grass RNA and the synthesis of cDNA
1.5.1 improved Trizol method extracts total serum IgE
(1) leaf tissue is clayed into power in liquid nitrogen, 1ml Trizol reagents is added in being organized to every 50-100mg,
Sample volume is not to be exceeded 100 μ l and is stored at room temperature 5min.
(2) 0.2ml chloroforms are added into every pipe sample, cover tightly pipe cap, violent oscillation sample 15 seconds is stablized at room temperature
5min。
(3) sample centrifuges 15min at 12000xg, 4 DEG C
(4) supernatant is taken to enter new pipe, often pipe plus 0.5ml isopropanols, mixing are placed at room temperature for 5min.
(5) sample centrifuges 10min at 12000xg, 4 DEG C.
(6) supernatant is abandoned, RNA precipitate, vortex mixing are washed with 75% ethyl alcohol of 1ml.
(7) 12000xg, centrifugation 5min. (if being used for reverse transcription PCR, repeating step 6 twice) at 4 DEG C.
(8) surface object is abandoned, drying at room temperature 5-10min (not be such that precipitation is completely dried up), go TE to dissolve with 30-60 μ l.
1.5.2RNA purity analysis
(1) 10 to 20 μ lRNA samples are taken from original stock, go RNA enzyme water dissolution micro- in 1.5ml with 990 or 980 μ l
In type centrifuge tube, the RNA sample of 100 or 200 times of dilution is obtained.
(2) 1ml is drawn to go RNA enzyme water to be added in clean cuvette and read the absorptance of blank.
(3) diluted RNA sample is drawn to be added in clean cuvette and read the absorptance under 260,280 and 230nm.With
Following formula measures the RNA concentration of primary sample：L=A260 × 33 RNA μ g/ μ × extension rate/1000.
1.5.3RNA integrity analysis
With 1.0% Ago-Gel under 90V voltages electrophoresis 45min, in gel imager observe RNA bands it is clear
Clear degree and integrality.
The design of 1.6 Sucrose Phosphate Synthases (SPS) primer
According to the gene coding regions known one section of sugar grass SPS3-1 cDNA sequence, using Oligo6 softwares and Primer
Primier softwares are designed primer.
Reaction system, response procedures and the detection method of 1.7PCR
1.7.1PCR the foundation of reaction system and response procedures
Reverse transcription is carried out with QuantiTect Reverse Transcription Kid, 0.2ml Eppendorf is taken to manage,
Often pipe is by being added 2 μ l, Template RNA total amounts of following ingredient gDNA Wipeout Buffer in 10pg to 1 after sample number into spectrum
Between μ g, RNase-free water is added to make reaction total volume greatly to 14 μ l.It is put into water-bath after sample-adding, removes RNA
In 42 DEG C of 10min of genomic DNA.QuantiTect Reverse Transcriptase 1ul are added into pipe again,
4 μ l, RT Primer Mix of QuantiTect RT Buffrt, 1 μ l, this reaction volume for being reach 20 μ l.42 DEG C of water-baths
15min, 95 DEG C of 3min.
The foundation of reaction system
After RNA passes through reverse transcription into cDNA, 0.2ml Eppendorf is taken to manage, often pipe is as follows by being added after sample number into spectrum
Ingredient：Reaction system is 25 μ l, including 10 × Buffer, 2.5 μ l, 25mg/ml MgCl22.5 μ l, 100 μm of ol/l
0.5 μ l, 5U/ μ l Taq enzymes of dNTP 0.5 μ l, 0.2 μm of ol/l Forward Primer 1.0 μ l, 0.2 μm of ol/l Reverse
1.0 μ l, cDNA templates of Primer, 1.0 μ l, use ddH2O supplies 25 μ l；
Response procedures：
The Eppendorf pipes that above-mentioned sample-adding is finished, are put into progress PCR reactions in EN61010-1PCR amplification instruments, reaction
Program is：94 DEG C of 2min of pre-degeneration → (94 DEG C of 53 DEG C of 30s → annealing, 72 DEG C of 20s → extension 1min 20s of denaturation) 32 are followed
72 DEG C of 10min → 4 DEG C of ring → extension preserve.
1.7.2PCR product analysis
PCR product is detected with 1% agarose gel electrophoresis, amplified production is further analyzed and determines production
Object specificity.
The expression quantity of 1.8 fluorescence quantitative PCR detection sugar grass sucrose phosphate synthetases (SPS)
1.8.1 the foundation of standard curve
CDNA is diluted into 5 gradients (22,24,25,26,27), carries out target gene SPS and reference gene β-respectively
The SYBR Green I real-time quantitative PCRs of actin react, and prepare the standard curve of two genes and detect amplification efficiency.
1.8.2 the reaction system and condition of quantitative fluorescent PCR
The reaction system of SYBR quantitative fluorescent PCRs：20μl：2 × SYBR Premix ExTaq II 10.0 μ l, ddH2O
6.0 μ l, Forward Primer, 0.8 μ l, Reverse Primer, 0.8 μ l, ROX Reference DYE 0.4 μ l, cDNA
2.0 μ l of template.
Response procedures：95 DEG C of 15min of pre-degeneration → (94 DEG C of 53 DEG C of 20s → annealing, 72 DEG C of 20s → extensions of denaturation
1min 20s) 35 cycles → 53-95 DEG C of solubility curve program reaction terminates instrument and automatically generates CT (threshold
Cycles) value and melting curve figure.Each sample sets 3 repeating pipes, while setting no Template-negative controls.
Embodiment 1：Sugar grass sucrose phosphate synthetase (SPS) activity and cane sugar content correlation analysis
The F that the present invention passes through measurement different times male parent, female parent and their hybridization5Gao Tang groups and low sugar colony leaves
In SPS activity, understand between different times different groups SPS changing rule.Parent and F5Gao Tang groups and low sugar
The activity of SPS in colony leaves is being stepped up.Maternal, Gao Tang groups and these three sugar grasses of low sugar group at the maturity period
It is respectively 14.86,17.63,13.76 μm of ol/h.g.FW that the activity of kind, which all reaches maximum value,；Work of the male parent in maturity period SPS
Property is slightly declined than heading stage.The activity of the SPS of four strains is without too big difference when in seedling stage, and is opened from the jointing stage
There is gap in the activity of the SPS of beginning four strains.The activity of two Hybrid SPS of Gao Tang groups and low sugar group is linear
Increase, the speed that activity increases is faster than parent, and the enzymatic activity highest of SPS of the high sugar products kind in the maturity period.To sorghum and sweet tea
Sucrose in Sorghum Leaves carries out correlation analysis with SPS activity, the results showed that sucrose is in significant positive correlation with SPS activity
(R=0.763, P<0.01).
Embodiment 2：The extraction interpretation of result of sugar grass RNA
Agarose gel electrophoresis detection shows that the RNA extracted using improved Trizol method has 3 bands, in total serum IgE
The brightness of 28S bands is approximately 2 times of 18S bands, illustrates that it degrades less in extraction process, the RNA integralities extracted are very
It is good.For 5S bands than shallower, illustrating RNA, there is no pollutions, can be used as template in next step is tested.
Embodiment 3：Sucrose Phosphate Synthase (SPS) design of primers
Primer in table 1 is according to the gene coding regions known one section of sugar grass SPS3-1 cDNA sequence, using Oligo6
Software and Primer primier softwares are designed primer.It is named as SPS-1, β-Actin are house-keeping gene, and two pairs of primers expand
The length of the target fragment of increasing is respectively 121bp and 210bp.
Table 1SPS gene magnification the primers
Embodiment 4：The qualitative analysis of Sucrose Phosphate Synthase (SPS) gene expression
Seedling stage parent, F5The expression of SPS genes in Gao Tang groups and low sugar colony leaves is as shown in Figure 1, in figure
1-4 bands of a spectrum be male parent, female parent, Gao Tang groups and low sugar group cDNA, using the PCR product that β-Actin go out as primer amplification, piece
Segment length is 210bp or so；5-8 bands of a spectrum be male parent, female parent, Gao Tang groups and low sugar group cDNA, using SPS-1 as primer
The PCR product amplified, fragment length 121bp.The result shows that SPS genes have begun to express in seedling stage.
The expression of jointing stage SPS gene is shown in that Fig. 2,1-4 bands of a spectrum are male parent, female parent, Gao Tang groups and low sugar group
CDNA, using the PCR product that β-Actin go out as primer amplification, fragment length 210bp；5-8 bands of a spectrum are male parent, female parent, high sugar
The cDNA of group and low sugar group, using the PCR product that SPS-1 goes out as primer amplification, fragment length is 121bp or so.As a result table
The bright SPS gene expressions in jointing stage male parent are weaker, and the SPS gene expressions of Gao Tang groups are stronger.
The expression of heading stage SPS gene is shown in that Fig. 3,1-4 bands of a spectrum are male parent, female parent, Gao Tang groups and low sugar group
CDNA, using the PCR product that β-Actin go out as primer amplification, fragment length 210bp；5-8 bands of a spectrum be with heading stage male parent,
Maternal, Gao Tang groups and low sugar group cDNA, using the PCR product that SPS-1 goes out as primer amplification, fragment length is 121bp left
It is right.The result shows that brightness and fineness from heading stage purpose piece degree all than the first two period reinforced, illustrate SPS bases
The amount when expression quantity of cause is than seedling stage and jointing stage is big.
The expression of maturity period SPS gene is shown in that Fig. 4,1-4 bands of a spectrum are male parent, female parent, Gao Tang groups and low sugar group
CDNA, using the PCR product that β-Actin go out as primer amplification, fragment length 210bp；5-8 bands of a spectrum are male parent, female parent, height
The cDNA of sugared group and low sugar group, using the PCR product that SPS-1 goes out as primer amplification, fragment length is 121bp or so.As a result
Show that SPS genes reach big value in maturity period expression quantity.
In conclusion can amplify target fragment by primer of β-Actin and SPS-1, bands of a spectrum are clear and without miscellaneous band.From
Fig. 1-4 is it is found that SPS gene expression amounts are smaller in seedling stage, and with the variation of growth period, SPS gene expression amounts are stepped up,
Reach maximum to the maturity period.This is consistent with the changing rule of cane sugar content, can influence sugarcane with the expression of preliminary proof SPS genes
The accumulation of sugar.
Embodiment 5：The quantitative analysis of Sucrose Phosphate Synthase (SPS) gene expression
5.1 seedling stage SPS Quantitative analysis of gene expression
Quantitative analysis results show that with SPS genes relative concentration in maternal blade be control, in seedling stage Gao Tang groups
SPS genes relative concentration is 1.59 times of maternal SPS gene expression amounts to big.The relative concentration of male parent SPS genes is minimum, only
It is the 1/2 of maternal SPS gene expression amounts.(being shown in Table 2)
Table 2 seedling stage SPS Quantitative analysis of gene expression
5.2 jointing stage SPS Quantitative analysis of gene expression
Jointing stage parent, F5Gao Tang groups and low sugar group are in this period expression difference very little, maximum high sugared group
The expression quantity of body SPS genes is 1.25 times of minimum low sugar group SPS gene expression amounts.(being shown in Table 3)
3 jointing stage of table SPS Quantitative analysis of gene expression
5.3 heading stage SPS Quantitative analysis of gene expression
The relative concentration of SPS genes is minimum in heading stage male parent blade, and the expression difference of other three groups is little, says
It is bright since heading stage, between sugar grass and sorghum SPS genes expression difference increase.The expression quantity of sugar grass SPS genes
About 2 times of sorghum gene expression amount.(being shown in Table 4)
Table 4 heading stage SPS Quantitative analysis of gene expression
5.4 maturity period SPS Quantitative analysis of gene expression
Maturity period parent, F5Gao Tang groups and low sugar group are larger in this period expression difference, Gao Tang groups SPS bases
The expression quantity of cause is 15 times of minimum low sugar group SPS gene expression amounts.(being shown in Table 5)
5 maturity period of table SPS Quantitative analysis of gene expression
In conclusion in each period with the relative concentration of maternal SPS genes to compare, SPS gene expression amounts are by height
It is followed successively by Gao Tang groups, female parent, low sugar group and male parent to low.Maturity period this variation tendency and sucrose accumulation law and
SPS activity change rules are consistent, it can be seen that the content that SPS gene expression amounts get over high-sucrose is also more.
SEQUENCE LISTING
<110>Shenyang Normal University
<120>The detection of expression method and amplimer of sugar grass Sucrose phosphate synthase gene
<130> 2018
<160> 4
<170> PatentIn version 3.3
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<211> 20
<212> DNA
<213>Artificial sequence
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<210> 4
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<212> DNA
<213>Artificial sequence
<400> 4
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Claims (6)
1. sugar grass Sucrose phosphate synthase gene amplimer, which is characterized in that including primer SPS-1 and primer β-Actin,
Primer SPS-1 includes SPS-1 forward primers and SPS-1 reverse primers, and nucleotide sequence is respectively such as SEQ ID NO：1 and SEQ ID
NO：Shown in 2；Primer β-Actin include β-Actin forward primers and β-Actin reverse primers, and nucleotide sequence is respectively such as SEQ
ID NO：3 and SEQ ID NO：Shown in 4.
2. sugar grass Sucrose phosphate synthase gene amplimer as described in claim 1, which is characterized in that the primer
The length of the target fragment of SPS-1 amplifications is 121bp；The length of the target fragment of the primer β-Actin amplifications is 210bp.
3. a kind of fast PCR reagent of detection sugar grass Sucrose phosphate synthase gene expression, which is characterized in that the PCR examinations
Agent includes 10 × Buffer, 2.5 μ l, 25mg/ml MgCl22.5 μ l, 100 μm of 0.5 μ l, 5U/ μ l Taq enzymes of ol/l dNTP
0.5 μ l, 0.2 μm of 1.0 μ l of ol/l Forward Primer, 0.2 μm of 1.0 μ l, cDNA template of ol/l Reverse Primer
1.0 μ l, use ddH2O supplies 25 μ l；
Primer in the PCR reagent is primer SPS-1 described in claim 1 or primer β-Actin.
4. the method for carrying out PCR reactions using reagent as claimed in claim 3, which is characterized in that including following response procedures：
94 DEG C of 2min → 32 amplification cycles of pre-degeneration → 72 DEG C of extension 10min → 4 DEG C preserve, and amplification cycles include the following steps：Become
94 DEG C of 53 DEG C of 30s → annealing, 72 DEG C of 20s → extension 1min 20s of property.
5. a kind of rapid fluorescence quantitative PCR reagent of detection Sucrose phosphate synthase gene expression, which is characterized in that the fluorescence
20 μ l of quantitative PCR reagent volume, including：2 × SYBR Premix ExTaq II 10.0 μ l, ddH2O 6.0 μ l, 0.2 μm of ol/l
Forward Primer 0.8 μ l, 0.2 μm of 0.8 μ l, ROX Reference DYE of ol/l Reverse Primer, 0.4 μ l,
2.0 μ l of cDNA templates；
Primer in the quantitative fluorescent PCR reagent is primer SPS-1 described in claim 1 or primer β-Actin.
6. the method for carrying out rapid fluorescence quantitative PCR using reagent as claimed in claim 5, which is characterized in that including as follows
Response procedures：95 DEG C of 15min → 35 amplification cycles → 53-95 DEG C of solubility curve programs of pre-degeneration；Amplification cycles include as follows
Step：It is denaturalized 94 DEG C of 53 DEG C of 20s → annealing, 72 DEG C of 20s → extension 1min 20s.
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| CN110592266A (en) * | 2019-11-01 | 2019-12-20 | 天津农学院 | Sweet sorghum stalk juice yield related gene and application thereof |
Citations (2)
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|---|---|---|---|---|
| WO2007022318A2 (en) * | 2005-08-17 | 2007-02-22 | Cornell Research Foundation | Nucleic acids and proteins associated with sucrose accumulation in coffee |
| CN102753688A (en) * | 2009-09-17 | 2012-10-24 | 斯泰伦博斯大学 | A method of modifying the carbohydrate content of a plant |
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2018
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007022318A2 (en) * | 2005-08-17 | 2007-02-22 | Cornell Research Foundation | Nucleic acids and proteins associated with sucrose accumulation in coffee |
| CN102753688A (en) * | 2009-09-17 | 2012-10-24 | 斯泰伦博斯大学 | A method of modifying the carbohydrate content of a plant |
Non-Patent Citations (2)
| Title |
|---|
| HILAL AHMAD QAZI等: "Stem sugar accumulation in sweet sorghum–activity and expression of sucrose metabolizing enzymes and sucrose transporters", 《J PLANT PHYSIOL》 * |
| 魏鑫: "甜高粱SPS基因表达及其对糖分含量积累的影响", 《中国优秀硕士学位论文全文数据库农业科技辑》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110592266A (en) * | 2019-11-01 | 2019-12-20 | 天津农学院 | Sweet sorghum stalk juice yield related gene and application thereof |
| CN110592266B (en) * | 2019-11-01 | 2022-03-29 | 天津农学院 | Sweet sorghum stalk juice yield related gene and application thereof |
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