CN108315394A - The detection of expression method and amplimer of sugar grass sucrose synthase gene - Google Patents
The detection of expression method and amplimer of sugar grass sucrose synthase gene Download PDFInfo
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Abstract
The present invention has chosen 10 sugar grass main breeds, in seedling stage, the jointing stage, heading stage, the maturity period sampling.Compare the sugared content of different cultivars different times and the activity change of sucrose synthase using high performance liquid chromatography and spectrophotometry, and cane sugar content and the correlation of SS enzymatic activitys are analyzed, the results showed that cane sugar content and SS enzymatic activity positive correlations；Compare the difference of SS gene expressions between 4 periods, 10 sugar grass kinds using Real-Time Fluorescent Quantitative PCR Technique, analyze the relationship between sugar Accumulation and SS gene expression amounts in sugar grass body, the results showed that SS gene expression amounts and cane sugar content positive correlation.Result according to the present invention establishes the reaction system of SS quantitative gene expressions detection and the research method of sugar grass SS gene expression rules, 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 synthase (SS) gene, especially sugar grass sucrose
The detection of expression method and amplimer of synthase gene.
Background technology
As the fossil energies such as oil, coal are increasingly exhausted and the deterioration of the ecological environment, energy security and Environmental security have become
The severe challenge faced for the whole world.Develop and use novel renewable energy has weight for society, economic sustainable development
Want strategic importance (the Ministry of Agriculture, 2007；People's Republic of China's national development and reform committee member, 2007).China is the energy
Big country is consumed, greatly develops that energy density is high, resistance is strong, the biomass energy crop of " not striving ground with grain, do not strive grain with people "
The structure of agricultural production can be expanded, promotes Regional Economic Development, alleviate energy supply anxiety situation, greenhouse gas emission is reduced, improves life
State environment, be ensure national energy security and Environmental security two-win behave (Zhang Zhipeng etc., 2005；Zou Jianqiu etc., 2007).
Currently, in numerous biomass energy crops, sugar grass is with its unique high sugar content, high biological yield, Gao Yi
Alcohol conversion and high resistance to cold and diseases, which become, to tally with the national condition, is suitble to China territory, weather conditions, the not only reason of production capacity source but also production grain
Thought of as one of object, its utilization are that the diversification of fuel ethanol production raw material opens the roads Tiao Xin.Sugar grass
(Sorghum bicolor L.Moench) stalk succulence is rich in sugar, also known as sugared sorghum or sorgo, with other energy crop phases
Than sugar grass has biological yield is high (to belong to C4Plant), sugar content is high, multiple resistance (drought resisting, waterlogging, saline-alkali tolerant) and second
The advantages that alcohol conversion is high, at low cost.In June, 2007, after the grain alcohol fuel project based on corn is halted by country, sweet tea
Sorghum as " the most competitive person in bioenergy system " (Li great Jue, 2003；Yang Wenhua, 2004), become China most
One of promising green energy resource crop (dawn cyanines etc. perhaps, 2008), the core of alcohol fuel is converted into using sweet sorghum stalk
Heart technology has been broken through, and gain the national patent, sweet sorghum stalk sugar content are to determine the principal element of ethanol production to scientific and technological achievement,
It is one of most important research contents of current Sweet Sorghum Breeding to improve sweet sorghum stalk sugar content.
Sucrose is most important energy carrier in higher plant body, is the prevailing traffic form of carbohydrate, or thin
The regulatory factor of born of the same parents' metabolism.In the growth and development process of sugar grass, there are three types of enzyme regulation and control Sucrose Metabolism, plant cell decide
The content of middle sucrose, respectively sucrose synthase (SS), Sucrose Phosphate Synthase (SPS) and invertase (Inv).These three
The content of glucose, fructose and sucrose in enzyme co- controlling plant, co-catalysis sucrose enter various metabolic pathways.Three kinds of enzymes exist
Play the role of very important in plant growth and development process, therefore, it is most important with the relevant enzyme of Sucrose Metabolism to study these.
The activity of SS directly reflects the ability of Sucrose synthesis in plant.It is to verify stem from analysis SS gene expression amounts
Stalk Sugar Accumulation explains sugared content difference between different cultivars and establishes the effective way of molecular regulation system do not have also at present
There is the report of related fields.
Invention content
The purpose of the present invention is in view of the above shortcomings of the prior art, having chosen domestic and international 10 sugar grass main breeds,
Compare the sugared content of different cultivars sugar grass different times and the activity change of sucrose synthase using high performance liquid chromatography,
Compare the difference of SS gene expressions between 4 periods, 10 sugar grass kinds using Real-Time Fluorescent Quantitative PCR Technique, understands sweet tea
Sugar Accumulation in sorghum body establishes a kind of sugar grass sucrose synthase (SS) gene expression law study method.
The object of the invention one：4 periods (seedling stage, jointing stage, pumpings are measured using high performance liquid chromatography and spectrophotometry
Ear period, maturity period) 10 sugar grass kinds (Tianjin be 53, sweet tea GL, LTR108, LTR168, HT, collier, M-81E, Rio,
Honey, umbrella) cane sugar content and sucrose synthase (SS) activity, analysis sucrose synthase (SS) and sucrose accumulation
Correlation.
The object of the invention two：Using fluorescent quantitative PCR technique, 10 sugar grass kinds of quantitative analysis (Tianjin be 53, sweet tea GL,
LTR108, LTR168, HT, collier, M-81E, Rio, Honey, umbrella) 4 growth periods (seedling stage, the jointing stage,
Heading stage, maturity period), SS expression conditions, the results showed that different cultivars SS gene expression relative concentrations have differences, and its
Otherness is consistent with different cultivars cane sugar content variation tendency, and then establishes a kind of sugar grass sucrose synthase (SS) gene table
Up to law study method, this method can be as the assisted Selection means of the high sorghum variety containing sugar of screening, to realize sugar grass
Assistant breeding provides technical guarantee.
(1) present invention is with 10 sugar grass kinds of sugar grass：Tianjin be 53, sweet tea GL, LTR108, LTR168, HT, collier,
M-81E, Rio, Honey, umbrella be examination material, in seedling stage, the jointing stage, heading stage, the maturity period sampling, carry out cane sugar content,
SS enzymatic activitys and SS gene expression quantitative fluorescence analysis.
(2) high effective liquid chromatography for measuring cane sugar content is applied；SS activity is measured using spectrophotometry, to cane sugar content
Correlation analysis is carried out with SS activity.
(3) extracting method for establishing sugar grass RNA is extracted using the RNA extracts kits of Promega companies
RNA integralities are fine, and there is no pollutions by RNA, can be used as template in next step is tested.
(4) sucrose synthase (SS) gene primer screens：According to the gene coding regions known one section of sugar grass SS cDNA sequences
Row, screening primer is designed using Oligo6 softwares and Primer softwares.It is named as SS-1, β-Actin.
(5) apply fluorescent quantitative PCR technique to SS gene expressions carry out quantitative analysis, the results showed that SS gene expression amounts compared with
High kind is LTR108.The changing rule basic one of the changing rule and sugar grass cane sugar content and SS enzymatic activitys of SS genes
It causes, and is all to reach maximum value in the maturity period, be the selection and breeding of the high sugar products kind of sugar grass and determine the best harvest of sugar grass
Period provides theoretical foundation.
The invention is realized in this way on the one hand providing sugar grass sucrose synthase gene amplimer, including primer SS-
1 and primer Actin, primer SS-1 include SS-1 forward primers and SS-1 reverse primers, and nucleotide sequence is respectively such as SEQ ID
NO：1 and SEQ ID NO：Shown in 2；Primer Actin includes Actin forward primers and Actin reverse primers, nucleotide sequence point
Not such as SEQ ID NO：3 and SEQ ID NO：Shown in 4.
On the other hand, a kind of synthetic method of sugar grass sucrose synthase gene cDNA is provided, is included the following steps：
1) synthesis of first chain of cDNA
Using TaKaRa RT-PCR kits, reaction system is 10 μ l, including：5 × PRT Mix, 2 μ l, RNA templates, 8 μ
l；Reaction condition is：37 DEG C of 15min, 85 DEG C of 5s, reverse transcription product are preserved in -20 degree；
2) amplification of cDNA
PCR reaction systems are 25 μ l, including：10 × Buffer 2.0 μ l, 100 μm of ol/l dNTPs 0.5 μ l, 0.2 μ
Mol/l F-Primer 0.8 μ l, 0.2 μm of ol/l R-Primer 0.8 μ l, cDNA 0.5 μ l, Taq enzyme 0.5 μ l, ddH2O
19.4μl；The primer is primer SS-1 described in claim 1 or primer Actin.
PCR reaction conditions are:94 DEG C of pre-degeneration 3min → 30 time amplification cycles → 72 DEG C extend 10min, amplification cycles packet
It includes：94 DEG C of denaturation 30s → 58 DEG C annealing 30s → 72 DEG C extend 45s；
3) detection of cDNA
Pcr amplification product is detected with 1% agarose gel electrophoresis, 100V electrophoresis 20min are detected in gel imager
The synthesis situation of cDNA.
In another aspect, a kind of rapid fluorescence quantifying PCR method of sugar grass sucrose synthase gene expression is provided, including such as
Lower step：
Real-time PCR amplifications are carried out with SYBR Premix ExTaq II, using the cDNA of sugar grass as template, are carried out
The amplification of target gene, reference gene, 3 repetitions are arranged in each sample, while blank control is arranged；
20 μ l of reaction system, including：2 × SYBR Premix ExTaq II 10.0 μ l, ddH2O 6.0 μ l, Forward
0.4 μ l, cDNA templates of Primer 0.8 μ l, Reverse Primer, 0.8 μ l, ROX Reference DYE, 2.0 μ l；It is described
Primer is primer SS-1 described in claim 1 or primer Actin；
PCR response procedures are：95 DEG C of pre-degeneration 30s → 40 amplification cycles → 72 DEG C extend 10min, amplification cycles packet
It includes：95 DEG C of denaturation 5s → 60 DEG C annealing 20s → 72 DEG C extend 30s；
60-95 DEG C of solubility curve program, after reaction is completed, computer automatically generates CT values, melting curve.
Compared with the prior art, the advantages of the present invention are as follows：Apply the present invention to sorghum sugared content, SS enzymatic activitys, SS
The quick analysis of gene expression is the selection of high sugar content kind, eliminates low sugar variety and genetype, accelerate breeding process, determines
Best water content in harvest, practice ground and the cost etc. saved needed for selection and breeding have larger practical value, and then overcome
Those are due to the phenotypic evaluation difficulty very huge difficulty of usual way assisted Selection workload, to shorten breeding cycle
Provide technical guarantee.Fill up the state of sugar grass SS gene expression assisted Selection researchs, inside and outside blank simultaneously.
Description of the drawings
Fig. 1 is that material RNA extraction purification electrophoresis detection results are tried in the part of the present invention.
Fig. 2 is 10 different cultivars cDNA electrophoresis detection results of the present invention.
Fig. 3 is 10 different cultivars seedling stage SS gene expression amounts of the present invention.
Fig. 4 is 10 different cultivars jointing stage SS gene expression amounts of the present invention.
Fig. 5 is 10 different cultivars heading stage SS gene expression amounts of the present invention.
Fig. 6 is 10 different cultivars maturity period SS gene expression amounts of the present invention.
Specific implementation mode
Referring to Fig.1, as shown, 1-8, it is 53, sweet tea GL, LTR108, LTR168, HT, collier, M- to be followed successively by Tianjin
81E、Rio。
With reference to Fig. 2, as shown, M, 1-10, be followed successively by Marker (DL 2000), Tianjin be 53, sweet tea GL, LTR108,
LTR168、HT、collier、M-81E、Rio、Honey、umbrella。
With reference to Fig. 3-6, as shown, abscissa be followed successively by M-81E, umbrella, LTR108, sweet tea GL, Honey, HT,
Collier, LTR168, Rio, Tianjin are 53.
1. material and method
1.1 test material
The present invention is with 10 sugar grass kinds：Tianjin be 53, sweet tea GL, LTR108, LTR168, HT, collier, M-81E,
Rio, Honey, umbrella be examination material, in seedling stage, the jointing stage, heading stage, the maturity period sampling.1.2 drugs, instrument and equipment
Glucose, fructose, sucrose, acetonitrile, anthrone, the concentrated sulfuric acid, EDTA, BSA, PVP, MgCl2, fructose-1, 6-diphosphate, UDPG,
NaOH, resorcinol, agarose, chloroform, Tris, isopropanol, RNA extracts kits (Promega), SYBR Premix Ex
Taq II (TaKaRa), PrimeScript RT Master Mix (TaKaRa).
Quantitative fluorescent PCR, temperature gradient PCR, high performance liquid chromatograph, high speed freezing centrifuge, electronic balance, electrophoresis
Instrument, electrophoresis tank, refrigerator, Sanyo's ultra low temperature freezer, micro-wave oven, ultra-pure water instrument, ultraviolet-uisible spectrophotometer, ice machine, water
Bath, pipettor.
1.3 experiment master-plans
Respectively in seedling stage, jointing stage, heading stage, maturity period take 10 sugar grass kinds (Tianjin be 53, sweet tea GL, LTR108,
LTR168, HT, collier, M-81E, Rio, Honey, umbrella) materials, 3 repetitions, materials position are sugar grass at random
2 leaf, -80 DEG C of refrigerators are put into after liquid nitrogen flash freezer preserve → uses high performance liquid chromatography and measure cane sugar content, using spectrophotometric
It is cDNA → sucrose synthase (SS) gene that method, which measures SS enzymatic activitys while extracting mRNA (RNA extracts kits) → reverse transcription,
The screening of amplimer → fluorescent quantitative PCR technique is applied to carry out quantitative analysis → analysis sucrose sugar amount and SS to SS gene expressions
The research method of the quick SS gene expressions rule of expression correlation → foundation → be the high sugar products kind of sugar grass selection and breeding and determination
Best water content in harvest provides theoretical foundation.
1.4 Grape by HPLC sugar, fructose, cane sugar content
It is Platisil μm of NH that this experiment, which uses Agilent liquid chromatograph, Agilent Composition distribution, chromatographic column,2,
250×4.6mm.Mobile phase is acetonitrile and ultra-pure water (70:30), flow velocity 1.0ml/min, column temperature are 30 DEG C, 20 μ of each sample introduction
L is repeated three times, and guarantee is repeated as an independent sample every time.
1.5 sugar grass sucrose synthase (SS) determinations of activity
The extraction of enzyme solution carries out under the conditions of 0-4 DEG C, weighs 0.5g Sorghum Leaves and is put in mortar, be added zyme extract and
A small amount of quartz sand, is fully ground on ice, ground sample is gone in centrifuge tube, 12000r/min, 4 DEG C of centrifugation 20min,
It is crude enzyme liquid to take supernatant, using the activity of resorcinol colorimetric method for determining enzyme.
The active measurement of SS：Take 50 μ l of crude enzyme liquid in test tube, 50 μ l HEPES-NaOH buffer solutions (PH 7.5) of addition,
20 μ l 100mmol/L UDPG, 20 μ l 100mmol/L fructose, 20 μ l 50mmol/L MgCl2,30 DEG C of water-bath 30min are added
200 μ l 2mmol/L NaOH, 100 DEG C of water-bath 10min.Flowing water cools down, and hydrochloric acid, resorcinol, fully shaking are added in test tube
It shakes up, is detected with spectrophotometer, repeated three times, is averaged.
The extraction purification of 1.6 sugar grass RNA detects
1.6.1RNA extraction
With reference to the operation instructions of promega RNA extracts kits, the sugar grass blade of 4 periods, 10 kinds is extracted
In RNA, steps are as follows：
(1) liquid nitrogen grinding 30-50mg sugar grass blade 30min are used, are sampled with the centrifuge tube of 2ml.
(2) 175 μ l RNA Lysis Buffer are added, blow and beat mixing several times repeatedly.
(3) 350 μ l RNA Dilution Buffer are added, overturn mixing.
(4) 70 DEG C of water-bath 3min, 12000r/min is centrifuged 10min minutes after taking-up
(5) in pipette tips slowly Aspirate supernatant to new 1.5ml centrifuge tubes, 200 μ l, 95% ethyl alcohol mixings are added, it will
Mixed liquid moves on in adsorption column, and 12000r/min centrifuges 1min, abandons waste liquid.
(6) 600 μ l RNA Wash Solution, 12000r/min centrifugation 1min are added, abandon waste liquid.
(7) match 50 μ l reaction solutions：40μl Yellow Core Buffer、5μl MnCl2, 5 μ l DNase I, it is small after mixing
The heart is added into adsorption column center, stationary incubation 15min.
(8) 200 μ l DNase Stop Solution, 12000r/min are added and centrifuge 1min.
(9) 600 μ l RNA Wash Solution, 12000r/min centrifugation 1min are added, abandon waste liquid.
(10) 250 μ l RNA Wash Solution, 12000r/min centrifugation 2min are added, abandon waste liquid.
(11) adsorption column is put into a new 1.5ml centrifuge tube, 100 μ l nuclease frees is added to adsorption column center
Water, then 12000r/min centrifuge 1min.The RNA of extraction is in -80 DEG C of preservations.
1.6.2RNA detection
RNA Concentration Testings
It draws diluted RNA sample to be added in clean cuvette, reads the light absorption value at 260,280 and 230nm,
A260/A280>1.8, A260/A230>2.0 prove that the RNA purity of extraction is qualified, can be used for subsequent experimental.The quality of RNA is dense
Degree calculates：=A260 × 33 × extension rate/1000 RNA (μ g/ μ L).RNA electrophoresis detections
Electronic balance weighs 0.2g agaroses and is dissolved in mixing in 1 × tbe buffer liquids of 20ml, is put into micro-wave oven and heats
2min when being cooled to 50 DEG C or so, is added 1 μ l EB mixings, pours into the lacquer disk(-sc) for being inserted into comb, after standing waits until glue solidification
It is careful to extract comb, gel is placed in electrophoresis tank, 1 × tbe buffer liquid is poured into and did not had glue surface.Take 5 μ L RNA and 1 μ l 6 ×
Loading buffer sample-loading buffers mix, addition loading wells, electrophoresis 7min under 300V burning voltages, in gel imager
It observes, take pictures, detect RNA extraction purification results.
The screening of 1.7 sucrose synthases (SS) primer
According to the gene coding regions known one section of sugar grass SS cDNA sequence, using Oligo6 softwares and Primer softwares into
Row design primer, and detect screening and obtain two pairs of SS gene magnification primers.
1.8 sugar grass cDNA synthesis and detection
The synthesis of first chain of cDNA：
The synthesis of first chain of cDNA uses TaKaRa RT-PCR kits, reaction system and operation as follows：
Reaction system is 10 μ l：5 × PRT Mix, 2 μ l, RNA templates, 8 μ l.Reaction condition is：37 DEG C of 15min, 85 DEG C of 5s,
Reverse transcription product is preserved in -20 degree.
The amplification and detection of cDNA：
PCR reaction systems are 25 μ l：10 × Buffer 2.0 μ l, 100 μm of ol/l dNTPs 0.5 μ l, 0.2 μm of ol/l F-
Primer 0.8 μ l, 0.2 μm of ol/l R-Primer 0.8 μ l, cDNA 0.5 μ l, Taq enzyme 0.5 μ l, ddH2O 19.4μl。
PCR reaction conditions are：94 DEG C of pre-degeneration 3min → 30 time amplification cycles → 72 DEG C extend 10min, amplification cycles packet
It includes：94 DEG C of denaturation 30s → 58 DEG C annealing 30s → 72 DEG C extend 45s；
Pcr amplification product, 100V electrophoresis 20min, using gel image analyser are detected with 1% agarose gel electrophoresis
Detect the synthesis situation of cDNA.
The expression quantity of 1.9 fluorescence quantitative PCR detection sucrose synthases (SS)
The present invention carries out Real-time PCR amplifications using SYBR Premix ExTaq II (TaKaRa, DRR081A).
It is template with 10 cDNA with kind sugar grass, carries out the amplification of target gene, reference gene, 3 weights is arranged in each sample
It is multiple, while blank control is set.
Reaction system is 20 μ l：2 × SYBR Premix ExTaq II 10.0 μ l, ddH2O 6.0 μ l, Forward
0.4 μ l, cDNA templates of Primer 0.8 μ l, Reverse Primer, 0.8 μ l, ROX Reference DYE, 2.0 μ l.
PCR response procedures：95 DEG C of pre-degeneration 30s → 40 amplification cycles → 72 DEG C extend 10min, and amplification cycles include：
95 DEG C of denaturation 5s → 60 DEG C annealing 20s → 72 DEG C extend 30s；
60-95 DEG C of solubility curve program, reaction complete after, computer automatically generate CT (threshold cycles) value,
Melting curve.
1.10 data analysis
Processing analysis is carried out to experimental data using Origin and SPSS softwares.
Embodiment 1：Sugar grass SS and sucrose accumulation correlation research
The present invention determines the enzymatic activity in 10 kind sugar grass blades of different growing stage, the results showed that：In 4 lifes
In long-term, the variation tendency of the enzymatic activity of SS is similar to sucrose in blade, the trend of rising is all manifested on the whole, in the maturity period
When all reach top value as cane sugar content, and be much larger than seedling stage, jointing stage and heading stage, this kind of wherein M-81E is from seedling
The Enzyme activities of phase to maturity period SS are maximum, and the enzymatic activity of maturity period SS is 10.14 times of the enzymatic activity of seedling stage SS.Than less
With the enzymatic activity of sugar grass SS in phase same time blade of kind, the enzymatic activity of seedling stage difference sugar grass kind SS is without too big
Difference, content is between 20.92-44.22mg/g FWh；Since the jointing stage the activity of different sugar grass kind SS
It is variant, but be not very significantly；Difference is most apparent when the maturity period, the enzymatic activity of this kind of wherein LTR108 SS is maximum, is
252.26mg/g FWh, this more minimum than SS enzymatic activity 149.78mg/g FWh wide in variety.
The correlation analysis of sugar grass SS enzymatic activitys and cane sugar content is the results show that cane sugar content and SS enzymatic activitys are in positive
Close (R=0.860, P<0.05), illustrate that SS enzymatic activitys influence the content of sugar grass sucrose.
Embodiment 2：The extraction interpretation of result of sugar grass RNA
The sugar grass total serum IgE agarose gel electrophoresis of extraction is detected into (see Fig. 1), from left to right (1-8) is followed successively by：Tianjin
It is 53, sweet tea GL, LTR108, LTR168, HT, collier, M-81E, Rio.It can be seen that from picture, band clearly becomes clear, wherein
The brightness of 28S bands is approximately 2 times of 18S bands, illustrates that its integrality and quality in extraction process are fine, after being used in
It is used as template in continuous experiment.
Embodiment 3：The synthesis and detection of cDNA
Using first chain of cDNA as template, PCR amplification is carried out by primer of target gene SS and reference gene Actin, so
Be detected afterwards (see Fig. 2) with gel electrophoresis, Marker (DL 2000) is followed successively by from M to 1-10, Tianjin be 53, sweet tea GL,
As a result LTR108, LTR168, HT, collier, M-81E, Rio, Honey, umbrella show that band clearly becomes clear, it was demonstrated that
Through successfully converting RNA to cDNA, the experiment of next quantitative fluorescent PCR can be carried out.
Embodiment 3：Sucrose synthase (SS) primer screening
Primer in table 1 is according to the gene coding regions known one section of sugar grass SS cDNA sequence, using Oligo6 softwares
It is designed screening primer with Primer primier softwares.It is named as SS-1, Actin is house-keeping gene.
1 SS gene magnification the primers of table
Embodiment 4：The research method of sucrose synthase (SS) gene expression rule
The comparative analysis of 4.1 seedling stage SS gene expression rules
Relative quantitative assay is carried out to the SS gene expressions of 10 kind sugar grass seedling leafs, it is pair that the present invention, which chooses HT,
According to (result is shown in Fig. 3), between seedling stage difference sugar grass kind SS gene expressions have differences, wherein M-81E, LTR108, HT
SS gene expression amounts are relatively high.
The comparative analysis of 4.2 jointing stage SS gene expression rules
SS gene expressions have differences (see Fig. 4) between jointing stage difference sugar grass kind, wherein LTR108, sweet tea GL,
The SS gene expression amounts of Honey are higher.
The comparative analysis of 4.3 heading stage SS gene quantification expression rules
SS gene expressions have differences (see Fig. 5) between heading stage difference sugar grass kind, and wherein Tianjin is 53, LTR108
SS gene expression amounts are higher.
4.4 maturity period SS gene quantification expressions are analyzed
SS gene expressions have differences (see Fig. 6) between maturity period difference sugar grass kind, and gene expression amount is from high to low
Be followed successively by LTR108, HT, LTR168, Honey, Rio, Tianjin are 53, sweet tea GL, Collier, M-81E, SS gene expression amounts are maximum
Kind LTR108.
In conclusion the present invention is control with the relative expression quantity of the SS genes of HT this kind in different times, lead to
It crosses to sugar grass the SS relative expression quantities in different times different cultivars blade and carries out analysis and find, SS genes are in different cultivars
There are difference for relative expression quantity between sugar grass, and wherein kind LTR108SS gene expression amounts are all highest in each period,
It is consistent with the variation tendency of cane sugar content, and then it is concluded that LTR108 should be and most have in 10 main cultivation sugar grass kinds
Germ plasm resource, the selection and breeding for sugar content sugar grass kind provide rapid screening method and theoretical foundation.
SEQUENCE LISTING
<110>Shenyang Normal University
<120>The detection of expression method and amplimer of sugar grass sucrose synthase gene
<130> 2018
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 19
<212> DNA
<213>Artificial sequence
<400> 1
gtccctcaag acactccct 19
<210> 2
<211> 19
<212> DNA
<213>Artificial sequence
<400> 2
attggattgg gcaaagtag 19
<210> 3
<211> 24
<212> DNA
<213>Artificial sequence
<400> 3
acggcctgga tggcgacgta catg 24
<210> 4
<211> 25
<212> DNA
<213>Artificial sequence
<400> 4
gcagaaggac gcctacgttg gtgac 25
Claims (3)
1. sugar grass sucrose synthase gene amplimer, which is characterized in that including primer SS-1 and primer Actin, primer SS-
1 includes SS-1 forward primers and SS-1 reverse primers, and nucleotide sequence is respectively such as SEQ ID NO：1 and SEQ ID NO：Shown in 2；
Primer Actin includes Actin forward primers and Actin reverse primers, and nucleotide sequence is respectively such as SEQ ID NO：3 and SEQ ID
NO：Shown in 4.
2. the synthetic method of sugar grass sucrose synthase gene cDNA, which is characterized in that include the following steps：
1) synthesis of first chain of cDNA
Using TaKaRa RT-PCR kits, reaction system is 10 μ l, including：5 × PRT Mix, 2 μ l, RNA templates, 8 μ l；Instead
The condition is answered to be：37 DEG C of 15min, 85 DEG C of 5s, reverse transcription product are preserved in -20 degree；
2) amplification of cDNA
PCR reaction systems are 25 μ l, including：10 × Buffer 2.5 μ l, 100 μm of ol/l dNTPs 0.5 μ l, 0.2 μm of ol/l
F-Primer 0.8 μ l, 0.2 μm of ol/l R-Primer 0.8 μ l, cDNA 0.5 μ l, Taq enzyme 0.5 μ l, ddH219.4 μ l of O, institute
It is primer SS-1 described in claim 1 or primer Actin to state primer；
PCR reaction conditions are：94 DEG C of pre-degeneration 3min → 30 time amplification cycles → 72 DEG C extend 10min, and amplification cycles include：94
DEG C denaturation 30s → 58 DEG C annealing 30s → 72 DEG C extend 45s；
3) detection of cDNA
Pcr amplification product is detected with 1% agarose gel electrophoresis, 100V electrophoresis 20min detect cDNA in gel imager
Synthesis situation.
3. the rapid fluorescence quantifying PCR method of sugar grass sucrose synthase gene expression, which is characterized in that include the following steps：
Real-time PCR amplifications are carried out with SYBR Premix ExTaq II, using the cDNA of sugar grass as template, carry out purpose
The amplification of gene, reference gene, 3 repetitions are arranged in each sample, while blank control is arranged；
20 μ l of reaction system, including：2 × SYBR Premix ExTaq II 10.0 μ l, ddH2O 6.0 μ l, Forward
0.4 μ l, cDNA templates of Primer 0.8 μ l, Reverse Primer, 0.8 μ l, ROX Reference DYE, 2.0 μ l；It is described
Primer is primer SS-1 described in claim 1 or primer Actin；
PCR response procedures are：95 DEG C of pre-degeneration 30s → 40 amplification cycles → 72 DEG C extend 10min, and amplification cycles include：95
DEG C denaturation 5s → 60 DEG C annealing 20s → 72 DEG C extend 30s；
60-95 DEG C of solubility curve program, after reaction is completed, computer automatically generates CT values, melting curve.
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