CN104894285A - Method for detecting genes Cf-9 and I-2 according to dual-PCR (polymerase chain reaction) technology - Google Patents
Method for detecting genes Cf-9 and I-2 according to dual-PCR (polymerase chain reaction) technology Download PDFInfo
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Abstract
The invention discloses a method for detecting genes Cf-9 and I-2 according to a dual-PCR (polymerase chain reaction) technology. The method includes the steps of tomato plant leaf DNA (deoxyribose nucleic acid) extraction, dual-PCR primer synthesis, single-tube PCR amplification, dual-PCR amplification and the like. I-2 and Cf-9 are subjected to multi-PCR marking in a reaction system. Different molecular markers are applied to auxiliary selection of anti-disease genes, genes Cf-D1/D2 and I-2/5F/R are amplified at the same time in a same tube by the system, and accordingly time and detection cost are greatly saved, and a feasible molecular marking detection method is provided for tomato breeding for disease resistance.
Description
Technical field
The invention provides a kind of quick, simple and direct, efficient method simultaneously detecting disease-resistant gene Cf-9 and I-2, platymiscium resist technology field.
Background technology
Tomato is vegetable crop important in the world, from the eighties of last century tomato wilt fifties (
fusarium oxysporumf. sp
. lycoper-sici) by since reported first, now various places all over the world, and become one of Major Diseases of harm tomato production.The gene of the anti-blight found at present mainly contains three and is respectively I-1, I-2 and I-3.In tomato breeding for disease-resistance, due to
i-2gene is anti-microspecies 1 and 2 simultaneously, thus especially come into one's own.
About the molecule marking research of I-2 gene, as far back as eighties of last century nineties, the RFLP molecule marker of just external report I-2 gene.Because RFLP marking operation is complicated, cost is high, and generally there is radiocontamination.Thus, this mark is not suitable for marker-assisted breeding.Along with the development of Protocols in Molecular Biology, the tomato disease-resistant gene be separated at present reaches 9, which includes I-2 gene and the I-2C gene of anti-blight, and this research is that good basis has been established in the exploitation of anti-blight functional label.In fastening the base sequence of the people such as storehouse according to tomato wilt disease-resistant gene I-2, design specific amplification primer, develops the functional label based on gene sequence label, for the molecular mark of tomato wilt disease-resistant gene I-2 lays the foundation.
In tomato production, recurrent another kind of disease is exactly leaf muld of tomato.Leaf mold is particularly outstanding to tomato in greenhouse disserve to produce.Along with expansion and the prolongation of continuous cropping time of tomato in greenhouse cultivated area, the harm of this disease is also on the rise.Leaf muld of tomato sickness rate is very high, and this is sick once occur, bamboo telegraph spreads.Leaf muld of tomato brings immeasurable impact to tomato production.
In breeding, I-2 and Cf-9 is considered to major gene, and in order to improve tomato to these two kinds sick resistances, these two genes can be imported certain kind by the mode of sexual hybridization by breeder simultaneously.And for the individuality meeting breeding objective selecting the unification of two genes in colony be the process of consuming time a, effort after hybridization.Due to by time, environment and region less-restrictive, so apply different molecule marker enantiopathy genes to carry out assisted Selection, accuracy and the efficiency of selection of required Fruit variety can be increased substantially, shortening the breeding cycle.
Develop the molecule marker detecting these two genes respectively at present, disease-resistant, susceptible material can have been distinguished.Have no the report I-2 and Cf-9 mark being carried out to multiplex PCR in a reaction system.
Summary of the invention
This experimental exploration by multiplex PCR complete in same reaction two mark amplifications, for the correlative study of tomato resistance breeding molecule marker provide one more to save time, laborsaving, cost-effective method.
For achieving the above object, the invention discloses following technical scheme:
Utilize a method for double PCR technology for detection Cf-9 and I-2 gene, it is characterized in that being undertaken by following step:
(1) DNA extracts: the blade getting tomato plant, extracts STb gene;
(2) Design and synthesis of primer: double PCR the primer I-2/5 primer comes from the primer (in fastening storehouse, heredity, in July, 2008,30 (7): 926-932) of fastening storehouse design; Cf-9D1/D2 according to Sun Yalin (Sun Yalin. the establishment of the genetic marker of 2008. tomatoes, four disease-resistant genes and assisted Selection [Master's thesis]. Wuhan: Hua Zhong Agriculture University) primer that designs
(3) one tube PCR amplification
PCR cumulative volume is 25 μ L, comprising 10 × reaction buffer (containing Mg
2+) 2.5 μ L, dNTP(each 10mmol/L) 1 μ l, upstream primer (10 μm of ol/L) 2.5 μ L, downstream primer (10 μm of ol/L) 2.5 μ L, template DNA (1 μ l/ μ L) 0.5-2 μ L, Taq enzyme (5 U/ μ L) 0.5 μ L, all the other are supplied with water.
Response procedures: 94 DEG C of sex change 2 min; 94 DEG C of 30 s, 58 DEG C of 30 s, 72 DEG C of 1. 5 min, 35 circulations; 72 DEG C extend 10 min.
(4) double PCR amplification: each system primer volume ratio
PCR reacts other parameter and adopts following system to increase:
Template DNA (1 μ l/ μ L) 2 μ l, dNTP(each 10mmol/L) 1 μ l, 10 × Buffer 2.5 μ l, Taq enzyme (5U/ μ L) 0.5 μ l, adds ddH
2o to 20 μ l.
Amplification PCR response procedures is: 94 DEG C of sex change 5min; Then 94 DEG C, 30sec, 57 DEG C, 1min, 72 DEG C, 2min 30sec, carries out 30 circulations; Last 72 DEG C extend 10min, 4 DEG C of insulations.
(5) double PCR amplification system:
Experiment has carried out primer proportioning to Cf-D1/D2 and I-2/5F/R two pairs of primers, finally determines that system is:
Template DNA (1 μ l/ μ L) 2 μ l, dNTP(each 10mmol/L) 1 μ l, 10 × Buffer 2.5 μ l, Taq enzyme (5U/ μ L) 0.5 μ l, primer concentration is respectively Cf-D1/D2(10 μM) be 2 μ l, I-2/5F/R(10 μM) be 0.5 μ l, add ddH2O to 20 μ l, amplification PCR response procedures is: 94 DEG C of sex change 5min; Then 94 DEG C, 30sec, 57 DEG C, 1min, 72 DEG C, 2min 30sec, carries out 30 circulations; Last 72 DEG C extend 10min, 4 DEG C of insulations.
The present invention is more detailed to be described below:
1 materials and methods
1.1 material
Experimental cultivar is the F2 individual plant of the HL108 of the anti-blight of Seed Co., Ltd. of roc of money of Xi'an and the kind M158 of leaf mold and Beijing silver moon Science and Technology Ltd..
method
1.2.1 DNA gets the blade of tomato plant, extracts STb gene (Wang Guanlin, plant genetic engineering, the 2nd edition, Beijing: Science Press, 2002) by the CTAB method of Wang Guanlin.
1.2.2 the Design and synthesis of primer is pursuant to the (heredity such as fasten people from storehouse, in July, 2008,30 (7): 926-932) pair of primers designed is used for detecting I-2 gene, according to Sun Yalin (Sun Yalin. the establishment of the genetic marker of 2008. tomatoes, four disease-resistant genes and assisted Selection [Master's thesis]. Wuhan: Hua Zhong Agriculture University) primer that designs, for the Cf-9 molecule marker fragment that increases.Primer sequence is respectively as table 1.
Table 1 double PCR the primer
Note: the material containing Cf-9 gene can expand the band of 514bp size, the material not containing this gene can not expand this band; Material containing I-2 gene can expand the band of 630bp, and the material not containing I-2 gene can expand the band of 690bp.
1.2.3 one tube PCR amplification
PCR cumulative volume is 25 μ L, comprising 10 × reaction buffer (containing Mg
2+) 2.5 μ L, dNTP(each 10mmol/L) 1 μ l, upstream primer (10 μm of ol/L) 2.5 μ L, downstream primer (10 μm of ol/L) 2.5 μ L, template DNA (1 μ l/ μ L) 0.5-2 μ L, Taq enzyme (5 U/ μ L) 0.5 μ L, all the other are supplied with water.Response procedures: 94 DEG C of sex change 2 min; 94 DEG C of 30 s, 58 DEG C of 30 s, 72 DEG C of 1. 5 min, 35 circulations; 72 DEG C extend 10 min.
1.2.4 double PCR amplification
The factor affecting double PCR system is a lot, and wherein topmost is exactly the proportioning of primer.
Utilize the primer proportioning that orthogonal is different in this experiment.Other factors is consistent, just the concentration of amendment primer in system.Final selection be that system 4 i.e. Cf and I2 primer volume is respectively (concentration is 10 μMs) 2 μ L and 0.5 μ L.
Table 2: each system primer volume ratio ratio
Double PCR increases: each system primer volume ratio
System name Primer volume ratio (10 μMs) μ L
PCR reacts other parameter and adopts following system to increase:
Template DNA (1 μ l/ μ L) 2 μ l, dNTP(each 10mmol/L) 1 μ l, 10 × Buffer 2.5 μ l, Taq enzyme (5U/ μ L) 0.5 μ l, adds ddH2O to 20 μ l.
Amplification PCR response procedures is: 94 DEG C of sex change 5min; Then 94 DEG C, 30sec, 57 DEG C, 1min, 72 DEG C, 2min 30sec, carries out 30 circulations; Last 72 DEG C extend 10min, 4 DEG C of insulations.
results and analysis
2.1Cf-9 amplification
To two commercially available kinds for examination, Cf-D1/D2 is utilized to carry out single-primed PCR
,these 2 parts of materials of result have all amplified the about band of 500bp (Fig. 1).
amplification
Same to these 2 parts of materials, utilize I-2/5F/R to carry out single-primed PCR
,result shows that early chief has expanded the band of 690bp; M158 has amplified the band (Fig. 2) of 630bp.This illustrates HL108-F2 not containing I-2; M158 contains I-2 gene.
double PCR amplification system amplification:
Can simultaneously to the pcr amplification reaction system that these two genes screen in order to obtain, with golden canopy 158 for material, on the basis of the PCR amplification system of former respective single primer, Cf-D1/D2 and I-2/5F/R two pairs of primers are combined in same pipe, result has only amplified larger band I-2/5F/R, and less band does not obtain increasing (Fig. 3 swimming lane 2).Given this, on the basis that other reaction parameter is constant, the proportioning of adjustment primer, to have done Cf and I2 primer concentration be respectively 10 μm of ol/L volumes is be the double PCR amplification system of 2 μ L:1.5 μ L and 2 μ L:1 μ L.Can find out the change along with two pairs of primer concentration proportionings by electrophorogram, the amplification of this band also displays (Fig. 3, swimming lane 3 swimming lane 4) gradually.Namely when in amplification system 4, primer proportioning is 2 μ L:1 μ L, the band of two entries all obtain amplification but less band still more shallow (Fig. 3, swimming lane 4).So I-2/5F/R primer amount reduces by this basis, i.e. Cf-D1/D2(10 μm of ol/L) concentration is 2 μ L, I-2/5F/R(10 μm of ol/L) concentration is 0.5 μ L.Electrophoresis result shows that two object bands can carry out increase (Fig. 4) by this system effectively in same pipe.
Experiment has carried out the test of many times such as primer proportioning, the adjustment of dNTP concentration to Cf-D1/D2 and I-2/5F/R two pairs of primers, finally determine that system is: template DNA (1 μ l/ μ L) 2 μ l, dNTP(each 10mmol/L) 1 μ l, 10 × Buffer 2.5 μ l, Taq enzyme (5U/ μ L) 0.5 μ l, primer concentration is respectively Cf-D1/D2(10 μM) be 2 μ l, I-2/5F/R(10 μM) be 0.5 μ l, add ddH2O to 20 μ l.
Amplification PCR response procedures is: 94 DEG C of sex change 5min; Then 94 DEG C, 30sec, 57 DEG C, 1min, 72 DEG C, 2min 30sec, carries out 30 circulations; Last 72 DEG C extend 10min, 4 DEG C of insulations
Utilize this system in same pipe, can amplify Cf-D1/D2 and I-2/5F/R two pairs of genes (Fig. 3), greatly save time and testing cost, the breeding for disease resistance for tomato provides practicable molecular mark detection method simultaneously.
Accompanying drawing illustrates:
Fig. 1 is Cf-9 amplification figure; M:DL2000; 1: golden canopy M158; 2:HL108-F2.
Fig. 2 is I25F/R primer amplification result figure; M:DL2000; 1:HL108-F2; 2: golden canopy M158.
The amplification of Fig. 3 system 123 multiplex PCR; Wherein 1.DL2000; 2. system 1; 3. system 2; 4. system 3
Fig. 4 is Cf-9/I2 multiplex PCR figure; M:DL2000; Gold canopy M158; 2:HL108-F2.
Embodiment
Below in conjunction with embodiment, the present invention is described further, and embodiment is only indicative, never means that it limits the scope of the invention by any way, and the raw material used by the present invention is by commercially available.
Embodiment 1
Utilize the method for double PCR technology for detection Cf-9 and I-2 gene:
(1) DNA extracts: with commercially available Jin Za 213(Tianjin Kerun Agricultural Science & Technology Co., Ltd.) for material, get the blade of tomato plant, extract STb gene;
(2) Design and synthesis of primer: double PCR the primer:
(3) one tube PCR amplification
PCR cumulative volume is 25 μ L, comprising 10 × reaction buffer (containing Mg
2+) 2.5 μ L, dNTP(each 10mmol/L) 1 μ l, upstream primer (10 μm of ol/L) 2.5 μ L, downstream primer (10 μm of ol/L) 2.5 μ L, template DNA 0.5 μ L, archaeal dna polymerase (5 U/ μ L) 0.5 μ L, all the other are supplied with water;
Response procedures: 94 DEG C of sex change 2 min; 94 DEG C of 30 s, 58 DEG C of 30 s, 72 DEG C of 1. 5 min, 35 circulations; 72 DEG C extend 10 min;
(4) double PCR amplification: Cf and I2 primer volume is respectively (concentration is 10 μMs) 2 μ L and 0.5 μ L.
PCR reacts other parameter and adopts following system to increase:
Template DNA (1 μ l/ μ L) 0.5 μ l, dNTP(each 10mmol/L) 1 μ l, 10 × Buffer 2.5 μ l, Taq enzyme (5U/ μ L) 0.5 μ l, adds ddH2O to 20 μ l.
Amplification PCR response procedures is: 94 DEG C of sex change 5min; Then 94 DEG C, 30sec, 57 DEG C, 1min, 72 DEG C, 2min 30sec, carries out 30 circulations; Last 72 DEG C extend 10min, 4 DEG C of insulations.
Experimental result:
Be 0.5 μ L(10 μM in template concentrations) time can amplify this two genes simultaneously, prove that Tianjin assorted 213 is containing these two resistant genes.
Embodiment 2
Utilize the method for double PCR technology for detection Cf-9 and I-2 gene:
(1) DNA extracts: with commercially available Tianjin assorted 213 for material, get the blade of tomato plant, extract STb gene;
(2) Design and synthesis of primer: double PCR the primer:
(3) one tube PCR amplification
PCR cumulative volume is 25 μ L, comprising 10 × reaction buffer (containing Mg
2+) 2.5 μ L, dNTP(each 10mmol/L) 1 μ l, upstream primer (10 μm of ol/L) 2.5 μ L, downstream primer (10 μm of ol/L) 2.5 μ L, template DNA 2 μ L, archaeal dna polymerase (5 U/ μ L) 0.5 μ L, all the other are supplied with water;
Response procedures: 94 DEG C of sex change 2 min; 94 DEG C of 30 s, 58 DEG C of 30 s, 72 DEG C of 1. 5 min, 35 circulations; 72 DEG C extend 10 min;
(4) double PCR amplification: Cf and I2 primer volume is respectively (concentration is 10 μMs) 2 μ L and 0.5 μ L.
PCR reacts other parameter and adopts following system to increase:
Template DNA (1 μ l/ μ L) 2 μ l, dNTP(each 10mmol/L) 1 μ l, 10 × Buffer 2.5 μ l, Taq enzyme (5U/ μ L) 0.5 μ l, adds ddH2O to 20 μ l.
Amplification PCR response procedures is: 94 DEG C of sex change 5min; Then 94 DEG C, 30sec, 57 DEG C, 1min, 72 DEG C, 2min 30sec, carries out 30 circulations; Last 72 DEG C extend 10min, 4 DEG C of insulations.
Experimental result:
Be 2 μ L(10 μM in template concentrations) time utilize this system also can amplify the band of two entries, prove that Tianjin assorted 213 is containing these two resistant genes.
Embodiment 3
Simultaneous test:
Conclusion:
Utilize the present invention to detect the cost of the gene I-2 of leafmold resistance gene C f-9 and anti-blight and time to detect than single tube and save half, for the screening in enormous quantities in tomato breeding process provides economy, efficiently detection method.
SEQUENCE LISTING
<110> Tianjin Agricultural Biotechnology Research Center
<120> mono-kind utilizes the method for double PCR technology for detection Cf-9 and I-2 gene
<160> 4
<170> PatentIn version 3.5
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<211> 21
<212> DNA
<213> artificial sequence
<400> 1
gttcttatcc tttaacaccc a 21
<210> 2
<211> 21
<212> DNA
<213> artificial sequence
<400> 2
tccctccaaa ttattacttc c 21
<210> 3
<211> 20
<212> DNA
<213> artificial sequence
<400> 3
caaggaactg cgtctgtctg 20
<210> 4
<211> 20
<212> DNA
<213> artificial sequence
<400> 4
atgagcaatt tgtggccagt 20
Claims (1)
1. utilize a method for double PCR technology for detection Cf-9 and I-2 gene, it is characterized in that being undertaken by following step:
(1) DNA extracts: the blade getting tomato plant, extracts STb gene;
(2) Design and synthesis of primer: double PCR the primer:
Primer sequence (5 '-3 ')
Cf-D1 GTTCTTATCCTTTAACACCCA
Cf-D2 TCC CTCCAA ATTATTACTTCC
I-2/5F CAAGGAACTGCGTCTGTCTG-3′
I-2/5R ATGAGCAATTTGTGGCCAGT-3′
(3) one tube PCR amplification
PCR cumulative volume is 25 μ L, comprising 10 × reaction buffer (containing Mg
2+) 2.5 μ L, dNTP mixture (2.5mmol/L) 0.5 μ L, upstream primer (10 μm of ol/L) 2.5 μ L, downstream primer (10 μm of ol/L) 2.5 μ L, template DNA 0.5-2 μ L, archaeal dna polymerase (5 U/ μ L) 0.5 μ L, all the other are supplied with water;
Response procedures: 94 DEG C of sex change 2 min; 94 DEG C of 30 s, 58 DEG C of 30 s, 72 DEG C of 1. 5 min, 35 circulations; 72 DEG C extend 10 min;
(4) double PCR amplification: each system primer proportioning
System name Primer volume proportion (10 μMs) μ L
System 1 Cf:I2 2:2
System 2 Cf:I2 2:1.5
System 3 Cf:I2 2:1
System 4 Cf:I2 2:0.5;
PCR reacts other parameter and adopts following system to increase:
Template DNA 2 μ l, dNTP1 μ l, 10 × Buffer 2.5 μ l, Taq enzyme 0.5 μ l, adds ddH2O to 20 μ l;
The PCR response procedures of amplification containing disease-resistant gene is 94 DEG C of sex change 5min; Then 94 DEG C, 30sec, 57 DEG C, 1min, 72 DEG C, 2min 30sec, carries out 30 circulations; Last 72 DEG C extend 10min, 4 DEG C of insulations, and amplified production is-20 DEG C of preservations;
(5) double PCR amplification system:
Experiment has carried out primer proportioning to Cf-D1/D2 and I-2/5F/R two pairs of primers, finally determines that system is:
Template DNA 2 μ l, dNTP1 μ l, 10 × Buffer 2.5 μ l, Taq enzyme 0.5 μ l, primer concentration is respectively Cf-D1/D2(10 μM) be 2 μ l, I-2/5F/R(10 μM) be 0.5 μ l, add ddH
2o to 20 μ l, amplification PCR response procedures is: 94 DEG C of sex change 5min; Then 94 DEG C, 30sec, 57 DEG C, 1min, 72 DEG C, 2min 30sec, carries out 30 circulations; Last 72 DEG C extend 10min, 4 DEG C of insulations.
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| CN109536631A (en) * | 2018-12-19 | 2019-03-29 | 河北科技大学 | Multiple PCR method that is a kind of while detecting tomato disease-resistant gene Sw-5 and I-2 |
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