CN101818197A

CN101818197A - Optimized Agi ISSR molecular marking method

Info

Publication number: CN101818197A
Application number: CN201010108950A
Authority: CN
Inventors: 刘越; 申刚义; 冯金朝; 周宜君; 王俊丽; 张淑萍; 孙洪波; 张琳霞; 韦善君; 高飞; 王斌; 王真
Original assignee: Minzu University of China
Current assignee: Minzu University of China
Priority date: 2010-02-08
Filing date: 2010-02-08
Publication date: 2010-09-01
Anticipated expiration: 2030-02-08
Also published as: CN101818197B

Abstract

The invention relates to the field of molecular biology, in particular to an optimized Agi (artemisia frigida willd) inter-simple sequence repeat (ISSR) molecular marking method. The optimized Agi ISSR molecular marking method comprises the following steps of: 1) extracting a genome DNA of Agi; and 2) performing ISSR amplification, wherein a reaction system of the ISSR amplification is that: 20 mu L of the reaction system contains 2 mu L of 10*PCR buffer solution, 50 ng of a template DNA, 1.5 mmol.L<-1> of Mg<2+>, 0.25 mmol.L<-1> of dNTP, 1.5 U of Taq enzyme, and 0.75 mu mol.L<-1> of a primer. The conditions for the ISSR amplification comprise: performing annealing for 5 minutes at 94 DEG C, then performing the annealing for 1 minute at 94 DEG C, 40 seconds at 56 DEG C and 1 minute and 30 seconds at 72 DEG C for 40 circulations, and finally performing extension for 5 minutes at 72 DEG C. Straps amplified by an Agi ISSR reaction system established by the method have the advantages of high polymorphism, high specificity, clear background and high stability.

Description

A kind of Agi ISSR molecular marking method of optimization

Technical field

The present invention relates to biology field, particularly, relate to a kind of Agi ISSR molecular marking method of optimization.

Background technology

Anaesthetic Ah Gei [Agi]---prairie sagewort (Artemisia frigida Willd.) has another name called small sievers wormwood herb, Tu hair wormwood artemisia, is the undershrub of composite family artemisia ^[1]Ah Gei has the effect of hemostasis, detumescence for Mongolian medicine's medicine commonly used, cures mainly various hemorrhage, arthroncuss, kidney heat, menoxenia, sore pain etc. clinically, also is that the Mongolian medicine goes up " one of artificial holy water composition " of using always.Along with the development of dna molecular marker technology, people have been widely used in this technology aspects such as traditional herb resource, evaluation, cultivation.

The research of Ah give'ing molecular biology level and is not to study as medicine seldom.Only obtained 5 dna sequence dnas at present, they are the fragment and the rrna rRNA gene fragment of the leu-tRNA gene of small sievers wormwood herb chloroplast(id), are mainly used to differentiate the difference between each plant.The genetic diversity variation of Ah Gei that the king has waited people detection quietly as herbage colony under different pasture pressure.Because Ah Gei is the anemophily allogamy, in time with on the space distribution is widely arranged, the environmental difference that is faced is big, individuality and population number are many, gene exchange is frequent, and the chance that therefore produces new heritable variation is many, and this helps it increases level of genetic diversity, and make it the level (the polymorphic site percentage is 94.49%) that keeps higher, being higher than the average level of genetic diversity of higher plant is 70%.Genetic diversity is along with the difference of grazing intensity is also different.Strengthen the research of anaesthetic Ah give'ing molecular biology level, provide solid theory for its seed seedling research, conservation of resources, effect application etc. undoubtedly.

ISSR (Inter-Simple Sequence Repeat, simple repeated sequence) molecule marker is a kind of new technology that grows up the nineties in 20th century, ultimate principle is to utilize normal SSR itself the design primer that occurs in Plant Genome, and the dna sequence dna between the SSR of reversed arrangement is carried out pcr amplification.ISSR is Mendelian inheritance, is the dominant marker, and stability is high, and technology is simple, quick, with low cost.Because it is very general that SSR distributes in eukaryote, and series connection multiple number is variable, be highly polymorphism, so the ISSR labeling technique can detect the difference in the many sites of genome.This technology has obtained widespread use in finger printing, genetic diversity, genetic mapping and the assignment of genes gene mapping research of plant at present.

Summary of the invention

The Agi ISSR molecular marking method that the purpose of this invention is to provide a kind of optimization.

The Agi ISSR molecular marking method of optimization of the present invention may further comprise the steps:

1) extraction Ah give'ing genomic dna;

2) carry out the ISSR amplification,

Wherein, the reaction system of described ISSR amplification is: contain 10 * PCR damping fluid, 2 μ L in the 20 μ L reaction systems, and template DNA 50ng, in addition, Mg ²⁺Be 1.5mmolL ^-1, dNTP is 0.25mmolL ^-1, the Taq enzyme is 1.5U, primer is 0.75 μ molL ^-1

The condition of described ISSR amplification is:

94 ℃ of annealing 5min, 94 ℃ of 1min, 56 ℃ of 40s, 40 circulations of 72 ℃ of 1min30s then, last 72 ℃ are extended 5min,

Wherein, described primer sequence as shown below.

Primer sequence (5 '-3 ')

UBC?807 AGA?GAG?AGA?GAG?AGA?GT

UBC?810 GAG?AGA?GAG?AGA?GAG?AT

UBC?811 GAG?AGA?GAG?AGA?GAG?AC

UBC?823 TCT?CTC?TCT?CTC?TCT?CC

UBC?834 AGA?GAG?AGA?GAG?AGA?GYT

UBC?840 GAG?AGA?GAG?AGA?GAG?AYT

UBC?841 GAG?AGA?GAG?AGA?GAG?AYC

UBC?845 CTC?TCT?CTC?TCT?CTC?TRG

UBC?857 ACA?CAC?ACA?CAC?ACA?CYG

UBC?864 ATG?ATG?ATG?ATG?ATG?ATG

UBC?873 GAC?AGA?CAG?ACA?GAC?A

UBC?887 DVD?TCT?CTC?TCT?CTC?TC

The band that the Agi ISSR reaction system that the present invention set up amplifies has polymorphism height, high specificity, background is clear and stable strong advantage.

Description of drawings

Fig. 1 is the amplification of orthogonal experimental design ISSR-PCR reaction system, and 1-9 is the treatment combination numbering of table 3; Primer is UBC807

Fig. 2 is the ISSR amplification of 12 different primers, M:100bp ladder; 1.UBC 807; 2.UBC810; 3.UBC 811; 4.UBC 823; 5.UBC 834; 6.UBC 840; 7.UBC 841,8.UBC 845; 9.UBC 857; 10.UBC 864; 11.UBC 873; 12.UBC 887.

Fig. 3 is the amplification of different annealing temperature in the ISSR-PCR reaction system, 1.52.0 ℃; 2.52.3 ℃; 3.52.9 ℃; 4.53.8 ℃; 5.55.0 ℃; 6.56.0 ℃; 7.56.6 ℃; 8.57.0 ℃; M:100bp ladder; Primer is UBC807.

The ISSR amplification of 2 different primers of Figure 41,1-12: selecting primer for use is respectively UBC807, UBC810, UBC811, UBC823, UBC834, UBC840, UBC841, UBC845, UBC857, UBC864, UBC873 and UBC887; M:100bp ladder.

The amplification of different cycle numbers in Fig. 5 ISSR-PCR reaction system, 1-4: represent that respectively used number of cycles is 30 circulations, 35 circulations, 40 circulations and 45 circulations; M:100bp ladder; Primer is UBC807.

Embodiment

Embodiment 1

Extract Ah 's genomic dna.

1, the screening of amplification system

The peak optimization reaction system (four factors, three levels) of orthogonal optimization design screening ISSR is selected L9 (3 for use ⁴) orthogonal table, the factor-horizontal quadrature contrived experiment table of each composition of design pcr amplification system (scheme see Table 2 and table 3).With UBC807 is primer, utilizes 9 individual system in the table 3, and Ah Gei carried out the ISSR amplification.Reaction system is 20 μ L, and the listed factor, every pipe contains 10 * PCR buffer, 2 μ L, template DNA 50ng in table.Just amplification program is: 4 ℃ of pre-sex change 5min; 94 ℃ of sex change 1min, 55 ℃ of annealing 1min, 72 ℃ are extended 1.5min, circulate 45 times; 72 ℃ are extended 5min, 4 ℃ of preservations.

Table 1 ISSR amplimer and sequence

Primer sequence (5 '-3 ')

Primer Primer?sequences(5’-3’)

UBC?807 AGA?GAG?AGA?GAG?AGA?GT

UBC?810 GAG?AGA?GAG?AGA?GAG?AT

UBC?811 GAG?AGA?GAG?AGA?GAG?AC

UBC?823 TCT?CTC?TCT?CTC?TCT?CC

UBC?834 AGA?GAG?AGA?GAG?AGA?GYT

UBC?840 GAG?AGA?GAG?AGA?GAG?AYT

UBC?841 GAG?AGA?GAG?AGA?GAG?AYC

UBC?845 CTC?TCT?CTC?TCT?CTC?TRG

UBC?857 ACA?CAC?ACA?CAC?ACA?CYG

UBC?864 ATG?ATG?ATG?ATG?ATG?ATG

UBC?873 GAC?AGA?CAG?ACA?GAC?A

UBC?887 DVD?TCT?CTC?TCT?CTC?TC

The factor and the level of table 2 ISSR-PCR reaction system

Orthogonal experimental design [the L of table 3 ISSR-PCR reaction system ₉(3 ⁴)]

With reference to the method for what text etc., to the direct analysis of how much doing of the power of electrophoretic band and assorted band.As can be seen from Figure 1, in 9 treatment combinations, because Mg ²⁺, 4 kinds of influence factors combinations of dNTP, Taq archaeal dna polymerase, primer difference, expanding effect exists notable difference.1st, it is relatively poor to handle expanding effect No. 6, and bands of a spectrum are weak and polymorphism is low, and No. 3 treatment effect is best, and bands of a spectrum are strong, the polymorphism height.Therefore the best ISSR amplification reaction system that obtains is: the reaction cumulative volume is 20 μ L, Mg ²⁺Be 1.5mmolL ^-1, dNTP is 0.25mmolL ^-1, the Taq archaeal dna polymerase is 1.5U, primer is 0.75 μ molL ^-1

Adopt the system after optimizing, with identical dna profiling, with UBC 807, UBC 810, and UBC 811, and UBC 823, UBC 834, and UBC 840, and UBC 841, UBC 845, and UBC 857, and UBC 864, UBC 873, and UBC 887 is a primer, carry out the ISSR amplification, can amplify the band of clear and good stability, polymorphism height (Fig. 2).Illustrate that this reaction system is suitable for Ah and reacts to ISSR-PCR.

2, the screening of amplification condition

1), select for use system 3 to be Ah Gei ISSR reaction conditions according to electrophoresis result.Promptly in 20 μ L reaction systems, Mg ²⁺Be 1.5mmolL ^-1, dNTP is 0.25mmolL ^-1, the Taq archaeal dna polymerase is 1.5U, primer is 0.75 μ molL ^-1, in addition, containing 2 μ L, 10 * PCR buffer, 2 μ L in each reaction system, template DNA is 50ng, not enough volume is supplied with the distilled water of sterilization.

2.1 the screening of annealing temperature

On the optimum response system basis that above-mentioned test is determined, annealing temperature is carried out the gradient test, optimize the screening optimum annealing temperature, according to fluctuate 2-4 ℃ principle up and down in the theoretical annealing temperature of primer, on PCR gradient amplification instrument minimum annealing temperature being set is 52 ℃, be 57 ℃ to the maximum, the PCR instrument forms 8 gradients, promptly 52.0 ℃, 52.3 ℃, 52.9 ℃, 53.8 ℃, 55.0 ℃, 56.0 ℃, 56.6 ℃, 57.0 ℃ automatically.

As can be seen from Figure 3: when annealing temperature was 52.0-53.8 ℃, the band of generation was unintelligible, and when annealing temperature is higher than 56.0 ℃, produced assorted band.Thereby think the annealing temperature of UBC807 primer be 56 ℃ comparatively suitable.

Use primer: UBC 810; 3.UBC 811; 4.UBC 823; 5.UBC 834; 6.UBC 840; 7.UBC841 8.UBC 845; 9.UBC 857; 10.UBC 864; 11.UBC 873; 12.UBC 887, increase with 56 ℃ of annealing temperatures, amplification as shown in Figure 4, the band of generation is clear.

2) cycle number determines

According to orthogonal experiment and annealing temperature results of screening, carry out determining of cycle number.Cycle number is followed successively by 30,35, and 40,45.2 repetitions are established in each circulation.

According to the result of orthogonal test and annealing temperature, select optimum annealing temperature to carry out the detection of cycle number.As can be seen from Figure 5: when cycle number is 30,35 o'clock, band is unintelligible, and cycle number is 40,45 o'clock, and band is more clear, and does not have assorted band, considers from the angle that saves time and cost, and selects cycle number 40 comparatively suitable.

Conclusion

Utilize orthogonal experimental design, from Mg ²⁺Concentration, dNTP concentration, Taq DNA enzyme concn and primer concentration 4 factors 3 levels are optimized analysis to Agi ISSR-PCR reaction system, and on this basis cycle number and the annealing temperature of PCR are tested.The result shows, in the 20 μ L reaction systems, and Mg ²⁺Be 1.5mmolL ^-1, dNTP is 0.25mmolL ^-1, Taq DNA enzyme is 1.5U, primer is 0.75 μ molL ^-1The time, and the suitableeest cycle number of ISSR is 40, and the suitableeest annealing temperature is 56 ℃, and expanding effect is best.For technical foundation is established in the further research of Ah 's genetic diversity.

Sequence table

＜110〉Central University for Nationalities

＜120〉a kind of Agi ISSR molecular marking method of optimization

<160>12

<210>1

<211>17

<212>DNA

＜213〉synthetic primer

<400>1

agagagagag?agagagt 17

<210>2

<211>17

<212>DNA

＜213〉synthetic primer

<400>2

gagagagaga?gagagat 17

<210>3

<211>17

<212>DNA

＜213〉synthetic primer

<400>3

gagagagaga?gagagac 17

<210>4

<211>17

<212>DNA

＜213〉synthetic primer

<400>4

tctctctctc?tctctcc 17

<210>5

<211>18

<212>DNA

＜213〉synthetic primer

<400>5

agagagagag?agagagyt 18

<210>6

<211>18

<212>DNA

＜213〉synthetic primer

<400>6

gagagagaga?gagagayt 18

<210>7

<211>18

<212>DNA

＜213〉synthetic primer

<400>7

gagagagaga?gagagayc 18

<210>8

<211>18

<212>DNA

＜213〉synthetic primer

<400>8

ctctctctct?ctctctrg 18

<210>9

<211>18

<212>DNA

＜213〉synthetic primer

<400>5

acacacacac?acacacyg 18

<210>10

<211>18

<212>DNA

＜213〉synthetic primer

<400>10

atgatgatga?tgatgatg 18

<210>11

<211>16

<212>DNA

＜213〉synthetic primer

<400>11

gacagacaga?cagaca 16

<210>12

<211>17

<212>DNA

＜213〉synthetic primer

<400>12

dvdtctctct?ctctctc 17

Claims

1. the Agi ISSR molecular marking method of an optimization said method comprising the steps of:

1) extraction Ah give'ing genomic dna;

2) carry out the ISSR amplification.

Wherein, the reaction system of described ISSR amplification is: contain 10 * PCR damping fluid, 2 μ L, wherein Mg in the 20 μ L reaction systems ²⁺Be 1.5mmolL ^-1, template DNA 50ng, dNTP are 0.25mmolL ^-1, the Taq enzyme is 1.5U, primer is 0.75 μ molL ^-1

The condition of described ISSR amplification is:

94 ℃ of annealing 5min, 94 ℃ of 1min, 56 ℃ of 40s, 40 circulations of 72 ℃ of 1min30s then, last 72 ℃ are extended 5min.

Wherein, described primer sequence as shown below

Primer sequence 5 '-3 '

UBC?807 AGA?GAG?AGA?GAG?AGA?GT

UBC?810 GAG?AGA?GAG?AGA?GAG?AT

UBC?811 GAG?AGA?GAG?AGA?GAG?AC

UBC?823 TCT?CTC?TCT?CTC?TCT?CC

UBC?834 AGA?GAG?AGA?GAG?AGA?GYT

UBC?840 GAG?AGA?GAG?AGA?GAG?AYT

UBC?841 GAG?AGA?GAG?AGA?GAG?AYC

UBC?845 CTC?TCT?CTC?TCT?CTC?TRG

UBC?857 ACA?CAC?ACA?CAC?ACA?CYG

UBC?864 ATG?ATG?ATG?ATG?ATG?ATG

UBC?873 GAC?AGA?CAG?ACA?GAC?A

UBC?887 DVD?TCT?CTC?TCT?CTC?TC。