CN101643784A - Molecule marking method for identifying paddy low glutelin gene Lgcl - Google Patents

Abstract

The invention relates to a molecule marking method for paddy low glutelin mutant gene Lgcl, which belongs to the technical field of agricultural biological engineering. Two pairs of Insert/Deletion marks, namely InDel-Lgcl-1 and InDel-Lgcl-2, are designed and synthesized according to the difference between the normal paddy variety and the low glutelin variety of LGC-1 on the nucleotide sequence ofthe low glutelin gene Lgcl. Two pairs of primers are added to the same PCR reaction system; the DNA of a paddy plant to be measured is enlarged and a marked characteristic strip is analyzed for correctly identifying whether a paddy low glutelin germ resource or a breeding group contains Lgcl genes or not. The marking method can distinguish the pure junction and the mixed junction of the Lgcl genes for forecasting offspring gene types, largely enhance the selection efficiency of the low glutelin genes and accelerate the breeding process.

Description

A kind of molecule marking method of differentiating paddy low glutelin gene Lgcl

One, technical field

The present invention relates to a kind of molecule marking method of differentiating paddy low glutelin gene Lgcl, belong to agricultural biotechnology engineering, be exclusively used in the evaluation and the seed selection of the low glutelin rice varieties that contains the Lgc1 gene.

Two, background technology

Protein is second largest reserve substance in the rice endosperm, also is simultaneously the main source of human vegetable-protein consumption.Difference according to its dissolution characteristics can be divided into 4 kinds: gluten, prolamine, sphaeroprotein and white protein, wherein glutelin content is higher, account for the proteic 60-80% of seed, it is can be for main component (the Shewryand Casey of absorption of human body in the rice protein, 1999, Seed proteins, In:Shewry P.R., Casey R. (eds.), Kluwer academicpublishers, Dordrecht, The Netherlands, pp.1-10), thus the improvement of rice nutritive value can be achieved by improving glutelin content.Yet for the nephropathy patient, a large amount of absorptions of gluten can cause the metabolic disorder of human body protein, and then cause that sb.'s illness took a turn for the worse.Therefore, can absorb proteic particular demands for satisfying the kidney patient to low, cultivate important directions (the Iida etal. that the good low glutelin new variety of comprehensive proterties have become current functional rice breeding, A rice mutant having a low content of glutelin and a high content of prolamine, Theor.Appl.Genet., 1993,87 (3): 374-378).

The low glutelin mutant is the important genetic resources that carries out the low glutelin rice variety selective, and its principal feature is the raising of reducing greatly of glutelin content and content of prolamine.(Iida et al. such as Iida, A rice mutant having alow content of glutelin and a high content of prolamine, Theor.Appl.Genet., 1993,87 (3): 374-378) handle Japanese excellent seed by the chemical mutagen ethyleneimine, obtain low glutelin mutant NM67, utilized it and original parent to backcross, selected first low glutelin rice varieties LGC-1.(Miyahara et al., Analysis of glutelin gene in rice low glutelin line LGC-1, Breeding Sci., 1996,46 (Supply.1): 42 such as Miyahara; Miyahara et al., Analysis of LGC-1, low glutelin mutant of rice, Gamma Field Symposia, 1999,38:43-52) LGC-1 is furtherd investigate, find that the low glutelin proterties among the LGC-1 is controlled by single dominant gene, and be located between paddy rice the 2nd chromosomal marker XNpb243 and G365.Method by similar physical and chemomorphosis, (Iida et al. such as Iida and Qu Leqing, Mutants lackingglutelin subunits in rice:mapping and combination of mutated glutelin genes, Theor.Appl.Genet., 1997,94 (2): 177-183; Qu Leqing etc., rice paddy seed storage gluten 2 subunits reduce mutant, Botany Gazette, 2001,4311): 1167-1171) also obtained a plurality of low glutelin mutant in succession, wherein type-1, type-2 and type-3 are controlled by recessive single-gene glu-1, glu-2 and glu-3 respectively, and the result of rflp analysis further shows, glu-1 be positioned at paddy rice the 2nd karyomit(e) and with the Lgc1 equipotential, glu-2 and glu-3 then lay respectively at the 10th and the 1st karyomit(e).

Along with the fast development of modern molecular biology theory and technology, the genetic mechanism of LGC-1 low glutelin variation is illustrated.(Kusaba et al. such as Kusaba, Low glutelin content1:A dominant mutation that suppressesthe glutelin multigene family via RNA silencing in rice, The plant cell, 2003,15 (6): 1455-1467) research thinks that the low glutelin sudden change takes place LGC-1 is the result of RNA silence, in normal japonica rice variety Japan was excellent, two nucleotide sequence similaritys reached GluB subtribe gene-GluB4 of 99.8% and link to each other by section of DNA sequence tail tail with GluB5; And in the LGC-1 mutant, 3 ' end of GluB5 gene and the DNA of the one section 3.5Kb in downstream thereof lack, the GluB5 that has lost terminator reads over when transcribing, thereby form the hairpin structure of RNA with GluB4, and the glutenin gene by RNAi degrade specifically B subtribe, cause LGC-1 performance low glutelin characteristic.

At present, LGC-1 uses one of successful low glutelin mutant.Japan rice breeding man has utilized this mutant to breed a large amount of best in quality, rice varieties that glutelin content is on the low side, as (Nishimura et al. such as LGC-Katsu and LGC-Jun, New rice varieties with low levels of easy-to-digest protein, ' LGC-Katsu ' and ' LGC-Jun ', Breeding Sci., 2005,55:103-105).Along with development of molecular biology, molecule marker based on the DNA variation has been brought into play enormous function in the rice genetic improvement, and important progress (Deng Huabing etc. have been obtained at aspects such as output, quality, resistance breedings, molecular marker assisted selection and the breeding effect thereof of Malaysia common wild-rice volume increase QTL, the rice in China science, 2007,21 (6): 605-611; Wang Cailin etc. cultivate excellent flavour rice new variety, rice in China science, 2009,23 (1): 25-30 by molecular marker assisted selection; All space torch etc. utilize four rice restorers of MAS technique improvement and disease resistance analysis thereof, Molecular Plant Breeding, 2008,6 (3): 480-490).Therefore,, can not only effectively identify the low glutelin rice germplasm resource that contains the Lgc1 gene, also can quicken the seed selection of the special-purpose low glutelin rice varieties of kidney patient simultaneously if can obtain with the goal gene close linkage or be divided into to separate sub-mark.

Three, summary of the invention

Technical problem:

The present invention is directed to above-mentioned situation, the base difference design that between two glutenin gene GluB4 and GluB5, exists according to LGC-1 mutant and normal water rice varieties synthetic with goal gene isolating molecule marker altogether, and by simple detection method, Rapid identification contains the low glutelin rice germplasm resource of Lgc1 mutator gene, can also further be applied to molecular mark simultaneously.

Technical scheme:

1, a kind of molecule marker primer of identifying paddy low glutelin gene Lgcl comprises:

Primer I nDel-Lgc1-1:

The forward primer sequence: 5 '-TTCTACAATGAAGGCGATGC-3 '

The reverse primer sequence: 5 '-CTGGGCTTTAACGGGACT-3 '

With primer I nDel-Lgc1-2:

The forward primer sequence: 5 '-ACCGTGTTATGGCAGTTT-3 '

The reverse primer sequence: 5 '-ATTCAAGGGCTATCGTCT-3 '

2, according to the described a kind of molecule marker primer method of identifying paddy low glutelin gene Lgcl of claim 1, its principal character step comprises:

(1) extraction of oryza sativa genomic dna;

(2) described two couples of molecule marker primer I nDel-Lgc1-1 of claim 1 and InDel-Lgc1-2 are added same PCR reaction system, and the DNA of rice germplasm resource or breeding population plant is increased.

20 μ LPCR reaction systems comprise: the DNA 2.0 μ L of 10ng/ μ L, the primer 2 .0 μ L of 4pmol/ μ L (each 1.0 μ L of primer I nDel-Lgc1-1 and InDel-Lgc1-2), 10 * Buffer, 2.0 μ L, the dNTP 0.4 μ L of 2.5mmol/L, the MgCl of 25mmol/L ₂1.2 μ L, Taq 0.2 μ L and the ddH of 5U/ μ L ₂O 12.2 μ L;

The PCR response procedures comprises: 95 ℃ of pre-sex change 5min; 95 ℃ of sex change 30s, 56 ℃ of renaturation 30s, 72 ℃ of extension 1min circulate 35 times then; 72 ℃ are extended 7min then, behind 10 ℃ of cooling 10min, amplified production are added sample damping fluid termination reaction.

(3) reaction product is electrophoresis on 1.2% the sepharose than concentration in quality, observes record down through ethidium bromide staining and in gel imaging system.If can only amplify the feature band of 881bp, illustrate that this plant is the homozygote of low glutelin mutator gene Lgc1; If can only amplify the feature band of 509bp, illustrate that this plant is the homozygote that does not contain the Lgc1 mutator gene; If can amplify two feature bands of 881bp and 509bp simultaneously, illustrate that then this plant is the heterozygote of low glutelin mutator gene Lgc1.

Beneficial effect

The present invention utilizes molecular biological method to obtain two insertion directly related with paddy low glutelin gene Lgcl-disappearance marks, and its advantage specifically is summarized as follows:

(1) molecule marker that the present invention obtained is the base deletion design synthetic that exists between two glutenin gene GluB4 and GluB5 according to the LGC-1 mutant, does not therefore have the exchange of heredity, does not also need the further checking of phenotype.

(2) clear and definite mutator gene source is the important prerequisite of carrying out the low glutelin rice variety selective, two genetic markers that utilize the present invention to obtain can screen a large amount of variety resources of rice, thereby accurately identify the low glutelin material that contains the Lgc1 mutator gene.

(3) the definite of low glutelin proterties must wait until that seed maturity just can carry out later on usually, and utilization and the directly related molecule marker of Lgc1 gene detect its leaf DNA, can identify the glutenin gene type in Lgc1 site in seedling stage, and then improve the efficient that kind is selected greatly.

Four, description of drawings

Fig. 1 is used for the PCR design of primers strategy that low glutelin mutator gene Lgc1 detects

(annotate: black box is represented the exon of gene; Dotted line is represented the DNA disappearance of 3.5Kb in the LGC-1 low glutelin mutant; Single arrow is represented the gene transcription direction; Double-headed arrow is represented the zone of design of primers)

Different parents of Fig. 2 and F ₁The whole protein SDS-PAGE of cross-fertilize seed detects

(1: protein molecular weight standard, 14.3-97.2KDa; 2-7:W3360, fine, the force of Japan is educated round-grained rice No. 3,02428,9311 and Nanjing 11; 8-11:W3660/ Japan fine, Japanese fine/W3660, W3660/02428,02428/W3660

Fig. 3 InDel-Lgc1-1 mark is to different parents and hybrid F ₁The Molecular Detection of plant

(1:DNA molecular weight standard, 100-2,000bp; 2-7:W3660, fine, the force of Japan is educated round-grained rice No. 3,02428,9311 and Nanjing 11; 8-11:W3660/ Japan fine, Japanese fine/W3660, W3660/02428,02428/W3660

Fig. 4 InDel-Lgc1-2 mark is to different parents and hybrid F ₁The Molecular Detection of plant

(annotate: 1:DNA molecular weight standard, 100-2,000bp; 2-7:W3360, fine, the force of Japan is educated round-grained rice No. 3,02428,9311 and Nanjing 11; 8-11:W3660/ Japan fine, Japanese fine/W3660, W3660/02428,02428/W3660

Fig. 5 InDel-Lgc1-1 and InDel-Lgc1-2 mark are to different parents and hybrid F ₁The Molecular Detection of plant

(1:DNA molecular weight standard, 100-2,000bp; 2-7:W3360, fine, the force of Japan is educated round-grained rice No. 3,02428,9311 and Nanjing 11; 8-11:W3660/ Japan fine, Japanese fine/W3660, W3660/02428,02428/W3660

Five, embodiment

Test materials comprises the japonica rice variety W3660 that contains low glutelin gene Lgcl, and this kind is to do maternal and the light hybridization more of Japanese main breed with low glutelin kind LGC-1, and is that recurrent parent carries out the continuous backcross transformation and forms precocious japonica rice variety with light more.The normal water rice varieties comprises that conventional japonica rice kind Japan is fine, force educate round-grained rice No. 3, the wide affine kind 02428 of round-grained rice type and conventional rice variety 9311 and Nanjing 11.F ₁Be to be 4 F that the parent obtains with the normal rice varieties of glutelin content Japan warm and fine 02428 reciprocal cross respectively with W3660 ₁

Above material is public material, and country or Jiangsu Province's germ plasm resource platform all can provide free.Concrete reference is: Wan Jianmin etc., the seed selection of functional rice special kind W3660, crop magazine, 2004,5:58; Http:// ineweb.narcc.affrc.go.jp/; Jiang Qixiang etc., intermediate keng rice new variety force is educated the seed selection and the utilization thereof of No. 3, round-grained rice, Jiangsu agricultural sciences, 1993,3:8-10; Zou Jiangshi etc., wide affine choosing system " 02428 " is in the Preliminary Exploitation of Hybrids of Indica and Japonica, Scientia Agricultura Sinica, 1989,22 (1): 6-14; Wear positive element etc., utilize nuclear radiation to breed the good quality and high output new rice variety and raise rice No. 6, nuclear agricultural science newspaper, 1999,13 (6): 377-379; Lin Shicheng, Min Shaokai chief editor: rice in China kind and pedigree thereof, Shanghai science tech publishing house, 1991,56-58

(1) with the acquisition of the directly related molecule marker of low glutelin gene Lgcl

Result of study (Kusaba et al. according to Kusaba etc., Low glutelin content1:A dominant mutationthat suppresses the glutelin multigene family via RNA silencing in rice, The plant cell, 2003,15 (6): 1455-1467), utilizing the nucleotides sequence of GluB5 gene complete to be listed in rice genome note website (http://rice.plantbiology.msu.edu/) carries out BLAST and analyzes, obtain the site title LOC_Os02g16820.1 of this gene and phage artificial chromosome clone (P1-derived artificialchromosome, PAC) the sequence number P0693E08 at place; Utilize acquired pac clone sequence number to download corresponding nucleotide sequence simultaneously, and sequence is analyzed at Gene Bank (www.ncbi.nlm.nih.gov/); Then, utilize near PrimerPremier 5.0 (http://www.premierbiosoft.com) corresponding molecule marker of the design zone of LGC-1 mutant 3.5Kb disappearance, and with synthetic labeled primer difference called after InDel-Lgc1-1 and InDel-Lgc1-2.The forward sequence of InDel-Lgc1-1 is 5 '-TTCTACAATGAAGGCGATGC-3 ', reverse sequence is 5 '-CTGGGCTTTAACGGGACT-3 ', the forward sequence of InDel-Lgc1-2 is 5 '-ACCGTGTTATGGCAGTTT-3 ', reverse sequence is 5 '-ATTCAAGGGCTATCGTCT-3 ', annealing temperature is 56 ℃ (Fig. 1).

(2) extraction of genomic dna

The water intaking rice seedling phase blade, in the mortar of-20 ℃ of precoolings with the liquid nitrogen grinding and the 1.5ml centrifuge tube of packing into; Add 600ul extracting solution (20%SDS, 1M Tris-HCl, 0.5M EDTA, 5M NaCl, 65 ℃ of preheatings), shake up, 65 ℃ of temperature are bathed 30min, middle vibration 3～4 times; Add 1/4 volume 5M KAC, shake up postposition 30min on ice; Add chloroform-primary isoamyl alcohol (24: 1) 300～400ul, fully vibration on shaking table, 120rpm, 30min; Centrifugal 15 minutes of 8000～10000rpm, the liquid level layering, lower floor's color is darker, and the little band yellow-green colour in upper strata is got supernatant (about 400ul) to another centrifuge tube; Add equal-volume chloroform-primary isoamyl alcohol (24: 1), fully vibration on the shaking table, 80～90rpm, 30min; Centrifugal 15 minutes of 8000rpm shifts supernatant (about 400ul) to new centrifuge tube; The dehydrated alcohol that adds 2 times of volume-20 ℃ precoolings shakes up gently up to there being floss to produce the centrifugal 6min of 12000rpm; Abandon dehydrated alcohol, add 4 ℃ of 70% ethanol, place 10min, abandon supernatant, air-dry 1h on the Bechtop; Add 100～200ulTE ,-20 ℃ of preservations.

(3) pcr amplification and agarose electrophoresis

20 μ L PCR reaction systems comprise DNA (10ng/ μ L) 2.0 μ L, Primer (4pmol/ μ L) 2.0 μ L (each 1.0 μ L of primer I nDel-Lgc1-1 and InDel-Lgc1-2), 10 * Buffer (free MgCl ₂) 2.0 μ L, dNTP (2.5mmol/L) 0.4 μ L, MgCl ₂(25mmol/L) 1.2 μ L, Taq (5U/ μ L) 0.2 μ L and ddH ₂O 12.2 μ L.Response procedures comprises 95 ℃ of pre-sex change 5min; Then 95 ℃ of sex change 30s, 56 ℃ of renaturation 30s, 72 ℃ extend 1min, circulate after 35 times, 72 ℃ are extended 7min, 10 ℃ cool off 10min after, amplified production is added sample damping fluid termination reaction.Reaction product is electrophoresis on 1.2% the sepharose than concentration in quality, observes record down through ethidium bromide staining and in gel imaging system.

(4) extraction of mature seed total protein and SDS-PAGE electrophoresis detection

The extraction of mature seed total protein is with reference to method (the Cagampan et al. of Cagampang etc., Studies on theextraction and composition of rice proteins, Cereal Chem, 1966,43:145-55), the single seed shelling goes to grind to form superfine powder with mortar behind the embryo, the eppendorf pipe of the 1.5mL that packs into, add 700uL SDS-UREA and extract damping fluid (8mol/L urea, 4%SDS, 5% beta-mercaptoethanol, 20% glycerol, 50mmol/L Tris-HCl, PH6.8), the vibration mixing is after 25 ℃ of incubated overnight, centrifugal 5 minutes of 7000g, and get 5 μ L supernatant liquors and be used for SDS-PAGE.

SDS-PAGE carries out (Laemmli U.K. according to the method for Laemmli, Cleavage of structural proteinsduring the assembly of the head of bacteriophage T4, Nature, 1970,227 (5259): 680-685), the separation gel of employing 15% and 7.5% concentrated glue adopt coomassie brilliant blue R250 dyeing behind the electrophoresis.

(5) the whole protein SDS-PAGE of paddy rice mature seed analyzes

From the electrophoretic band of rice paddy seed total protein SDS-PAGE as can be seen,, force fine with the normal rice varieties of glutelin content Japan educate round-grained rice No. 3,02428,9311 and Nanjing 11 compare, the content of low glutelin kind W3660 prolamine (13KDa) is higher relatively, and the content of acid subunit (22-23KDa) of ripe gluten and alkaline subunit (37-39KDa) is then relatively low; With W3660 is father, female parent, respectively with Japan warm and fine 02,428 4 F obtaining of mutual cross mutually ₁Protein band and the W3660 of cross-fertilize seed are identical, this shows that the low glutelin proterties among the W3660 is dominant inheritance, and the dosage effect (Fig. 2) that does not have endosperm, this and former study conclusion (Miyaharaet al. in full accord, Analysis of LGC-1, low glutelin mutant of rice, Gamma Field Symposia, 1999,38:43-52).

(6) PCR of molecule marker InDel-Lgc1-1 detects

Utilize design synthetic InDel-Lgc1-1 labeled primer to 10 parts of parents and hybrid F ₁Material carries out PCR and detects, and the result shows: at LGC-1 deutero-low glutelin kind W3660 and by W3660 is father, female parent, respectively with Japan warm and fine 02,428 4 F of mutual cross mutually ₁All can stablize the dna fragmentation that amplifies a treaty 881bp size in the plant,, force fine in 5 normal valley albumen rice varieties Japan educate round-grained rice No. 3,02428,9311 and Nanjing 11 in then can not effectively increase (Fig. 3).The such detected result and the notional result of primer amplification are also not quite identical, because there is not the disappearance of 3.5Kb in the normal water rice varieties between GluB4 and GluB5 gene, one treaty 4, the band of 381bp, and F should appear so these materials are carried out pcr amplification ₁Also 881bp and 4 should appear simultaneously, 381bp two bands.Yet in this process of the test, 4, the dna fragmentation of 381bp size is not effectively increased all the time, and analyzing its reason may be because general T aq enzyme causes the amplification of lengthy motion picture segment DNA is limited in one's ability.Thereby InDel-Lgc1-1 here is a dominant marker.

(7) PCR of molecule marker InDel-Lgc1-2 detects

Isozygoty and heterozygosis low glutelin genotype for distinguishing better, a pair of InDel-Lgc1-2 labeled primer has been synthesized in design in the disappearance zone of 3.5Kb simultaneously, and utilizes above-mentioned identical parent and hybrid F ₁Material is verified it.The result shows: at 5 normal gluten rice varieties and 4 F ₁In all can stablize the dna fragmentation that amplifies a treaty 509bp size, in LGC-1 deutero-low glutelin rice varieties W3660, then can not effectively increase (Fig. 4).Therefore, this dominant marker can be used as effectively replenishing of InDel-Lgc1-1, is used for accurately identifying the glutenin gene type in Lgc1 site.

(8) double PCR of molecule marker InDel-Lgc1-1 and InDel-Lgc1-2 detects

Because InDel-Lgc1-1 has identical annealing temperature and different product length with InDel-Lgc1-2, be the short form test operation, save cost, two pairs of primers are added same PCR reaction system, and expectation just reaches by once using of two dominant markers, and differentiation is isozygotied and the purpose of heterozygosis glutenin gene type.From electrophoresis detection we are not difficult to find out as a result, in LGC-1 deutero-low glutelin rice varieties W3660, can stablize the single band that amplifies a treaty 881bp, equally fine, force in 5 normal rice varieties Japan educate round-grained rice No. 3,02428,9311 and Nanjing 11 in also can amplify the band of a 509bp, and be father, female parent by W3660, respectively with Japan warm and fine 02,428 4 hybrid F of mutual cross mutually ₁In but can amplify the band (Fig. 5) of two clauses and subclauses of 509bp and 881bp simultaneously.This conforms to fully with expected results, and the double PCR of two pairs of primers detects and can reach the purpose of identifying Lgc1 low glutelin germ plasm resource and marker-assisted breeding.

Sequence table

＜110〉Jiangsu Province Agriculture Science Institute

＜120〉a kind of molecule marking method of differentiating paddy low glutelin gene Lgcl

＜130〉specification sheets

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<141>2009-09-01

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<170>PatentIn?version?3.1

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ttctacaatg?aaggcgatgc 20

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attcaagggc?tatcgtct 18

Claims (3)

1, a kind of molecule marker primer of identifying paddy low glutelin gene Lgcl comprises:

Primer I nDel-Lgc1-1:

The forward primer sequence: 5 '-TTCTACAATGAAGGCGATGC-3 '

The reverse primer sequence: 5 '-CTGGGCTTTAACGGGACT-3 '

With primer I nDel-Lgc1-2:

The forward primer sequence: 5 '-ACCGTGTTATGGCAGTTT-3 '

The reverse primer sequence: 5 '-ATTCAAGGGCTATCGTCT-3 '

2, a kind of molecule marking method of identifying paddy low glutelin gene Lgcl is characterized in that:

Described two couples of molecule marker primer I nDel-Lgc1-1 of claim 1 and InDel-Lgc1-2 are added same PCR reaction system, DNA to rice germplasm resource or breeding population plant increases, reaction product is electrophoresis on 1.2% the sepharose than concentration in quality, observe down through ethidium bromide staining and in gel imaging system, record, if can only amplify the feature band of 881bp, illustrate that this plant is the homozygote of low glutelin mutator gene Lgc1; If can only amplify the feature band of 509bp, illustrate that this plant is the homozygote that does not contain the Lgc1 mutator gene; If can amplify two feature bands of 881bp and 509bp simultaneously, illustrate that then this plant is the heterozygote of low glutelin mutator gene Lgc1.

3, according to the molecule marking method of the described evaluation paddy low glutelin gene Lgcl of claim 2, it is characterized in that: 20 μ LPCR reaction systems comprise: the DNA 2.0 μ L of 10ng/ μ L, the primer I nDel-Lgc1-21.0 μ L of the primer I nDel-Lgc1-11.0 μ L of 4pmol/ μ L and 4pmol/ μ L, 10 * Buffer, 2.0 μ L, 2.5mmol/L dNTP0.4 μ L, the MgCl of 25mmol/L ₂1.2 μ L, Taq 0.2 μ L and the ddH of 5U/ μ L ₂O 12.2 μ L;

The PCR response procedures comprises: 95 ℃ of pre-sex change 5min; 95 ℃ of sex change 30s, 56 ℃ of renaturation 30s, 72 ℃ of extension 1min circulate 35 times then; 72 ℃ are extended 7min then, behind 10 ℃ of cooling 10min, amplified production are added sample damping fluid termination reaction.

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