CN101429561B

CN101429561B - Method for detecting rice gene with PCR and PCR buffer solution employing the method

Info

Publication number: CN101429561B
Application number: CN2008103063577A
Authority: CN
Inventors: 罗天宽; 张小玲; 朱世杨; 陈海英; 杨文清; 卢华金; 唐征; 刘庆
Original assignee: WENZHOU INSTITUTE OF AGRICULTURAL SCIENCES
Current assignee: WENZHOU INSTITUTE OF AGRICULTURAL SCIENCES
Priority date: 2008-12-18
Filing date: 2008-12-18
Publication date: 2011-08-10
Anticipated expiration: 2028-12-18
Also published as: CN101429561A

Abstract

The invention relates to a method for detecting paddy rice genes by utilizing PCR and a PCR buffer solution for the method. The method for detecting the paddy rice genes by utilizing the PCR comprises the following steps; (1) acquiring a paddy template for PCR, namely a paddy rice leaf with a length between 0.5 and 3 mm<2> is cut and directly used as the template; (2) preparing a PCR reaction liquid which comprises a primer, the PCR buffer solution, DNTP and enzyme; (3) performing PCR amplification; and (4) analyzing the gene detection result by means of gel electrophoresis. Compared with theprior art, the method does not need to extract DNA of the paddy rice and directly uses a small quantity of leaves (without any treatment) for PCR amplification to obtain a stable PCR amplification system, so as to save a large amount of time, labor cost and cost of medicines and reagents. Moreover, the required instruments and equipment are single (equipment such as a centrifuge required for extracting the DNA is saved), and the process is simple, so that each person can perform PCR detection on thousands of samples per day.

Description

Detect the method for paddy gene and be used for the PCR damping fluid of this method with PCR

Technical field

The present invention relates to a kind of method that detects gene, relate in particular to and a kind ofly detect the method for paddy gene and be used for the PCR damping fluid of this method with PCR.

Background technology

Polymerase chain reaction (Polymerase Chain Reaction, be called for short PCR) is external, amplification target dna segmental a kind of method synthetic by enzyme reaction, also is one of the most frequently used, most important Protocols in Molecular Biology.The detection that traditional application round pcr carries out paddy gene comprises the extraction of oryza sativa genomic dna, carry out pcr amplification, the PCR product carries out three steps of gel electrophoresis analysis, detection wherein, analysis phase operating automation degree are higher, cost reduces significantly, need to be resolved hurrily most also be most possible improved be the DNA extraction technology.Since Marmur set up the classical way of using sodium laurylsulfonate (SDS) and chloroform separation and Extraction DNA sample from biomass cells in 1961, people are devoted to improving of DNA method for extracting always and simplify, and comparatively ripe mostly, but program is loaded down with trivial details, wastes time and energy very much.B.C.Y.Collard etc., the article of delivering on Plant Breeding is analyzed the cost of multiple paddy DNA extracting method (comprising the extracting method of multiple simplification), and analytical results is presented at the cost that each sample of the simplest method extracts in a small amount in the several method of comparison also needs 0.48 dollar.Therefore, DNA extraction has become a big bottleneck of labeling technique and transgenosis detection widespread use.

In view of the vital role of paddy rice to the mankind, the dna molecular marker technology of PCR-based is used by having in fields such as the breeding of rice molecular marker assisted selection, germ plasm resource evaluation, sibship analysis, the assignment of genes gene mapping, genetic map constructions widely.So it is significant to carry out efficient, cheap PCR molecular detection technology.The existing PCR damping fluid component prescription that is applied to has a variety ofly, contains the KC1 of different concns, MgCl mostly ₂, Tris-Cl (different PH), etc., because different organization structure of the plant differences, so different PCR damping fluid composition is also different to the effect of the pcr amplification of the DNA of different plants or extracting method extraction.Existing PCR damping fluid composition commonly used is difficult to directly rice leaf be carried out pcr amplification.

Directly report is also being arranged with blade or plant tissue as the research that template is carried out PCR, but its method need to the blade of plant tissue is handled or very high to the requirement of operation, operation easier is very big.As Lu Xueying etc. (arid biogeographic zone grinds, 2007), do not give birth to 0.2mm then with what accurate Ge Er had leaf beans ²Organize and successfully carried out pcr amplification, as a rule 0.2mm ²The sampling ratio difficulty of tissue because 0.2mm ²Tissue to put into PCR pipe back difficult to observe, so the 0.2mm that takes off ²Tissue put in the PCR pipe also difficult, need according to this kind method operation very skilled, therefore difficult application the in actually operating.(rice in China science such as Wang Xiufeng, 2002), used after the alkaline purification with blade directly as the carrying out of pcr template success amplification and succeedd, the process of its alkaline purification is: cut the long rice leaf of 1-2cm, put into the Eppendorf pipe that contains 200 μ l 0.25mol/L NaOH, this pipe inserted boil 30s in people's boiling water and add people 200 μ l0.25mol/L HCl and 100 μ l 5mol/LTris-HCl then (pH 8.0, contain volume integral and cause P40) into 0.25%Nonidet, once more this pipe is put in people's boiling water and hatched 2min, last picking one small pieces directly carry out pcr amplification reaction as template through the blade of above-mentioned processing.The process of finding out alkaline purification thus still needs expensive time and certain processing cost, has therefore also limited detection speed significantly.

Summary of the invention

The objective of the invention is to overcome the defective of prior art, a kind of efficient that paddy gene detects that improves is provided, reduce the method that detects cost with PCR detection paddy gene, and the PCR damping fluid that is used for this method.

For achieving the above object, the present invention adopts a kind of method of extracting paddy gene, is made up of following step:

(1) obtains PCR paddy rice template: cut 0.5-3mm ²Long rice leaf directly is a template;

(2) configuration of PCR reaction solution comprises primer, PCR damping fluid, DNTP, enzyme;

(3) carry out pcr amplification;

(4) gel electrophoresis analysis gene test result.

The rice leaf source that the present invention cuts can be that fresh blade also can be a cryopreservation rice leaf for many years.Wherein obtain the process of fresh blade: for planting experimentally son at the 1/4MS liquid nutrient medium, be cultured to for 3 leaf phases at 25-30 ℃ then, directly sowing and land for growing field crops to 3 leaf phase, Direct Sampling rice leaf at last; Obtain the process of cryopreservation rice leaf for many years: 1) for planting experimentally the blade of son in 1/4MS liquid nutrient medium, 25-30 ℃ cultivation; 2) directly be seeded in the blade in land for growing field crops; 3) after the sampling blade is loaded in the paper bag, places-70 ℃ of cryogenic refrigerators to preserve the blade of 1-3; 4) after the rice leaf that cuts used, remainder can use for continuing next time in-20 ℃ or-70 ℃ of cryopreservation, realizes the primary sample life-time service.

The present invention is used for the PCR damping fluid that above-mentioned PCR detects the method for paddy gene, and its composition comprises:

(1)KC1 10-20mmol/L；

(2)MgCl ₂ 2-2.5mmol/L；

(3)(NH ₄) ₂SO ₄ 10-30mmol/L；

(4) Tris-Cl, pH value are 9.0 10-50mmol/L;

(5) PCR increased response agent 1-5%.

PCR increased response agent among the present invention comprises DMSO, NP40, BSA.

The present invention compares with prior art, need not paddy DNA is extracted, and is directly used in pcr amplification with a small amount of blade (need not any processing), obtains stable pcr amplification system, has saved a large amount of time, the cost of artificial and medicine and reagent.Required plant and instrument single (save extract DNA required equipment such as whizzer), step process are simple, and the PCR that can make thousands of samples for each person every day detects (under the sufficient situation of PCR instrument).Present method greatly reduces cost for the pcr analysis that can carry out a large amount of samples at short notice provides possibility.Present method can effectively be applied to aspects such as the screening in a large number of paddy rice transgenic line, molecular marker assisted selection, variety authentication Rapid identification, germ plasm resource selection, can carry out high-throughout analysis and detection, only needing to be particularly suitable for the paddy rice PCR experiment of qualitative evaluation, to provide strong auxiliary for utilizing dna molecular marker to accelerate the rice breeding process.

Description of drawings

Fig. 1 directly is used as the pcr amplification figure of pcr template for the blade of embodiment of the invention different varieties different sources.

Fig. 2 be the embodiment of the invention in the PCR reaction solution through 94 ℃, behind the 5min with the blade photo (the ultraviolet reflectance photo-beat is taken the photograph) after the EB dyeing

Embodiment

The present invention detects the method specific embodiment of paddy gene with PCR:

1 materials and methods

1.1 material material

The KR117 that experiment material provides for IRBB21 that rice in China provided and academy of agricultural sciences, Wenzhou.

1.2 experimental technique

1) ℃ was cultured to for 3 leaf phases for planting experimentally son at 1/4MS liquid nutrient medium, 25-30; 2) directly sowing and land for growing field crops to 3 leaf phase; 3) after the sampling blade is loaded in the paper bag, places-70 ℃ of cryogenic refrigerators to preserve 1-2.Cutting the long blade of 2-3mm2 is template, the oryza sativa genomic dna that contrast is extracted with the SDS method.

1.3 primer

By last maritime works's synthetic PTA248 (Forward:5 '-aga cgc gga agg gtg gttcccgga-3 ', Reverse:S '-aga cgc ggt aat cga aag atg aaa-3 ')..

1.4PCR reaction process

PCR reaction buffer composition: 50mmol/LKCL, 2.5mmol/L MgCl ₂, 30mmol/L (NH ₄) ₂SO ₄, 20mmol/L Tris-Cl, pH value are 9.0,0.2mmol/LDNTP, and DMSO 2%, BSA 20 μ g/mL, the forward and reverse primer of the PCR of 0.5 μ mol/L, the Tag archaeal dna polymerase of 2U.Dna profiling is directly used the 2-3mm2 blade, contrasts the genomic dna (50ng) that extracts into the SDS method.PCR is reflected on the U.S. product IBA9700 type DNA cloning instrument and carries out, and response procedures is: 94 ℃, and after the 5min sex change, by 94 ℃ of sex change 1min, 58 ℃ of annealing 30s, 72 ℃ of extension 2min carry out 35 circulations, and 10min, 4 ℃ of preservations are extended at 72 ℃ in reaction end back.Amplified production is electrophoresis 1h (5V/cm) on 1.0% sepharose, and (ultraviolet lamp is observed the analysis of taking pictures on the U.S. SIM BEST-200E gel imaging system down to ethidium bromide for ethidium bromide, EB) dyeing.

1.5 experimentation

1.5.1 experimentation of the present invention:

1) saves the DNA extraction process, saved required time, instrument, medicine and the consumables cost of this process.

2)PCR

---the 20ul system adds each required composition of PCR reaction respectively, and---getting the 2-3mm2 blade puts into wherein, and---put into the PCR instrument and carry out pcr amplification---the PCR product carries out gel electrophoresis---analyzes at gel imaging system preparation PCR damping fluid.

2 interpretations of result:

As shown in Figure 1, the fresh blade 3-4 that M:Marker Bio DL 2000,1-2:1/4MS cultivate: the field freezing blade of the freezing blade 9-10:2008 of the freezing blade 7-8:2007 of fresh blade 5-6:2006 of taking a sample.11-12:CK (DNA that blade extracts with the SDS method is a template).Wherein 1,3,5,7,9,11 is IRBB21, and 2,4,6,8,10,12 is KR117.0.2mmol/L dNTP, the forward and reverse primer of the PCR of 0.5 μ mol/L, the Tag archaeal dna polymerase of 2U.Dna profiling is directly used 1-2mm ²Blade contrasts the genomic dna (50ng) that extracts into the SDS method.The blade of presentation of results different varieties, different sources under the situation of any processing, all can amplify band and with the contrast no significant difference.

1 through PCR94 ℃, the mensuration of the dna content of the different treatment blade behind the 5min

Table 1 is through PCR94 ℃, the result that the dna content of different treatment blade is measured through U-0080D nucleic acid-protein detector behind the 5min, wherein: 1: be field in 2006 sampling (70 ℃ freezing preservation 3 years), 2: be field in 2007 sampling (70 ℃ freezing preservation 2 years), 3: be in February, 2008 field sampling (70 ℃ freezing preservation 9 months).The fresh blade that 4:1/4MS cultivates, 5: be the fresh blade of field sampling, the template of table 1 explanation PCR reaction has just had when pre-sex change.In the process of PCR reaction, will constantly have the reaction that participates in PCR that discharges of dna profiling.

As shown in Figure 2, blade directly use EB after-dyeing K R117 blade in the PCR reaction solution through PCR94 ℃, behind the 5min with the blade photo (the ultraviolet reflectance photo-beat is taken the photograph) after the EB dyeing, the fresh blade that 1:1/4MS cultivates, 2: be the fresh blade of field sampling, 3: be field in 2006 sampling (70 ℃ freezing preservation 3 years), 4: be field sampling in 2007 (70 ℃ freezing preservation 2 years), 5: be in February, 2008 field sampling (70 ℃ freezing preservation 9 months).As seen from the figure: all there is DNA in the surface of the blade of different sources.

A kind of PCR damping fluid that is used for the method for PCR detection paddy gene of the specific embodiment of the invention, its composition comprises:

(1)KC1 10-20mmol/L；

(2)MgCl ₂ 2-2.5mmol/L；

(3)(NH ₄) ₂SO ₄ 10-30mmol/L；

(4) Tris-Cl, pH value are 9.0 10-50mmol/L;

(5) PCR increased response agent 1-5%.

PCR increased response agent wherein comprises DMSO, NP40, BSA.

Claims

1. method that detects paddy gene with PCR, form by following step:

(1) obtains PCR paddy rice template: cut 0.5-3 mm ²The rice leaf of size directly is a template;

(3) carry out pcr amplification;

(4) gel electrophoresis analysis gene test result;

Wherein, PCR reaction solution composition is: 50 mmol/L KCl, 2.5 mmol/L MgCl ₂, 30mmol/L (NH ₄) ₂SO ₄, 20 mmol/L Tris-Cl, pH value are 9.0,0.2mmol/L DNTP, and DMSO 2%, BSA20 μ g/mL, the forward and reverse primer of the PCR of 0.5 μ mol/L, the Tag archaeal dna polymerase of 2U.

2. the method that detects paddy gene with PCR according to claim 1 is characterized in that: the described rice leaf source that cuts is fresh blade or cryopreservation rice leaf for many years.