CN103882126B - For detecting the primer, method and the test kit that turn mCry1Ac gene pest-resistant corn strain - Google Patents

Abstract

The invention belongs to technical field of biological, providing a kind of for detecting the primer and probe that turn mCry1Ac gene pest-resistant corn strain, is forward primer nucleotide sequence as SEQ? shown in IDNO.1; Is reverse primer nucleotide sequence as SEQ? ID? shown in NO.2; Is probe nucleotide sequence as SEQ? ID? shown in NO.3, or its complementary strand, reporter fluorescence group is connected to 5 ' end of probe, and quenching fluorescence group is connected to 3 ' end of probe.Further providing a kind of detection method, is template with sample total DNA, utilizes above-mentioned primer and probe to carry out real-time fluorescent PCR amplification to it, each loop ends image data, and reaction terminates rear according to amplification curve result of determination.Above-mentioned primer and probe in detecting specificity good, for detecting real-time fluorescence PCR, susceptibility is high; Detection method accuracy is high, is quick on the draw, for Transgene-safty provides guarantee.

Description

For detecting the primer, method and the test kit that turn mCry1Ac gene pest-resistant corn strain

Technical field

The invention belongs to technical field of biological, being specifically related to a kind of for detecting the primer, method and the test kit that turn mCry1Ac gene pest-resistant corn strain.

Background technology

Corn (ZeamaysL.) is important food, feed and industrial raw material, and cultivated area is only second to wheat and paddy rice, occupies an important position in world's grain-production.And insect is one of Main Factors causing corn yield to lose.In world wide, Corn Pests reaches more than kind more than 350, wherein serious with the European corn borer and Ostrinia furnacalis that eat into stem and leaf-feeding.Pyrausta nubilalis (Hubern). popular name borer, become Eimeria lepidopteran Pyralidae, be global moth feeding habits major pest, affect the important factor of corn yield and quality, the loss that the world causes because of insect pest every year accounts for more than 15% of crop yield.Pyrausta nubilalis (Hubern). can endanger milpa each position on the ground, makes some lost function of being injured, and reduces grain yield.Current prophylactico-therapeutic measures mainly uses chemical insecticide in a large number, and this has not only had a strong impact on ecotope and species diversity, adds production cost and labour intensity, also increases the poisoning probability of human body.One of effective way solved the problem improves the insect-resistance of corn itself.Traditional breeding method, owing to being subject to the restriction of many factors, is difficult to cultivate pest-resistant corn.The transgenic insect-resistant corn produced under this background then can efficient single-minded control Pyrausta nubilalis (Hubern)., is the new way of control of maize snout moth's larva.Conventional method is separated to obtain goal gene from microorganism, transfers in the genome of ordinary maize self-mating system by method for transformation such as particle guns by goal gene, makes it genetic stability thus give corn new economical character-insect-resistance.

Bt is the abbreviation of Tribactur (Bacillusthuringiensis), is a kind of Gram-positive bacillus agri.This bacteriogenic Bt insecticidal proteins can destroy the Premeabilisation of cells pressure in insect gut, causes insect death.Bt gene is the Typical Representative of first-generation anti insect gene, and the albumen expressed by it is referred to as Cry crystallin.According to the difference of insecticidal spectrum and amino acid identity, Cry gene is divided into different types, and wherein the lepidoptera pest such as Cry1Ab and Cry1Ac gene pairs Pyrausta nubilalis (Hubern). has specificity toxicity.Be no matter the anti insect gene in which kind of source for genetically engineered, the problem of overriding concern improves its expression amount exactly.In general, expression amount is higher, and insect resistant effect is better.The expression amount of insecticidal proteins on plant very low (accounting for less than 0.001% of whole soluble proteins) of early stage transform insect-resistant gene plant, insect resistant effect is undesirable.From existing result of study, by the modification of Cry gene and transformation, the crop of trans Bt gene can obtain good insect resistant effect.

Along with the commercialization of genetically modified crops develops; the safety issue of transgenic crop; namely there is potential harm to environment protection, the eubiosis in it, and the edible safety caused containing new genetic material and protein in the genetically modified food processed by it is paid close attention to more and more widely.Most countries such as the states such as European Union, Russia, Japan, Korea S, Australia, New Zealand advocate to stick on limitation label to transgenic product in the world at present, namely the particle ratio of regulation transgenic product exceedes certain threshold value (as 1%, namely having 1 in 100 for transgenic corns) has then needed mandatory mark.China has promulgated agriculture GMO bio-safety management rules May 23 calendar year 2001, highlights the management to genetically modified organism and products thereof and control.No matter be carry out mark management to genetically modified food, or the conveying respectively to transgenosis and non-transgenic raw material, the analysis and detection technology of transgenosis raw material and food is absolutely necessary and has more realistic meaning and application prospect, and this is the basis that transgenic product is carried out to safety evaluation and implemented supervision.

Lai Jinsheng etc. are optimized transformation to Cry1Ac gene, by the method for particle gun cotransformation, mCry1Ac gene are transferred in the cross combination of corn inbred line HiIIA and HiIIB with high transformation efficiency, screen with riddled basins bar.Research, interim test and Environment release carry out the qualification of molecular level, protein level and field insect-resistance to the transformation event obtained by experiment, transformation event excellent for resistance and Zheng 58 are carried out many for backcrossing and selfing, finishing screen selects 1 excellent transgenic strain BT-799.Experimental result is presented at the offspring of molecular level transgenic line can genetic stability anti insect gene mCry1Ac, each generation all express target protein and from generation to generation between expression amount basically identical, have obvious insect-resistance from the every Dai Jun of field insect-resistance, pest-resistant grade is basically identical.

Summary of the invention

Not enough for prior art, the object of the present invention is to provide a kind of for detecting the primer and probe that turn mCry1Ac gene pest-resistant corn strain,

Its forward primer nucleotides sequence is classified as: Bt-799-F:5 '-GGAACAAACGATGATTCA-3 ' (SEQIDNO.1);

Its reverse primer nucleotides sequence is classified as: Bt-799-R:5 '-CTTCCCTTTTAGTTGTGTC-3 ' (SEQIDNO.2);

Its probe nucleotide sequence is: Bt-799-P:5 '-AGCCTCTCATTTTCTTCTTTGAGCT-3 ' (SEQIDNO.3) or its complementary strand, and reporter fluorescence group is connected to 5 ' end of probe, and quenching fluorescence group is connected to 3 ' end of probe.

While adding pair of primers, add a specific fluorescent probe during pcr amplification, when probe is complete, the fluorescent signal that reporter group is launched is quenched group absorptions; During pcr amplification, probe enzyme is cut degraded by 5 '-3 ' 5 prime excision enzyme activity of Taq enzyme, reporter fluorescence group is separated with quenching fluorescence group, thus fluorescence monitoring system can receive fluorescent signal, namely often increase a DNA chain, just have a fluorescence molecule to be formed, the accumulation and the PCR primer that achieve fluorescent signal form Complete Synchronization.By this method, can detect in sample whether to have and turn mCry1Ac gene pest-resistant corn strain Bt-799.

Preferably, described reporter fluorescence group is Fam, Hex, Tet, Joe, Vic, Fite, Cy3 or Cy5; Described quenching fluorescence group is Tamra, Rox, Dabcy, Bhq1 or Bhq2.

Another object of the present invention is to provide a kind of test kit containing above-mentioned primer and probe.

Another object of the present invention is to provide a kind of for detecting the method turning mCry1Ac gene pest-resistant corn strain, take sample total DNA as template, utilize above-mentioned primer and probe, or mentioned reagent box carries out real-time fluorescent PCR amplification to it, each loop ends image data, reaction terminates rear according to amplification curve result of determination.

Test sample foreign gene detects Ct value between 36 ~ 40, should adjust template concentrations, real-time fluorescence PCR of reforming.Foreign gene again after amplification detects Ct value and is still less than 40, then can be judged to be that this sample is containing the foreign gene examined to some extent.Foreign gene again after amplification detects Ct value and is more than or equal to 40, then can be judged to be that this sample does not contain examined foreign gene.

Preferably, the present invention take sample total DNA as the optimization system that template carries out real-time fluorescent PCR amplification is 25 μ l, and reaction conditions is Mix12.5 μ l, primer final concentration 0.2 μm of ol/L, and probe final concentration is 0.1 μm of ol/L.Taq DNA polymerase used and damping fluid thereof are ABTaqManGeneExpressionMasterMix.

Preferably, PCR reaction parameter is 95 DEG C of 30sec; 95 DEG C of 5sec, 60 DEG C of 30sec, totally 4 circulations.

Preferably, the annealing temperature in the reaction process of real-time fluorescence PCR is 55 DEG C.

Beneficial effect of the present invention: primer of the present invention, probe are according to the design of Transgenic corn lines flanking sequence, and detection specificity is good, and for detecting real-time fluorescence PCR, susceptibility is high.Detection method accuracy is high, is quick on the draw, can rapid detection sample whether containing turning mCry1Ac gene pest-resistant corn strain Bt-799, for Transgene-safty provides guarantee.

Accompanying drawing explanation

Figure 1B t-799 real-time PCR detection result.

Embodiment

Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.

The extraction of embodiment 1 sample total DNA

1. sample thief blade is about 0.1g in mortar, adds liquid nitrogen and is ground to rapidly Powdered.

2. blade powder is transferred to rapidly in the 2ml centrifuge tube adding 700 μ lCTAB damping fluids in advance, gently after mixing, 65 DEG C of water bath heat preservations 30 minutes.Carefully mixing is rocked every ten minutes.

3. take out centrifuge tube, after being chilled to room temperature (25 DEG C), add equal-volume phenol (350 μ l)/chloroform (350 μ l) 700 μ l.Turn upside down and fully mix, extracting 5 minutes.

4., under room temperature, centrifugal 10 minutes of 12000rpm, transfers to supernatant in another new centrifuge tube with the 1ml rifle head cutting off head.Add 2 μ lRNaseA(10mg/ml).

5. add the chloroform 700 μ l isopyknic with supernatant, fully mixing of turning upside down, extracting 5 minutes.

6., under room temperature, centrifugal 10 minutes of 12000rpm, draws supernatant in another new centrifuge tube.

7. add isopyknic Virahol (700 μ l), fully mix, normal temperature places visible precipitate after 10 minutes.For precipitating completely, can place 1 ~ 2 hour at-20 DEG C.

8., under room temperature, centrifugal 10 minutes of 12000rpm, sinks and is deposited at the bottom of pipe.Abandon most supernatant liquor.

9. add 700 μ l70% ethanol purge 30 minutes.

10. remove the ethanol in centrifuge tube, DNA is deposited in pipe and naturally dries.

11. add appropriate TE(50 μ l) dissolve, put into-20 DEG C of Refrigerator stores for subsequent use.

Embodiment 2 primed probe designs

Turn on the basis of mCry1Ac gene pest-resistant corn strain Bt-799 complete sequence at acquisition foreign gene, design a pair transgenic corns Bt-799 foreign gene Bt-799 and detect PCR primer and TaqMan probe.Design software Primer5.0.Primer sequence is:

Bt-799-F(forward): 5 '-GGAACAAACGATGATTCA-3 ';

Bt-799-R(is reverse): 5 '-CTTCCCTTTTAGTTGTGTC-3 ';

Bt-799-P(probe): 5 '-FAM-AGCCTCTCATTTTCTTCTTTGAGCT-Eclipse-3 '.

Probe 5 ' end reporter fluorescence group FAM used marks, 3 ' end quenching fluorescence group Eclipse mark.

The foundation of embodiment 3 real-time fluorescent PCR amplification method

1. real-time fluorescence PCR reaction system

Be that template carries out real-time fluorescence PCR reaction with sample total DNA, the real-time fluorescence PCR reaction system after optimization is 25 μ l, and reaction conditions is Mix12.5 μ l, primer final concentration 0.2 μm of ol/L, and probe final concentration is 0.1 μm of ol/L.

2. real-time fluorescence PCR reaction parameter

Real-time fluorescence PCR reaction parameter is 95 DEG C of 30sec; 95 DEG C of 5sec, 60 DEG C of 30sec, totally 4 circulations.Reaction terminates rear according to amplification curve result of determination.

The specific detection of embodiment 4 turns of mCry1Ac gene pest-resistant corn strain Bt-799

To turn mCry1Ac gene pest-resistant corn strain Bt-799(GMO+), non-transgenic corn Zheng 58(GMO-), transgenic Fructus Lycopersici esculenti (China kind No. 1), transgenic paddy rice Bt63, transgene cotton (turning Bt anti insect gene) and GM Maize mon810, Mon88017, nk603, rape RT73, T45, soybean GST40-3-2 STb gene for template, carry out real-time fluorescence PCR reaction, detect the specificity of primed probe.

As shown in Figure 1, below baseline be negative control: Bt63, No. KF6, China kind No. 1, Mon810, Mon88017, nk603, rape RT73, T45, soybean GST40-3-2, detect the primed probe turning mCry1Ac gene pest-resistant corn strain Bt-799 and only have signal at strain Bt-799, equal no signal in other material.

Although above with general explanation, embodiment and test, the present invention is described in detail, and on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.

Claims (1)

1. one kind for detecting the method turning mCry1Ac gene pest-resistant corn strain, it is characterized in that, be template with sample total DNA, utilizes primer and probe to carry out real-time fluorescent PCR amplification, each loop ends image data, reaction terminates rear according to amplification curve result of determination;

Its forward primer nucleotides sequence is classified as: Bt-799-F:5 '-GGAACAAACGATGATTCA-3 ' (SEQIDNO.1);

Its reverse primer nucleotides sequence is classified as: Bt-799-R:5 '-CTTCCCTTTTAGTTGTGTC-3 ' (SEQIDNO.2);

Its probe nucleotide sequence is: Bt-799-P:5 '-AGCCTCTCATTTTCTTCTTTGAGCT-3 ' (SEQIDNO.3) or its complementary strand, and reporter fluorescence group is connected to 5 ' end of probe, and quenching fluorescence group is connected to 3 ' end of probe;

Wherein, described reporter fluorescence group is Fam, Hex, Tet, Joe, Vic, Fite, Cy3 or Cy5; Described quenching fluorescence group is Tamra, Rox, Dabcy, Bhq1 or Bhq2;

Wherein, PCR reaction parameter is 95 DEG C of 30sec; 95 DEG C of 5sec, 60 DEG C of 30sec, totally 4 circulations.