CN103882016B

CN103882016B - Primer and its purposes and the method for detection wheat vernalization gene promoter region insertion variation

Info

Publication number: CN103882016B
Application number: CN201410026128.5A
Authority: CN
Inventors: 王翔; 尹钧; 郭总总; 尹飞
Original assignee: Henan Agricultural University
Current assignee: Henan Agricultural University
Priority date: 2014-01-14
Filing date: 2014-01-14
Publication date: 2018-04-24
Anticipated expiration: 2034-01-14
Also published as: CN103882016A

Abstract

The invention discloses a kind of primer, it includes sense primer VRN D1cF and anti-sense primer VRN D1cR, and primer pair wheat VRN D1 promoter sequences provided by the invention have very strong specificity, can distinguish wheat A B chromosome group interference.Easy to operate, the quick, high specificity and testing cost being detected using primer provided by the invention are relatively low, and available for the MITE transposons insertion of detection VRN D1 promoter regions, the precise Identification for wheat Growth habit provides scientific basis.

Description

What primer and its purposes and the promoter region insertion of detection wheat vernalization gene made a variation Method

Technical field

The present invention relates to field of biological detection, and in particular to a kind of primer and its purposes and detection wheat vernalization gene open The method of mover area insertion variation.

Background technology

Wheat vernalization developmental characteristic is to determine wheat planting zoning, introduce a fine variety the principal element for using kind, cultivation technique etc..Wheat Vernalization developmental characteristic is mainly what is regulated and controled by vernalization gene VRN1.The composition of vernalization gene not iso-allele determines wheat product Adaptability of the kind in different ecological environment all over the world.

Wheat vernalization effect key gene VRN-A1, VRN-B1, VRN-D1 are respectively positioned at chromosome of wheat 5A, 5B, 5D On long-armed.Additive effect, Ren Heyi are had no between VRN1 sites different dominant allele VRN-A1, VRN-B1, VRN-D1 A dominance at locus allele can determine that the kind is spring habit.From effect analysis VRN-A1, VRN-B1, VRN-D1 effect according to Just energy normal mature, VRN-D1 need part vernalization completely without vernalization for secondary reduction, the i.e. wheat breed comprising VRN-A1, VRN-B1 then falls between vernalization demand.Correlative study finds that the VRN1 of one grained wheat encodes a MADS-box base Cause, is the ortholog of arabidopsis AP1 (apetalal), it is expressed as the vernalization time is stepped up.In common wheat In, VRN-A1 is respectively Vrn-Ala, Vrn-Alb, Vrn-Alc and vrn-Al there are 4 kinds of locis, wherein Vrn-Ala, Vrn-Alb, Vrn-Alc performance are dominant, are spring varieties, vrn-Al is recessive, Performance of cultivar winter habit.VRN-B1 is due to first Caused by the insertion mutation of introne, and corresponding wild type is vrn-B1.There are 4 kinds of allelic variations on D1 sites, wherein, three Kind dominant mutation, when VRN-Dla (First Intron has large fragment deletion), second, (First Intron lacks and point by VRN-Dlb Mutation exists at the same time), the 3rd is our newfound VRN-Dlc (there are the insertion of class MITE transposons for promoter region), in addition one Kind is recessive genotype vrn-D1.

The content of the invention

Present invention aims at provide a kind of primer.

Specifically scheme is：

A kind of primer, including sense primer VRN-D1cF and anti-sense primer VRN-D1eR, its nucleotide sequence are specifically as follows：

Sense primer VRN-D1cF：5′-CGACCCGGGCGGCACGAGTG-3′；

Anti-sense primer VRN-D1cR：5′-GCGGGACGAAAGAGGAAATGC-3′.

The primer is used to detect whether wheat vernalization gene VRN-D1 promoter region MITE transposons is inserted into variation, is identified Whether wheat shows semi-winterness feature.

Using the method for primer detection wheat vernalization gene promoter region insertion variation, include the following steps：

S1：Extract the genomic DNA of wheat leaf blade or tissue；

S2：PCR amplification is carried out as template with above-mentioned primer using the DNA extracted in S1, recycling pcr amplification product is stand-by；

S3：Take the pcr amplification product in S2 to be detected with agarose gel electrophoresis, after Gelred is dyed, use gel imaging Network analysis result：If have at 225bp band for recessiveness, band is produced at 399bp, then wheat vernalization gene VRN-D1 Promoter region MITE transposons insertion mutations, are VRN-Dlc types.

PCR amplification condition is in step S2：PCR amplification uses 20 μ L reaction systems, contains 2 μ L10 × buffering in reaction system Liquid, 1.5 μ L (25mmol.L^-1)MgCl₂, 0.2 μ L (5U) rTaqDNA polymerases, 1.6 μ L (2.5 μm of ol L^-1) dNTP, every is drawn 1 μ l of thing (10 μm of ol L^-1), 1 μ L of DNA profiling；

PCR amplification program：94 DEG C of pre-degeneration 5min；94 DEG C of denaturation 30s, 65 DEG C of annealing 30s, 72 DEG C of extension 40s, 35 are followed Ring；72 DEG C extend 10min eventually.

The concentration of agarose gel is 1.5% in step S3, and buffer system is 1 × TAE solution, 5V/cm electrophoresis 30min。

Primer provided by the invention has the effect that：

1：Primer pair wheat VRN-D1 promoter sequences provided by the invention have very strong specificity, can distinguish wheat The interference of A \ B chromosome group.

2：Easy to operate, the quick, high specificity that is detected using primer provided by the invention and testing cost compared with It is low, available for detection VRN-D1 promoter regions MITE transposons insertion, for wheat Growth habit precise Identification provide science according to According to.

3：The foundation of the present invention makes the identification of wheat Growth habit more accurate, and base is provided for the popularization and cultivation of wheat breed Plinth, is conducive to promote genetic research and the breeding work of wheat adaptability.

Analyzed by the allelic variation that national 200 parts of wheat breeds and material have been carried out with vernalization gene, in Yellow River-Huai River region 100 parts of wheat breeds and material be found that the MITE transposons insetion sequences of 4 parts of material VRN-D1 promoter regions.According to insertion The flanking sequence in site devises the primer that energy specificity differentiates promoter region insertion mutation, establishes the Molecular Detection of the present invention Primer and system.

Brief description of the drawings

Fig. 1 is VRN-D1 partial promoters and its MITE transposon sequences of insertion；

Fig. 2 is the insertion mutation of the VRN-D1 promoter regions of the part kind of detection.

Wherein, 1：marker；2：Safety 6；3：All wheats 23；4：In educate 12；5：Jining 9903；6：Yanzhan4110；7：Henan Wheat 56；8：Hundred agricultures 3217；9：Place 042；10：Flower training 6；11：Henan agriculture 202；12：Place 553.

Embodiment

In order to which objects and advantages of the present invention are more clearly understood, the present invention is carried out with reference to embodiments further Describe in detail.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to limit this hair It is bright.

Test method used in embodiment is conventional method unless otherwise specified；Used material, reagent Deng unless otherwise specified, being bought by commercial sources from biological reagent company.

The wheat breed and material being related to include：Safety 6, all wheats 23, in educate 12, Jining 9903, Yanzhan4110, Henan wheat 56th, hundred agricultures 3217, place 042, flower training 6, Henan agriculture 202, place 553.

Used primer is：

Sense primer VRN-D1cF：5′-CGACCCGGGCGGCACGAGTG-3′；

Anti-sense primer VRN-D1cR：5′-GCGGGACGAAAGAGGAAATGC-3′.

1 wheat vernalization site VRN-D1 promoter region MITE transposons insertion mutation detection of embodiment

Above-mentioned primer is used to detect wheat vernalization site VRN-D1 promoter region insertion mutation detection methods, including following step Suddenly：

Step 1: the preparation of Wheat volatiles DNA

STb gene is carried out using the wheat breed of CTAB methods 11 parts of known types to more than to extract, concrete operations are：

a：0.25g blades are taken, liquid nitrogen grinding is added into powder (not making material melts) in the centrifuge tube of 2ml；

b：Add 700ul2X CTAB buffer fully to mix, during which shake 3-5 times, 65 DEG C of water-bath 1hr；

2X CTAB buffer：

c：It is cooled to room temperature, adds 700ul chloroforms/isoamyl alcohol (24：1), rotation mixes that (or to be placed in rotation mixed to emulsion Clutch), 15-60min is handled at room temperature；

d：Emulsion is poured into centrifuge tube, 10000rpm centrifuges 15min at room temperature；

e：Supernatant is transferred to clean centrifuge tube, adds 2/3 volume isopropanol, turns upside down for several times, ticks DNA, is put into another One clean 1.5ml centrifuge tubes；

f：70% alcohol washes DNA, is repeated 2 times；Alcohol is poured out, is air-dried 1hr；

g：After adding 500 μ l TE dissolving DNAs, add 5 μ l RNase A (10mg/ml), 4 DEG C of conditions overnight or 37 ℃1hr。

Step 2: PCR amplification

PCR amplification uses the reaction system of 20ul：Specifically include 2 μ L10 × buffer solution, 1.5 μ L (25mmolL^-1)MgCl₂、 0.2 μ L (5U) rTaqDNA polymerases, 1.6 μ L (2.5 μm of ol L^-1) dNTP, every 1 μ L of primer (10 μm of ol L^-1), DNA profiling 1 μL.PCR amplification program is：PCR reaction conditions are：94 DEG C of pre-degeneration 5min；94 DEG C of denaturation 30s, 65 DEG C of annealing 30s, 72 DEG C are prolonged Stretch 40s, 35 circulations；72 DEG C extend 10min eventually.

Step 3: electrophoresis detection

The above-mentioned PCR products of 8ul are taken, are separated with the agarose gel electrophoresis that concentration is 1.5%, buffer system is molten for 1 × TAE Liquid, 5V/cm electrophoresis 30min, after Gelred is dyed, is analyzed with gel imaging system.Testing result is shown in attached drawing 1,2 and table Shown in 1.

Table 1 is the result using 11 wheat breeds of primer detection provided by the invention

The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art For member, without departing from the principle of the present invention, some improvements and modifications can also be made, these improvements and modifications also should It is considered as protection scope of the present invention.

Claims

1. whether a kind of primer is inserted into variation in detection wheat vernalization gene VRN-D1 promoter region MITE transposons, so as to identify Purposes in wheat Growth habit feature, it is characterised in that the nucleotide sequence of the primer is specifically as follows：

Sense primer VRN-D1cF：5′-CGACCCGGGCGGCACGAGTG-3′；

Anti-sense primer VRN-D1cR：5′-GCGGGACGAAAGAGGAAATGC-3′；

The identification includes the following steps：

S1：Extract the genomic DNA of wheat leaf blade or tissue；

S3：Take the pcr amplification product in S2 to be detected with agarose gel electrophoresis, after Gelred is dyed, use gel imaging system Analysis result：Have at 225bp band for recessiveness；The wheat vernalization gene VRN-D1 promoters of band are produced at 399bp Area MITE transposons insertion mutations, are VRN-D1c types；

PCR amplification condition is in step S2：PCR amplification uses 20 μ L reaction systems, in reaction system containing 2 μ L10 × buffer solution, 1.5μL 25mmol.L^-1MgCl₂, 0.2 μ L5U rTaqDNA polymerases, 1.6 μ L, 2.5 μm of ol L^-1DNTP, every 1 μ l of primer 10μmol L^-1, 1 μ L of DNA profiling；

PCR amplification program：94 DEG C of pre-degeneration 5min；94 DEG C of denaturation 30s, 65 DEG C of annealing 30s, 72 DEG C of extension 40s, 35 circulate； 72 DEG C extend 10min eventually；

The concentration of agarose gel is 1.5% in step S3, and buffer system is 1 × TAE solution, 5V/cm electrophoresis 30min.