CN101565751B - Development of wide stress protein family gene specific molecular marker and application thereof - Google Patents

Abstract

The invention is based on partial DNA sequences of USP genes of Steptoe and Morex, which are two varieties of hordeum vulgare. DNA MAN 5.11 software is used for comparing the gene sequences of the twomaterials to find base mutation, insertion and deletion domains; the different domains are used for designing a primer so as to cause only one of Steptoe and Morex to be capable of amplifying a targe t strip; PCR amplification is utilized to detect the condition of the target strip amplified by a DH population material to be detected and data is sorted out according to the requirement of Mapmaker 3.0 software. The Mapmaker 3.0 software is used for positioning the USP genes on a chromosome; a hordeum vulgare DH population resistance QTL map and a real-time fluorescent quantization PCR technology are utilized to analyze the relation between the USP genes and resistance; and the genes are led into crops by a transgenic technology or a chromosome engineering technology, thus effectively enhancing the external pressure resisting capability of living beings and remarkably improving the resisting capability of the crops.

Description

A kind of exploitation of wide stress protein family gene specific molecular marker and application

Technical field

The invention belongs to technical field of biological genetic engineering, specially refer to a kind of exploitation and application that utilizes the wide stress protein family gene specific molecular marker.

Background technology

(Universal Stress Protein, USP) gene family is the bacterial protein of one type of minicell matter to wide stress protein.The USP gene is identified at first in intestinal bacteria, in archeobacteria and bacterium, extensively exists.When cell under most pressure condition (nutritive deficiency, heat, osmotic stress and ultraviolet damage etc.), the expression of USP just strengthens thereupon.In the stronger individuality of resistance capacity, the height that its expression amount is more individual than susceptibility.Significant effect is also arranged in dormant cell.Mainly make tryptophane and serine/threonine protein phosphorylation through tyrosine phosphorylated proteins, the pair cell stage of stopping growing plays important effect.But the accurate role that it was played the part of in this stage does not know as yet.According to whether combining, be divided into two types to the USP of bacterium with ATP.One type is to contain the MJ0577 albumen that ATP combines the territory methanococcus., another kind of is not contain protoheme influenza virus and the colibacillary UspA albumen that ATP combines the territory.In plant, only isolate a spot of homologous protein.

According to existing structural analysis, there is homodimer in USP, and genetics research shows that its effect in cell is widely, comprises and resistance, carbon metabolism, cell adhesion, function that motion is relevant with bacterial virulence.Utilize the model protein of intestinal bacteria USP, confirmed the existence of mutual transformation of homodimer and the heterodimer of USP, this possibly be a kind of explanation to the effect of USP expanded functionality.The discovery of USP and research mainly are present in bacterium and the fungi, and the correlative study in plant is seldom only isolated the USP homologous protein of seldom counting at present in wheat, corn, barley, Arabidopis thaliana and plant of Solanaceae.Research in eukaryote focuses mostly in aspect protein and the RNA, mainly is its amino acid is formed, coerced and express abundance and coerce the research of expressing aspects such as position.And to the exploitation of Auele Specific Primer, the report of aspects such as chromosomal localization is very few, and special this type of research aspect farm crop remains in very big shortcoming.

Summary of the invention

The object of the present invention is to provide the exploitation and the application thereof of a kind of wide stress protein (USP) family gene specific molecular marker.Wide stress protein family gene specific molecular marker through the present invention's exploitation; Can use Mapmaker3.0 software the USP assignment of genes gene mapping to karyomit(e), utilize getting in touch between barley DH population resistance QTL collection of illustrative plates and real-time fluorescence quantitative PCR technical Analysis USP gene and resistance.Simultaneously, with in this gene transferred crop, can effectively strengthen the ability of biological opposing ambient pressure itself, the resistivity of crop self is significantly strengthened through transgenic technology or chromosome engineering technology.

To achieve these goals, the technical scheme of the present invention's employing is following:

The present invention is the basis with the partial dna sequence of the USP gene of barley variety Steptoe and Morex; Seek the zone of base mutation, insertion and disappearance through the gene order of these two kinds of materials of DNAMAN 5.11 softwares comparison; Utilize these difference zone design primers; Making in these two parts of materials of Steptoe and Morex has only a material can amplify target stripe; Detect the situation (having/do not have) of the amplification target stripe of detected materials through pcr amplification, then according to the disposal data that requires of Mapmaker3.0 software.Through the Mapmaker3.0 processing data, selecting LOD value is 3.0, with the data that obtain again with the processing of winQTLCart2.0 drawing tool, in conjunction with existing mark spectrum data, with the USP assignment of genes gene mapping on karyomit(e).

Particular content of the present invention is:

1, gene order

Adopt the inventor to clone the nucleotide sequence of these 5 wide stress protein genes of hv.3739, hv.7364, hv.23267, hv.11351 and hv.11741 of barley variety Steptoe and the Morex of acquisition; Utilize software DNAMAN 5.11 comparison nucleotide sequences, obtain sequence alignment figure.The nucleotide sequence of this wide stress protein gene is specially:

The hv.3739 nucleotide sequence of Steptoe is shown in SEQ ID No.1;

The hv.3739 nucleotide sequence of Morex is shown in SEQ ID No.2.

The hv.7364 nucleotide sequence of Steptoe is shown in SEQ ID No.3;

The hv.7364 nucleotide sequence of Morex is shown in SEQ ID No.4.

The hv.23267 nucleotide sequence of Steptoe is shown in SEQ ID No.5;

The hv.23267 nucleotide sequence of Morex is shown in SEQ ID No.6.

The hv.11351 nucleotide sequence of Steptoe is shown in SEQ ID No.7;

The hv.11351 nucleotide sequence of Morex is shown in SEQ ID No.8.

The hv.11741 nucleotide sequence of Steptoe is shown in SEQ ID No.9;

The hv.11741 nucleotide sequence of Morex is shown in SEQ ID No.10.

Utilize software DNAMAN 5.11 comparison nucleotide sequences, obtain sequence alignment figure.

2, extract genomic dna

Adopt the SDS method to extract barley variety Morex and Steptoe, with and the genomic dna of DH colony.

3, designs specificity amplification difference primer

The dna sequence dna of the hv.3739 gene of employing Morex is compared with the sequence of Steptoe and has been lacked the GAGGGA base sequence; And inserted the GCAGCAGCGCCGATTTCG base sequence; The utilization insertion sequence has designed downstream primer 5 '-CCATATGGCGAACCGAAATC-3 ', designs a upstream primer 5 '-GTCCTTAATTGTTCCTGCGTC-3 ' and its pairing at random.

The present invention also can adopt the dna sequence dna of the hv.7364 gene of Morex to compare with the sequence of Steptoe and insert the GGCCGGGAA base sequence; Same this section of utilization insertion sequence has designed upstream primer 5 '-CTCTGGCAGACATGGCAGC-3 ', designs downstream primer 5 '-AGAGATAACTGAGGAAAACACACTG-3 ' and its pairing at random.

The present invention also can adopt the dna sequence dna of the hv.11351 gene of Morex to compare with the sequence of Steptoe and lack the CCCTGAAT base sequence, and another place's diversity sequence is: the sequence of Morex is ACTACGAG, and the sequence of Steptoe is AGTAGTAGTAGCAC.Utilization diversity sequence zone downstream primer 5 '-GCTCGTAGTCGATCCATCAAC-3 ' designs a upstream primer 5 '-TTAAAGCCAAAGCAAGCCGT-3 ' and its pairing at random.

The present invention also can adopt the dna sequence dna of the hv.11741 gene of Morex to compare with the sequence of Steptoe, and the base that wherein has a place that transversion: Morex takes place is C, and the base of Steptoe is G.The characteristics of utilization sequence base generation transversion have designed upstream primer 5 '-AGAAGGGAGGAGGAGGTGATG-3 ', design downstream primer 5 '-GCTTTTATAGAAACACGGTCG-3 ' and its pairing at random.Wherein 3 ' end bit base third from the bottom is provided with a base mismatch in the upstream primer, makes the mispairing rate of dna profiling of itself and Morex higher.

The present invention also can adopt the dna sequence dna of the hv.23267 gene of Morex to compare with the sequence of Steptoe and lack the AACT base sequence; Utilization difference sequences Design upstream primer 5 '-CTTAACCAACTAACCAAGGAGAT-3 ', design downstream primer 5 '-CATGAGTACAGTAGATGGTTGGG-3 ' and its pairing at random.

4, pcr amplification and electrophoresis detection:

DNA with Steptoe and Morex is a template, and applying step 3 designed primer are carried out pcr amplification and detected.

The pcr amplification reaction system is following: (20 μ l): 1 * PCR damping fluid

10 * PCR damping fluid, 2.0 μ l

dNTPs 100μM

Taq polysaccharase 1U

Primer 10pmol

Dna profiling 30-50ng

Add water to 20 μ l

The pcr amplification program is:

1) 94 ℃ of preparatory sex change are 5 minutes

2) 94 ℃ of sex change are 45 seconds

3) annealed 35 seconds for 58 ℃-62 ℃

4) 72 ℃ were extended 1 minute

5) 72 ℃ were extended 7 minutes

Circulation step is 2), 3), 4), 35 circulations

Electrophoresis detection: 1.5% agarose gel electrophoresis detects, 130V, 30 minutes.

5, the screening of specific amplification difference primer

Expanding fragment length (hv.3739:689bp according to the gene order information calculations; Hv.7364:1013bp; Hv.11351:365bp; Hv.11741:187bp hv.23267:271bp) requires (these 3 gene PCRs amplification Morex of hv.3739, hv.7364 and hv.11351 have target stripe and Steptoe does not have, and these 2 gene Steptoe of hv.11741 and hv.23267 have target stripe Morex not have) to judge the exactness of the electrophorogram detection primer of pcr amplification with the purpose of design of primers.Screen the primer that occurs amplified fragments difference in these two parent materials then, the required Auele Specific Primer that is of this species diversity occurs.Through detecting, the described 5 pairs of primers of step 3 all reach the screening requirement.Be specially:

1. adopt the upstream and downstream primer of hv.3739 gene design, amplification Morex fragment length is 689bp, and Steptoe does not amplify target fragment, and its marker number is designated as BUG1.

2. adopt the upstream and downstream primer of hv.7364 gene design, the fragment length of amplification Morex is 1013bp, and Steptoe amplification place purpose fragment not, its marker number is BUG2.

3. adopt the upstream and downstream primer of hv.11351 gene design, amplification Morex fragment length is 365bp, and Steptoe does not amplify target fragment, and its marker number is designated as BUG3.

4. adopt the upstream and downstream primer of hv.11741 gene design, amplification Steptoe fragment length is 187bp, and Morex does not amplify target fragment, and its marker number is designated as BUG4.

5. adopt the upstream and downstream primer of hv.23267 gene design, amplification Steptoe fragment length is 271bp, and Morex does not amplify target fragment, and its marker number is designated as BUG5.

6, the acquisition of genetic mapping data

5 pairs of specificity difference amplimers according to obtaining carry out pcr amplification according to amplification system of primer PCR separately in the screening process and pcr amplification program respectively, and are consistent with step 4.The note that the situation of amplified band is consistent with Steptoe is made " A ", and the note consistent with Morex made " B ", and the missing data note is done "-".

7, construction of genetic atlas

Put the amplification data of the DH colony that obtains in order according to the data processing form of software Mapmaker3.0, in conjunction with existing mark spectrum data, setting the LOD value is 3.0 analytical data, with software winQTLCart2.0 it is mapped then, obtains genetic map.

Described 5 the specific amplification difference primers of the application of the invention; DNA with the DH population material of 150 parts of Steptoe * Morex is a template respectively; Detect the situation (having/do not have) of the amplification target stripe of its every part material through pcr amplification, then according to the disposal data that requires of Mapmaker3.0 software.In conjunction with existing mark spectrum data, through the Mapmaker3.0 software processes, selecting the LOD value is 3.0, and the data that obtain are handled with the winQTLCart2.0 drawing tool again, and genetic map draws.

Beneficial effect of the present invention is:

Through molecule marker according to the invention with the wide stress protein assignment of genes gene mapping on the karyomit(e) of barley DH colony, in conjunction with the relevant QTL location collection of illustrative plates of barley resistance, can effectively judge the contact of this gene and anti-coercive (salt, arid, hot and cold etc.).Through salt tolerant coercive experiment proof; This gene distribution is in the main effect QTL zone that salt tolerant is coerced; Can judge tentatively that this gene is relevant with salt resistance, use salt tolerance material and salt sensitive material then, behind salt stress; Divide time point (0 hour, 1 hour, 3 hours, 8 hours, 24 hours, 72 hours) to extract the RNA of these two kinds of materials respectively; In two parts of resistance difference materials, have rna expression difference through this gene of fluorescence quantitative PCR detection, and this expression of gene amount is much larger than the salt sensitive material in the salt tolerance material, this gene length time high abundance is expressed simultaneously.Through transgenic technology, forward this gene to quality better, in the material of salt stress-resistant property difference; The result shows; The salt susceptibility of wheat is brought up to 150-200mmol/l from 50-150mmol/l, and quality and output kept the wheat lines of good proterties basically, accelerates salt tolerant breeding of wheat speed.

Therefore through the specific marker of exploitation, utilize Mapmaker software the USP assignment of genes gene mapping to karyomit(e), it and resistance QTL are connected, can analyze getting in touch between USP gene and resistance.The overexpression of USP gene can strengthen the ability of biological opposing ambient pressure own, and imports in the crop through the method for transgenic technology or chromosome engineering, makes it strengthen the resistivity of self, and this has been well-known technology.

Description of drawings

Fig. 1: the partial sequence comparison figure of hv.3739 gene M orex and Steptoe, M representes Morex, S representes Steptoe.With "-" expression design of primers reference area, F representes upstream primer among the figure, and R representes downstream primer.

Fig. 2: the partial sequence comparison figure of hv.7364 gene M orex and Steptoe, M representes Morex, S representes Steptoe.With "-" expression design of primers reference area, F representes upstream primer among the figure, and R representes downstream primer.

Fig. 3: the partial sequence comparison figure of hv.11351 gene M orex and Steptoe, M representes Morex, S representes Steptoe.With "-" expression design of primers reference area, F representes upstream primer among the figure, and R representes downstream primer.

Fig. 4: the partial sequence comparison figure of hv11741 gene M orex and Steptoe, M representes Morex, S representes Steptoe.With "-" expression design of primers reference area, F representes upstream primer among the figure, and R representes downstream primer.

Fig. 5: the partial sequence comparison figure of hv.23267 gene M orex and Steptoe, M representes Morex, S representes Steptoe.With "-" expression design of primers reference area, F representes upstream primer among the figure, and R representes downstream primer.

Fig. 6: Auele Specific Primer electrophorogram.The every couple of primer Morex and Steptoe are spaced successively, middle additional Maker I and Maker II.Order is successively: 1-hv.7364,2-Maker II, 3-Maker I, 4-hv.23267,5-hv.11351,6-hv.3739,7-Maker II, 8-hv.11741.

Mark with " _ " below the genetic map of Fig. 7: BUG1, BUG2, BUG3, BUG4 and BUG5, its mark.Be respectively 1-karyomit(e) 1H, 2-karyomit(e) 2H, 3-karyomit(e) 3H and 4-karyomit(e) 6H.

Embodiment

Below in conjunction with embodiment the present invention is made further detailed description, but is not limitation of the present invention, all any this areas of doing according to the disclosure of invention be equal to replacement, all belong to protection scope of the present invention.

Embodiment 1:

1, gene order:

The nucleotide sequence of hv.3739 wide stress protein gene that at first utilizes the inventor to clone barley variety Steptoe and the Morex of acquisition, through software DNAMAN 5.11 than nucleotide sequence result shown in accompanying drawing 1, obtain the sequence comparison diagram.

2, extract genomic dna:

Adopt the SDS method to extract barley variety Morex and Steptoe, with and the genomic dna of DH colony.The steps include:

(1) the fresh young leaflet tablet that takes a morsel, liquid nitrogen grinding become the centrifuge tube of putting into 2.0ml behind the fine powder, add the SDS extracting solution (100mMTris-Cl PH5.8,100mMNaCl, 2%SDS) the 1ml mixing that are preheated to 65 ℃;

(2) 65 ℃ of water-baths 50 minutes, jog mixing therebetween.Add isopyknic chloroform after being cooled to room temperature: primary isoamyl alcohol (24: 1), mixing to supernatant is the milk shape gently, centrifugal 10 minutes of 4000rpm.

(3) get supernatant, add the chloroform of 600 μ l, mixing was placed 10 minutes gently, centrifugal 10 minutes of 4000rpm

(4) Virahol of 2/3 volume such as add, 1 hour deposit D NA of ice bath;

(5) tick DNA, wash 2 times with 70% alcohol, absolute ethyl alcohol is washed and is once dried up DNA, is dissolved in 1 * TE solution of an amount of pH8.0.Add the RNA enzyme to final concentration 100 μ g/ μ l;

(6) under the 100V constant voltage, 1% agarose gel electrophoresis 30 minutes detects DNA concentration and quality.

3, according to the difference partial design primer of known correlated series:

Through the Morex of DNAMAN5.11 software comparison hv.3739 gene and the partial dna sequence of Steptoe; The insertion base sequence GCAGCAGCGCCGATTTCG zone that uses the dna sequence dna of the hv.3739 gene of Morex to Duo than the sequence of Steptoe then, the design primer:

F：5’-GTCCTTAATTGTTCCTGCGTC-3’；

R：5’-CCATATGGCGAACCGAAATC-3’。

4, pcr amplification:

DNA with Steptoe and Morex is a template, and applying step 3 designed primer are carried out pcr amplification and detected.

The pcr amplification reaction system is following: (20 μ l): 1 * PCR damping fluid

10 * PCR damping fluid, 2.0 μ l

dNTPs 100μM

Taq polysaccharase 1U

Primer 10pmol

Dna profiling 30-50ng

Add water to 20 μ l.

The pcr amplification program is:

1) 94 ℃ of preparatory sex change are 5 minutes

2) 94 ℃ of sex change are 45 seconds

3) annealed 35 seconds for 58 ℃

4) 72 ℃ were extended 1 minute

5) 72 ℃ were extended 7 minutes

Circulation step is 2), 3), 4), 35 circulations.

Electrophoresis detection: 1.5% agarose gel electrophoresis detects, 130V, 30 minutes.

5, the screening of specific amplification difference primer:

The purpose that goes out expanding fragment length (hv.3739:689bp) and design of primers according to the gene order information calculations requires (hv.3739 gene PCR amplification Morex target stripe is arranged and Steptoe do not have) to judge that the electrophorogram of pcr amplification detects the exactness of primer.Screen the primer that occurs amplified fragments difference in these two parent materials then, the required Auele Specific Primer that is of this species diversity occurs.Through detecting, the primer that step 3 is described reaches the screening requirement.

6, the acquisition of genetic mapping data:

Specificity difference amplimer according to obtaining carries out pcr amplification according to primer PCR amplification system in the screening process and pcr amplification program, and reaction process is consistent with step 4 with condition.The note that the situation of amplified band is consistent with Steptoe is made " A ", and the note consistent with Morex made " B ", and the missing data note is done "-".

7, construction of genetic atlas:

Put the amplification data of the DH colony that obtains in order according to the data processing form of software Mapmaker3.0, in conjunction with existing mark spectrum data, setting the LOD value is 3.0 analytical data, with software winQTLCart2.0 it is mapped then, obtains genetic map.

Embodiment 2:

1, gene order:

At first utilize the inventor to clone the nucleotide sequence of hv.7364 wide stress protein gene of barley variety Steptoe and the Morex of acquisition, shown in accompanying drawing 2, obtain the sequence comparison diagram through software DNAMAN 5.11 comparison nucleotide sequence results.

2, with the 2nd step of embodiment 1.

3, according to the difference partial design primer of known correlated series:

Through the Morex and the partial dna sequence of Steptoe of DNAMAN5.11 software comparison hv.3739 gene, the insertion base sequence GGCCGGGAA that uses the dna sequence dna of the hv.7364 gene of Morex Duo than the sequence of Steptoe then is regional, designs primer:

F：5’-CTCTGGCAGACATGGCAGC-3’；

R：5’-AGAGATAACTGAGGAAAACACACTG-3’。

4, pcr amplification:

DNA with Steptoe and Morex is a template, and applying step 3 designed primer are carried out pcr amplification and detected.

The pcr amplification reaction system is following: (20 μ l): 1 * PCR damping fluid

10 * PCR damping fluid, 2.0 μ l

dNTPs 100μM

Taq polysaccharase 1U

Primer 10pmol

Dna profiling 30-50ng

Add water to 20 μ l.

The pcr amplification program is:

1) 94 ℃ of preparatory sex change are 5 minutes

2) 94 ℃ of sex change are 45 seconds

3) annealed 35 seconds for 60 ℃

4) 72 ℃ were extended 1 minute

5) 72 ℃ were extended 7 minutes

Circulation step is 2), 3), 4), 35 circulations.

Electrophoresis detection: 1.5% agarose gel electrophoresis detects, 130V, 30 minutes.

5, the screening of specific amplification difference primer

The purpose that goes out expanding fragment length (hv.7364:1013bp) and design of primers according to the gene order information calculations requires (hv.7364 gene PCR amplification Morex target stripe is arranged and Steptoe do not have) to judge that the electrophorogram of pcr amplification detects the exactness of primer.Screen the primer that occurs amplified fragments difference in these two parent materials then, the required Auele Specific Primer that is of this species diversity occurs.Through detecting, the primer that step 3 is described reaches the screening requirement.

6, with the 6th step of embodiment 1.

7, with the 7th step of embodiment 1.

Embodiment 3:

1, gene order:

At first utilize the inventor to clone the nucleotide sequence of hv.23267 wide stress protein gene of barley variety Steptoe and the Morex of acquisition, shown in accompanying drawing 3, obtain the sequence comparison diagram through software DNAMAN 5.11 comparison nucleotide sequence results.

2, with the 2nd step of embodiment 1.

3, according to the difference partial design primer of known correlated series:

Through the Morex and the partial dna sequence of Steptoe of DNAMAN5.11 software comparison hv.23267 gene, the dna sequence dna of hv.23267 gene that uses Morex then designs primer than the base sequence AACT zone of the sequence deletion of Steptoe:

F：5’-CTTAACCAACTAACCAAGGAGAT-3’；

R：5’-CATGAGTACAGTAGATGGTTGGG-3’。

4, pcr amplification:

DNA with Steptoe and Morex is a template, and applying step 3 designed primer are carried out pcr amplification and detected.

The pcr amplification reaction system is following: (20 μ 1): 1 * PCR damping fluid

10 * PCR damping fluid, 2.0 μ l

dNTPs 100μM

Taq polysaccharase 1U

Primer 10pmol

Dna profiling 30-50ng

Add water to 20 μ l.

The pcr amplification program is:

1) 94 ℃ of preparatory sex change are 5 minutes

2) 94 ℃ of sex change are 45 seconds

3) annealed 35 seconds for 61 ℃

4) 72 ℃ were extended 1 minute

5) 72 ℃ were extended 7 minutes

Circulation step is 2), 3), 4), 35 circulations.

Electrophoresis detection: 1.5% agarose gel electrophoresis detects, 130V, 30 minutes.

5, the screening of specific amplification difference primer

The purpose that goes out expanding fragment length (hv.23267:271bp) and design of primers according to the gene order information calculations requires (hv.23267 gene Steptoe target stripe is arranged and Morex do not have) to judge that the electrophorogram of pcr amplification detects the exactness of primer.Screen the primer that occurs amplified fragments difference in these two parent materials then, the required Auele Specific Primer that is of this species diversity occurs.Through detecting, the primer that step 3 is described reaches the screening requirement.

6, with the 6th step of embodiment 1.

7, with the 7th step of embodiment 1.

Embodiment 4:

1, gene order:

At first utilize the inventor to clone the nucleotide sequence of hv.11351 wide stress protein gene of barley variety Steptoe and the Morex of acquisition, shown in accompanying drawing 4, obtain the sequence comparison diagram through software DNAMAN 5.11 comparison nucleotide sequence results.

2, with the 2nd step of embodiment 1.

3, according to the difference partial design primer of known correlated series:

Through the Morex of DNAMAN5.11 software comparison hv.11351 gene and the partial dna sequence of Steptoe; Use the diversity sequence between the sequence of dna sequence dna and Steptoe of hv.11351 gene of Morex then: the sequence of Morex is ACTACGAG, and the sequence of Steptoe is AGTAGTAGTAGCAC.The design primer:

F：5’-TTAAAGCCAAAGCAAGCCGT-3’；

R：5’-GCTCGTAGTCGATCCATCAAC-3’。

4, pcr amplification:

DNA with Steptoe and Morex is a template, and applying step 3 designed primer are carried out pcr amplification and detected.

The pcr amplification reaction system is following: (20 μ l): 1 * PCR damping fluid

10 * PCR damping fluid, 2.0 μ l

dNTPs 100μM

Taq polysaccharase 1U

Primer 10pmol

Dna profiling 30-50ng

Add water to 20 μ l.

The pcr amplification program is:

1) 94 ℃ of preparatory sex change are 5 minutes

2) 94 ℃ of sex change are 45 seconds

3) annealed 35 seconds for 60 ℃

4) 72 ℃ were extended 1 minute

5) 72 ℃ were extended 7 minutes

Circulation step is 2), 3), 4), 35 circulations.

Electrophoresis detection: 1.5% agarose gel electrophoresis detects, 130V, 30 minutes.

5, the screening of specific amplification difference primer

The purpose that goes out expanding fragment length (hv.11351:365bp) and design of primers according to the gene order information calculations requires (hv.11351 gene PCR amplification Morex target stripe is arranged and Steptoe do not have) to judge that the electrophorogram of pcr amplification detects the exactness of primer.Screen the primer that occurs amplified fragments difference in these two parent materials then, the required Auele Specific Primer that is of this species diversity occurs.Through detecting, the primer that step 3 is described reaches the screening requirement.

6, with the 6th step of embodiment 1.

7, with the 7th step of embodiment 1.

Embodiment 5:

1, gene order:

At first utilize the inventor to clone the nucleotide sequence of hv.11741 wide stress protein gene of barley variety Steptoe and the Morex of acquisition, shown in accompanying drawing 5, obtain the sequence comparison diagram through software DNAMAN 5.11 comparison nucleotide sequence results.

2, with the 2nd step of embodiment 1.

3, according to the difference partial design primer of known correlated series:

Through the Morex of DNAMAN5.11 software comparison hv.11741 gene and the partial dna sequence of Steptoe; Use the transversion site, a place between the sequence of dna sequence dna and Steptoe of hv.11741 gene of Morex then: the base of Morex is C and the base of Steptoe is G, the design primer:

F：5’-AGAAGGGAGGAGGAGGTGATG-3’；

R：5’-GCTTTTATAGAAACACGGTCG-3’。

Wherein 3 ' end bit base third from the bottom is provided with a base mismatch in the upstream primer, makes the mispairing rate of dna profiling of itself and Morex higher.

4, pcr amplification:

DNA with Steptoe and Morex is a template, and applying step 3 designed primer are carried out pcr amplification and detected.

The pcr amplification reaction system is following: (20 μ l): 1 * PCR damping fluid

10 * PCR damping fluid, 2.0 μ l

dNTPs 100μM

Taq polysaccharase 1U

Primer 10pmol

Dna profiling 30-50ng

Add water to 20 μ l.

The pcr amplification program is:

1) 94 ℃ of preparatory sex change are 5 minutes

2) 94 ℃ of sex change are 45 seconds

3) annealed 35 seconds for 62 ℃

4) 72 ℃ were extended 1 minute

5) 72 ℃ were extended 7 minutes

Circulation step is 2), 3), 4), 35 circulations.

Electrophoresis detection: 1.5% agarose gel electrophoresis detects, 130V, 30 minutes.

5, the screening of specific amplification difference primer

The purpose that goes out expanding fragment length (hv.11741:187bp) and design of primers according to the gene order information calculations requires (hv.11741 gene Steptoe target stripe is arranged and Morex do not have) to judge that the electrophorogram of pcr amplification detects the exactness of primer.Screen the primer that occurs amplified fragments difference in these two parent materials then, the required Auele Specific Primer that is of this species diversity occurs.Through detecting, the primer that step 3 is described reaches the screening requirement.

6, with the 6th step of embodiment 1.

7, with the 7th step of embodiment 1.

Embodiment 6,

It is that example is introduced application process of the present invention that present embodiment is coerced with salt tolerant.

1, the preliminary screening of gene

Utilize the BUG1, BUG2, BUG3, BUG4 and the BUG5 mark that have been positioned the USP gene on the karyomit(e),, find the USP gene that is distributed in the salt tolerant QTL zone in conjunction with the salt tolerant QTL of barley DH colony collection of illustrative plates.

2, screening material

Plantation is from Israel in the greenhouse, and Iran and Steptoe and Morex be totally 138 parts of barley materials, and when plant grew to 2 leaves, 1 core, the salt stress that adds 250mmol/l was handled, and contrast is set, and passed through the material phenotype after 21 days, and propanedioic acid in the plant body, Na ⁺And K ⁺Ratio, salt tolerant indexs of correlation such as biological yield filter out a salt tolerant material and a salt sensitive material.

3, the further screening of gene

Plant these two parts of materials in the greenhouse; When plant grows to 2 leaves, 1 core; Adding concentration is that the NaCl salts solution of 250mmol/l is handled, and the RNA that divides time point (0 hour, 1 hour, 3 hours, 8 hours, 24 hours, 72 hours) to extract these two kinds of materials respectively is according to the fluorescent quantitation primer of this gene of requirement of experiment design; Analyze the expression amount of these two parts of materials through fluorescent quantitative PCR technique at each this gene RNA of time point; If the expression amount of this gene in the salt tolerant material is far above the salt sensitive material, and long-time high abundance expression, judge that tentatively its gene and salt tolerance are closely related.

4, make up transgene carrier

The design two ends have the Auele Specific Primer of restriction enzyme site, RT-PCR amplification amino acid coding region total length.Be connected to then on the PGEX-4T-1 carrier; Correct clone is building up on the intermediate carrier through restriction enzyme site with order-checking; With the double digestion method gene is downcut from middle carrier, be building up on the transgene carrier, PCR or enzyme are cut the exactness of identifying that transgene carrier makes up.

5, particle bombardment transgenic wheat

1., to get wheat back about the 14 days rataria of pollinating be explant, is seeded in SD ₂Or on the MM substratum, (26 ℃) evoked callus concentrated on the petridish middle section with callus after 7-10 days under the dark condition, through 0.4mol/l high osmotic pressure substratum (SD ₂Substratum adds 0.2mol/l N.F,USP MANNITOL, 0.2mol/l sorbyl alcohol) bombardment of processing 4-6h preparation particle gun.

2., an amount of bronze (1.0 μ m) suspension-s (60 μ g/ rifle), the centrifugal 30s of 14000rpm removes supernatant, adds the 1ml vaal water, centrifugal 14000rpm/5min removes supernatant, adds 220 μ l sterilized waters, 250 μ lCaCl successively ₂(2.5M), 50 μ l spermidines (Spermidine) (0.1M), an amount of DNA (1 μ gDNA/ rifle); With the mixed solution of bronze and DNA at 4 ℃ of vibration 10min; Centrifugal 14000rpm/5min removes supernatant; Add 600 μ l absolute ethyl alcohols and break up deposition, remove ethanol behind the centrifugal 14000rpm/1min, add absolute ethyl alcohol (10 μ l/ rifles add ethanol) and prepare to be used for the particle gun bombardment.

3., adopt particle gun bombardment wheat immature embryo inductive callus, the callus after the bombardment continues on former osmotic pressure substratum, to cultivate 16-18h, changes the SD that does not add selective agent then over to ₂Or in the MM substratum under the dark condition (26 ℃) recover to cultivate for 2 weeks.

4., recover to cultivate for 2 weeks after, changing for the first time on the screening culture medium callus over to (1/2MS+NAA1mg/l+KT 1mg/l+Bialaphos 3mg/l), under 24 ℃ of illumination 10h conditions, screen differentiation culture 3-4 all.

5. by the time callus differentiate after the green bud; Change the green bud of differentiation over to no hormone culture-medium (1/2MS+Bialaphos 3mg/l) upward up to seedling elongation (illumination and temperature are the same); By the time seedling when being elongated to 1-2em, moves into strong seedling culture base (1/2MS+IAA 0.5mg/l) strong sprout of lasting, regeneration plant grows into suitable big or small (root system is better during height of seedling 6-8cm) time shift and goes in the Nursery; 15 ℃ of left and right sides illumination cultivation, treat the strong greenhouse that is placed on of seedling.

6., the detection of transgenic wheat plant

Promotor and the target gene that changes over to are carried out pcr amplification, the double check transfer-gen plant, with detect the copy number of positive plant through Southern blot technical Analysis gene.

7., resistance is identified

Salt concn (200mmol/l) is set the T1 of detected positive plant is carried out salt stress for seed and the plant that do not change target gene over to; T1 is for plant normal growth under salt stress; And the adjoining tree growth that does not change target gene over to is suppressed, and leaf jaundice committee here, explains that this gene can improve the salt tolerance of plant; Breeding T1 seed is cultivated the salt tolerant wheat lines.

SEQUENCE?LISTING

< 110>Sichuan Agricultural University

< 120>a kind of exploitation of wide stress protein family gene specific molecular marker and application

<130>090518

<160>20

<170>PatentIn?version?3.3

<210>1

<211>689

<212>DNA

< 213>barley (Morex)

<400>1

gtccttaatt?gttcctgcgt?cttccaccac?caccaccacc?tccaccacct?gtcaagccga 60

gcgacgagaa?gagaagggaa?gagagaagag?atcgaccatg?gccgcggaag?gcgagaggtg 120

ggtgggcctg?gcggtggact?tctcggaggg?gagccgcgcg?gcgctgcagt?gggcggcgga 180

caacctgctc?cgctccggcg?acaacctgcc?cctcctccac?gtcctcaagg?accccgacta 240

cgagcagggc?gagaccctcc?tctgggaggc?ctccggctcg?cgtgcgtacc?aaccaaactc 300

atcatcctct?ccctccctag?tcccccttct?cttgctactg?taaccgcagc?cagttgcgtg 360

cctgtctgcc?cgcttgctgc?tcttgatctt?agtttcttgt?cgcgcccacc?aatcgccccg 420

acccgtgcta?aaattccgac?ctgtaacagc?cgcgtatgtt?cggcgcaagg?ggtagcagtt 480

gccatggtcc?ctgcgcgtgt?gttcgtgctg?attaccttgc?ttgtcagctg?ccttgttacc 540

gtgcttggtt?ttgattcaat?ttaagagggg?gggggggggg?gcggttcgca?accgatgttc 600

gctcatcctg?ctctgtccat?ggggctttat?taggtgtcgt?ttttaactct?gggagagagc 660

agcagcgccg?atttcggttc?gccatatgg 689

<210>2

<211>677

<212>DNA

< 213>barley (Steptoe)

<400>2

gtccttaatt?gttcctgcgt?cttccaccac?caccaccacc?tccaccacct?gtcaagccga 60

gcgacgagaa?gagaagggaa?gagagaagag?atcgaccatg?gccgcggaag?gcgagaggtg 120

ggtgggcctg?gcggtggact?tctcggaggg?gagccgcgcg?gcgctgcagt?gggcggcgga 180

caacctgctc?cgctccggcg?acaacctgct?cctcctccac?gtcctcaagg?accccgacta 240

cgagcagggc?gagaccctcc?tctgggaggc?ctccggctcg?cgtgcgtacc?aaccaaactc 300

atcatcctct?ccctccctag?tcccccttct?cttgctactg?taaccgcagc?cagttgcgtg 360

cctgtctgcc?cgcttgctgc?tcttgatctt?agtttcttgt?cgcgcccacc?aatcgccccg 420

acccgtgcta?aaattccgac?ctgtaacagc?cgcgtatgtt?cggcgcaagg?ggtagcagtt 480

gccatggtcc?ctgcgcgtgt?gttcgtgctg?attaccttgc?ttgtcagccg?ccttgttacc 540

gtgcttggtt?ttgattcaat?ttaagaggga?gggagggggg?gggggggcgg?ttcgcaaccg 600

atgttcgctc?atcctgctct?gtccatgggg?ctttattagg?tgtcgttttt?aactctggga 660

gagagttcgc?catatgg 677

<210>3

<211>1043

<212>DNA

< 213>barley (Morex)

<400>3

ctctggcaga?catggcggcc?gggaagcgca?ccatcggcct?ggcgatggac?tactcgccgt 60

ccagcaaggc?cgcgacgagg?tgggagatcg?agaacctggt?gaaggccggc?gaccggatca 120

tcctcatcca?cgtcctcccc?aagggagcag?acgccagcca?caaggggctc?tggaagagca 180

ccggttcacg?tatgtgtcaa?gacactgaat?tctctgttct?gttgtgtagc?tccctcgctc 240

ttatctttcc?gtttcgcaaa?gattgcaact?gacaccgtcg?tttgtgattg?tttacctggg 300

actgaagcgc?tgatccctct?gctggagttc?atggagatga?acgtgcaggc?gaggtacggg 360

gtcaaccccg?acaaggacgt?gctggagatc?ctgcaagctg?agtccaagtc?caagcaggta 420

atagcctaca?tcaactactg?aattgtccat?acaatgatgc?aattatcttt?gttgaattca 480

taaggccagt?tcaggcaatg?aaaatgtgct?gctaactaga?gatcgattcc?acgttggttg 540

aatcaggtgg?aaatactggc?aaaaatatat?tggggtgatg?caagggagaa?actttgtgag 600

gcagtagatg?atctcaaggt?ggactctgtt?gtgcttggct?gcaggggctt?agggccgttg 660

aaaaggtagt?acatctgaac?ctctttctga?ctaaactatg?aggatctagt?tcttggctaa 720

tcagagtttt?ctcatatttc?tacatctcct?tggacctcga?tgcagggcac?tccttggaag 780

cgtcagcaat?tatgtcgtca?acaatgcagc?ctgccccgtc?acagtggtgc?gcggaccaaa 840

tggatcactt?gcttgaagat?gtcacttatg?gattgatggg?ctagtttgtg?tgttcattgg 900

tccgtcagca?taggtgcaaa?tcgtgctatt?taagactata?gtgatatttt?tcctttttgg 960

attcctggct?atccttgtag?tgcaaattca?gtgtgttttc?ctcagttatc?tctttagtta 1020

tactatattt?ctgttgtttc?tta 1043

<210>4

<211>1034

<212>DNA

< 213>barley (Steptoe)

<400>4

ctctggcaga?catggcgcgc?accatcggcc?tggccatgga?ctactcgccg?tccagcaagg 60

ccgcgacgag?gtgggtggtc?gagaacctgg?tgaaggccgg?cgaccggatc?atcctcatcc 120

acgtcctccc?caagggagca?gacgccagcc?acaaggggct?ctggaagagc?accggttcac 180

gtatgtgtca?agacactgaa?ttctctgttc?tgttgtgtag?ctccctcgct?cttatctttc 240

cgcttcgcaa?agattgcaac?tgacaccgtc?gtttgtgatt?gtttacctgg?gactgaagcg 300

ctgatccctc?tgctggagtt?catggagatg?aacgtgcagg?cgaggtacgg?ggtcaacccc 360

gacaaggacg?tgctggagat?cctgcaagct?gagtccaagt?ccaagcaggt?aatagcctac 420

atcaactact?gaattgtcca?tacaatgatg?caattatctt?tgttgaattc?ataaggccag 480

ttcaggcaat?gaaaatgtgc?tgctaactag?agatcgattc?cacgttggtt?gaatcaggtg 540

gaaatactgg?caaaaatata?ttggggtgat?gcaagggaga?aactttgtga?ggcagtagat 600

gatctcaagg?tggactctgt?tgtgcttggc?tgcaggggct?tagggccgtt?gaaaaggtag 660

tacatctgaa?cctctttctg?actaaactat?gaggatctag?ttcttggcta?atcagagttt 720

tctcatattt?ctacatctcc?ttggtcctcg?atgcagggca?ctccttggaa?gcgtcagcaa 780

ttatgtcgtc?aacaatgcaa?cctgccccgt?cacagtggtg?cgcggaccaa?atggatcact 840

tgcttgaaga?tgtcacttat?ggattgatgg?gctagtttgt?gtgttcattg?gtccgtcagc 900

ataggtgcaa?atcgtgctat?ttaagactat?agtgatattt?ttcctttttg?gattcctggc 960

tatccttgta?gtgcaaattc?agtgtgtttt?cctcagttat?ctctttagtt?atactatatt 1020

tctgttgttt?ctta 1034

<210>5

<211>293

<212>DNA

< 213>barley (Morex)

<400>5

ttgattgttc?ggagcactag?cctgtactta?accaaccaag?gagatgataa?gagctactac 60

actgctagaa?ctgctacctg?gttattatgc?atagcatctc?agacacattt?gacaggatgg 120

tacaaatcat?catggggatc?aaagtttctt?cacctccaac?taatctgatt?gcttccatgg 180

tcactgcagg?ttcttccttg?gcagtgtgag?caactactgt?agccaccatg?caaagtgccc 240

ggttcttgtt?gtgaagaaga?aagaatgaaa?cccaaccatc?tactgtactc?atg 293

<210>6

<211>297

<212>DNA

< 213>barley (Steptoe)

<400>6

ttgattgttc?ggagcactag?cctgtactta?accaactaac?caaggagatg?ataagagcta 60

ctacactgct?agaactgcta?cctggttatt?atgcatagca?tctcagacac?atttgacagg 120

atggtacaaa?tcatcgtggg?aatcaaagtt?tcttcacctc?caactaatct?gattgctgcc 180

atggtcactg?caggttcttc?cttggcagtg?tgagcaacta?ctgtagccac?catgcaaagt 240

gcccggttct?tgttgtgaag?aagaaagaat?gaaacccaac?catctactgt?actcatg 297

<210>7

<211>383

<212>DNA

< 213>barley (Morex)

<400>7

agccaaagca?agccctcccc?tttctgctgg?taagcatatc?tcctactgct?gctgctatac 60

gtgcacacct?gtttcccttt?gtcacggaat?cgtcctgtac?aaagttactc?taaaaaggaa 120

gcagagaatc?ttattataca?ctacatcgct?ggcacggccc?atcacgctgt?aaagcccact 180

gtcgtccttt?gcaaacaaac?atcctgagag?ctttgggcca?aataatgtta?taagagttca 240

gagaacttac?tgtagtgagt?aagttgatgg?gaagagagag?ccatgtgcca?tccataaacc 300

agagatgcct?ttccctgtcc?tctcagctta?aactctacta?gttgatggat?cgactacgag 360

caagaaacca?tgatgggagg?agt 383

<210>8

<211>387

<212>DNA

< 213>barley (Steptoe)

<400>8

agccaaagca?agccgtcccc?tttctgctgg?taagcatatc?tcctactgct?gctgctatgc 60

gtgcacacct?gtttcccttt?gtcacggaat?cgtcctgtac?aaagttactc?taaaaaggat 120

gcagagaatc?ttattataca?ctacatcgct?ggcacggccc?atcacgctgt?aaagcccact 180

gtcgtccttt?gcaaacaaac?atcctgagag?ctttgggcca?aataatgtta?taagagttca 240

gagaacttac?tgtagtgagt?aagttgatgg?gaagagagag?ccatgtgcca?tccataaatc 300

agagatgcct?ttccctgtcc?tctcagctca?aaccctgaat?ctcaagtagt?agtagtagta 360

gcaccaagaa?gccatgatgg?gaggagt 387

<210>9

<211>203

<212>DNA

< 213>barley (Morex)

<400>9

agcttggcgc?cgagcagagg?ggaggaggag?gtggtcgagc?agtgcatcaa?ccaggccgag 60

tgcctgacgc?tggcggtgag?gaagcagagc?aagggcgtcg?gcggatacct?cgtcagcacc 120

cggtggcaga?agaacttctg?gctcctcgct?tgaccgtccg?tcgtccccgt?ccgccatgtc 180

cacgaccgtg?tttctataaa?agc 203

<210>10

<211>202

<212>DNA

< 213>barley (Steptoe)

<400>10

agcttggcgc?cgagcagagg?ggaggaggag?gtggtggagc?agtgcatcaa?ccaggccgag 60

tgcctgacgc?tggcggtgag?gaagcagagc?aagggcgtcg?gcggatacct?cgtcagcacc 120

cggtggcaga?agaacttctg?gctcctcgct?tgaccgtccg?tcgtcccgtc?cgccatgtcc 180

acgaccgtgt?ttctataaaa?gc 202

<210>11

<211>21

<212>DNA

<213>Unknown

<220>

< 223>artificial sequence

<400>11

gtccttaatt?gttcctgcgt?c 21

<210>12

<211>20

<212>DNA

< 213>artificial sequence

<400>12

ccatatggcg?aaccgaaatc 20

<210>13

<211>19

<212>DNA

<213>Unknown

<220>

< 223>artificial sequence

<400>13

ctctggcaga?catggcagc 19

<210>14

<211>25

<212>DNA

<213>Unknown

<220>

< 223>artificial sequence

<400>14

agagataact?gaggaaaaca?cactg 25

<210>15

<211>23

<212>DNA

<213>Unknown

<220>

< 223>artificial sequence

<400>15

cttaaccaac?taaccaagga?gat 23

<210>16

<211>23

<212>DNA

<213>Unknown

<220>

< 223>artificial sequence

<400>16

catgagtaca?gtagatggtt?ggg 23

<210>17

<211>20

<212>DNA

<213>Unknown

<220>

< 223>artificial sequence

<400>17

ttaaagccaa?agcaagccgt 20

<210>18

<211>21

<212>DNA

<213>Unknown

<220>

< 223>artificial sequence

<400>18

gctcgtagtc?gatccatcaa?c 21

<210>19

<211>21

<212>DNA

<213>Unknown

<220>

< 223>artificial sequence

<400>19

agaagggagg?aggaggtgat?g 21

<210>20

<211>21

<212>DNA

<213>Unknown

<220>

< 223>artificial sequence

<400>20

gcttttatag?aaacacggtc?g 21

Claims (2)

1. wide stress protein family gene specific molecular marker; It is that partial dna sequence with the USP gene of barley variety Steptoe and Morex is the basis; Specificity difference amplimer according to the difference zone design of base mutation, insertion and disappearance in the sequence; It is characterized in that: said primer is to be the basis with the dna sequence dna of gene hv.3739, hv.7364, hv.23267, hv.11351 and the hv.11741 of barley Steptoe and Morex respectively, according to the particular sequence oligomer with following based composition of difference zone design:

(1) forward: 5 '-GTCCTTAATTGTTCCTGCGTC-3 ',

Oppositely: 5 '-CCATATGGCGAACCGAAATC-3 ';

Or (2) forward: 5 '-CTCTGGCAGACATGGCAGC-3 ',

Oppositely: 5 '-AGAGATAACTGAGGAAAACACACTG-3 ';

Or (3) forward: 5 '-CTTAACCAACTAACCAAGGAGAT-3 ',

Oppositely: 5 '-CATGAGTACAGTAGATGGTTGGG-3 ';

Or (4) forward: 5 '-TTAAAGCCAAAGCAAGCCGT-3 ',

Oppositely: 5 '-GCTCGTAGTCGATCCATCAAC-3 ';

Or (5) forward: 5 '-AGAAGGGAGGAGGAGGTGATG-3 ',

Oppositely: 5 '-GCTTTTATAGAAACACGGTCG-3 '.

2. wide stress protein family gene specific molecular marker application method; It is characterized in that: utilize the described specificity difference of claim 1 amplimer right; The gene that it is corresponding is labeled as BUG1, BUG2, BUG3, BUG4 and BUG5 respectively; Detect the situation of amplification target stripe of DH population material to be measured through pcr amplification after; Use Mapmaker3.0 software the USP assignment of genes gene mapping to karyomit(e), utilize getting in touch between barley DH population resistance QTL collection of illustrative plates and real-time fluorescence quantitative PCR technical Analysis USP gene and resistance, through transgenic technology or chromosome engineering technology with in this gene transferred crop; Effectively the ability of enhancing crop opposing salt stress itself significantly strengthens the salt resistance ability of crop self.

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