CN101054595A - Gene molecule marking method for grape anthracnose disease resistant breeding - Google Patents

Abstract

The invention relates to a gene molecular marker method for grape black pox breeding, which uses the parents, _F1 generation and _F2 generation of the interspecific hybrid combination Baihe-35-1×Carigny as test materials, and uses random primers OPS03 was amplified to obtain a deoxyribonucleic acid fragment of about 1300 base pairs, which was ligated with pGEM-T vector, and then transformed into Escherichia coli DH5α to clone the fragment. After sequencing, the fragment was 1365 base pairs. According to the sequence, an artificially synthesized oligonucleotide can detect the existence of the anti-black pox gene and the expression of the anti-black pox traits of parents, hybrids, European grapes and wild grapes, and is used as a method for detecting the anti-black pox gene of grapes. probe. The molecular marker of the present invention can be used for early screening and identification of grape black pox-resistant breeding; its base sequence provides a basis for the study of grape black pox-resistant genes; the artificially synthesized oligonucleotide sequence can detect grape black pox The function of the presence and expression of disease genes.

Description

A gene molecular marker method for grape black pox breeding

                      

The present invention relates to a gene molecular marker method, in particular to a gene molecular marker method for grape black pox breeding, and the sequence of deoxyribonucleic acid (DNA) fragments and as a probe for detecting grape black pox resistance genes The oligonucleotide sequence belongs to the field of plant genetic engineering.

Background technique

Grape is a worldwide fruit, its area and output both rank second among all kinds of fruits. Grape black pox is a fungal disease that seriously harms grapes, especially in areas with more rain and high humidity during the grape growing season. In these regions, the damage is serious, often causing the grapes to drop flowers and fruits, the leaves are perforated and fall off, and there are bird's-eye black spots on the fruit grains, which seriously affect the growth and yield of grapes. The traditional method of preventing and controlling grape black pox is to spray grapes repeatedly to prevent the disease, but chemical control will cause residual poison in the fruit and pollute the environment, which is not conducive to people's health. Utilizing the disease resistance of the grape resources itself, cross breeding is the fundamental way to cultivate new varieties of black pox resistant grapes, but the cycle of breeding new varieties resistant to black pox through cross breeding is relatively long, and generally identification of hybrid resistant Black pox performance needs 3-5 years, this manpower, material resources, time-consuming and efficient breeding method are far from being able to meet the actual needs of production.

Randomly amplified polymorphic deoxyribonucleic acid (RAPD) is a molecular marker technology, which has the characteristics of rapidity, simplicity, low cost, rich polymorphism detection, no isotope tracer, and safety and reliability. This technology was first applied to the identification of genus, strain and intraspecific differences in 1990, and then it was widely used in the field of molecular biology research and crop breeding, and achieved remarkable results, but it has not been used in grape anti-black pox disease breeding.

Contents of invention

The purpose of the present invention is to solve the problem of long period and low efficiency in traditional hybrid breeding of grapes against black pox, and to disclose a gene molecular marker method for grapes against black pox breeding.

The technology that the present invention adopts and operation step are:

a. Using the parents, F ₁ generation and F ₂ generation of the interspecific hybrid combination Baihe-35-1×Carigny of grape black pox breeding as test materials, extract, separate and purify deoxyribonucleic acid (DNA)

b. RAPD amplification using deoxyribonucleic acid (DNA) as a template

c. Obtain a fragment of deoxyribonucleic acid (DNA) linked to the grape black pox gene

d. Extract and purify the deoxyribonucleic acid (DNA) fragments from the agarose gel with the UNIQ-10 Column DNA Gel Recovery Kit

e. Link the deoxyribonucleic acid fragments with the pGEM-T vector

f. Transform Escherichia coli DH5α, extract plasmid DNA for enzyme digestion

g. Determining the base composition and sequence of the DNA fragment from the bacteria solution identified as a positive clone

h. Obtain the entire base composition and sequence of the DNA fragment of 1365 base pairs

i. Further based on the complete base composition and sequence of 1365 base pairs

j. Artificially synthesize 4 oligonucleotides, one of which (5'ACAATCACCCAACTCCTC 3') can be used as a primer to perform PCR amplification on deoxyribonucleic acid (DNA) as a template

k. Amplify the parents of the original hybrid combination and its F ₁ generation, F ₂ generation, Chinese wild grape, European grape, American wild grape, and the parent of the hybrid combination Guangxi-1×Jingkejing and its F ₁ generation

l. Obtain specific deoxyribonucleic acid (DNA) fragments

m. Reclaim, clone and sequence the DNA fragments by the above methods

n. Obtained another deoxyribonucleic acid (DNA) sequence measuring 1110 base pairs for the black pox resistance trait of grapes

o. Determine the obtained oligonucleotide sequence (5'ACAATCACCCAACTCCTC 3') as a probe for detecting grape black pox resistance gene.

The anti-black pox gene RAPD marker OPS03-1354 of the present invention has an anti-black pox breeding hybrid combination Guangxi-1×Jingkejing F ₁ generation 339 strains, and the coincidence rate of detection and field plant disease resistance performance is 98.53%, and the combination Baihe- The coincidence rate between the detection of 207 strains of 35-1× Carignan F ₂ generation and field plant resistance was 96.17%; oligonucleotide (5′ACAATCACCCCAACTCCTC 3′) was used as a probe pair to detect grape black pox resistance gene _The coincidence rate of the 339 plants of the hybrid combination Guangxi-1×Jingkejing F ₁ generation and the disease resistance performance of the field plants was 83.57%. The performance compliance rate was 87%.

Detailed ways

The present invention uses deoxyribonucleic acid (DNA) as template to carry out the concrete steps of RAPD amplification as:

b.1 Using polymerase chain reaction (PCR)

b.1.1 The volume of the mixed solution is 25 microliters

b.1.2 Contains 2.5 μl of 10× PCR buffer

b.1.3 Magnesium chloride 1.5 mmol/L

b.1.4 dNTP 200 μmol/L

b.1.5 Taq DNA polymerase 1 unit

b.1.6 Random primer 1 microliter

b.1.7 45 nanograms of template deoxyribonucleic acid (DNA);

b.2 Overlay the reaction solution with 25 microliters of mineral oil

b.2.1 PCR instrument is Eppendorf-AG22331

b.2.2 Polymerase chain reaction Melting at 94°C for 5 minutes;

b.3 Perform 45 cycles

b.3.1 Each cycle includes melting at 94°C for 1 minute

b.3.2 Primers attach to template DNA for 2 minutes at 36°C

b.3.3 Elongation of deoxyribonucleic acid (DNA) polymer chain at 72°C for 2 minutes

b.3.4 The last cycle is an extension at 72°C for 10 minutes;

b.4 Store the amplified product at 4°C or use agarose gel electrophoresis directly

b.4.1 Agarose gel concentration is 1.2%

b.4.2 Add ethidium bromide 0.5 ng/ml

b.4.3 The electrophoresis voltage is 110 volts

b.4.4 Electrophoresis with water plate electrophoresis tank for 1-1.5 hours;

b.5 After electrophoresis, take pictures under ultraviolet light.

The present invention uses artificially synthesized oligonucleotides (5'ACAATCACCCAACTCCTC 3') as primers, and deoxyribonucleic acid (DNA) is used as a template to carry out the specific steps of PCR amplification as follows:

j.1 Using polymerase chain reaction (PCR)

j.1.1 The volume of the mixed solution is 25 microliters

j.1.2 Contains 2.5 μl of 10× PCR buffer

j.1.3 Magnesium chloride 1.5 mmol/L

j.1.4 dNTP 200 μmol/L

j.1.5 Taq DNA polymerase 1 unit

j.1.6 oligonucleotide (5′ACAATCACCCAACTCCTC 3′) 1 microliter

j.1.7 45 nanograms of template deoxyribonucleic acid (DNA);

j.2 Overlay the reaction solution with 25 microliters of mineral oil

j.2.1 PCR instrument is Eppendorf-AG22331 type

j.2.2 Polymerase chain reaction, melt at 94°C for 5 minutes;

j.3 Perform 40 cycles

j.3.1 Each cycle includes melting at 94°C for 30 seconds

j.3.2 Primers attach to template DNA for 30 seconds at 62°C

j.3.3 Elongation of deoxyribonucleic acid (DNA) polymer chain at 72°C for 1 minute

j.3.4 The last cycle is extended at 72°C for 5 minutes;

j.4 Store the amplified product at 4°C or use agarose gel electrophoresis directly

j.4.1 Agarose gel concentration is 1.2%

j.4.2 Add ethidium bromide 0.5 ng/ml

j.4.3 The electrophoresis voltage is 110 volts

j.4.4 Electrophoresis with water plate electrophoresis tank for 1-1.5 hours;

j.5 Take pictures under ultraviolet light after electrophoresis.

Using random amplified polymorphic deoxyribonucleic acid (RAPD) technology, a molecular marker linked to the grape anti-black pox gene was obtained. The molecular marker was cloned and sequenced, and the deoxyribose sugar of the molecular marker of the grape anti-black pox gene was obtained. The composition and sequence of nucleic acid (DNA), the presence or absence of grape black pox resistance gene can be detected with this specific DNA sequence, to speed up the breeding process and improve the accuracy of black pox resistance breeding; further through the chromosome walking method, Mapping and positioning of the anti-black pox gene, based on the obtained molecular marker sequence of the grape anti-black pox gene, the artificially synthesized oligonucleotide sequence (5'ACAATCACCCAACTCCTC 3') also has the ability to detect the existence and identification of the grape anti-black pox gene. expressive function.

The present invention has the following purposes: can be used as deoxyribonucleic acid (DNA) probe, ribonucleic acid (RNA) probe, gene localization mapping, gene transfer, the DNA molecule basis of early screening and identification in grape anti-black pox breeding, as Deoxynucleic acid (DNA) fingerprinting of black pox resistant grape varieties and lines and rationale for registration of plant patents.

The grape black pox resistant gene molecular marker of the present invention provides the DNA basis for identification at the seedling stage for grape hybrid breeding, will accelerate the process of black pox resistant breeding and improve breeding efficiency, and will provide a new method for rapid selection of black pox resistant grape varieties , further cloning the anti-black pox gene and gene transfer provides a method and material basis, and also provides a scientific basis for the study of the genetic law of grape anti-black pox and the linkage genetic mapping of molecular markers. The invention detects the deoxyribonucleic acid (DNA) sequence and the artificially synthesized oligonucleotide sequence of grape black pox resistance gene, fundamentally solves and accelerates the breeding process of grape black pox resistance.

Claims (3)

1. gene molecule marking method that is used for grape anthracnose disease resistant breeding is characterized in that adopting following technological operation step to carry out:

1.a make up the parents and the F of the Baihe-35-1 * Carignan with the species hybridization of grape anthracnose disease resistant breeding ₁Generation, F ₂On behalf of examination material, extraction, separation, purifying DNA (DNA)

1.b carry out the RAPD amplification as template with thymus nucleic acid (DNA)

1.c the fragment of the thymus nucleic acid (DNA) that acquisition and spot anthracnose resisting gene of grape are chain

From sepharose, extract this thymus nucleic acid of purifying (DNA) fragment 1.d reclaim test kit with UNIQ-10 pillar DNA glue

1.e connect deoxyribonucleic acid fragment with the pGEM-T carrier

1.f transformed into escherichia coli DH5 α, the extraction plasmid DNA is carried out enzyme and is cut

1.g will be accredited as base formation and sequence thereof that the bacterium liquid of positive colony is measured this dna fragmentation

1.h obtain the whole based compositions and the sequence thereof of the dna fragmentation of 1365 base pairs

1.i further according to the whole based compositions and the sequence thereof of 1365 base pairs

1.j 4 oligonucleotides of synthetic, one of them (5 ' ACAATCACCCAACTCCTC 3 ') can be used as primer, and (DNA) carries out pcr amplification as template to thymus nucleic acid

1.k to former cross combination parents and F thereof ₁Generation, F ₂Generation, Chinese wild grape, vitis vinifera, the America Wild Grape, and cross combination Guangxi-1 * capital can brilliant parent and F thereof ₁In generation, increase respectively

1.l obtain specificity thymus nucleic acid (DNA) fragment

1.m with aforesaid method this dna segment that reclaims, clones, checks order

Detect thymus nucleic acid (DNA) sequence of grape anthracnose disease resistant proterties 1.n obtained another, its size is 1110 base pairs

1.o the oligonucleotide sequence of determining to obtain (5 ' ACAATCACCCAACTCCTC 3 ') is as the probe that detects spot anthracnose resisting gene of grape.

2. a kind of gene molecule marking method that is used for grape anthracnose disease resistant breeding according to claim 1 is characterized in that with the concrete steps that thymus nucleic acid (DNA) carries out the RAPD amplification as template being:

2.b.1 adopt polymerase chain reaction (PCR)

2.b.1.1 mixeding liquid volume is 25 microlitres

2.b.1.2 contain 10 * PCR damping fluid, 2.5 microlitres

2.b.1.3 magnesium chloride 1.5 mmoles/liter

2.b.1.4dNTP 200 micromoles per liter

2.b.1.5Taq 1 unit of archaeal dna polymerase

2.b.1.6 random primer 1 microlitre

2.b.1.745 the template DNA of nanogram(ng) (DNA);

2.b.2 the mineral oil with 25 microlitres covers reaction solution

2.b.2.1PCR instrument is the Eppendorf-AG22331 type

2.b.2.2 unwind 5 minutes under 94 ℃ of the polymerase chain reactions;

2.b.3 carry out 45 circulations

2.b.3.1 comprising, each circulation unwind under 94 ℃ 1 minute

2.b.3.236 a ℃ following primer adhered to template DNA 2 minutes

2.b.3.372 the elongation of ℃ following thymus nucleic acid (DNA) polymeric chain 2 minutes

Extend after 10 minutes down 2.b.3.4 last circulation is 72 ℃;

2.b.4 amplified production is preserved or directly uses agarose gel electrophoresis down at 4 ℃

2.b.4.1 sepharose concentration is 1.2%

2.b.4.2 add ethidium bromide 0.5 millimicro grams per milliliter

2.b.4.3 electrophoretic the electricity is at 110 volts of pressure

2.b.4.4 water disk electrophoresis groove electrophoresis 1-1.5 hour;

2.b.5 electrophoresis is taken a picture under ultraviolet lamp after finishing.

3. a kind of gene molecule marking method that is used for grape anthracnose disease resistant breeding according to claim 1, it is characterized in that making primer with artificial synthetic oligonucleotide (5 ' ACAATCACCCAACTCCTC 3 '), the concrete steps that thymus nucleic acid (DNA) is carried out pcr amplification as template are:

3.j.1 adopt polymerase chain reaction (PCR)

3.j.1.1 mixeding liquid volume is 25 microlitres

3.j.1.2 contain 10 * PCR damping fluid, 2.5 microlitres

3.j.1.3 magnesium chloride 1.5 mmoles/liter

3.j.1.4dNTP 200 micromoles per liter

3.j.1.5Taq 1 unit of archaeal dna polymerase

3.j.1.6 oligonucleotide (5 ' ACAATCACCCAACTCCTC 3 ') 1 microlitre

3.j.1.745 the template DNA of nanogram(ng) (DNA);

3.j.2 the mineral oil with 25 microlitres covers reaction solution

3.j.2.1PCR instrument is the Eppendorf-AG22331 type

3.j.2.2 unwind 5 minutes under 94 ℃ of the polymerase chain reactions;

3.j.3 carry out 40 circulations

3.j.3.1 comprising, each circulation unwind under 94 ℃ 30 seconds

3.j.3.262 a ℃ following primer adhered to template DNA 30 seconds

3.j.3.372 the elongation of ℃ following thymus nucleic acid (DNA) polymeric chain 1 minute

3.j.3.4 last circulation was extended 5 minutes down for 72 ℃;

3.j.4 amplified production is preserved or directly uses agarose gel electrophoresis down at 4 ℃

3.j.4.1 sepharose concentration is 1.2%

3.j.4.2 add ethidium bromide 0.5 millimicro grams per milliliter

3.j.4.3 electrophoretic the electricity is at 110 volts of pressure

3.j.4.4 water disk electrophoresis groove electrophoresis 1-1.5 hour;

3.j.5 finishing the back, electrophoresis under ultraviolet lamp, takes a picture.