CN1814810A - Molecule label related to soybean resistant germplasm gene and its obtaining method and use - Google Patents
Molecule label related to soybean resistant germplasm gene and its obtaining method and use Download PDFInfo
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- CN1814810A CN1814810A CN 200510134363 CN200510134363A CN1814810A CN 1814810 A CN1814810 A CN 1814810A CN 200510134363 CN200510134363 CN 200510134363 CN 200510134363 A CN200510134363 A CN 200510134363A CN 1814810 A CN1814810 A CN 1814810A
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Abstract
This invention relates to a molecular label linkaged with the gene of the soya anti-utricle roundworm disease and a method, which applies a RAPD molecular labeling method to select the resistance labels to the small sized black soya beans of anti-soya utricle roundworms, Liao-bean 10 and its later generation 10 x small size black beans, taking 10bp oligonucleotide as the random primer to select a molecular label S 11700 linkaged with the anti-utricle roundworm disease gene of soyas.
Description
Technical field:
The invention belongs to agricultural biotechnology engineering, particularly relevant molecule marker and preparation method and application with the soybean cyst nematode resistant gene.
Technical background:
(Hederodera glycines Ichinohe, SCN) disease is one of important disease that threatens world soybean production to the soybean Cyst nematode.Along with manufacturing soybean year after year, heavily meeting a batch area constantly increases, and the generation of soybean Cyst nematode is also more and more serious.China northeast and soybean producing region, the Yellow River and Huai He River Haiti district almost generally take place, and annual harm reaches more than 2,000,000 hectares, from world wide, and the harm of soybean cyst nematode and spread the trend that increases the weight of is day by day arranged.With the Heilongjiang Province that accounts for China's soybean area 1/3rd is example, and all there is the generation of this disease in nearly all inside the province soybean planting district, estimates 400,000,000 kilograms of the annual underproduction, causes 900,000,000 yuans loss, has seriously restricted soybean production.
Domestic and international research and facts have proved, utilizing good disease-resistant variety control soybean cyst nematode is most economical, safety and valid approach.The germ plasm resource of the anti-soybean cyst nematode of a collection of height has been screened and identified in China, but these resources mostly are Semen glycines sojae, economical character and the cultivated soybean are far apart, rely on conventional Phenotypic Selection and traditional soybean cyst nematode resistance authentication method, with the beans Cyst nematode ospc gene transformation of the anti-source of the Semen glycines sojae Chinese People's Anti-Japanese Military and Political College in the cultivated soybean, cultivate the good disease-resistant variety of economical character, need the cost lot of manpower and material resources, and breeding cycle is long.In recent years, the continuous development of molecular marking technique is for new thinking has been expanded in the auxiliary nematicide breeding of molecule.Utilize and to discern different disease-resistant genes easily and accurately with the closely linked molecule marker of disease-resistant gene.Utilize Markers for Detection not to be subjected to the restriction of time and trait expression, can carry out Molecular Identification, solved the difficult problem that traditional breeding method must be identified in the specific period, thereby shortened breeding cycle greatly in any period of soybeans they grow.
Along with molecular biological continuous development, many scholars both domestic and external are devoted to the sick Study on Molecular Marker of the anti-Cyst nematode of soybean.It is that segregating population fractional analysis method is studied the soybean varieties of different resistances that BSA (Bulked Segregates Analysis) is adopted in this research, be intended to seek and the closely linked RAPD mark of soybean Cyst nematode resistant gene, set up the molecular mark technical system, for the blindness that reduces in the sick breeding of the anti-Cyst nematode of soybean, improve efficiency of selection and accelerate breeding process significant.
Summary of the invention:
The present invention be directed to above-mentioned situation, be material screening with the soybean varieties granule black soya bean of high Chinese People's Anti-Japanese Military and Political College beans Cyst nematode and seek new and stable existence and Cyst nematode disease closely-related molecule marker of resistant gene and method thereof with molecular biology method, be used for the soybean cyst nematode assisted selection, for the anti-Cyst nematode gene of rear clone soybean and carry out sequential analysis and lay a good foundation.
The present invention relates to a kind of acquisition and the linked molecular marker method of the anti-Cyst nematode ospc gene of soybean, comprise the following steps:
(1) the disease-resistant gene donor is the Semen glycines sojae kind granule black soya bean of No. 3 physiological strains of the distinctive high Chinese People's Anti-Japanese Military and Political College's beans Cyst nematode of China, and itself and high soybean varieties the Liao Dynasty beans 10 of feeling Cyst nematode are hybridized, and cross combination is distant beans 10 * granule black soya bean, resists, feels individual plant by F
2Obtain for segregating population;
(2) extract soybean parents and filial generation leaf DNA with phenol-chloroform method, from F
2Constitute disease-resistant pond for the anti-individual plant DNA of 10 plant heights of picked at random in segregating population balanced mix, the DNA balanced mix of 10 plant height sense individual plants constitutes susceptible pond;
(3) adopt the RAPD molecule marking method to carry out the screening of soybean cyst nematode resistance molecule marker;
(4) filter out a RAPD molecule marker S11
700, find that after testing this mark and the susceptible gene of soybean Cyst nematode are closely related.
Wherein, adopting the RAPD technology to screen the molecular marker method relevant with the anti-Cyst nematode ospc gene of soybean comprises: adopt the 10bp oligonucleotide as random primer, carry out pcr amplification, analyze the RAPD primer and resisting, feeling the dna polymorphism between parent and their filial generation, preferred random primer is S11, and its sequence is CCCAGCTGTG.Also can comprise and use F
2Further verify the step of the polymorphism mark that between parent and anti-sense pond, produces for the individual plant of segregating population.
With RAPD labeling technique screening, adopt 10bp oligonucleotide that Shanghai gives birth to the exploitation of worker Bioisystech Co., Ltd as random primer, in the enterprising performing PCR amplification of PTC-200 type PCR instrument, the PCR reaction system is: template 20ng μ L wherein
-1Soybean gene group DNA 2 μ L, 5U μ L
-1Taq archaeal dna polymerase 0.2 μ L, 10 * PCR Buffer, 2 μ l, 2.5mM MgCl
22 μ L, 2.5mM dNTP 1.6 μ L, random primer 1 μ L, ddH
2O 11.2 μ L totally are 20 μ L.
The PCR response procedures: 94 ℃ of pre-sex change 5min of the 1st circulation, the 2nd to be circulated to 35 cycling programs be 94 ℃ of sex change 1min, 36 ℃ of annealing 1min, 72 ℃ are extended 1min, and last circulates in 72 ℃ insulation and extends 5min.Amplified production is electrophoresis on 1.2% sepharose, and voltage is 4Vcm
-1, the 30min that in EB, dyes after electrophoresis finishes, check and analysis in the gel imaging instrument.
Also comprise the evaluation to molecule marker: soybean filial generation soybean cyst nematode resistance is identified and is carried out in the field, the resistance standard of perfection is pressed (" plant nematology investigative techniques " such as Liu Weizhi, Liaoning science and technology press, 1995, p202-204) the standard of perfection method of the similar U.S. of Ti Chuing: with the cyst number is disease-resistant index, be divided into five ranks, 0=does not have cyst (immunity); 1=1-5 (disease-resistant); 2=6-10 (in anti-); 3=11-30 (susceptible); 4=30 above (high sense).Extract total DNA disease-resistant, susceptible individual plant respectively, use the reaction system same with the front, primer and program to carry out the pcr amplification of individual plant, statistics is anti-, the appearance of sense individual plant indicia band.
The invention still further relates to the RAPD molecule marker S11 linked that obtains according to above-mentioned method with the anti-Cyst nematode ospc gene of soybean
700, it is the dna fragmentation of about 700bp.
The molecule marker that the present invention obtained can be used for the assisted selection of the sick soybean of anti-Cyst nematode.
Soybean cyst nematode is to influence a kind of important disease that soybean produces.The present invention utilizes molecule marking method to obtain new and the closely-related molecule marker of soybean cyst nematode resistant gene.Utilize this method, not only overcome shortcomings such as the conventional breeding method required time cycle is long, targetedly disease-resistant gene selected to obtain in the laboratory, also on purpose a plurality of anti-Cyst nematode ospc genes of polymerization, cultivate new soybean varieties with stable resistance, simultaneously also can utilize the anti-Cyst nematode ospc gene of molecular marker clone of this resistant gene and it is carried out the 26S Proteasome Structure and Function analysis, this molecular genetics mechanism for the anti-Cyst nematode disease of further understanding soybean has positive effect.Therefore, this result of study is all very valuable in soybean breeder practice and disease-resistant theoretical investigation.Its advantage specifically is summarized as follows:
(1) the of the present invention and closely-related molecule marker of soybean cyst nematode resistant gene, be the new mark that in the filial generation individual plant that high anti-soybean varieties granule black soya bean and resistance thereof are stable to No. 3 physiological strains of soybean Cyst nematode, obtains, can be used for soybean cyst nematode cultivar identification and the disease-resistant offspring's of soybean assisted selection.
(2) this research material therefor is the soybean varieties high anti-to No. 3 physiological strains of soybean Cyst nematode (physiological strain that the antagonism gene is the most responsive, virulence is the most weak), can be according to the molecular marker clone of the gained new sick resistant gene of Cyst nematode.
(3) for cloning the anti-Cyst nematode ospc gene of soybean, gene sequencing and changeing the soybean research of anti-Cyst nematode ospc gene and established good basis.
Description of drawings:
Fig. 1: primer S11 is at distant beans 10 * granule black soya bean F
2RAPD amplification in the part plant, arrow is depicted as S11
700Special band, wherein: 4: the granule black soya bean; 5: distant beans 10; 6: susceptible pond; 7: disease-resistant pond; 1,2,10,17,18,21,23 is F
2For disease-resistant individual plant; 3,8,9,11-16,20,22,24 is F
2For susceptible individual plant;
Fig. 2: different resistant soybean kinds are to mark S11
700Detection (arrow is depicted as S11
700Special band), wherein: M: λ DNA/EcoRI+HindIII; Numbering 1-21 represents different soybean varieties (seeing Table 1) respectively; P
SRepresent the compound sample of the susceptible DNA of colony; P
RRepresent the compound sample of disease-resistance population DNA.
Embodiment
Embodiment 1: with the relevant molecule marker S11 of the anti-Cyst nematode ospc gene of soybean
700Acquisition
Adopt the RAPD molecule marking method, specific practice is:
The disease-resistant gene donor parents is the soybean varieties granule black soya bean of high Chinese People's Anti-Japanese Military and Political College beans Cyst nematode, susceptible parent feels Cyst nematode for height but the good soybean varieties the Liao Dynasty beans 10 of economical character are hybridized, cross combination is distant beans 10 * granule black soya bean, obtains the anti-sense of 85 strains individual plant by hybridization.
One, extract DNA:
Get fresh and tender soybean leaves 0.1g, grind into powder in liquid nitrogen adds SDS rapidly and extracts damping fluid (50mMTris-HCl, pH 8.0,20mM EDTA, pH 8.0,50mM NaCl) 500 μ L, and 65 ℃ of water-bath 40min rotate mixing gently every 10min therebetween.The centrifugal 20min of 10000rpm gets supernatant liquor, adds equal-volume phenol: chloroform (1: 1), chloroform: each extracting of primary isoamyl alcohol (24: 1) 1 time.The centrifugal 8min of 10000rpm gets supernatant liquor, adds the dehydrated alcohol of 2 times of volume precoolings, puts upside down mixing gently, places more than the 1h for-20 ℃; The centrifugal 10min of 10000rpm, the supernatant that inclines stays precipitation; Use 70% ethanol washing and precipitating twice then, air-dry after, be dissolved among the 50 μ L TE.Ultraviolet spectrophotometry detects the concentration of DNA sample, and 0.8% agarose gel electrophoresis detects the integrity of DNA.
Two, randomly amplified polymorphic DNA (RAPD) is analyzed:
The RAPD primer is given birth to worker bio-engineering corporation (totally 143 of 10bp Oligonucleolide primers) available from Shanghai.
1.PCR amplification
(1) reaction system:
20ng μ L
-1Soybean gene group DNA 2 μ L, 5U μ L
-1Taq archaeal dna polymerase 0.2 μ L, 10 * PCR Buffer, 2 μ L, 2.5mM MgCl
22 μ L, 2.5mM dNTP 1.6 μ L, random primer 1 μ L, ddH
2O 11.2 μ L totally are 20 μ L.On PTC-200 type PCR instrument, react.
(2) response procedures:
94 ℃ of pre-sex change 5min; 94 ℃ of sex change 1min, 36 ℃ of annealing 1min, 72 ℃ are extended 1min, 35 circulations of increasing; Last 72 ℃ are extended 5min.
2. electrophoresis detection:
Get amplified production 6 μ L, carry out electrophoresis on 1.2% sepharose, electrophoresis finishes the back at 0.5 μ g μ L
-1EB in dye, detect in the Ultraviolet Detector and take pictures and write down the result.
3. segregating population fractional analysis method (BSA)
With reference to (Michelmore R.W. such as Michelmore, Paran I and Kesseli R.V.1991.Identification of markers linkedto disease-resistance genes by using segregating analysis:A rapid method to detect markers in specific genomicregions by using segregating populations.Proc Natl Acad Science USA, method 88:9828-9832) is at F
2Constitute disease-resistant pond for the disease-resistant individual plant DNA of 10 strains of picked at random in segregating population balanced mix, the DNA balanced mix of the susceptible individual plant of 10 strains constitutes susceptible pond.Random primer is screened with anti-, sense pond with the parent.Between parent and anti-sense pond, produce polymorphism mark and further use F
2Individual plant checking for segregating population.
Three, the RAPD mark relevant with soybean Cyst nematode resistant gene:
Utilizing 143 random primers that distant beans 10 * granule black soya bean filial generation is carried out RAPD analyzes, discovery has 92 to produce amplified production, the random primer that produces the RAPD polymorphism has 25, wherein primer S11 (sequence is CCCAGCTGTG) can amplify polymorphic bands (susceptible pond have and disease-resistant pond do not have) between anti-/ sense pond, the length of polymorphic bands is about 700bp (as shown in Figure 1), contrast as anti-sense pond, disease-resistant parent does not have this bar segment, and susceptible parent has this bar segment, through repeated authentication repeatedly, this specific fragment all can be amplified out in susceptible parent and susceptible pond, and does not all have this segmental generation in disease-resistant parent and disease-resistant pond.
With primer S11 distant beans 10 and granule black soya bean are hybridized the F that obtains
2Anti-sense of generation individual plant carries out RAPD to be analyzed, and is divided into and analyses 85 individual plants.This is marked in all susceptible individual plants and occurs, and does not produce and all there is this fragment in all disease-resistant individual plants, proves that this fragment is the peculiar specific fragment of susceptible variety, infers that tentatively this specific fragment and soybean Cyst nematode resistant gene are closely related.Do not influence with the amplification of different batches Taq enzyme then, and in revision test, S11 amplification good reproducibility can confirm mark S11 primer S11
700Accurately and reliably.
Embodiment 2: different soybean varieties are to RAPD mark S11
700Evaluation and assisted Selection
1: " open and educate 10 " of No. 3 physiological strains of high sense soybean Cyst nematode
" opening and educate 10 " with No. 3 physiological strains of height sense soybean Cyst nematode is experiment material, and specific practice is with embodiment 1.As table 1 and shown in Figure 2, in gel imaging system, to observe, the specific band that size is about 700bp appears, and bands of a spectrum 1 are the susceptible gene molecule marker band of soybean Cyst nematode.
2: " Lee " of No. 3 physiological strains of high sense soybean Cyst nematode
With height sense soybean No. 3 physiological strains of Cyst nematode " Lee " is experiment material, and specific practice is with embodiment 1.As table 1 and shown in Figure 2, in gel imaging system, to observe, the specific band that size is about 700bp appears, and bands of a spectrum 17,18 are the susceptible gene molecule marker band of soybean Cyst nematode.
3: " the grey cutaneous branch black soya bean " of No. 3 physiological strains of high Chinese People's Anti-Japanese Military and Political College's beans Cyst nematode
With No. 3 physiological strains of high Chinese People's Anti-Japanese Military and Political College beans Cyst nematode " grey cutaneous branch black soya bean " is experiment material, and specific practice is with embodiment 1.As table 1 and shown in Figure 2, in gel imaging system, to observe, the specific band of 700bp does not appear, and bands of a spectrum 5 are soybean cyst nematode resistance genes molecule marker band.
4: " Peking " of No. 3 physiological strains of high Chinese People's Anti-Japanese Military and Political College's beans Cyst nematode
With the high Chinese People's Anti-Japanese Military and Political College No. 3 physiological strains of beans Cyst nematode " Peking " is experiment material, and specific practice is with embodiment 1.As table 1 and shown in Figure 2, in gel imaging system, to observe, the specific band of 700bp does not appear, and bands of a spectrum 10 are soybean cyst nematode resistance genes molecule marker band.
Table 1. experimental cultivar is to anti-sense reaction and the RAPD mark S11 of No. 3 physiological strains of soybean Cyst nematode
700Evaluation and assisted Selection
Sequence number | Kind | Resistance | S11
700Having or not of |
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 | Hold and educate even | + + + + - - - - - - - - - - - - + + - + + | Having or not have to have or not has |
Annotate :+susceptible ,-disease-resistant
Claims (8)
1. acquisition and the linked molecular marker method of the anti-Cyst nematode ospc gene of soybean, described method comprises the following steps:
(1) the disease-resistant gene donor parents is a Semen glycines sojae kind granule black soya bean, and itself and susceptible variety the Liao Dynasty beans 10 preparing hybrids make up, and are resisted, feel individual plant by F2 for segregating population;
(2) extract soybean parents and filial generation leaf DNA with phenol-chloroform method, from F
2Constitute disease-resistant pond for the anti-individual plant DNA of 10 plant heights of picked at random in segregating population balanced mix, the DNA balanced mix of 10 plant height sense individual plants constitutes susceptible pond;
(3) adopt the RAPD molecule marking method to carry out the screening of soybean cyst nematode resistance molecule marker;
(4) filter out a RAPD molecule marker S11
700, find that after testing this mark and the susceptible gene of soybean Cyst nematode are closely related.
2. in accordance with the method for claim 2, it is characterized in that adopting the RAPD molecule marker to screen the molecular marker method sick relevant with the anti-Cyst nematode of soybean comprises: adopt the 10bp oligonucleotide as random primer, carry out pcr amplification, analyze the RAPD primer and resisting, feeling the dna polymorphism between parent and their filial generation.
3. according to claim 1 or 2 described methods, it is characterized in that described method also comprises: use F
2Further verify the polymorphism mark that between parent and anti-sense pond, produces for the individual plant of segregating population.
4. in accordance with the method for claim 2, it is characterized in that the PCR reaction system is: template 20ng μ L wherein
-1, soybean gene group DNA 2 μ L, 5U μ L
-1Taq archaeal dna polymerase 0.2 μ L, 10 * PCR Buffer, 2 μ L, 2.5mMMgCl
22 μ L, 2.5mM dNTP 1.6 μ L, random primer 1 μ L, ddH
2O 11.2 μ L totally are 20 μ L.
5. according to claim 2 or 4 described methods, it is characterized in that the response procedures of pcr amplification is: 94 ℃ of pre-sex change 5min of the 1st circulation, the 2nd to be circulated to 35 cycling programs be 94 ℃ of sex change 1min, 36 ℃ of annealing 1min, 72 ℃ are extended 1min, and last circulates in insulation extension 5min in 72 ℃.
6. according to claim 2 or 4 described methods, it is characterized in that described random primer is S11, its sequence is CCCAGCTGTG.
7. obtain according to each described method of claim 1-6 with the linked molecule marker of the anti-Cyst nematode ospc gene of soybean, it is characterized in that described molecule marker is the dna fragmentation of about 700bp.
8. the application of the described molecule marker of claim 7 in the assisted selection of the sick soybean of anti-Cyst nematode.
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Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
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CN102206651A (en) * | 2011-04-27 | 2011-10-05 | 东北农业大学 | Soybean cyst nematode resistance gene and application thereof |
CN102690824A (en) * | 2012-06-14 | 2012-09-26 | 中国农业科学院植物保护研究所 | Rapid molecular detection method of heterodera elachista by specific RAPD (Randomly Amplified Polymorphic DNA) and SCAR (Sequence-characterized Amplified Region) markers |
CN103347382A (en) * | 2010-11-05 | 2013-10-09 | 农业基因遗传学有限公司 | Soybean markers linked to SCN resistance |
CN111471689A (en) * | 2019-01-23 | 2020-07-31 | 东北农业大学 | Gene for improving resistance of soybean to cyst nematode disease and application thereof |
CN116445441A (en) * | 2022-11-30 | 2023-07-18 | 东北农业大学 | Soybean glycosyltransferase and encoding gene and application thereof |
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2005
- 2005-12-16 CN CN 200510134363 patent/CN1814810A/en active Pending
Cited By (9)
Publication number | Priority date | Publication date | Assignee | Title |
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CN103347382A (en) * | 2010-11-05 | 2013-10-09 | 农业基因遗传学有限公司 | Soybean markers linked to SCN resistance |
CN103347382B (en) * | 2010-11-05 | 2016-09-14 | 农业基因遗传学有限公司 | Sex-linked Semen sojae atricolor mark is resisted with SCN |
CN102206651A (en) * | 2011-04-27 | 2011-10-05 | 东北农业大学 | Soybean cyst nematode resistance gene and application thereof |
CN102690824A (en) * | 2012-06-14 | 2012-09-26 | 中国农业科学院植物保护研究所 | Rapid molecular detection method of heterodera elachista by specific RAPD (Randomly Amplified Polymorphic DNA) and SCAR (Sequence-characterized Amplified Region) markers |
CN102690824B (en) * | 2012-06-14 | 2013-06-05 | 中国农业科学院植物保护研究所 | Rapid molecular detection method of heterodera elachista by specific RAPD (Randomly Amplified Polymorphic DNA) and SCAR (Sequence-characterized Amplified Region) markers |
CN111471689A (en) * | 2019-01-23 | 2020-07-31 | 东北农业大学 | Gene for improving resistance of soybean to cyst nematode disease and application thereof |
CN111471689B (en) * | 2019-01-23 | 2022-12-27 | 东北农业大学 | Gene for improving resistance of soybean to cyst nematode disease and application thereof |
CN116445441A (en) * | 2022-11-30 | 2023-07-18 | 东北农业大学 | Soybean glycosyltransferase and encoding gene and application thereof |
CN116445441B (en) * | 2022-11-30 | 2023-11-03 | 东北农业大学 | Soybean glycosyltransferase and encoding gene and application thereof |
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