CN108165648A - A kind of molecule labelling method for differentiating rice blast broad-spectrum resistance gene Bsr-d1 - Google Patents
A kind of molecule labelling method for differentiating rice blast broad-spectrum resistance gene Bsr-d1 Download PDFInfo
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Abstract
The present invention relates to a kind of molecule labelling methods for differentiating rice blast broad-spectrum resistance gene Bsr d1, belong to technical field of bioengineering.A pair of positive and negative primer of design, expands different rice plant DNA, if the characteristic bands that amplified production can be cut into 99bp, 214bp are the homozygote of the genes of d1 containing Bsr；If can not digestion, contain only 313bp characteristic bands be without Bsr d1 genes homozygote；If exist simultaneously three characteristic bands of 99bp, 214bp and 313bp, the heterozygote for Bsr d1 genes.The present invention can not only quickly and accurately identify in Rice Germplasm Resources or its breeding population whether contain rice blast broad-spectrum resistance gene Bsr d1, the homozygote and heterozygote of Bsr d1 genes can be further discriminated between simultaneously, predict offspring's genotype, the efficiency of selection to Rice Resistance To Rice Blast is greatly improved, accelerates breeding process.
Description
First, technical field
The present invention relates to a kind of molecule labelling methods for differentiating rice blast broad-spectrum resistance gene Bsr-d1, belong to biology
Engineering field is exclusively used in the germplasm identification of genotypic rice containing Bsr-d1 and breed breeding.
2nd, background technology
Rice is the most important cereal crops in China, and there is the population of more than half in the whole nation using rice as staple food.Influence rice
There are many factor of production safety, and wherein disease problem is one of significant obstacle for restricting Rice Production.According to statistics, hazard rice
Infectious disease has more than 300 kinds, some are distributed in the whole world, some are confined to some areas.In China, the rice that occurs throughout the year
Disease has 29 kinds of (thunder wealth woods etc., biology notification, 2004,39 (11):4-7).Wherein, by Pyricularia oryzae
(Magnaportheoryzae；Invisible element, Pyriculariaoryzae) caused by rice blast be most destructive on rice
One of disease is commonly called as " cancer " (Ou S H Rice Diseases, 2nd edn UK of rice:CAB International,
1985,109-201).Rice blast has generation in global 85 countries and regions for having Rice Cropping.According to statistics, 1975~
Between 16 years of nineteen ninety, the global Rice Yield Loss Caused as caused by rice blast be up to 1.57 hundred million t (Baker B, et al.,
Science, 1997,276：726-733).Primary fairly large rice blast harm just occurred every 7~10 years for China, aggrieved
Area loses 70~1,250,000 t (Shen M G, et al.Rice Blast Disease.UK up to 300~6,000,000 hm2, paddy：
Int Rice ResInst, 1994：321-331.)
The controlling way of rice blast is mainly chemical prevention and plantation disease-resistant variety, and long-term practice proves, selection and breeding and popularization
Disease-resistant variety is that rice blast is most economical, method effectively and safely for prevention.It is but general since Pyricularia oryzae has the variability of height
Resistance will gradually be lost after logical disease-resistant variety is planted 3~5 years.Therefore, select anti-spectrum is wide, resistance is lasting disease-resistant gene as
Selection and breeding new varieties are carried out in anti-source, have become one of most economical effective strategy of breeding for disease resistance.Traditional rice blast is disease-resistant
Breeding depends on the identification of resistant phenotype, and not only the period is long and is easily influenced by environmental condition and human factor, so as to
Reduce the efficiency of selection of target gene.In addition, due to being limited by the discriminating fungus strain that can differentiate different genes, merely rely on
Phenotypic evaluation is difficult to realize the polymerization of multiple resistant genes.Molecular marker assisted selection be utilize with target gene close linkage or
The molecular labeling isolated selects genotype, can be in rice at whole growth periods even Seed Identification target gene and its homozygosis
Property, and it is not affected by environment, thus substantially increase the efficiency and accuracy of selection.Exploitation closely connects with rice blast resistance gene
The molecular labeling of lock, the gene function that the DNA sequence dna difference of resistant variant is caused to be developed in itself in particular according to resistant gene
Label carries out molecular labeling and carries out assisted Selection, can not only greatly improve resistance screening, Resistance genes and breeding selection
Efficiency, and to cultivate lasting, broad-spectrum disease resistance breeding by gene pyramiding means.
Since the 1960s, Japanese system carried out the genetic research of blast resistant gene, domestic and international researcher is
More than 30 anti-rice blasts of a rice blast resistance gene, Pi-b, Pi-ta, Pi-d2, Pi9 etc. more than 80 are identified from different disease-resistant varieties
Ospc gene has been cloned (Yang Qinzhong etc., Scientia Agricultura Sinica, 2009,42 (5)：1601-1615).Li(Cell,2017,
170:66 parts of non-broad-spectrum disease resistance rice that 114-126) etc. using wide spectrum highly resistance rice paddy has been sequenced with genome carry out
GWAS (full-length genome association) is analyzed, and application highly resistance rice Di Gu feels the weight that material Lijiang xintuanheigu is parent's structure with high
Group self-mating system (disease resistance is identified through many years of field spontaneous induction and Seedling Inoculation) carries out common correlation analysis, it was found that coding C2H2
After the promoter natural variation of the gene Bsr-d1 of class transcription factor to rice blast there is the disease resistance of broad spectrum durable, further divide
Analysis finds that in ground paddy the promoter regions of Bsr-d1 genes leads to upstream myb transcription factor pair because a critical base makes a variation
The promoter of Bsr-d1 combines enhancing, so as to inhibit the expression of Bsr-d1 response Pyricularia oryzae inductions, and causes BSR-D1 direct
The H of regulation and control2O2Degrading enzyme gene expression is lowered, and makes H2O2Degradation weakens, intracellular H2O2Enrichment, improves the immune response of rice
And disease resistance.The allelic variation has not significant impact yield traits and rice quality while disease resistance is improved, thus has
There is highly important application value.
Therefore, isobase is become according to the promoter region key of Bsr-d1 genes and is designed what is isolated with target gene
PCR molecular labelings carry out quick, precise Identification Bsr-d1 genes different genotype, are beneficial to wide spectrum anti-rice blast rice new product
The selection and breeding of kind, so as to improve the technical merit of rice blast resistance breeding.
3rd, invention content
Technical problem：The present invention for the identification of Rice Resistance To Rice Blast improvement Breeding Process resistant phenotype easily by environment and
Human factor influences, so as to reduce the technical barrier of target gene selection material rate, according to rice blast broad-spectrum resistance gene Bsr-d1
Single base difference existing for functional area, the functional label that design, synthesis are isolated with target gene, and pass through and simply detect
Method, the Rice Germplasm Resources of Rapid identification Bsr-d1 containing broad-spectrum resistance gene, while molecular labeling can also be further applied
Assistant breeding.
Technical solution：
Kind differentiates the molecule labelling method of rice blast broad-spectrum resistance gene Bsr-d1, which is characterized in that used special
Property molecular labeling primer CAPs5-1 sequences are：
Forward primer CAPs5-1-F sequences are 5'-AGTCTAGCATCCACCGTTCCAC-3'
Reverse primer CAPs5-1-R sequences are 5'-GTAGGCAGGCAGTGGGATGA-3'.
With the genomic DNA of the Bsr-d1 gene-specific primers CAPs5-1 amplifying rice plant, amplified production passes through
Digestion with restriction enzyme, if it is possible to which the characteristic bands for being cut into 99bp, 214bp are the homozygote of the gene containing Bsr-d1；Such as
Fruit can not digestion, contain only 313bp characteristic bands be without Bsr-d1 genes homozygote；If it exists simultaneously
Three characteristic bands of 99bp, 214bp and 313bp, the then heterozygote for Bsr-d1 genes.
20 μ LPCR reaction systems include：10×PCR Buffer(Mg2+) 0.5 μ L, 5U/ μ of 2.0mL, dNTP (10mmol/L)
Reverse primer 1.0 μ L, DNA2.0 the μ L, ddH of forward primer 1.0 the μ L, 10pmol/L of Taq enzyme 0.2 the μ L, 10pmol/L of L2O
13.3μL。
PCR reaction conditions include：94 DEG C of pre-degeneration 5min, then 94 DEG C of denaturation 1min, 58 DEG C of annealing 1min, 72 DEG C of extensions
After 10 DEG C cool down 10min, amplified production plus sample-loading buffer are terminated by 1.5min, 32 cycles, last 72 DEG C of extensions 10min
Reaction；Amplified production adds in indicator after reaction, and amplified production is carried out electrophoresis, DuRed dyes on 1.5% agarose
Color, ultraviolet gel preserve image into phase system；
I digestions of AIU are carried out to the PCR product of CAPs5-1 amplifications, endonuclease reaction system is 10 μ L, and respectively PCR reactions are produced
5 μ L, 10 × Buffer R of object, 1 μ L, AIU I (10U/ μ L) 0.1 μ L, ddH23.9 μ L of O, again in 37 DEG C of thermostat water baths after mixing
Digestion 5-8h, digestion products carry out electrophoresis on 1.5% agarose, and DuRed dyeing is imaged through ultraviolet gel into phase system.
Advantageous effect：
A kind of molecule labelling method for differentiating rice blast broad-spectrum resistance gene Bsr-d1 provided by the invention, have with
Lower advantage：
(1) molecular labeling provided by the invention is the based on PCR amplification and limit designed according to the functional area difference of gene
The functional indicia of property restriction endonuclease processed, genotype can directly reflect the phenotype of plant, not only be not present due to heredity exchange and
Caused by mistake identification, and operationally it is accurate, efficiently, it is quick.
(2) molecule labelling method provided by the invention can realize the Rapid identification to paddy gene resource, can be from a large amount of water
The kind containing rice blast broad-spectrum resistance gene Bsr-d1 is accurately filtered out in rice resource, the rice product of these resistant genes
Kind can be used as excellent parent to be applied in Rice Resistance To Rice Blast improvement breeding in breeding.
(3) molecule labelling method provided by the invention can be widely used in the assistant breeding of Rice Resistance To Rice Blast improvement.It can
Effectively to carry out genotype selection to rice blast broad-spectrum resistance gene Bsr-d1 in segregating population, and heterozygosis base can be distinguished
Because of type and homozygous genotype, the foresight of breeding work is greatly improved.
Therefore, molecule labelling method provided by the invention, for rice blast broad-spectrum resistance gene Bsr-d1 is accelerated to exist
Application in rice breeding is of great significance.
4th, it illustrates
Fig. 1 detects the layout strategy of resistance gene of rice blast Bsr-d1 functional labels
(dash area represents primer position, and Blocked portion represents the digestion position for the AIU I that single base difference generates
Point)
Agarose gel electrophoresis result (M of the grads PCR amplified production of Fig. 2 Bsr-d1 gene functions label 1.5%：
DNA molecular marker；a：100bp；b：250bp；c：500bp；d：750bp；e；1kb, f；2kb；It is CAPs5-1 grads PCRs on the left of M
Amplified production；It is CAPs5-2 grads PCR amplified productions on the right side of M；1-12 annealing temperatures are respectively：45.1℃、45.3℃、45.9
℃、46.8℃、47.9℃、49.1℃、50.4℃、51.7℃、52.8℃、53.8℃、54.5℃、54.8℃)
Fig. 3 CAPs5-1PCR amplified productions after digestion 1.5% agarose gel electrophoresis result (M：DNA molecular mark
Note；a：100bp；b：250bp；c：500bp；d：750bp；e；1kb, f；2kb；1-6：Ground paddy, Nipponbare, southern round-grained rice 9108,
Guanglu ai 4, Wu-Yu-Geng 3, southern round-grained rice 45；It is pcr amplification product on the left of M；It is the product after digestion on the right side of M)
The sequence alignment result of Fig. 4 Some Rice Varieties Bsr-d1 gene locis functional areas
Fig. 5 Bsr-d1 gene functions mark Molecular Detection result (Ms of the CAPs5-1 to Some Rice Varieties：DNA molecular mark
Note；a：100bp；b：250bp；c：500bp；d：750bp；e；1kb, f；2kb；1-24：Ground paddy, Nipponbare；Nanjing 1；
Mianhui 501；Zhejiang extensive 7954；Yanhui 559；Zhenhui 084；BG90-2；Gui 630；Bright extensive 63；Four short 16B of blueness；Spoke extensive 718；Raise rice 6
Number；Ridge 46B；Land wealth number；Bright extensive 78；IRBB21；Another name for Sichuan Province extensive 527；It is No. 1 mostly extensive；Osmanthus is towards No. 2；Mianhui725；Happy extensive 188；It is wide extensive
128；Zhenshan 97B)
5th, specific embodiment
Fully to disclose a kind of molecule labelling method for differentiating rice blast broad-spectrum resistance gene Bsr-d1 of the present invention, with
Lower combined method verification and embodiment are illustrated.Its specific implementation step is as follows：
(1) test material
Be Rice Production commercial variety paddy (Fujian), Nipponbare (Japan), southern round-grained rice 9108 (Jiangsu), wide land it is short
No. 4 (Guangdong), Wu-Yu-Geng 3 (Jiangsu), southern round-grained rice 45 (Jiangsu), Nanjing No. 1 (Jiangsu), Mianhui 501 (Sichuan), Zhejiang extensive 7954
(Zhejiang), Yanhui 559 (Jiangsu), Zhenhui 084 (Jiangsu), BG90-2 (Sri Lanka), Gui 630 (Guyana), bright extensive 63 (good fortune
Build), it is green four short 16B (Guangdong), spoke extensive 718 (Sichuan), Yang No.6 rice (Jiangsu), ridge 46B (Sichuan), land wealth number (Fujian), bright extensive
78 (Fujian), IRBB21 (Philippine), another name for Sichuan Province extensive 527 (Sichuan), how extensive No. 1 (Sichuan), osmanthus are towards No. 2 (Guangdong), Mianhui725 (four
River), happy extensive 188 (Sichuan), wide extensive 128 (Guangdong), Zhenshan 97B (Zhejiang).
More than material is public material, and Jiangsu Province agricultural germ plasm resource mid-term library can provide free.With specific reference to
Document is：You Nianshun etc., the selection and breeding of ground paddy sterile line and its main feature, Fujian agriculture science and technology, 1991,1：5-6；Jiang Yunhong,
Nipponbare high-yielding rice cultivation technique, Shandong agricultural sciences, 1981,2：28-29；Wang Cailin etc., excellent flavour japonica rice new varieties
The selection and breeding and utilization of southern round-grained rice 9108,2013,41 (9)：86-88；Huangyan county Institute of agricultural sciences, the volume increase of early rice Guanglu ai 4
The discussion of approach, Zhejiang Agriculture science, 1980,2：59-64；Zou Jiangshi etc., the cultivation design of force fortune round-grained rice 3 and production performance,
Jiangsu's agriculture science, 1994,6：7-9；Zhong Weigong etc., the selection and breeding of japonica rice new varieties south round-grained rice 45 and cultivation technique, Jiangsu's agriculture section
It learns, 2009,5：123-125；Lin Shicheng etc., rice in China kind and its pedigree, Shanghai science tech publishing house, 1991:57；It thanks
Chong Hua etc., the selection and breeding of rice restorer Mianhui 501 and the application of series of combination, hybrid rice, 1997,12 (3)：8-10；Huang Yi
Peak etc., the key technology of the extensive 7954 realization high-yield seeds production in Zhejiang, Zhejiang Agriculture science, 2009,2：339-340；Yao Lisheng etc., wide spectrum
Property restorer Yanhui 559 and its serial hybridization rice combination selection and breeding, Jiangsu's agriculture journal, 2009,25 (3)：469-473；Sheng Shenglan
Deng, the selection and breeding and utilization of Indica Rice Restorer Line Zhenhui 084, hybrid rice, 2002,17 (2)：6-7；Li Yuhong etc., rice backbone parent
This BG90-2 raise rice series cultivate in effect and to bacterial leaf spot resistance, 2011,25 (4)：439-442；Wang Deshi, Gui
630 and its hybrid rice variety of combo, Fujian agriculture science and technology, 1979,357-59；Wu Fangxi etc., indica hybrid rice restorer
Bright extensive 63 utilization and innovation, Fujian agriculture science, 2011,26 (6)：1101-1112；Wan Jianmin, rice in China genetic breeding
With kind pedigree, Chinese agriculture publishing house, 2009,624；Huo Erwei etc., the double spokes excellent 718 of high-yield disease resisting New Hybrid Rice Combination,
Hybrid rice, 2011,26 (5)：88-89；Xu Maolin etc., the selection and breeding and utilization of the disease-resistant middle Xian new varieties Yang No.6 rice of good quality and high output,
Chinese rice, 2001,1：24-26；Peng Huabi etc., the Techniques for Gang are summarized, hybrid rice, and 2002,17
(6)：28-29；Xu Xuzeng, early Indica Lotus pools morning, land wealth number, southern No. 1 clear, the identification of short-foot south top grade kind typicalness discussion, Zhejiang
River agricultural sciences, 1964,9：436-438；Zheng Jiatuan, restorer " selection and breeding of bright extensive 78 " and its research of feature, Fujian
Agricultural science and technology, 1995,2：2-3；Once it arranged and first waits Resistant reactions of the IRBB21 (Xa21) to 5 microspecies of Guangdong rice bacterial leaf spot pathogenic bacteria,
Plant protection journal, 2002,29 (2)：97-100；Wang Yu equalitys, the selection and breeding in high-combining ability high-grade rice restorer another name for Sichuan Province extensive 527 with
It utilizes, hybrid rice, 2004,19 (4)：12-14；Guo Futai etc., special excellent polyphyly 1, hybrid rice, 1998,13 (4)：32；Liu
Latitude, new rice variety " osmanthus is towards No. 2 ", Wenzhou agricultural science and technology, 1980,3：13-14；Huang Tingyou etc., Mianhui725 and its related parent
ISSR analysis, Xinan Science and Technology Univ.'s journal, 2008,23 (1)：87-90；Li Qianan etc., stable high yield New Hybrid Rice Combination
Rong 18 excellent 188, hybrid rice, 2013,28 (3)：77-78；Guo Guoqiang etc., wide extensive 128 are measured F with different type sterile line1Generation
Performance research, Guangxi Agricultural science, 2004,35 (4)：279-281；Luo Tiankuan etc., long-grained nonglutinous rice Zhenshan 97B mature embryo regeneration induction body
The foundation and optimization of system, hybrid rice, 2013,28 (6)：69-72.
(2) differentiate the acquisition of resistance gene of rice blast Bsr-d1 genotype molecules label
The exploitation of 1 molecular labeling
(1) nucleotide sequence analysis of resistance gene of rice blast Bsr-d1 variant sites
According to Li (Cell, 2017,170:Result of study 114-126) etc., Bsr-d1 disease-resistant genes are in LOC-
Os03g32230 promoter 618bp sites are G bases, and susceptible gene bsr-d1 is A bases in the site, should in disease-resistant variety
Variation causes upstream myb transcription factor to combine enhancing to the promoter of Bsr-d1, so as to which Bsr-d1 response Pyricularia oryzaes be inhibited to lure
The expression led, and lead to the H of BSR-D1 direct regulations and controls2O2Degrading enzyme gene expression is lowered, and makes H2O2Degradation weakens, intracellular H2O2
Enrichment improves immune response and the disease resistance of rice.Bioinformatic analysis is carried out to the functional area, finds disease-resistant gene
The variation of this single base produces an AIU I limitation restriction enzyme site (AG ∧ CT), and susceptible gene does not contain AIU I digestions
Site (AACT).(Fig. 1)
(2) design of primers
Using the nucleotides sequence of Bsr-d1 genes be listed in rice genome website (Gene Bank,
Www.ncbi.nlm.nih.gov/ pac clone (P1-derived where it is located at the 3rd chromosome of rice) is downloaded
Artificial chromosome, PAC) nucleic acid sequence (AP014959), and correlated series are analyzed；It utilizes
Primer Premier 5.0(http://www.premierbiosoft.com) it is designed in the both sides of single base variable region
Primer comprising the variant sites, and ensure that the segment after digestion can divide electrophoresis preferably on the Ago-Gel of low concentration
Analysis.The specific amplification of DNA fragmentation that PCR product for Bsr-d1 functional labels extension in this clear and definite research is Bsr-d1, if
The functional area for measuring sequence primer pair part kind Bsr-d1 genes carries out PCR amplification, sequencing, further authentication function label
Accuracy.This research institute synthetic primer is shown in Table 1, and primer is synthesized by Invitrogen Chinese companies.
The CAPs primers and sequencing primer that table 1 is designed based on variant sites
The verification of 2 molecular labelings
(1) rice plants extracting genome DNA
The extraction of rice plant genomic DNA is with reference to SDS methods (Dellaporta S L, et al., PlantMolB
iolRep,1983,1(1):19221.).The specific steps are：Rice plant of tillering stage blade 1g or so is taken, the mortar of the precooling at -20 DEG C
It is middle with liquid nitrogen grinding and to be packed into 2.0mL centrifuge tubes；Addition 600uL extracting solutions (20%SDS, 1M Tris-HCl, 0.5M EDTA,
5M NaCl, 65 DEG C of preheatings), it shakes up, 65 DEG C of warm bath 30min, centre oscillation 3~4 times；1/4 volume 5M KAC are added in, after shaking up
Put 30min on ice；Add in chloroform-isoamyl alcohol (24:1) 300~400uL fully vibrates on shaking table, 120rpm, 30min；8,
000~10,000rpm centrifuge 15min, and liquid level layering, lower floor's color is deeper, upper strata micro-strip yellow green, and taking supernatant, (400uL is left
It is right) to another centrifuge tube；Add in isometric chloroform-isoamyl alcohol (24:1) it, is fully vibrated on shaking table, 80~90rpm, 30min；8,
000rpm centrifuges 15min, transfer supernatant (400uL or so) to new centrifuge tube；Add in the anhydrous second of -20 DEG C of precoolings of 2 times of volumes
Alcohol is gently shaken up until having floccule generation, 12,000rpm centrifugation 6min；Absolute ethyl alcohol is abandoned, adds in 4 DEG C of 70% ethyl alcohol, is placed
10min abandons supernatant, 1h is air-dried on superclean bench；Add in 100~200uLTE, -20 DEG C of preservations.
(2) amplification of molecular labeling and electrophoresis detection
20 μ LPCR reaction systems include：10×PCR Buffer(Mg2+) 2.0mL, dNTP (10mmol/L) 0.5 μ L, Taq
Enzyme (5U/ μ L) 0.2 μ L, 1.0 μ L of forward primer (10pmol/L), reverse primer (10pmol/L) 1.0 μ L, DNA2.0 μ L, ddH2O
13.3μL。
PCR reaction conditions include：94 DEG C of pre-degeneration 5min, then 94 DEG C of denaturation 1min, 58 DEG C of annealing 1min, 72 DEG C of extensions
After 10 DEG C cool down 10min, amplified production plus sample-loading buffer are terminated by 1.5min, 32 cycles, last 72 DEG C of extensions 10min
Reaction.
Amplified production adds in indicator (0.25% bromjophenol blue, 0.25% dimethylbenzene blueness FF, 40% sucrose water after reaction
Solution), amplified production is subjected to electrophoresis, DuRed dyeing on 1.5% agarose, ultraviolet gel preserves image into phase system.
(3) screening and verification of molecular labeling
Ground paddy (Digu), Nipponbare (Nip), southern round-grained rice 9108 (NJ9108), Guanglu ai 4 are extracted in SDS methods
(GLA4), Wu-Yu-Geng 3 (WYJ3) and the DNA of southern round-grained rice 45 (NJ45) are template, and gradient is carried out using the CAPs primers that table 1 designs
PCR amplification, gradient scope are 45-55 DEG C, and amplified production is at 1.5% agarose electrophoretic analysis (Fig. 2).As seen from Figure 2,
CAPs5-1 is the most clear, single and without miscellaneous band for the PCR product bands of 50 DEG C of amplifications in annealing temperature.
I digestions of AIU are carried out to the PCR product of CAPs5-1 amplifications.Endonuclease reaction system is 10 μ L, and respectively PCR reactions are produced
5 μ L, 10 × Buffer R of object, 1 μ L, AIU I (10U/ μ L) 0.1 μ L, ddH2O 3.9μL.Again in 37 DEG C of thermostat water baths after mixing
Digestion 5-8h, digestion products carry out electrophoresis on 1.5% agarose, and DuRed dyeing (is schemed through ultraviolet gel into phase system imaging
3).PCR product in Fig. 3 on the left of M for CAPs5-1 amplifications, M right sides are the digestion of PCR product as a result, containing as seen from Figure 3
Paddy can be respectively formed the characteristic bands of 99bp, 214bp by I digestions of AIU with having the rice varieties of Bsr-d1 genes；And it is free of
The rice varieties Nipponbare for having Bsr-d1 genes cannot only form the characteristic bands of 313bp by I digestions of AIU；Wide land is short by 4
Number amplified production can illustrate that Guanglu ai 4 contains rice blast wide spectrum and resists by I digestions of AIU into the characteristic bands of 99bp, 214bp
Property gene Bsr-d1；The amplified production of 3 kinds such as southern round-grained rice 9108, Wu-Yu-Geng 3 and southern round-grained rice 45 cannot be said by I digestions of AIU
Bsr-d1 genes are not contained in these bright kinds.
In order to further verify Bsr-d1 gene functions label CAPs5-1 testing results accuracy, we over the ground paddy,
6 rice varieties such as Nipponbare, southern round-grained rice 9108, Guanglu ai 4, Wu-Yu-Geng 3 and southern round-grained rice 45 have carried out Bsr-d1 genes
The overall length sequencing of functional site, is found by sequence alignment, can be by the Guanglu ai 4 of I digestions of AIU and Bsr-d1 work(in ground paddy
Energy regional sequence is completely the same, i.e., is G bases at the 618bp of Bsr-d1 gene promoters；Southern round-grained rice 9108, Wu-Yu-Geng 3 and
Bsr-d1 sequences and Nipponbare are completely the same in 3 kinds that can not be digested of southern 45 grade of round-grained rice, i.e., in Bsr-d1 genes
It is A bases (Fig. 4) at the 618bp of promoter.It therefore, can using CAPs5-1 I digestion methods of PCR amplification combination AIU marked
Go out in rice varieties whether contain rice blast broad-spectrum resistance gene Bsr-d1 with precise Identification.
(4) to the Bsr-d1 genotype identifications of rice pest insects
22 parts of rice varieties from different regions are carried out with PCR amplification, PCR amplification production using molecular labeling CAPs5-1
Object is detected through I digestion rear electrophoresis of AIU.As a result it shows：Mianhui 501, Zhejiang are extensive 7954, Zhenhui 084, BG90-2, Gui 630, it is bright extensive 63,
Spoke is extensive 718, Yang No.6 rice, bright extensive 78, IRBB21, another name for Sichuan Province are extensive 527, No. 1 how extensive, Mianhui725, happy extensive 188 and extensive 128 etc. 15 wide
Kind CAPs5-1PCR products can not form the characteristic bands of 313bp by I digestions of AIU, consistent with Nipponbare, therefore
Bsr-d1 genes are not contained in these kinds；Nanjing 1, Yanhui 559, four short 16B of blueness, ridge 46B, land wealth number, osmanthus towards No. 2 and
The feature item that the CAPs5-1 PCR products of 7 kinds such as Zhenshan 97B by I digestions of AIU, can form 99bp, 214bp is special, with ground
The banding pattern of paddy is completely the same, illustrates in this 7 rice varieties containing rice blast broad-spectrum resistance gene Bsr-d1, these rice product
Kind can improve (Fig. 5) as excellent parent in breeding for the resistance of rice blast.Therefore, it is marked by CAPs5-1
I digestion energy precise Identification Rice Resistance To Rice Blast germ plasm resources of PCR amplification combination AIU.
Sequence table
<110>Jiangsu Province Agriculture Science Institute
<120>A kind of molecule labelling method for differentiating rice blast broad-spectrum resistance gene Bsr-d1
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 22
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<221> 5’UTR
<222> (1)..(22)
<400> 1
agtctagcat ccaccgttcc ac 22
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<221> 5’UTR
<222> (1)..(20)
<400> 2
gtaggcaggc agtgggatga 20
<210> 3
<211> 25
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<221> 5’UTR
<222> (1)..(25)
<400> 3
ttttatagga cagagggaat atgta 25
<210> 4
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<221> 5’UTR
<222> (1)..(20)
<400> 4
gcagtgggat gaacctgtac 20
<210> 5
<211> 22
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<221> 5’UTR
<222> (1)..(20)
<400> 5
agtctagcat ccaccgttcc ac 22
<210> 6
<211> 22
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<221> 5’UTR
<222> (1)..(22)
<400> 6
atgatttgat gggattgatt gc 22
<210> 7
<211> 25
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<221> 5’UTR
<222> (1)..(22)
<400> 7
ttttatagga cagagggaat atgta 25
<210> 8
<211> 23
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<221> 5’UTR
<222> (1)..(23)
<400> 8
gcgaggtact cctccttgtt gat 23
Claims (5)
1. a kind of molecule labelling method for differentiating rice blast broad-spectrum resistance gene Bsr-d1, which is characterized in that used special
Property molecular labeling primer CAPs5-1 sequences are：
Forward primer CAPs5-1-F sequences are 5'-AGTCTAGCATCCACCGTTCCAC-3'
Reverse primer CAPs5-1-R sequences are 5'-GTAGGCAGGCAGTGGGATGA-3'.
2. a kind of molecule labelling method for differentiating rice blast broad-spectrum resistance gene Bsr-d1 according to claim 1,
It is characterized in that, described in claim 1 Bsr-d1 gene-specific primers CAPs5-1 amplifying rice plant genomic DNA,
Amplified production passes through digestion with restriction enzyme, if it is possible to which the characteristic bands for being cut into 99bp, 214bp are gene containing Bsr-d1
Homozygote；If can not digestion, contain only 313bp characteristic bands be without Bsr-d1 genes homozygote；If
Three characteristic bands of 99bp, 214bp and 313bp are existed simultaneously, then the heterozygote for Bsr-d1 genes.
3. a kind of molecular labeling side for differentiating rice blast broad-spectrum resistance gene Bsr-d1 according to claim 1 or 2
Method, which is characterized in that 20 μ L PCR reaction systems include：10×PCR Buffer(Mg2+) 2.0mL, dNTP (10mmol/L) 0.5
1.0 μ L, the DNA2.0 μ of reverse primer of forward primer 1.0 the μ L, 10pmol/L of Taq enzyme 0.2 the μ L, 10pmol/L of μ L, 5U/ μ L
L, ddH2O 13.3μL。
PCR reaction conditions include：94 DEG C of pre-degeneration 5min, then 94 DEG C of denaturation 1min, 58 DEG C of annealing 1min, 72 DEG C of extensions
After 10 DEG C cool down 10min, amplified production plus sample-loading buffer are terminated by 1.5min, 32 cycles, last 72 DEG C of extensions 10min
Reaction；Amplified production adds in indicator after reaction, and amplified production is carried out electrophoresis, DuRed dyes on 1.5% agarose
Color, ultraviolet gel preserve image into phase system；
4. a kind of molecular labeling side for differentiating rice blast broad-spectrum resistance gene Bsr-d1 according to claim 1 or 2
Method, which is characterized in that I digestions of AIU are carried out to the PCR product of CAPs5-1 amplifications, endonuclease reaction system is 10 μ L, respectively PCR
5 μ L, 10 × Buffer R of reaction product, 1 μ L, AIU I (10U/ μ L) 0.1 μ L, ddH23.9 μ L of O, again in 37 DEG C of constant temperature after mixing
Water-bath digestion 5-8h, digestion products carry out electrophoresis on 1.5% agarose, DuRed dyeing, through ultraviolet gel into phase system into
Picture.
5. a kind of molecule labelling method for differentiating rice blast broad-spectrum resistance gene Bsr-d1 according to claim 3,
It is characterized in that, carrying out I digestions of AIU to the PCR product of CAPs5-1 amplifications, endonuclease reaction system is 10 μ L, and respectively PCR is anti-
Answer 5 μ L, 10 × Buffer R of product, 1 μ L, AIU I (10U/ μ L) 0.1 μ L, ddH23.9 μ L of O, again in 37 DEG C of thermostatted waters after mixing
Bath digestion 5-8h, digestion products carry out electrophoresis on 1.5% agarose, DuRed dyeing, through ultraviolet gel into phase system into
Picture.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109486854A (en) * | 2018-11-29 | 2019-03-19 | 宜春学院 | A kind of rice blast resistance of wide spectrum Rice Germplasm Resources method for creating |
| CN109652439A (en) * | 2018-12-27 | 2019-04-19 | 宜春学院 | Utilize the method for the CRISPR/Cas9 adenine base editing system improvement rice blast resistance of wide spectrum mediated |
| CN112522432A (en) * | 2020-12-17 | 2021-03-19 | 华智生物技术有限公司 | Molecular marker for assisted breeding of rice blast resistance gene Bsr-d1 and application thereof |
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| CN103866036A (en) * | 2014-04-02 | 2014-06-18 | 江苏省农业科学院 | Method for identifying genes of 1000-grain weight of rice TGW6 by molecular markers |
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| CN103866036A (en) * | 2014-04-02 | 2014-06-18 | 江苏省农业科学院 | Method for identifying genes of 1000-grain weight of rice TGW6 by molecular markers |
| CN107022634A (en) * | 2017-05-25 | 2017-08-08 | 江苏省农业科学院 | A kind of molecule labelling method for differentiating rice ear sprouting period gene qHD7.4 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109486854A (en) * | 2018-11-29 | 2019-03-19 | 宜春学院 | A kind of rice blast resistance of wide spectrum Rice Germplasm Resources method for creating |
| CN109652439A (en) * | 2018-12-27 | 2019-04-19 | 宜春学院 | Utilize the method for the CRISPR/Cas9 adenine base editing system improvement rice blast resistance of wide spectrum mediated |
| CN112522432A (en) * | 2020-12-17 | 2021-03-19 | 华智生物技术有限公司 | Molecular marker for assisted breeding of rice blast resistance gene Bsr-d1 and application thereof |
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