CN101240346A - Method for analyzing rice blast resisting gene - Google Patents
Method for analyzing rice blast resisting gene Download PDFInfo
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- CN101240346A CN101240346A CNA2008100581786A CN200810058178A CN101240346A CN 101240346 A CN101240346 A CN 101240346A CN A2008100581786 A CNA2008100581786 A CN A2008100581786A CN 200810058178 A CN200810058178 A CN 200810058178A CN 101240346 A CN101240346 A CN 101240346A
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Abstract
The invention relates to a method for analyzing rice blast resistance gene. The technical scheme of the invention is: 1)searching a property-stable avirulent strain; 2)conversing the strain by agrobacterium-mediated method, inoculating the transformant on near-isogenic lines of rice, getting single-ascospore strain having mutated pathogenicity from susceptible type spot; and 3)return inoculating, identifying and confirming the pathogenicity of the single-ascospore strain, and getting a set of near strains for identifying resistance gene of varieties. The invention can construct a set of rice blast resistance near-isogenic gene stains to deduce resistance gene, directly inoculate stable variety by mutant without using resistance after rice hybridization and inoculation hybridization, as well as allelism test, find out the resistance gene of variety according to the resistance type; and can be used for deducing resistance gene of rice variety containing single or multiple resistance genes.
Description
Technical field:
The present invention relates to closely wait bacterial strain to carry out a kind of method that blast resistant gene is analyzed platymiscium protection field with Pyricularia oryzae.
Background technology:
Rice blast is a kind of global disease, facts have proved for many years: cultivate and the plantation disease-resistant variety is that this disease of control is the safest, economy, effectively preventing measure.But rice blast fungus is one is easy to the cause of disease colony that morphs, often makes a new kind of promoting just lose resistance in plantation after 3～5 years.In order to give stable high yields irrespective of drought or water logging, breeding man has to utilize new disease-resistant gene or adopts the incompatible seed selection new disease-resistant varieties of genome, and therefore, blast resistant gene analysis accurately and reliably is significant to the paddy disease-resistant breeding.Up to the present, people have approximately identified more than 40 blast resistant gene.Yet these disease-resistant genes are by different laboratories, and different year, different location adopt different strains to identify out.Because it is strict to onset condition that rice blast resistance is identified, have only the genetic background of seedling age when inoculation, growth potential, bacterial strain uses therefor, under all consistent condition of the temperature during inoculation, humidity, used spore amount or the like, the inoculation result who is obtained is just reliably.At present, the bacterial strain difference that adopt in each laboratory, the genetic background of each bacterial strain is inconsistent, and inoculates under different inoculation conditions, and the result comparison that is obtained is relatively poor.The disease-resistant gene that to be each laboratory derive according to the performance of paddy rice segregating generation resistance does not have comparability, different disease-resistant genes may be named to be identical gene, identical gene is named be different genes.In order to prove the real relation of each gene inter-entity, carry out cloned resistance gene and functional analysis, then need to do a large amount of test crosses and allelomorphism analysis.This is a very heavy work, and the relation between different disease-resistant genes all analyzed clear is difficult to realize.
Summary of the invention:
Main purpose of the present invention is the deficiency that overcomes existing method, and a kind of accurate, efficient, easy, method of analyzing of blast resistant gene fast is provided, for rice blast breeding for disease resistance and cloned resistance gene and functional analysis lay the foundation.
The technical scheme of present method:
1 utilizes one to containing the rice near isogenic line of more than 30 disease-resistant gene, comprise all strains that generally believe the Lijiang xintuanheigu that does not have disease-resistant gene, all can not morbific bacterial strain CY2, adopt agrobacterium mediation method, and obtain a large amount of transformants;
2 are inoculated into the transformants that obtain on the rice near isogenic line of known disease-resistant gene, obtain single-ascospore strain from susceptible type scab, and tieback is fastened near isogene, confirm pathogenicly, have obtained 35 pathogenic bacterial strains that make a variation that taken place;
3 divide the bunch planting kind rice varieties to be measured in seedling pan, and with Lijiang xintuanheigu as susceptible contrast, manage with ordinary method, standby when treating rice shoot length to 3 leaves, 1 heart;
4 cultivate 35 bacterial strains respectively on oat medium, prepare spore suspension behind the product spore;
5 are inoculated into the spore suspension for preparing respectively on the rice shoot to be measured, inoculum density, and inoculation method is with conventional;
6 after preserving moisture 7 days under 25-28 ℃, observes the record state of an illness according to a conventional method, determines anti-sense response type, the genotype of the kind to be measured of deriving.
Rice blast fungus and paddy rice are the disease systems of a gene pairs gene relationship.The evaluation of paddy disease-resistant gene and avirulence gene of rice blast is a kind of relation of interdependence with deriving.Promptly utilize the disease-resistant gene of the pathogenic type derivation rice varieties of rice blast fungi isolates to form, utilize the nontoxic gene or the Disease-causing gene of the disease-resistant gene derivation bacterial strain of paddy rice to form.Based on this theoretical basis, structure one cover Pyricularia oryzae that just can be artificial closely waits bacterial strain to carry out resistant gene derivation work.If utilize an on all four bacterial strain of genetic background, the different pathogenic mutant of screening, make up near grade for bacterial strain, can realize then that it goes without doing paddy rice cross breeding and inoculation filial generation disease resistance, also need not pass through allelism test, just can according to disease-resistant type, understand the resistant gene of kind and form by directly utilizing the stable kind of mutant inoculation.
Variation has taken place in what 35 pathogenic bacterial strains that morph that we obtain had on a site, what have on a plurality of sites variation has taken place, each bacterial strain has caused a disease type constitution pathotype matrix.Therefore, utilize this cover Disease-causing gene closely to wait bacterial strain not only to can be used for containing the disease-resistant gene derivation of single disease-resistant gene water kind kind, also can be used for containing the derivation of a plurality of disease-resistant gene rice varieties.The particularly application of this cover bacterial strain, can change the traditional method of analyzing disease-resistant gene in the past by paddy rice cross breeding, test cross, make the result who identifies disease-resistant gene accurate more, reliable, resulting result has eurytropy, avoids the inconsistent situation of result of different experiments chamber inoculation to occur; Utilize this cover bacterial strain can also find new disease-resistant gene.
Embodiment:
Be described in further detail the present invention with examples of implementation below.Varietal resistance adopts seedling pest identification method to carry out in the usual way.
Embodiment one
1 adopts agrobacterium mediation method, transforms extensive avirulent bacterial strain and obtains CY2, obtains a large amount of transformants;
2 transformants with acquisition are inoculated on the rice near isogenic line of known disease-resistant gene, obtain single-ascospore strain from susceptible type scab, tieback is fastened near isogene, confirms pathogenic, 3 pathogenic bacterial strain T1 (VirPi-ta) that variation takes place have been obtained, T2 (VirPi-ta
2) and T3 (VirPi-a);
3 near isogenic lines that in seedling pan, divide bunch planting kind rice varieties to be measured and 3 corresponding known disease-resistant genes (A, B, C), and with Lijiang xintuanheigu as susceptible contrast, manage with ordinary method, standby when treating rice shoot length to 3 leaves, 1 heart;
4 cultivate 3 bacterial strains and wild type strain CY2 respectively on oat medium, prepare spore suspension behind the product spore;
5 are inoculated into the spore suspension for preparing respectively on the rice shoot to be measured, inoculum density, and inoculation method is with conventional;
6 after preserving moisture 7 days under 25-28 ℃, observes the record state of an illness according to a conventional method, determines anti-sense response type, the genotype of the kind to be measured of deriving.
Table 1 inoculation is table as a result
From the inoculation result of last table as can be seen, the genotype of kind to be measured is-Pi-a.
Embodiment two
1 adopts agrobacterium mediation method, transforms extensive avirulent bacterial strain and obtains CY2, obtains a large amount of transformants;
2 transformants with acquisition are inoculated on the rice near isogenic line of known disease-resistant gene, obtain single-ascospore strain from susceptible type scab, tieback is fastened near isogene, confirms pathogenicly, has obtained 4 pathogenic bacterial strain T1 (VirPi-5 that variation has taken place, Pi-7), T2 (VirPi-7), and T3 (VirPi-5, Pi-9), T4 (VirPi-7, Pi-12).
3 divide the bunch planting kind rice varieties to be measured and the near isogenic line of 4 corresponding known disease-resistant genes in seedling pan, and with Lijiang xintuanheigu as susceptible contrast, manage with ordinary method, standby when treating rice shoot length to 3 leaves, 1 heart;
4 cultivate 4 bacterial strains and wild type strain CY2 respectively on oat medium, prepare spore suspension behind the product spore;
5 are inoculated into the spore suspension for preparing respectively on the rice shoot to be measured, inoculum density, and inoculation method is with conventional;
6 after preserving moisture 7 days under 25-28 ℃, observes the record state of an illness according to a conventional method, determines anti-sense response type, the genotype of the kind to be measured of deriving.
Table 2 inoculation is table as a result
From the inoculation result of last table as can be seen, the genotype of kind to be measured is identical with B, is Pi-7 (t).
Embodiment three
1 adopts agrobacterium mediation method, transforms extensive avirulent bacterial strain and obtains CY2, obtains a large amount of transformants;
2 transformants with acquisition are inoculated on the rice near isogenic line of known disease-resistant gene, obtain single-ascospore strain from susceptible type scab, tieback is fastened to corresponding product, confirm pathogenic, 8 pathogenic bacterial strain T1 (VirPi-a that variation takes place have been obtained, Pi-i), T2 (VirPi-i), T3 (VirPi-K
s), T4 (VirPi-K
P, Pi-k
H), T5 (VirPi-k
H), T6 (VirPi-Z, Pi-ta), T7 (VirPi-ta), T8 (VirPi-b);
3 divide the bunch planting kind rice varieties to be measured and the near isogenic line of 8 corresponding known disease-resistant genes in seedling pan, and with Lijiang xintuanheigu as susceptible contrast, manage with ordinary method, standby when treating rice shoot length to 3 leaves, 1 heart;
4 cultivate 8 bacterial strains and wild type strain CY2 respectively on oat medium, prepare spore suspension behind the product spore;
5 are inoculated into the spore suspension for preparing respectively on the rice shoot to be measured, inoculum density, and inoculation method is with conventional;
6 heat and moisture preservings are observed the record state of an illness after 7 days according to a conventional method, determine anti-sense response type, the genotype of the kind to be measured of deriving.
Table 3 inoculation is table as a result
Note：A＝Pi-a；B＝Pi-i；C＝Pi-K
s；D＝Pi-k
P；E＝Pi-k
h；F＝Pi-Z；G＝Pi-ta；H＝Pi-b
From the inoculation result of last table as can be seen, the genotype of kind to be measured is identical with B, is Pi-i.
Embodiment four
1 adopts agrobacterium mediation method, transforms extensive avirulent bacterial strain and obtains CY2, obtains a large amount of transformants;
2 are inoculated into the transformants that obtain on the rice near isogenic line of known disease-resistant gene, obtain single-ascospore strain from susceptible type scab, and tieback is fastened to corresponding product, confirm pathogenicly, have obtained 5 pathogenic bacterial strain T1 (VirPi-k that make a variation that taken place
P), T2 (VirPi-k
h, Pi-Z), T3 (VirPi-Z), T4 (VirPi-Z
5), T5 (VirPi-k
t, VirPi-Z
5).
3 divide the bunch planting kind rice varieties to be measured and the near isogenic line of 5 corresponding known disease-resistant genes in seedling pan, and with Lijiang xintuanheigu as susceptible contrast, manage with ordinary method, standby when treating rice shoot length to 3 leaves, 1 heart;
4 cultivate 5 bacterial strains and wild type strain CY2 respectively on oat medium, prepare spore suspension behind the product spore;
5 are inoculated into the spore suspension for preparing respectively on the rice shoot to be measured, inoculum density, and inoculation method is with conventional;
6 heat and moisture preservings are observed the record state of an illness after 7 days according to a conventional method, determine anti-sense response type, the genotype of the kind to be measured of deriving.
Table 4 inoculation is table as a result
From the inoculation result of last table as can be seen, the genotype of kind to be measured is Pi-Z.
Claims (1)
1, a kind of method of analyzing rice blast resisting gene the steps include:
1) utilize one to containing the rice near isogenic line of 30 disease-resistant genes, comprise all strains that generally believe the Lijiang xintuanheigu that does not have disease-resistant gene, all can not morbific bacterial strain CY2, adopt agrobacterium mediation method, obtain a large amount of transformants;
2) transformant that obtains is inoculated on the rice near isogenic line of known disease-resistant gene, obtains single-ascospore strain from susceptible type scab, tieback is fastened to corresponding product, confirms pathogenicly, has obtained 35 pathogenic bacterial strains that make a variation that taken place;
3) in seedling pan, divide the bunch planting kind rice varieties to be measured, and with Lijiang xintuanheigu as susceptible contrast, manage with ordinary method, standby when treating rice shoot length to 3 leaves, 1 heart;
4) respectively 35 bacterial strains are cultivated on oat medium, prepared spore suspension behind the product spore;
5) spore suspension for preparing is inoculated into respectively on the rice shoot to be measured, inoculum density, inoculation method is with conventional;
6) heat and moisture preserving is observed the record state of an illness after 7 days according to a conventional method, determines anti-sense response type, the genotype of the kind to be measured of deriving.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101418332B (en) * | 2008-11-25 | 2011-04-20 | 云南农业大学 | Method for detecting rice blast pathogenic protein |
| CN103229674A (en) * | 2013-05-21 | 2013-08-07 | 江苏胜田农业科技发展有限公司 | Method for simply and rapidly identifying resistance of pyricularia grisea |
| CN106754612A (en) * | 2017-02-14 | 2017-05-31 | 云南农业大学 | A kind of method for suppressing to infect the rise of paddy rice SA approach related gene |
| CN106754614A (en) * | 2017-02-14 | 2017-05-31 | 云南农业大学 | A kind of method for suppressing to infect the rise of paddy rice PCD approach related gene |
| CN110257473A (en) * | 2019-07-01 | 2019-09-20 | 湖北省农业科学院植保土肥研究所 | Resistant rice varieties plant blast resisting identification method in specific rice region |
| CN112314613A (en) * | 2020-12-04 | 2021-02-05 | 福建农林大学 | Application of sanguinarine to excitation of rice resistance and rice blast resistance of rice |
-
2008
- 2008-03-13 CN CNA2008100581786A patent/CN101240346A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101418332B (en) * | 2008-11-25 | 2011-04-20 | 云南农业大学 | Method for detecting rice blast pathogenic protein |
| CN103229674A (en) * | 2013-05-21 | 2013-08-07 | 江苏胜田农业科技发展有限公司 | Method for simply and rapidly identifying resistance of pyricularia grisea |
| CN106754612A (en) * | 2017-02-14 | 2017-05-31 | 云南农业大学 | A kind of method for suppressing to infect the rise of paddy rice SA approach related gene |
| CN106754614A (en) * | 2017-02-14 | 2017-05-31 | 云南农业大学 | A kind of method for suppressing to infect the rise of paddy rice PCD approach related gene |
| CN110257473A (en) * | 2019-07-01 | 2019-09-20 | 湖北省农业科学院植保土肥研究所 | Resistant rice varieties plant blast resisting identification method in specific rice region |
| CN112314613A (en) * | 2020-12-04 | 2021-02-05 | 福建农林大学 | Application of sanguinarine to excitation of rice resistance and rice blast resistance of rice |
| CN112314613B (en) * | 2020-12-04 | 2021-09-07 | 福建农林大学 | Application of sanguinarine to excitation of rice resistance and rice blast resistance of rice |
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Open date: 20080813 |