CN104404052B - Rice blast resistance gene RMg39 and its application - Google Patents

Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to a rice blast resistance gene RMg39 and its application. The invention discloses a nucleotide sequence of a novel rice blast resistance gene RMg39 and an amino acid polypeptide sequence coded by the same. The gene belongs to a member of the NBS-LRR type plant disease resistance gene family. The rice blast resistance gene is cloned from a rice line showing high resistance to Magnaporthe grisea, and is converted to a variety susceptible to Magnaporthe grisea; Magnaporthe grisea invasion method is used to assess the disease resistance, so as to ultimately determine that the gene has resistance to rice blast.

Description

A kind of resistance gene of rice blast rmg39 and its application

Technical field

The invention belongs to gene engineering technology field and in particular to a kind of resistance gene of rice blast rmg39 and its should With.

Background technology

Plant disease resistance genes be plant with pathogen interact for a long time, mutually select, the result of coevolution, in plant Evolutionary process in play highly important role.Since first plant disease resistance genes hm1 1992 by johal and (tanksley et al. 2007 since briggs separation from Semen Maydiss；Dong Yuchen, 2001), up to the present, already exceed 60 plant disease resistance genes are cloned from different plants and are separated, and part disease-resistant gene is answered in molecular breeding With being that effective control plant disease provides new approach.In addition to minority disease-resistant gene, the structure of plant disease resistance genes is protected relatively Keep, main Types are nbs-lrr structure type (nucleotide-binding site and leucine-rich- Repeat, i.e. nucleotide binding site and full asphalt mixture albumen)；In addition small part disease-resistant gene is also had to be mainly elrr- The types such as tm-pkinase, elrr-tm, stk.

In a large number it was verified that development and utilization existing plant disease-resistant germ plasm resource is having the most of controlling plant diseases One of the method for effect (tanksley et al. 2007；Dong Yuchen, 2001).Traditional disease-resistant variety is cultivated, and generally comprises three Individual basic link: (1) extensively collects germplasm materials；(2) germplasm materials collected are preserved, identified and screened, therefrom To Resistant gerplasm；(3) the material hybridization strong with disease resistance of the kind of good quality and high output, then passes through continuous backcross breeding, finally Obtain disease-resistant and high-quality new varieties.This Breeding Process is not only loaded down with trivial details, and extremely time-consuming.Classical map based cloning method utilizes Disease-resistant and susceptible mixing breed, then separates offspring inoculation pathogenic bacteria, identification resistance, genetic mapping etc. to substantial amounts of, finally exists Cloned on the basis of precise genetic positioning.But because of its cycle length, waste time and energy, separate and equipotentiality detection is more difficult etc. former Cause, is not suitable for cloning the needs of disease-resistant gene in a large number.Meanwhile, because of the mode of inheritance that disease-resistant gene is unique, also make answering of the method With being limited by very large.Taking rice blast resistant gene as a example, this genoid usual cluster (gene in genome Cluster) it is distributed (wang et al. 1999;liu et al. 2002;Lin et al. 2007), in different cultivars Homologous chromosome between position and structure also more be in mal-distribution, allelic relationship is indefinite, the big (yang of copy number variation et al. 2007; ding et al. 2007a; sun et al. 2008; li et al. 2010).These heredity are existing As considerably increasing the difficulty of map based cloning, having had a strong impact on the cloning efficiency of disease-resistant gene.

In recent years, with the breakthrough progress of plant disease-resistant and disease-resistant related gene Molecular level study, make us right The understanding of the structure of plant disease-resistant and disease-resistant related gene, function, origin, variation and preservation has the change of essence.Plant Thing disease-resistant gene, mainly one class contains the gene of lrr domain, wherein combines-be rich in leucine with nbs-lrr(nucleic acid again) Based on type disease-resistant gene.Because these genes are in structure and the similarity functionally all with height, in conjunction with the something lost of its uniqueness Pass and Evolution, provide convenience with these genes of identification for quick clone, also for efficiently utilizing Resistant gerplasm resource Provide new opportunity.

Rice blast is to be distributed one of the widest, disease of hazard rice most serious in the world, badly influences the yield of Oryza sativa L., Produce huge loss to agricultural, the whole world accounts for 11-30% by the Rice Yield Loss Caused that rice blast causes every year and (is roughly equal to 1.57 hundred million Dollar, http://www.fungalgenomics.ncsu.edu), directly threaten rice agriculture and increase income and national grain security. Therefore, in countries in the world, solve the problem that a rice blast difficult problem is always a priority research ensureing Oryza sativa L. continuous production.But Due to pathogenic bacteria variation itself fast with differentiation speed (Ling Zhong specially etc., 2004), new physiological races of rice blast fungus emerges in an endless stream, and makes Some disease-resistant genes (plant disease resistance, r gene) lose resistance quickly, and the preventing and treating of rice blast just becomes More difficult.

Therefore, clone and directly convert the approach that disease-resistant gene possibly the most effectively cultivates disease-resistant variety.This The bright similarity making full use of in plant disease resistance genes structure and the inheritable variation and evolution feature of uniqueness, using bioinformatics Method screens potential disease-resistant gene, and using Protocols in Molecular Biology is a large amount of, quick clone Candidate Disease Resistant Genes, finally in water Multiple genes with rice blast resistance are obtained in rice.

Content of the invention

The purpose of the present invention is, the rice blast resistance gene rmg39 carrying in the high anti-kind of separating clone Oryza sativa L. and comprising Regulate and control the dna fragment of the promoter of this gene.

It is a further object to provide the protein sequence coded by above-mentioned rice blast resistance gene rmg39.

It is a further object to provide the carrier containing above-mentioned resistant gene.

It is a further object to provide the transfer-gen plant of above-mentioned carrier conversion.

It is a further object to provide application in preparing resisting rice blast bacteria medicine for the above-mentioned protein.

The present invention relates to cloning and identifying a kind of dna fragment comprising rmg39 gene, the albumen of these gene codes can make Oryza sativa L. produces specific disease resistance response to the disease caused by Pyricularia oryzae.Wherein, described fragment is respectively as sequence table seq Id no:l is shown or is approximately equivalent to the dna sequence shown in seq id no:l, or its function is equivalent to seq id The subfragrnent of sequence shown in no:l.A kind of these dna sequential codings nbs-lrr albuminoid, its aminoacid sequence such as seq id Shown in no:2.Separated, clone rmg39 resistant gene coding nbs-lrr albumen.Albumen comprises two main domains: Nbs and lrr region, the c- end of rmg39 albumen is that 17 lrr repeat.

The rmg39 gene sequence information (seq id no:l) being provided according to the present invention, those skilled in the art can pass through The gene that following methods acquisition is equal to rmg39: (1) is obtained by database retrieval；(2) with rmg39 genetic fragment as probe The genomic library of screening Oryza sativa L. or other plant or cdna library obtain；(3) few core is designed according to rmg39 gene sequence information Former times thuja acid primer, is obtained from the genome, mrna and cdna of Oryza sativa L. or other plant with the method for pcr amplification；(4) exist Transformed with gene engineering method on the basis of rmg39 gene order and obtain；(5) obtain this gene with the method for chemosynthesis.

The rice blast resistance gene rmg39 that the present invention provides has important using value.By described rmg39 gene sequence Row any method for transformation Introduced into Rice or other plant cell, can obtain the disease-resistant product of transgenic of expressing said gene Kind, thus being applied to produce.Of the present invention gene constructed in plant conversion carrier, can to described gene or its regulation and control Sequence is suitably modified it is also possible to replace the original promoter of described gene with other promoteres, thus widening anti-spectrum or strengthening anti- Property.

The invention has the following beneficial effects: proceeding to the blast resistant gene of clone in susceptible plant, contribute to obtaining Obtain disease-resistant plants newly.The disease-resistant gene of clone can shift and using such that it is able to overcome tradition disease-resistant between different species The difficulty of distant hybridization in breeding.Furthermore, it is possible to transgenic technology in plant add up multiple disease-resistant genes, shorten breeding week Phase.The present invention can also further provide for or apply disease-resistant transgenic plant and the corresponding seed that above-mentioned dna fragment obtains, with And the gene with the present invention or the recombinant based on this gene conversion plant or the seed being obtained by this kind of plant.Can be with having Property hybridization mode the gene of the present invention is proceeded to other plant.

Brief description

Fig. 1 is schematic flow sheet of the present invention.Fig. 1 a: the determination of blast resisting candidate locus；Fig. 1 b-e: blast resistant gene Separation and clone；The genetic transformation of Fig. 1 f-g: blast resistant gene；The identification of Fig. 1 h: transformant.

Fig. 2 is the hygromycin gene of transformant and the camv 35s promoter of rice blast resistance gene rmg39 Pcr detects electrophoretogram；Fig. 1 a is the pcr amplified production of camv 35s promoter, and swimming lane 1-6 is that the pcr amplification of transformant is produced Thing, swimming lane 7 is the pcr amplified production of carrier pcambia1300-asci plasmid, and swimming lane 8 is the non-transformed body of new No. 2 of receptor Pcr amplified production；Fig. 1 b is the pcr product of hygromycin gene, and swimming lane 1-6 is the pcr amplified production of transformant, swimming lane 7 For the pcr amplified production of carrier pcambia1300-asci plasmid, swimming lane 8 is that the pcr amplification of the non-transformed body of new No. 2 of receptor is produced Thing.

Fig. 3 is the transformed plant Resistance Identification figure of rice blast resistance gene rmg39.Fig. 3 a: rice blast resistance gene rmg39 Transformed plant Resistance Identification figure；The Resistance Identification figure of new No. 2 of Fig. 3 b: adjoining tree.

Specific embodiment

The determination of rice anti-rice blast candidate gene: by Oryza sativa L. genome sequencing kind 93-11 and Japan fine based on, The disease-resistant gene of all of nbs-lrr type first in identification genome, and constructing system cladogram.On this basis, we Have chosen the blast resistant gene site of 20 candidates, by design of primers, carry out lengthy motion picture in the rice varieties of blast resisting The processes such as section pcr, vector construction, transgenic, Resistance Identification, derive from China's Mainland finally by 10 and are distributed all Oryza sativa L. masters The Pyricularia oryzae system identification in producing region, identifies 1 and has resistance specified blast resistant gene.

In following embodiments, method therefor if no special instructions, is conventional method.

The clone of embodiment 1 rice blast resistance gene rmg39, vector construction, disease-resistant plant identification

The experiment process of the present embodiment is as shown in Figure 1.

1st, the determination of blast resisting candidate locus rmg39: (1) has 1 close copy in Japan is fine, has 2 in 93-11 Individual close copy；(2) in its lrr region, particularly xxlxlxx region has higher ka/ks value；

2nd, rice blast resistance gene rmg39(os 04g30930_tetep) separation and clone: using public database survey Sequence kind Japan fine (nipponbare) and 93-11 are reference sequences, and (primer two ends all carry restriction enzyme site to design primer Asci)., referring to sequence table, forward primer sequence is as shown in seq id no:4 for primer sequence；Reverse primer sequences such as seq id Shown in no:5.

With disease resisting rice kind tetep, Gumei2 and q2436 are template, using long segment pcr technology (long-pcr) Amplification candidate gene fragment.Pcr program is as follows: 95 DEG C of denaturations 5 minutes, 95 DEG C of degeneration 30 seconds, 60 DEG C of renaturation 45 seconds, and 68 DEG C are prolonged Stretch 7 minutes, totally 35 circulations, subsequent 72 DEG C are incubated 10 minutes, last 10 DEG C of constant temperature.Subsequently pcr product is carried out with rubber tapping reclaim.

3rd, the preparation of difunctional carrier is carrier: rare restriction enzyme site asci is imported bifunctional vector pcambia1300's Between multiple clone site bamhi and sali, replace restriction enzyme site xbai, formation base carrier pcambia1300-asci.

4th, the connection of candidate resistance gene and carrier is carrier: use restricted enzyme asci, simultaneously enzyme action pcr product and base Plinth vector plasmid, and purification.In the presence of t4 ligase, candidate gene fragment is connected into carrier is carrier pcambia1300- asci.

5th, the genetic transformation of candidate resistance gene: the bifunctional vector carrying candidate gene is imported Agrobacterium tumefaciems Eha105, using agrobacterium-mediated transformation, candidate gene is transformed into Oryza sativa L. new No. 2 of kind of general sense.Finally obtain altogether 20 plants solely Vertical transformant.

6th, pcr Molecular Detection: with transformant dna as template, started with hygromycin gene and its upstream camv35s The specific primer of son carries out pcr reaction to it.The primer sequence of hygromycin gene participates in sequence table, forward primer sequence As seq id no:6；Reverse primer sequences such as seq id no:7.The primer sequence of camv35s promoter participates in sequence table, just To primer sequence such as seq id no:8；Reverse primer sequences such as seq id no:9.

7th, the Resistance Identification of transformant: select 10 source place differences, the good independent physiological races of rice blast fungus of perspective Pathogen species as detection.Using Pyricularia grisea Race inoculation t2 for transfer-gen plant and check variety, screen disease-resistant The transformant sexually revising.Resistance Identification result shows candidate gene rmg39 under the genetic background of general new No. 2 of kind of sense, to 4 The resistance of pathogenic strains all showed different, shows that candidate gene rmg39 has resistance specified and by successful clone (Fig. 2 And Fig. 3).

8th, the gene structure of rice blast resistance gene rmg39 and protein structural analysis: using the dna to rmg39 for the walking method Sequence is sequenced.Rmg39 gene dna length is 6697bp, and containing 3 opening code-reading frames, 2 introns and 3 show outward Son.The protein sequence of rmg39 coding is as shown in seq id no:2.Rmg39 gene code 1 is by 1002 amino acid residue groups The protein polypeptide becoming.Rmg39 albumen belongs to nbs-lrr albumen, and the c- end of its albumen is that 17 lrr repeat.

Claims (9)

1. a kind of rice blast resistant gene rmg39, its nucleotides sequence is classified as shown in seq id no:1.

2. the albumen that gene order encodes according to claim 1.

3. albumen according to claim 2, its aminoacid sequence is as shown in seq id no:2.

4. the promoter of a kind of regulation and control rice blast resistant gene rmg39 as claimed in claim 1, its nucleotide sequence such as seq Shown in id no:3.

5. a kind of rice blast resistant gene rmg39 according to claim 1, is characterized in that rice blast anti-containing adjusting and controlling rice The promoter of ospc gene rmg39, the nucleotide sequence of described promoter is as shown in seq id no:3.

6. the conversion carrier containing gene described in claim 1.

7. the host cell Agrobacterium tumefaciems eha105 containing conversion carrier described in claim 6.

8. application in anti-rice blast rice breeding for the gene described in claim 1.

9. application in preparing resisting rice blast bacteria medicine for the albumen described in claim 2.