CN104293799B - Rice blast resistance gene RMg38 and application thereof - Google Patents

Abstract

The invention belongs to the field of a gene engineering technology and specifically relates to a rice blast resistance gene RMg38 and its application. The invention discloses a nucleotide sequence and its coding amino polypeptide sequence of the rice blast new resistance gene RMg38. The gene belongs to a member of NBS-LRR type resistance gene family. The rice blast resistance gene provided by the invention is cloned from the rice line which shows highly resistance to rice blast, and the rice blast resistance gene is transformed into rice varieties which are sensitive to rice blast pathogen. The disease resistance of the transformed rice is evaluated by infection of rice blast pathogen so as to finally determine that the gene has resistance to rice blast.

Description

A kind of resistance gene of rice blast RMg38 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 RMg38 and its should With.

Background technology

Plant disease resistance genes are the results that plant interacts for a long time with pathogen, are play in the evolutionary process of plant Highly important role.Therefore, the analysis of plant disease resistance genes structure and research, are the bases for understanding plant disease-resistant mechanism, right Also there is important directive significance in the prevention and control of plant disease.Since first plant disease resistance genes Hm1 was in 1992 (Tanksley et al. 2007 since being separated from Semen Maydiss by Johal and Briggs；Dong Yuchen, 2001), up till now for Only, more than 80 plant disease resistance genes are able to clone from different plants and separate, wherein major type of disease-resistant base Because NBS-LRR structure types (Nucleotide-binding site and leucine-rich-repeat, i.e. nucleotide Binding site and full asphalt mixture albumen) disease-resistant gene；There is small part disease-resistant gene to be mainly eLRR-TM- in addition The type of pkinase, eLRR-TM, STK etc. 6.These disease-resistant genes are separately encoded to virus, antibacterial, funguses, even oomycetes, line The resistance protein of worm and insecticide etc..

Plant disease every year to agriculture, woods production bring huge loss, rationally and effectively controlling plant diseases be ensure agriculture, One of woods sustainable development key issue to be solved.In a large number it was verified that the existing Genes For Plant Tolerance disease of development and utilization Matter resource is one of maximally efficient method of controlling plant diseases (Tanksley et al. 2007；Dong Yuchen, 2001). The method that traditional disease-resistant variety is cultivated, typically by the material hybridization that the kind of good quality and high output and disease resistance are strong, then by not Disconnected backcross breeding, finally obtains disease-resistant and high-quality new varieties.Although this method is largely effective, extremely take, when new After breed of variety out, the strain or microspecies of the generation that new to pathogen may evolve show as susceptible (Tanksley et al. 2007)；Classical map based cloning method utilizes disease-resistant and susceptible mixing breed, then to substantial amounts of separation offspring inoculation Pathogenic bacteria, identification resistance, genetic mapping etc., are finally cloned on the basis of precise genetic positioning.This method is achieved very Good effect, serves very important effect (such as dividing for Bacterial blight resistance gene Xa21 in the clone of plant disease resistance genes From and utilize, Song et al. 1995), but because of cycle length, waste time and energy, separate and the equipotentiality detection reason such as more difficult, no It is suitable to clone disease-resistant gene in a large number.Meanwhile, because the unique mode of inheritance of disease-resistant gene, the application for also making the method is received very Big restriction.By taking rice blast resistant gene as an example, the genoid usual cluster (gene cluster) distribution in genome (Wang et al. 1999;Liu et al. 2002;Lin et al. 2007), between the homologous chromosome of different cultivars Position and structure it is also more be in mal-distribution, allelic relationship is indefinite, big (the Yang et al. 2007 of copy number variation; Ding et al. 2007a; Sun et al. 2008; Li et al. 2010).These genetic phenomenons, considerably increase figure The difficulty of position clone, has had a strong impact on the cloning efficiency of disease-resistant gene.

In recent years, with plant disease-resistant and the breakthrough progress of disease-resistant related gene Molecular level study, make us right The structure of plant disease-resistant and disease-resistant related gene, function, origin, variation and the understanding for preserving have the change of essence.Plant Disease-resistant gene, mainly a class contain the gene of LRR domains, wherein again with NBS-LRR（Nucleic acid combination-be rich in leucine）Class Based on type disease-resistant gene.Because these genes all have the similarity of height in structure and functionally, with reference to the heredity of its uniqueness With Evolution, be quickly clone these genes are provided convenience with identification, also efficiently to be carried using Resistant gerplasm resource New opportunity is supplied.

Oryza sativa L. (Oryza sativa) it is one of most important cereal crops in the world, there are about the population one of more than half Rice is food, while Oryza sativa L. is also one of topmost raise crop of China, but it suffers from every year serious pest and disease damage Invade and harass, to agricultural production huge loss is brought.Wherein, rice blast be distributed most wide, hazard rice most serious disease it One, the yield of Oryza sativa L. is badly influenced, the Rice Yield Loss Caused that the whole world is caused every year by rice blast accounts for 11-30% and (is roughly equal to 1.57 Hundred million dollars, http://www.fungalgenomics.ncsu.edu), directly threaten rice agriculture and increase income and national grain peace Entirely.No matter in the world or in China, an a rice blast difficult problem always priority research of guarantee Oryza sativa L. continuous production is solved Problem.

One of greatest difficulty of rice blast preventing and treating is that pathogenic bacteria variation itself is fast with differentiation speed（Ling Zhong specially etc., 2004）.Newly Physiological races of rice blast fungus emerge in an endless stream, existing disease-resistant gene (Plant disease resistance, R genes) is very It is fast to lose resistance, and find new disease-resistant material and be increasingly difficult to, threat of the rice blast to Rice Production is also increasing.For The variable pathogen of reply, Oryza sativa L. must possess many disease-resistant genes.From the point of view of more than 80 gene locis after positioning, Oryza sativa L. Blast resistant gene is large number of.But, the blast resistant gene up to the present cloned only 23.Although located Gene can be used by the method for molecular marker assisted selection, this Breeding Process is loaded down with trivial details, time-consuming, when new varieties training Bring out after coming, susceptible, clone and directly to convert disease-resistant gene possibly most straight may be shown as to the new dissociant for producing Connect the approach for effectively cultivating disease-resistant variety.Therefore, using new thinking, the new side of exploitation energy quick clone blast resistant gene Method, is just particularly important.

The present invention is exactly to be combined using bioinformatics means and molecular engineering, isolating from Oryza sativa L. rapidly and efficiently Multiple genes with rice blast resistance.

The content of the invention

The purpose of the present invention is, the rice blast resistance gene RMg38 that carries in the high anti-kind of separating clone Oryza sativa L. and includes Regulate and control the DNA fragmentation of the promoter of this gene.

It is a further object to provide the protein sequence coded by above-mentioned rice blast resistance gene RMg38.

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 of the above-mentioned protein in resisting rice blast bacteria medicine is prepared.

The present invention relates to clone and identify a kind of DNA fragmentation comprising RMg38 genes, the albumen of the gene code can make water Rice produces the disease resistance response of specificity to the disease caused by Pyricularia oryzae.Wherein, the fragment is respectively such as sequence table SEQ ID NO:Shown in l or it is approximately equivalent to SEQ ID NO:DNA sequences shown in l, or its function is equivalent to SEQ ID NO:l Show the subfragrnent of sequence.These DNA sequence all encode a kind of NBS-LRR albuminoids, its aminoacid sequence such as SEQ ID NO:2 institutes Show.Separated, clone RMg38 resistant genes coding NBS-LRR albumen.Their albumen all includes two main structures Domain：NBS and LRR regions, the C- ends of RMg38 albumen are that 9 LRR repeat.

According to the RMg38 gene sequence informations that the present invention is provided（SEQ ID NO:L,）, those skilled in the art can lead to Cross following methods and obtain the gene being equal to RMg38：（1）Obtained by database retrieval；（2）With RMg38 genetic fragments to visit Pin screens Oryza sativa L. or the genomic library or cDNA library of other plants are obtained；（3）Few core former times is designed according to RMg38 sequence informations Thuja acid primer, the method expanded with PCR is obtained from the genome, mRNA and cDNA of Oryza sativa L. or other plants；（4） Obtained with gene engineering method transformation on the basis of RMg38 gene orders；（5）The gene is obtained with the method for chemosynthesis.

The rice blast resistance gene RMg38 that the present invention is provided has important using value.By described RMg38 gene sequences 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, so as to be applied to production.It is of the present invention gene constructed in plant conversion carrier, can be to the gene or its regulation and control Sequence is suitably modified, it is also possible to replace the original promoter of the gene with other promoteres, so as to widening anti-spectrum or strengthening anti- Property.

The present invention has the advantages that：The blast resistant gene of clone is proceeded in susceptible plant, contributes to obtaining Obtain disease-resistant plants newly.The disease-resistant gene of clone can be shifted and utilized between different species such that it is able to overcome tradition disease-resistant 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 be further provided for or the disease-resistant transgenic plant using the acquisition of above-mentioned DNA fragmentation and corresponding seed, with And the gene with the present invention or the plant for converting of the recombinant based on the gene or the seed obtained by this kind of plant.Can be with having Property hybridization mode by the present invention gene proceed to other plant.

Description of the drawings

Fig. 1 is schematic flow sheet of the present invention.Figure 1A：The determination of blast resisting candidate locus；Figure 1B-E：Blast resistant gene Separation with clone；Fig. 1 F-G：The genetic transformation of blast resistant gene；Fig. 1 H：The identification of transformant.

Fig. 2 is the hygromycin gene of the transformant of rice blast resistance gene RMg38 and CaMV 35S promoteres PCR detects electrophoretogram；Fig. 2A is the pcr amplification product of CaMV 35S promoteres, and swimming lane 1-6 is produced for the PCR amplifications of transformant Thing, swimming lane 7 is the pcr amplification product of carrier pCAMBIA1300-AscI plasmids, and swimming lane 8 is the non-transformed body of new No. 2 of receptor Pcr amplification product；Fig. 2 B for hygromycin gene PCR primer, swimming lane 1-6 for transformant pcr amplification product, swimming lane 7 For the pcr amplification product of carrier pCAMBIA1300-AscI plasmids, swimming lane 8 is that the PCR amplifications of the non-transformed body of new No. 2 of receptor are produced Thing.

Fig. 3 has RMg38 gene plant Resistance Identification figures for conversion.Fig. 3 A：The Resistance Identification figure of new No. 2 of adjoining tree；Figure 3B：The Resistance Identification figure of the transformed plant of rice blast resistance gene RMg38.

Specific embodiment

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

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

The clone of the rice blast resistance gene RMg38 of embodiment 1, 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 RMg38：（1）More in the form of gene family and gene cluster, in day Respectively there are 4 and 2 close copies in this warm and fine 93-11；（2）Have in its LRR region, particularly LxxLxLxx regions higher Ka/Ks values；

2nd, rice blast resistance gene RMg38（12g36730-Q2436）Separation with clone：It is sequenced using public database Kind Japan is fine（Nipponbare）It is reference sequences with 93-11, designs primer（Primer two ends all carry restriction enzyme site AscI）. Primer sequence referring to sequence table, forward primer sequence such as SEQ ID NO:Shown in 4；Reverse primer sequences such as SEQ ID NO:5 institutes Show.

With disease resisting rice kind Tetep, Gumei2 and Q2436 are template, using long-range PCR（Long-PCR） Amplification candidate gene fragment.PCR programs are as follows：95 DEG C of denaturations 5 minutes, 95 DEG C of degeneration 30 seconds, 60 DEG C of renaturation 45 seconds, 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 rubber tapping recovery is carried out to PCR primer.

3rd, the preparation of difunctional carrier is carrier：Rare restriction enzyme site AscI is imported into 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：Restriction enzyme A scI is used, while enzyme action PCR primer and base Plinth vector plasmid, and purification.In the presence of T4 ligases, candidate gene fragment is connected into into carrier is carrier pCAMBIA1300- AscI。

5th, the genetic transformation of candidate resistance gene:The bifunctional vector for carrying candidate gene is imported into Agrobacterium tumefaciems EHA105, using agrobacterium-mediated transformation, is transformed into the general sense kind of Oryza sativa L. new No. 2, in TP309 and Co39 by candidate gene.Finally 20 plants of independent transformants are obtained altogether.

6th, PCR Molecular Detection：With transformant DNA as template, started with hygromycin gene and its upstream CaMV35S The specific primer of son enters performing PCR reaction to it.The primer sequence canonical sequence table of hygromycin gene, forward primer sequence Such as SEQ ID NO:6；Reverse primer sequences such as SEQ ID NO:7.The primer sequence canonical sequence table of CaMV35S promoteres, just To primer sequence such as SEQ ID NO:8；Reverse primer sequences such as SEQ ID NO:9（Fig. 2 and Fig. 3）.

7th, the Resistance Identification of transformant：Select 10 source place differences, the good independent physiological races of rice blast fungus of perspective As the pathogen species of detection.Using Pyricularia grisea Race inoculation T2 for transfer-gen plant and check variety, screen disease-resistant The transformant for sexually revising.Resistance Identification result shows candidate gene RMg38 under the genetic background of general new No. 2 of kind of sense, to 4 The resistance of pathogenic strains all showed differents, shows that candidate gene RMg38 has resistance specified and by successful clone.

8th, the gene structure and protein structural analysis of rice blast resistance gene RMg38：Using DNA of the walking method to RMg38 Sequence is sequenced.RMg38 gene DNAs length is 3533bp, and containing 6 opening code-reading frames, 5 introns and 6 show outward Son.The protein sequence such as SEQ ID NO of RMg38 codings:Shown in 2.RMg38 gene codes 1 are made up of 748 amino acid residues Protein polypeptide.RMg38 albumen belongs to NBS-LRR albumen, and the C- ends of its albumen are that 8 LRR repeat.

Claims (9)

1. a kind of rice blast resistant gene RMg38, its nucleotides sequence is classified as such as SEQ ID NO:Shown in 1.

2. the albumen that according to claim 1 gene order is encoded.

3. albumen according to claim 2, its aminoacid sequence such as SEQ ID NO:Shown in 2.

4. the promoter of a kind of regulation and control rice blast resistant gene RMg38 as claimed in claim 1, its nucleotide sequence such as SEQ ID NO:Shown in 3.

5. a kind of rice blast resistant gene RMg38 according to claim 1, is characterized in that containing the anti-rice blast of adjusting and controlling rice The promoter of ospc gene RMg38, the nucleotide sequence such as SEQ ID NO of the promoter:Shown in 3.

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

7. the host cell Agrobacterium tumefaciems EHA105 containing conversion carrier described in claim 6.

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

9. application of the albumen described in claim 2 in resisting rice blast bacteria medicine is prepared.