CN104293799A - Rice blast resistance gene RMg38 and application thereof - Google Patents
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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
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Technical field
The invention belongs to gene engineering technology field, be specifically related to a kind of resistance gene of rice blast RMg38 and application thereof.
Background technology
Plant disease resistance genes is the long-term interactional result of plant and pathogenic bacteria, plays very important role in the evolutionary process of plant.Therefore, the analysis of plant disease resistance genes structure and research, be the basis understanding plant disease-resistant mechanism, the prevention and corntrol for Plant diseases also has important directive significance.(Tanksley et al. 2007 since first plant disease resistance genes Hm1 was separated by Johal with Briggs in 1992 from corn; Dong Yuchen, 2001), up to the present, more than 80 plant disease resistance genes has been had to be cloned from different plants and be separated, wherein the disease-resistant gene of main Types is the disease-resistant gene of NBS-LRR structure type (Nucleotide-binding site and leucine-rich-repeat, i.e. nucleotide binding site and full asphalt mixture albumen); Small part disease-resistant gene is also had to be mainly 6 type such as eLRR-TM-pkinase, eLRR-TM, STK in addition.These disease-resistant genes are encoded respectively to the resistance protein of virus, bacterium, fungi, oomycetes, even nematode and insect etc.
Plant diseases gives agriculture every year, woods produces and brings huge loss, and controlling plant diseases is one of key issue ensureing that agriculture, woods Sustainable development must solve rationally and effectively.Facts have proved in a large number, the existing plant disease-resistant germ plasm resource of development and utilization is (the Tanksley et al. 2007 of one of effective means the most of controlling plant diseases; Dong Yuchen, 2001).The method that tradition disease-resistant variety is cultivated, normally hybridizes material strong to the kind of good quality and high output and disease resistance, then passes through continuous backcross breeding, finally obtains the disease-resistant and new variety of high-quality.Although this method is very effective, extremely consuming time, after rearing new variety is out, may show as susceptible (Tanksley et al. 2007) newly the evolve strain that produces or microspecies of pathogenic bacteria; Classical map based cloning method utilizes disease-resistant and susceptible mixing breed, then inoculates germ, qualification resistance, genetic mapping etc. to a large amount of separation offsprings, finally clones on the basis of precise genetic location.This method achieves good effect, very important effect is served (as separation and the utilization of Bacterial blight resistance gene Xa21 in the clone of plant disease resistance genes, Song et al. 1995), but because the cycle is grown, wastes time and energy, to be separated and allelomorphism detects the reason such as more difficult, be unsuitable for cloning disease-resistant gene in a large number.Meanwhile, because the mode of inheritance of disease-resistant gene uniqueness, the application of the method is also made to be limited by very large.For rice blast resistant gene, this genoid is usual cluster (gene cluster) distribution (Wang et al. 1999 in genome; Liu et al. 2002; Lin et al. 2007), the position between the homologous chromosomes of different varieties and structure are also many, and in mal-distribution, allelic relationship is indefinite, large (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 the difficulty of map based cloning, have had a strong impact on the cloning efficiency of disease-resistant gene.
In recent years, along with the breakthrough progress of plant disease-resistant and disease-resistant related gene Molecular level study, us are made to have had the change of essence to the understanding of the structure of plant disease-resistant and disease-resistant related gene, function, origin, variation and preservation.Plant disease resistance genes, mainly a class contains the gene of LRR structural domain, wherein combines-be rich in leucine based on NBS-LRR(nucleic acid again) type disease-resistant gene.Because these genes all have the similarity of height, in conjunction with the genetics and evolution feature of its uniqueness, for these genes of cloning and identification are provided convenience, also for utilize Resistant gerplasm resource to provide new opportunity efficiently fast on structure and fuction.
Paddy rice (
oryza sativa) be one of most important food crop in the world, about there is population one rice over half for food, one of topmost raise crop of paddy rice Ye Shi China simultaneously, but it is all subjected to the invasion of serious disease and pest every year, brings huge loss to agriculture production.Wherein, rice blast is one of the widest, hazard rice the is the most serious disease that distributes, badly influence the output of paddy rice, the Rice Yield Loss Caused that the whole world is caused by rice blast every year 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 security.No matter in the world or in China, solve the problem that a rice blast difficult problem is the priority research ensureing paddy rice continuous production always.
One of greatest difficulty of rice blast control is germ variation itself and differentiation speed fast (Ling Zhongzhuan etc., 2004).New physiological races of rice blast fungus emerges in an endless stream, existing disease-resistant gene (Plant disease resistance, R gene) lose resistance very soon, and it is more and more difficult to find new disease-resistant material, rice blast is also increasing to the threat of Rice Production.For tackling variable pathogenic bacteria, paddy rice must have a lot of disease-resistant gene.From more than 80 gene locuss after positioning, rice blast resistant gene One's name is legion.But, the blast resistant gene of up to the present having cloned only 23.Although gene after positioning can be used by the method for molecular marker assisted selection, this Breeding Process is loaded down with trivial details, consuming time, after rearing new variety out, may show as susceptible to the new dissociant produced, clone also directly transforms the approach that disease-resistant gene may be the most effective cultivation disease-resistant variety.Therefore, use new thinking, the novel method of exploitation energy quick clone blast resistant gene, just seems particularly important.
The present invention utilizes information biology means and molecular engineering to combine exactly, rapidly and efficiently from paddy rice, isolate multiple gene with rice blast resistance.
Summary of the invention
The object of the invention is, the rice blast resistance gene RMg38 carried in separating clone paddy rice high resistance kind and comprise the DNA fragmentation of promotor of this gene of regulation and control.
Another object of the present invention is to provide the protein sequence coded by above-mentioned rice blast resistance gene RMg38.
Another object of the present invention is to provide the carrier containing above-mentioned resistant gene.
Another object of the present invention is to provide the transfer-gen plant of above-mentioned vector.
Another object of the present invention is to provide above-mentioned protein and is preparing the application in resisting rice blast bacteria medicine.
The present invention relates to a kind of DNA fragmentation comprising RMg38 gene of cloning and identification, the albumen of this genes encoding can make paddy rice produce specific disease resistance response to the disease caused by Pyricularia oryzae.Wherein, described fragment is respectively as shown in sequence table SEQ ID NO:l or be substantially equivalent to the DNA sequence shown in SEQ ID NO:l, or its function is equivalent to the subfragment that SEQ ID NO:l shows sequence.These DNA sequence dnas are all encoded a kind of NBS-LRR proteinoid, and its aminoacid sequence is as shown in SEQ ID NO:2.The RMg38 resistant gene coding NBS-LRR albumen being separated, cloning.Their albumen all comprises two main structural domains: NBS and LRR region, and the C-end of RMg38 albumen is that 9 LRR repeat.
According to RMg38 gene sequence information provided by the invention (SEQ ID NO:l), those skilled in the art can obtain the gene be equal to RMg38 by the following method: (1) is obtained by database retrieval; (2) obtain with the RMg38 gene fragment genomic library that is probe Screening of Rice or other plant or cDNA library; (3) design few core former times thuja acid primer according to RMg38 sequence information, obtain from the genome, mRNA and cDNA of paddy rice or other plant by the method for pcr amplification; (4) obtain with gene engineering method transformation on the basis of RMg38 gene order; (5) this gene is obtained by the method for chemosynthesis.
Rice blast resistance gene RMg38 provided by the invention has important using value.By described RMg38 gene order any one method for transformation Introduced into Rice or other plant cell, the transgenosis disease-resistant variety of expressing said gene can be obtained, thus be applied to production.Of the present invention gene constructed in plant conversion carrier, suitably can modify described gene or its regulating and controlling sequence, also can replace the original promotor of described gene by other promotor, thus widen anti-spectrum or strengthen resistance.
The present invention has following beneficial effect: proceeded in susceptible plant by the blast resistant gene of clone, contributes to obtaining new disease-resistant plants.The disease-resistant gene of clone can shift and utilize between different species, thus can overcome the difficulty of distant hybirdization in traditional breeding for disease resistance.In addition, with transgenic technology cumulative multiple disease-resistant gene in plant, breeding cycle can be shortened.The present invention can also provide or apply the disease-resistant transgenic plant and corresponding seed that above-mentioned DNA fragmentation obtains further, and with gene of the present invention or the plant transformed based on the recombinant chou of this gene or the seed obtained by this kind of plant.By the mode of sexual hybridization, gene of the present invention can be proceeded to other plant.
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Accompanying drawing explanation
Fig. 1 is schematic flow sheet of the present invention.Figure 1A: the determination of blast resisting candidate locus; Figure 1B-E: the separation of blast resistant gene and clone; Fig. 1 F-G: the genetic transformation of blast resistant gene; Fig. 1 H: the qualification of transformant.
Fig. 2 is that the hygromycin gene of the transformant of rice blast resistance gene RMg38 and the PCR of CaMV 35S promotor detect electrophorogram; Fig. 2 A is the pcr amplification product of CaMV 35S promotor, and swimming lane 1-6 is the pcr amplification product of transformant, and swimming lane 7 is the pcr amplification product of carrier pCAMBIA1300-AscI plasmid, and swimming lane 8 is the pcr amplification product of the non-transformed body of new No. 2 of acceptor; Fig. 2 B is the PCR primer of hygromycin gene, and swimming lane 1-6 is the pcr amplification product of transformant, and swimming lane 7 is the pcr amplification product of carrier pCAMBIA1300-AscI plasmid, and swimming lane 8 is the pcr amplification product of the non-transformed body of new No. 2 of acceptor.
Fig. 3 is that conversion has RMg38 gene plant Resistance Identification figure.Fig. 3 A: the Resistance Identification figure of new No. 2 of adjoining tree; The Resistance Identification figure of the transformed plant of Fig. 3 B: rice blast resistance gene RMg38.
Embodiment
The determination of rice anti-rice blast candidate gene: by paddy rice genome sequencing kind 93-11 and Japan fine based on, the disease-resistant gene of first all in identified gene group NBS-LRR types, and constructing system evolutionary tree.On this basis, we have chosen the blast resistant gene site of 20 candidates, by design of primers, in the rice varieties of blast resisting, carry out the processes such as LA-PCR, vector construction, transgenosis, Resistance Identification, derive from China's Mainland finally by 10 to distribute the Pyricularia oryzae system identification of all paddy rice main producing regions, identify 1 and there is resistance specified blast resistant gene.
In following embodiment, method therefor if no special instructions, is ordinary method.
the clone of embodiment 1 rice blast resistance gene RMg38, vector construction, disease-resistant plant qualification
The experiment process of the present embodiment as shown in Figure 1.
1, the determination of blast resisting candidate locus RMg38: (1) exists mainly with the form of gene family and gene cluster, respectively has the copy that 4 close with 2 in the warm and fine 93-11 of Japan; (2) in its LRR region, particularly LxxLxLxx region, there is higher Ka/Ks value;
2, rice blast resistance gene RMg38(12g36730-Q2436) separation and clone: utilize check order kind Japan fine (Nipponbare) and 93-11 of public database to be reference sequences, design primer (primer two ends are all with restriction enzyme site AscI).Primer sequence is see sequence table, and forward primer sequence is as shown in SEQ ID NO:4; Reverse primer sequences is as shown in SEQ ID NO:5.
With disease resisting rice kind Tetep, Gumei2 and Q2436 are template, utilize long-range PCR (Long-PCR) to increase candidate gene fragment.PCR program is as follows: 95 DEG C of denaturations 5 minutes, 95 DEG C of sex change 30 seconds, and 60 DEG C of renaturation 45 seconds, 68 DEG C extend 7 minutes, totally 35 circulations, and 72 DEG C are incubated 10 minutes, last 10 DEG C of constant temperature subsequently.Carry out rubber tapping to PCR primer subsequently to reclaim.
3, the preparation of difunctional carrier is carrier: between the multiple clone site BamHI that rare restriction enzyme site AscI is imported bifunctional vector pCAMBIA1300 and SalI, replace restriction enzyme site XbaI, formation base carrier pCAMBIA1300-AscI.
4, the connection of candidate resistance gene and carrier is carrier: use restriction enzyme A scI, enzyme cuts PCR primer and carrier is carrier plasmid simultaneously, and purifying.Under the effect of T4 ligase enzyme, candidate gene fragment is connected into carrier is carrier pCAMBIA1300-AscI.
5, the genetic transformation of candidate resistance gene: the bifunctional vector carrying candidate gene is imported agrobacterium tumefaciens EHA105, adopts agrobacterium-mediated transformation, candidate gene is transformed into new No. 2 of paddy rice general sense kind, in TP309 and Co39.Finally have acquisition 20 strain independently transformant altogether.
6, PCR Molecular Detection: with transformant DNA for template, carries out PCR reaction with the Auele Specific Primer of hygromycin gene and upstream CaMV35S promotor thereof to it.The primer sequence canonical sequence table of hygromycin gene, forward primer sequence is as SEQ ID NO:6; Reverse primer sequences is as SEQ ID NO:7.The primer sequence canonical sequence table of CaMV35S promotor, forward primer sequence is as SEQ ID NO:8; Reverse primer sequences is as SEQ ID NO:9(Fig. 2 and Fig. 3).
7, the Resistance Identification of transformant: select 10 source place differences, the good independent physiological races of rice blast fungus of perspective is as the pathogen species detected.Utilize Pyricularia grisea Race inoculation T2 for transfer-gen plant and check variety, the transformant that screening disease resistance changes.Resistance Identification result show candidate gene RMg38, under the genetic background of new No. 2 of general sense kind, to the resistance of 4 pathogenic strains showed different, shows that candidate gene RMg38 has resistance specified and by successful clone.
8, the gene structure of rice blast resistance gene RMg38 and protein structural analysis: adopt the DNA sequence dna of walking method to RMg38 to check order.RMg38 gene DNA length is 3533bp, containing 6 opening code-reading frames, and 5 introns and 6 exons.The protein sequence of RMg38 coding is as shown in SEQ ID NO:2.RMg38 genes encoding 1 protein polypeptide be made up of 748 amino-acid residues.RMg38 albumen belongs to NBS-LRR albumen, and the C-end of its albumen is that 8 LRR repeat.
Claims (10)
1. a rice blast resistant gene RMg38, its nucleotides sequence is classified as the DNA sequence dna as shown in SEQ ID NO:1, or its nucleotide sequence be to the DNA sequence dna shown in SEQ ID NO:1 in similarity >=95% similar DNA sequence dna, or the function of its nucleotide sequence is equivalent to the subfragment of DNA sequence dna shown in SEQ ID NO:1, or the DNA sequence dna as shown in SEQ ID NO:1 is through replacing, lacking or increase one or more Nucleotide and the nucleotide sequence of coding same acid sequence.
2. the albumen of gene sequences encode according to claim 1.
3. albumen according to claim 2, its aminoacid sequence is as shown in SEQ ID NO:2, or the aminoacid sequence with identical function that the sequence shown in SEQ ID NO:2 is formed through replacement, disappearance or adding portion amino acid.
4. regulate and control a promotor of rice blast resistant gene RMg38 as claimed in claim 1, its nucleotide sequence is as shown in SEQ ID NO:3.
5. a kind of rice blast resistant gene RMg38 according to claim 1, is characterized in that the promotor containing adjusting and controlling rice blast resistant gene RMg38, and the nucleotide sequence of described promotor is as shown in SEQ ID NO:3.
6. the conversion carrier containing gene described in claim 1.
7. the host cell agrobacterium tumefaciens EHA105 containing conversion carrier described in claim 6.
8. the transgenic plant containing conversion carrier described in claim 6.
9. the application of gene according to claim 1 in anti-rice blast rice breeding.
10. albumen described in claim 2 is preparing the application in resisting rice blast bacteria medicine.
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| CN115873867A (en) * | 2022-09-28 | 2023-03-31 | 云南省农业科学院农业环境资源研究所 | Rice blast resistant gene Pi69, and coding protein and application thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108753793A (en) * | 2017-10-30 | 2018-11-06 | 南京大学 | A kind of resistance gene of rice blast RMg42 and its application |
| CN108753793B (en) * | 2017-10-30 | 2021-06-22 | 南京大学 | Rice blast resistance gene RMg42 and application thereof |
| CN115873867A (en) * | 2022-09-28 | 2023-03-31 | 云南省农业科学院农业环境资源研究所 | Rice blast resistant gene Pi69, and coding protein and application thereof |
| CN115873867B (en) * | 2022-09-28 | 2023-06-23 | 云南省农业科学院农业环境资源研究所 | Rice blast resistance gene Pi69, and encoding protein and application thereof |
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