CN101974079B - avrXa23 protein activating disease-resistant reaction of rice and coding gene thereof - Google Patents

Abstract

The invention discloses an avrXa23 protein activating disease-resistant reaction of rice and coding gene thereof. The protein provided by the invention is (a) or (b), wherein (a) is protein composed of amino acid sequence shown as sequence 1 in sequence list, (b) is protein formed by the way that substitution and/or deletion and/or addition of one or more than one amino acid residues are carried out on amino acid sequence shown as the sequence 1 and derivation related to activating of disease-resistant reaction of rice is carried out on the sequence 1. Coding gene of avrXa23 protein obtained by the invention can be taken as gene resource of rice bacterial leaf blight resistant disease-resistant breeding, gene is introduced into rice xanthomonas oryzae pv. oryzae XM81 strain, and rice containing Xa23 disease-resistant gene can produce disease resistance, so that xanthomonas oryzae pv. oryzae is difficult to effectively infect rice. The invention is conductive to researching typical specificity interaction between plant and pathogenic bacteria and is conductive to clarifying broad spectrum pathogenicity mechanism of xanthomonas oryzae pv. oryzae.

Description

Excite avrXa23 albumen and the encoding gene thereof of rice to generate disease resistance response

Technical field

The invention belongs to the genetically engineered field, relate to a kind of avrXa23 albumen and encoding gene (pathogenic bacteria nontoxic gene) thereof that excites rice to generate disease resistance response, be specifically related to a kind of nontoxic gene of doing mutually with paddy rice host disease-resistant gene that be separated to from the former bacterium of bacterial blight of rice.

Background technology

Paddy rice is the important food crop of China, and approximately 60% population be take rice as staple food, and the Sustainable development of Rice Production is directly connected to the grain security of China.Bacterial blight of rice (Bacterial blight, BB) be one of Major Diseases in world's Rice Production, found in area, Japanese Fukuoka early than 1884, since the fifties, the morbidity scope constantly enlarges, at present occurrence scope each paddy rice producing region all over the world of bacterial leaf-blight.Bacterial blight of rice is by Gram-negative bacteria Xanthomonas campestris paddy rice mutation (Xannthomonas oryzae pv.oryzae, Xoo) a kind of vascular bundle diseases caused, generally can cause the paddy rice underproduction 20%～30%, even have no harvest when serious.

Facts have proved, the disease resistance of utilizing rice varieties is to prevent and treat the most economical and effective approach of bacterial blight of rice, so, from the sixties in 20th century, excavating and utilizing the new gene of bacterial leaf spot resistant is one of focus of research both at home and abroad always.The Gene For Resistance To Rice Bacterial Blight of having reported at present reaches 36, but their most of anti-spectrums are narrow or the resistance persistence is poor, on producing, be used effectively only have Xa3, Xa4, Xa7 and Xa21 etc. a few.Due to the variation of Pathogenic, the long-term extensively utilization of minority bacterial leaf spot resistant ospc gene always faces the risk of resistant lose.Harm for the long-term control bacterial blight of rice, generally take at present two strategies both at home and abroad: the one, the new gene of resisting bacterial leaf-blight of searching tool breeding utility value from the paddy rice resources such as wild-rice, another is the molecule mechanism of further investigation plant disease-resistant, to developing, can make the lasting new way of utilizing of more disease-resistant genes.

Along with molecular biological develop rapidly, particularly, in recent years to various plants pathogenic bacteria and the genomic order-checking of host plant thereof and to the analysis of various mutations body, greatly promoted the understanding of the mankind about the plant immunization system.The immunity system of known plants comprises the twice defence line generally at present: first is called as pathogen-associated molecular pattern (Pathogen/microbe-associated molecular patterns, PAMPs/MAMPs) bring out immunity (PAMPs triggered immunity, PTI), be that the pattern recognition receptors (Patternrecognition receptors, PRRs) that plant passes through its cell surface is also defended the accurate perception of the PAMPs molecule of pathogenic bacteria; The second defence line is called as the pathogenic bacteria effector and brings out immunity (Effector triggered immunity, ETI), this is because some pathogenic bacterias can suppress PTI by producing effector (Effectors), thereby break through the first line of defence of plant, so plant just for example, is scouted these effectors and starts defense response by new molecular receptor (the NBS-LRR protein of R genes encoding).Usually the gene pairs gene resistance of saying just belongs to the category of second defense mechanism.Over several hundred million years, the attack of pathogenic bacteria and the defence of plant hocket, thereby have promoted the coevolution of pathogenic bacteria and Plant Genome.

Bacterial blight of rice is research important food crop and the pathogenetic bacteria idealized system of work mutually, has become an international study hotspot, and has advanced the research of making mutually molecule mechanism about host and cause of disease.Both at home and abroad first rear clone disease-resistant (R) gene Xa1, xa5, xa13, Xa21, Xa23, Xa26 and the Xa27 etc. of paddy rice and nontoxic gene avrXa3, avrxa5, avrXa7, avrXa10, avrXa21 (Ax21) and the avrXa27 of bacterial leaf spot pathogenic bacteria.Already proved: avrXa21 (Ax21) 194 the amino acid whose albumen of encoding are PAMP molecules of Xanthomonas campestris, thereby proof Xa21 is the pattern recognition receptors PRR of paddy rice, and its resistance reaction belongs to the first line of defence of plant disease-resistant.And avrXa3, avrxa5, avrXa7, avrXa10 and avrXa27 all belong to avrBs3 gene family member, their encoding transcription factor type effector (Transcription activator-like effectors, TAL effectors), this effector albumen contains nucleus positioning sequence (NLS) and genetic transcription active region (AD), they inject host plant cell by pathogenic bacteria III type excretory system (TTS system), the genetic expression of then by nucleo-cytoplasmic transport albumen importin α and importin β, assisting to input nucleus and regulating and controlling the host, the resistance reaction excited belongs to the second defence line of plant disease-resistant.

The research in past also discloses, the iteron formed by 34 amino acid that contains different numbers in TAL effector effector albumen (for example AvrBs3 contains 17.5 repeating units), 34 amino acid of each repeating unit are almost completely identical, and just the 12nd and 13 amino acid wherein are alterable heights.The researchs such as Boch etc. and Moscou are found, the special DNA base of each repeating unit identification of AvrBs3, when each repeat type (the 12nd and the 13rd amino acid of each repeating unit) of AvrBs3 corresponds to the host and goes in by the UPA box of regulatory gene promotor, specific repeat type is corresponding with the special base on target dna.By the corresponding relation of analyzing many TAL effector effector albumen repeat types and being subject to the UPA box of its gene promoter area of inducing, build the specific molecular model of the different repeat type identification target dna of TAL effectors, thereby disclosed minute subcipher of TAL effector protein-specific identification host gene DNA.

Although in recent years about the making great progress of plant disease-resistant molecular biology research, because the plant immunization reaction is very complicated, at present the mankind about host plant, with pathogenic bacteria, on molecular level, the understanding of work is still very limited mutually.Wherein about the result of PTI and ETI Molecular interaction, mainly from Arabidopis thaliana and pseudomonas syringae (Pseudomonas syringae), make mutually system, be badly in need of having more at present making mutually the systematic study result from other pathogenic bacteria-host plant, so just more be conducive to disclose the panorama of pathogenic bacteria-host plant Molecular interaction.Molecular interaction research specific to Xanthomonas campestris TAL effectors and host plant R-gene, although the gene avrBs3 of first coding TAL effector just was cloned before 20 years, but its result is mainly from the system of doing mutually of capsicum and X.campegtris pv.vesicatoria, its ubiquity is still needed and will more results of study be verified, and the recognition process of TAL Effector albumen and target gene DNA base remains a unsolved mystery.Can estimate, in coming 10 years, about host plant and pathogenic bacteria, the identification on molecular level is done with mutual, will remain one of international study hotspot, and, likely by disclosing the molecule mechanism of plant disease-resistant, develops the new technology of preventing and treating crop pest.

Xanthomonas oryzae pv oryzae PXO99 ^apathogenic the strongest, the widest bacterial strain of the spectrum of causing a disease of finding so far, except to several resistant gene kinds as IRBB5 (containing resistant gene xa5), IRBB10 (Xa10), IRBB21 (Xa21), CBB23 (Xa23), almost all rice varieties are caused a disease.Identify in laboratory Zeng Cong China common wild-rice at contriver place and excavate out a dominant gene Xa23 to bacterial leaf-blight wide spectrum high resistance, it can resist China, Philippines and Japan all represent fungus strain.To more than 20 bacterial strain that can make Xa4, Xa21 cause a disease from states such as Vietnam, Korea S, Bangladesh, Xa23 all shows as high resistance, not yet finds to overcome so far the natural bacterial strain of Xa23 resistance.If find albumen and the encoding gene thereof that can excite Xa23 genetic expression, will provide genetic resources for the disease-resistant molecular breeding of rice bacterial blight resistance, be conducive to prevent and treat the harm of bacterial blight of rice.

Summary of the invention

The purpose of this invention is to provide a kind of avrXa23 albumen and encoding gene thereof that excites rice to generate disease resistance response.

Protein provided by the invention, derive from Xanthomonas oryzae pv oryzae, is following (a) or (b):

(a) protein that the aminoacid sequence shown in sequence 1 forms in sequence table;

(b) by the aminoacid sequence of sequence 1 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and by the sequence 1 derivative protein relevant to exciting rice to generate disease resistance response.

In order to make the protein in (a) be convenient to purifying, the N-terminal of the protein that can form at the aminoacid sequence shown in sequence in sequence table 1 or C-terminal connect label as shown in table 1.

The sequence of table 1 label

Label	Residue	Sequence
			Poly-Arg	5-6 (being generally 5)	RRRRR
Poly-His	2-10 (being generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

Above-mentioned (b) but in the protein synthetic, also can first synthesize its encoding gene, then carry out biological expression and obtain.The encoding gene of the protein in above-mentioned (b) can be by the codon by one or several amino-acid residue of disappearance in the DNA sequence dna shown in sequence in sequence table 2, and/or carry out the missense mutation of one or several base pair, and/or obtain at the encoding sequence that its 5 ' end and/or 3 ' end connects the label shown in table 1.

The gene of encoding said proteins also belongs to protection scope of the present invention.

Described gene can be following 1) or 2) or 3) DNA molecular:

1) DNA molecular shown in sequence 2 in sequence table;

2) under stringent condition with 1) the DNA sequence dna hybridization that limits and the coding DNA molecular that excites the rice to generate disease resistance response associated protein;

3) with 1) or 2) DNA sequence dna that limits has 90% above homology, and encode and excite the DNA molecular of rice to generate disease resistance response associated protein.

Described stringent condition is in the solution of 0.1 * SSPE (or 0.1 * SSC), 0.1%SDS, hybridizes under 65 ℃ of conditions and washes film.

The recombinant expression vector that contains described gene, expression cassette, transgenic cell line or recombinant bacterium all belong to protection scope of the present invention.

Described recombinant expression vector specifically can be in the multiple clone site of pHM1 carrier and inserts recombinant C osmid (Ke Si) plasmid that described gene obtains.

Described recombinant expression vector specifically can be pHM1/L, the colon bacillus that contains pHM1/L (Escherichiacoli) pHM1/L-2 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on August 04th, 2010 and (is called for short CGMCC, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), preserving number is CGMCCNo.4049.In pHM1/L-2, inserted the AvrXa23 gene shown in the sequence 2 of sequence table at the HindIII of pHM1 restriction enzyme site.

Recombinant expression vector pHM1/L and colon bacillus (Escherichia coli) pHM1/L-2 all belongs to protection scope of the present invention.

AvrXa23 albumen provided by the invention is comprised of 1483 amino-acid residues, and 34 amino acid repeating units of being encoded by 102bp repeat 26.5 times in this albumen.AvrXa23 mrna length provided by the invention is 4452bp, and the restriction enzyme site on gene is identical with the avrBs3/PthA family gene, and the repeat number in gene of 102bp repeating unit is 26.5, is a kind of nontoxic gene that excites rice to generate disease resistance response.The avrXa23 gene that the present invention obtains can be used as the genetic resources of rice bacterial blight resistance breeding for disease resistance, by after gene Introduced into Rice bacterial leaf spot pathogenic bacteria XM81 bacterial strain, can make the rice to generate disease resistance reaction containing the Xa23 disease-resistant gene, make bacterial leaf spot pathogenic bacteria be difficult to effectively infect paddy rice.This albumen and encoding gene thereof have potential breeding and are worth, and can be applied to cultivate the rice varieties of resisting bacterial leaf-blight.

In the present invention by anti-spectrum the widest paddy rice CBB23 (Xa23) and the widest bacterial leaf spot pathogenic bacteria PXO99 of spectrum that causes a disease ^afor material, typical specificity interaction between research plant and pathogenic bacteria, the clone has obtained PXO99 ^athe nontoxic gene avrXa23 of bacterial strain specific recognition host Xa23 gene, will contribute to illustrate the pathogenic mechanism of wide spectrum of bacterial leaf spot pathogenic bacteria.

The accompanying drawing explanation

Fig. 1 is pcr amplification wild-type PXO99 ^atn5 transposon Insert Fragment in bacterial strain and 14 mutant bacteria genomes, the about 569bp of object tape; Swimming lane is followed successively by M (Marker), PXO99 from left to right ^a, XM12, XM16, XM25, XM30, XM41, XM42, XM57, XM81, XM86, XM89, XM93, XM96, XM804, XM806; 14 disease cause mutation bacterial strains can amplify the target bands of a spectrum of 569bp, and PXO99 ^atarget stripe can not increase.

The Tn5 that Fig. 2 is Southern hybridization analysis part mutant strain inserts copy number; Swimming lane is followed successively by M (Marker), WT (PXO99 from left to right ^a), 16 (XM16), 86 (XM86), 25 (XM25), 41 (XM41), 42 (XM42), 30 (XM30), 57 (XM57), 81 (XM81); The pathogenic mutation bacterial strain is the mono-copy of Tn5 and inserts.

Fig. 3 is that rice varieties CBB23 and JG30 react the resistance of the former bacterium of bacterial leaf-blight; A is CBB23, and B is JG30; 1 is wild mushroom PXO99 ^a, 2 is mutant bacteria XM81, and 3 is XM81/45-1, and 4 is XM81/45-2; R is disease-resistant, and S is susceptible.

Fig. 4 is that restriction enzyme BamH1 enzyme is cut the P99ACo45 clone; M is Marker, and 1-3 is the P99ACo45 clone.

Fig. 5 is the pathogenic analysis of each bacterial strain on CBB23 and JG30, and X-coordinate is each bacterial strain, and ordinate zou is the ratio that scab length accounts for the blade total length; A is CBB23, and B is JG30; 1 is wild mushroom PXO99 ^a, 2 is mutant bacteria XM81, and 5 is XM81/45-1, and 6 is XM81/L-2, and 10 is the contrast bacterium.

Fig. 6 is that rice varieties CBB23 and JG30 react the resistance of the former bacterium of bacterial leaf-blight; A is CBB23, and B is JG30; 1 is wild mushroom PXO99 ^a, 2 is mutant bacteria XM81, and 5 is XM81/45-1, and 6 is XM81/L-2, and 10 is the contrast bacterium.

Embodiment

Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, be ordinary method.Conventional experimental methods of molecular biology can be referring to molecular cloning: experiment guide, and J.Sambrook, wait and write, second edition, cold spring harbor laboratory publishes, cold spring port, N.Y., 1989.Test materials used in following embodiment, if no special instructions, be and purchase available from routine biochemistry reagent shop.Quantitative test in following examples, all arrange repeated experiments three times, results averaged.

Association's Ben Zheshi substratum (XBZ): 300g potato, 5g peptone, 15g sucrose, 0.5g Ca (NO ₃) ₂4H ₂o, 2g Na ₂hPO ₄12H ₂o, solid medium adds 17g agar, and water is settled to 1L; PH=6.8-7.0.

Wild mushroom PXO99 ^a: the public can obtain from Institute of Crop Science, Chinese Academy of Agricultural Science; Reference: Hopkins C.M., White F.F., Choi S.-H., Guo A.and Leach J.E., Identificationof a family of avirulence genes from Xanthomonas oryzae pv.Oryzae, Molecularplant-microbe interactions vol.5, No.6.pp.451-459,1992.

Mutant strain XM81 belongs to rice Xanthomonas (Xanthomonas oryzae), on April 30th, 2010, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center and (be called for short CGMCC, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), preserving number is CGMCC No.3795.

Cosmid carrier pHM1: the public can obtain from Institute of Crop Science, Chinese Academy of Agricultural Science; Reference: Hopkins C.M., White F.F., Choi S.-H., Guo A.and Leach J.E., Identificationof a fami ly of avirulence genes from Xanthomonas oryzae pv.Oryzae, Molecularplant-microbe interactions vol.5, No.6.pp.451-459,1992.

Rice varieties JG30: the public can obtain from Institute of Crop Science, Chinese Academy of Agricultural Science; Reference: Wang Chunlian, Qi Huaxiong, Pan Haijun, lijin's ripple, Fan Yinglun, Zhang Qi, Zhao Kaijun, the EST mark of Gene For Resistance To Rice Bacterial Blight Xa23 and the utilization on molecular breeding thereof, Scientia Agricultura Sinica, 2005,38 (10): 1996-2001.

Rice varieties CBB23: the public can obtain from Institute of Crop Science, Chinese Academy of Agricultural Science; Reference: Wang Chunlian, Qi Huaxiong, Pan Haijun, lijin's ripple, Fan Yinglun, Zhang Qi, Zhao Kaijun, the EST mark of Gene For Resistance To Rice Bacterial Blight Xa23 and the utilization on molecular breeding thereof, Scientia Agricultura Sinica, 2005,38 (10): 1996-2001.

The discovery of embodiment 1, AvrXa23 gene

One, Xanthomonas oryzae pv oryzae PXO99 ^athe structure of mutant library

Utilize Tn5 transposon system [EZ-Tn5 ^tM<KAN-2>TnpTransposome ^tMkit (EpicentreTechnologies, Madison, WI)] structure PXO99 ^amutant library.

1, competent cell preparation

By PXO99 ^abacterium, the upper stroke bacterium of association's Ben Zheshi substratum (XBZ), is cultivated activation for 28 ℃.Picking mono-clonal bacterium colony coated plate on the XBZ solid medium, cultivate 2 days.With the transfering loop thalline that takes a morsel, be seeded in liquid nutrient medium 28 ℃, 220rpm shaking culture.Work as OD ₆₀₀the failure of oscillations immediately in=0.6 o'clock, take out to be put in frozen water and shake rapidly to fully cooling, is sub-packed in the 50ml centrifuge tube, and 2200g (g=rcf), 4 ℃ are centrifugal.Pour out supernatant, add 30ml 10% sterile glycerol, sway in frozen water to precipitation dissolving (ice 10-15 minute) fully.Recentrifuge, 2400g, 4 ℃ are centrifugal, then repeat once.Remove supernatant, in bottle during the glycerine of surplus have ± 0.5ml, and throw out fully mixes, and with the dosage of 60 μ l/ pipes, is distributed in the centrifuge tube of 1.5ml, places-80 ℃ of preservations.

2, Electroporation

By 1ul Tn5 transposon system DNA (establishing blank) and 60ul PXO99 ^athe competent cell mixing is carried out Electroporation, parameter E.coli-1mm, 1.8k υ: Voltage (v) 1800, Capacitance (μ F) 25, Resistance (Ω) 200, Cuvette (mm) 1.After Electroporation completes suspension cell in the XBZ substratum in 28 ℃ of shaking culture 1h, coat on the XBZ substratum that contains the 30ug/mL kantlex, cultivate about 2-4 days for 28 ℃, by the positive bacterium colony that grows (owing to only having positive colony just to there is kalamycin resistance, therefore the bacterium colony grown is mutant) be transferred in 384 well culture plates that XBZ substratum (containing the 30ug/mL kantlex) is housed, be stored in-80 ℃ of refrigerators, standby.Altogether picking 63 384 orifice plates, obtain 24192 mutants which hads.

3, the PCR of mutant library identifies

Random picking mutant bacterium carries out bacterium liquid PCR and detects, purpose checking Tn5 swivel base efficiency.With unconverted PXO99 ^adNA make negative control.Primer sequence is: TN5F:5 '-ATTCAACGGGAAACGTCTTG-3 '; TN5R:5 '-ACTGAATCCGGTGAGAATGG-3 ', the product size is 569bp.Amplification program is 95 ℃ of 3min, (95 ℃ of 30s, 58 ℃ of 30s, 72 ℃ of 50s), 35 circulations, 72 ℃ of 7min.Tn5 swivel base efficiency reaches 99%, illustrates that this mutant library is available.

Two, screening mutant library

Adopt the method for artificial leaf-cutting inoculation.Rice varieties JG30 and CBB23 are a pair of near isogenic lines, and JG30 is to PXO99 ^asusceptible, CBB23 is to PXO99 ^adisease-resistant.Be inserted into PXO99 by the TN5 swivel base ^athe genomic different positions of bacterium produces sudden change, therefrom finds and makes the susceptible mutant strain of CBB23.Therefore plant nearly 30,000 strains of CBB23, plant to be planted grows to 5 leaves and starts to carry out inoculated identification wholeheartedly the time.

1, the primary dcreening operation of mutants which had

At first will be stored in each sucking-off 3ul of mutant strain in 384 orifice plates of-80 ℃ of refrigerators in the 12ml culture tube in rejuvenation, every pipe adds 4ml XBZ liquid nutrient medium (containing the 30ug/mL kantlex), 28 ℃, 230rpm, shaking culture 2-3 days, bacterial concentration is at OD ₆₀₀during=0.8-1.0, take out, each pipe is put into a scissors, dips bacterium liquid and inoculates a strain, 24192 whole individual plant leaf-cutting inoculations of mutants which had.Within about 2 weeks, is investigated after inoculation, chosen the blade of the percentage that lesion area accounts for blade area>15%, through 20 of preliminary screening acquisitions, made the susceptible mutant bacterium of CBB23.

2, the multiple sieve of pathogenic mutation body

The pathogenic mutation body bacterium that preliminary screening goes out, further sieve again, to guarantee its accuracy.Multiple sieve carries out in the cement pit of paddy rice solarium.Plantation JG30 and CBB23, all divide individual plant to be seeded in respectively on these 2 kinds above-mentioned 20 mutant strains, and wild mushroom PXO99 is set simultaneously ^afor contrast, control time and method are the same.Result is therefrom selected 14 and is made the pathogenic bacterial strain of CBB23, and numbering is: XM12, XM16, XM25, XM30, XM41, XM42, XM57, XM81, XM86, XM89, XM93, XM96, XM804, XM806.

Three, the Molecular Detection of mutants which had

1, PCR detects

Make in order to verify whether the pathogenic bacterial strain of CBB23 is that real Tn5 insertion causes, with wild-type PXO99 ^abacterial strain and 14 mutants which had genomic dnas are done template.Carry out pcr amplification with primer TN5F/TN5R as primer pair, amplification program is the same.Result shows, can the increase target bands of a spectrum of a treaty 569bp of all disease cause mutation bodies that screen, and PXO99 ^athe target stripe that can not increase (Fig. 1).Illustrate that the mutant obtained is that the slotting people of transposon causes.

2, in the pathogenic mutation body, transposon insertion copy number is determined

Sudden change may be that the transposon insertion causes, is likely also that spontaneous mutation causes, therefore by Southern, hybridizes and gets rid of the sudden change that spontaneous mutation causes.In mutant, transposon insertion the definite of copy number is the follow-up requisite previous work of analysis of molecules.Because only had clearly mutant to insert copy number, the flanking sequence of insertion point in segregation mutant on purpose, and then carry out complementation and the functional analysis of gene.Therefore, in order to determine insertion copy number and the position of mutant, with ³²the PdCTP mark, the TNF/R fragment obtained by pcr amplification is probe, with the total DNA of mutant gene group of SphI complete degestion, carries out Southern hybridization (the wild-type PXO99 that same enzyme is cut ^agenome DNA compares).Result shows that 14 pathogenic mutation bodies are single copy and insert (Fig. 2), illustrates that only having a site has caused CBB23 susceptible because of the insertion of Tn5.

Four, in the pathogenic mutation body, the transposon flanking sequence is analyzed

Adopt the method for PCR walking (Cottage et al.2001) to separate the flanking sequence of TN5 insertion point.

PCR wal king method is to improve through the asymmetric mutual PCR of heat (Thermal Asymmetric Interlaced PCR, TAIL-PCR) a kind of round pcr that is applied to separate the T-DNA flanking sequence of coming.Flush end restriction enzyme digestion for the genome that will insert through TN5-DNA (this experiment adopts DraI/EcoRV), this Restriction Enzyme only has a point of contact on T-DNA, then this enzyme Qie Wenku that cuts rear formation to enzyme adds the specificity joint AP design, AP is a long-chain DNA sequence dna LAP (5 '-CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGGAGGT-3 ') and a short chain, with the long-chain complementation, and 3 ' end forms through amidized DNA sequence dna NAP (5 '-ACCTCCCC-H2N-3 '), what 3 ' amination assurance and enzyme cut that product is connected is not this 3 ' amination end, facilitate the nested Auele Specific Primer of designed joint.According to the nested Auele Specific Primer AP1 of joint design (AP1 (5 '-GGATCCTAATACGACTCACTATAGGGC-3 ') and AP2 (5 '-CTATAGGGCTCGAGCGGC-3 '), with the nested primers forward aligning primer TnFP1 of the upper design of TN5-DNA (5 '-GGCAGAGCATTACGCTGACT-3 ') and TnFP2 (5 '-ACCTACAACAAAGCTCTCATCAACC-3 ') and reverse sequence primer TnRP1 (5 '-CTGATTGCCCGACATTATCG-3 ') and TnRP2 (5 '-GCAATGTAACATCAGAGATTTTGAG-3 '), the two-wheeled pcr amplification is carried out in pairing respectively, the product of amplification is checked order, by the NCBI website, sequencing result and PXO99 ^athe whole genome sequence comparison, compare with KACC10331 and MAFF311018t simultaneously.Result shows that 14 mutant only have XM804, XM806 to insert PXO99 ^athe position that genome is identical, other 12 are all inserted different positions.4 mutant XM81 are arranged, the albumen that XM86, XM804, XM806 are identical by the genes encoding of Tn5 on position, belong to TAL effector AvrBs3/PthA family, a plurality of tumor-necrosis factor glycoproteinss are contained in this family, each repeats to contain 102 bases, about 34 amino acid, according to the repeat number difference, inducing plant produces the disease resistance difference.According to the result of former studies, infer that it is also due to this genoid that CBB23 produces disease-resistant.Wild mushroom PXO99 originally ^acan bring out CBB23 and produce disease resistance response, after being inserted by Tn5, some bacterial strain, as XM81, XM86, XM804 etc. make disease-resistant gene CBB23 produce susceptible reaction, has changed its virulence, and TN5 inserts and destroyed PXO99 ^athe expression of middle nontoxic gene.Want gram and fall this nontoxic gene, must be from mutants which had and wild mushroom PXO99 ^atwo aspects.

Five, the clone of the Cosmid that contains AvrXa23

According to forefathers research, show, the sequence of the nontoxic gene in Xanthomonas campestris is guarded very much, plant produce different disease-resistant genes mainly due to nontoxic gene tumor-necrosis factor glycoproteins unit the number or the amino acid whose variation of certain specific site cause.Bacterial blight of rice also causes by Xanthomonas campestris, and its pathogenic bacteria has typical above-mentioned characteristic.Therefore adopt the conserved sequence of bacterial blight of rice nontoxic gene, screening wild-type PXO99 ^athe Cosmid genomic library.

By the partial sequence of bacterial blight of rice nontoxic gene AvrXa7, be probe, Southern hybridization wild-type PXO99 ^athe Cosmid genomic library, obtain 36 positive colonies, respectively called after pHM1/PXO99lib1-36.From library, these 36 clones are picked out, on LB+SPE (spectinomycin) substratum, again shake bacterium, extract plasmid DNA.With the BamH1 enzyme, cut respectively, result shows that these clones contain the DNA fragmentation difference, vary in size.

Six, positive colony is proceeded to mutant strain

3 mutant strain XM81, XM86, XM806, although TN5 inserts the different positions of AvrBs3/PthA family conserved sequence, the identical albumen of encoding.Therefore only XM81 is carried out to competent preparation early stage, the preparation method is the same.After prepared by competence, above-mentioned 36 Cosmid are cloned to difference Electroporations (parameter is the same) in the XM81 competence.Get respectively resuscitation fluid and coat and contain on spectinomycin (100 μ g/ml) XBZ solid medium, cultivate 2-4 days for 30 ℃, obtain the transformant of different quantities, 4 ℃ of preservations.

Seven, inoculated identification

Will be to PXO99 ^asusceptible variety JG30 and disease-resistant near isogenic line CBB23 kind thereof are planted in the cement pit of paddy rice solarium, and plant to be planted grows to 5 leaves and carries out inoculated identification wholeheartedly the time.

36 Cosmid clones are transformed into respectively to (difference called after P99Aco31-66 after being transformed in XM81) 2 transformants of each clone's picking in XM81, rejuvenation on the XBZ+Spe solid medium, cultivate 2 days for 28-30 ℃, with sterilized water wash-out bacterium liquid, concentration OD ₆₀₀=1.0, carry out the leaf-cutting inoculation at JG30 and CBB23 respectively.Inoculate and investigated after 14 days, measure the length of scab length and whole blade.The investigation result demonstration, all transformants can make JG30 susceptible, and CBB23 is only disease-resistant to the P99Aco45 performance, all the other performances are susceptible.P99Aco45 clone has recovered the resistance (Fig. 3) of CBB23 in other words, illustrates that P99ACo45 contains the nontoxic gene with the Xa23 interaction of genes.2 transformant called after XM81/45-1, XM81/45-2.

Eight, P99ACo45 subclone and subclone expression vector establishment

In general, nontoxic gene 2 ends in Xanthomonas campestris have 2 conservative BamH1 restriction enzyme sites, if the P99ACo45 clone who makes CBB23 recover resistance is the nontoxic gene in typical Xanthomonas campestris, it just should have the BamH1 restriction enzyme site, therefore with the BamH1 enzyme, cuts the P99ACo45 clone.As Fig. 4: enzyme goes out 4 bands earnestly.In order to determine which band is and the nontoxic gene of Xa23 interaction of genes, carries out subclone to the P99ACo45 clone.The first band called after L, the second band called after M, the 3rd band called after S.

1, P99ACo45 subclone

The P99ACo45 clone is cut with the BamH1 enzyme, and enzyme is cut rear electrophoresis, cuts respectively three L, M, S sheet segment DNA (Fig. 4) under ultraviolet lamp, reclaims test kit with glue and reclaims respectively three DNA, standby.PBluescript II sk+ is cut with the BamH1 enzyme, after complete degestion, with alkaline phosphatase dephosphorization (method is shown in the CIAP dephosphorizing method of TAKARA company), dehydrated alcohol precipitation DNA after dephosphorization.Get purpose fragment and pBluescript II sk+ carrier (the BamH1 enzyme is cut) under the effect of T4 ligase enzyme, 16 ℃ of connections are spent the night.Connect the product electrode and proceed in DH5 α competent cell, containing on penbritin (100 μ g/ml) LB solid medium, 37 ℃ of cultivations, obtain single colony clone of different quantities.With and the several mono-clonals of picking, shake alkaline lysis upgrading grain after bacterium, enzyme is cut the checking junction fragment, obtains the subclone that contains purpose fragment (L, M).

The pBluescript II sk+ cloning and sequencing that will contain the L fragment, obtain AvrXa23DNA sequence and aminoacid sequence.The aminoacid sequence of AvrXa23 albumen is as shown in the sequence 1 of sequence table, and the nucleotide sequence of AvrXa23 gene is as shown in the sequence 2 of sequence table.The AvrXa23 gene contains 26.5 repetitions, each repeats to contain 102bp, and 34 amino acid of each repeated encoding contain nucleus positioning sequence (NLS) and genetic transcription active region (AD), full length gene 4452bp, the AvrXa23 albumen that 1483 amino-acid residues of encoding form.AvrXa23 albumen is a newcomer of the distinctive transcription factor analogue of Xanthomonas (transcription activator-like effector, TAL) gene family, different from all nontoxic genes of having cloned at present.

2, build the subclone expression vector

Cosmid carrier pHM1 (spectinomycin resistance) and pBluescriptII sk+ (amicillin resistance) carrier that contains external source fragment (L or M fragment) are cut with the HindIII enzyme respectively, and under the effect of T4 ligase enzyme, 16 ℃ connect 3 hours.Connect the product Electroporation in DH5 α, method is the same.Converted product is coated and is contained on two anti-(100 μ g/ml) the LB solid mediums of spectinomycin/penbritin, cultivates 8-12 hour for 37 ℃, and a small amount of single colony clone of picking, shake bacterium immediately, the upgrading grain, and the HindIII enzyme is cut checking.Cut with the BamH1 enzyme simultaneously, compare with the P99ACo45 clone, obtain the Cosmid expression vector that contains L, M DNA fragmentation.

The pHM1 called after pHM1/L that will contain the L fragment, colon bacillus (Escherichiacoli) the called after pHM1/L-2 that will contain pHM1/L, pHM1/L-2 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on August 04th, 2010 and (is called for short CGMCC, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), preserving number is CGMCC No.4049.In pHM1/L, inserted the AvrXa23 gene shown in the sequence 2 of sequence table at the HindIII of pHM1 restriction enzyme site.

The checking that has complementary functions of embodiment 2, AvrXa23 gene

One, the preparation of recombinant bacterium XM81/L-2

1, competent cell preparation

Mutant strain XM81 is drawn on the XBZ solid medium to bacterium, cultivate activation for 28 ℃.Picking mono-clonal bacterium colony coated plate on the XBZ solid medium, cultivate 2 days.With the transfering loop thalline that takes a morsel, be seeded in the XBZ liquid nutrient medium 28 ℃, 220rpm shaking culture.Work as OD ₆₀₀the failure of oscillations immediately in=0.6 o'clock, take out to be put in frozen water and shake rapidly to fully cooling, is sub-packed in the 50ml centrifuge tube, and 2200g, 4 ℃ are centrifugal.Pour out supernatant, add 30ml 10% sterile glycerol, sway in frozen water to precipitation and dissolve fully, 2400g, 4 ℃ are centrifugal.Remove supernatant, residue fully mixes during 0.5ml, with the dosage of 60 μ l/ pipes, is distributed in the centrifuge tube of 1.5ml, places-80 ℃ of preservations.

2, Electroporation and screening

Extract pHM1/L from pHM1/L-2, by electrization, import in mutant strain XM81 competent cell, it is E.coli-1mm that electricity swashs parameter, (electricity swashs gap 1mm), Voltage (v) 1800, Capacitance (μ F) 25, Resistance (Ω) 200, Cuvette (mm) 1; Containing on spectinomycin (100 μ g/ml) XBZ solid medium, cultivating 2-4 days, obtain recombinant bacterium, called after XM81/L-2 for 30 ℃; Rejuvenation on new XBZ solid medium, cultivate 2 days for 30 ℃, scrapes a little bacterium colony in the XBZ liquid nutrient medium that contains 40% glycerine, and-80 ℃ save backup.

Two, the preparation of contrast bacterium

Replace pHM1/L to import mutant strain XM81 competent cell Cosmid carrier pHM1, other same step 1, obtain contrasting bacterium.

Three, after inoculating each bacterial strain, the incidence of disease-resistant plant and disease plant

By XM81/L-2, wild mushroom PXO99 ^a, to adjust concentration be OD for mutant strain XM81, XM81/45-1 and contrast bacterium ₆₀₀=1.0, then carry out the leaf-cutting inoculation at rice varieties JG30 and rice varieties CBB23 respectively.Each inoculation 5 strain, 5 of every strain inoculations launch leaves, take pictures and incidence is investigated after inoculating 14 days.Measure the length of scab length and whole blade, calculate the ratio that scab length accounts for the blade total length.

Incidence the results are shown in Figure 5.Transfection XM81/L-2 and transfection wild mushroom PXO99 ^a(P6) the scab length of CBB23 rice plant accounts for the ratio of blade total length all below 0.05, and both approach, and the ratio that the scab length of the CBB23 rice plant of transfection mutant strain XM81 and contrast bacterium accounts for the blade total length is more than 0.6; The scab length of the JG30 rice plant of each bacterial strain of transfection accounts for the ratio of blade total length all more than 0.45, and each bacterial strain does not have marked difference.

Fig. 6 is shown in by photo.Result shows, containing the transformant of the AvrXa23 gene shown in the sequence 2 of ordered list, makes the resistance of CBB23 (Xa23) obtain recovery.Illustrate that the AvrXa23 gene shown in the sequence 2 of sequence table is the nontoxic gene of Xa23.

Claims (2)

1.avrXa23 the application of albumen in exciting rice to generate disease resistance response, described avrXa23 albumen is the protein of sequence as the 1 described amino acid residue sequence of sequence in sequence table; Described disease resistance response is the disease resistance response to bacterial blight of rice; Described paddy rice is rice varieties CBB23.

2.avrXa23 the application of the encoding gene of albumen in exciting rice to generate disease resistance response, the sequence of the encoding gene of described avrXa23 albumen is the nucleotide sequence shown in sequence 2 in sequence table; Described disease resistance response is the disease resistance response to bacterial blight of rice; Described paddy rice is rice varieties CBB23.

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