CN104004771B - A kind of avirulence gene of rice blast AvrPi9, the polypeptide of coding, polynucleotide and its application - Google Patents

Abstract

The invention discloses avirulence gene of rice blast AvrPi9, the polypeptide of coding, polynucleotide and its application.The invention provides Pyricularia oryzae (Magna porthe grisea) the newly nucleotide sequence of nontoxic gene Avr Pi9 and its an amino acid polypeptide sequence for coding, this gene is the gene of a special abduction delivering when infecting plant.Based on this gene, the enhanced transgenic plant of resistances against diseases can be prepared, or prepare the molecular marked compound following the tracks of the change of Pyricularia oryzae population.

Description

A kind of avirulence gene of rice blast AvrPi9, coding polypeptide, polynucleotide and its Application

Technical field

The present invention relates to gene engineering technology field is and in particular to a kind of rice blast fungus nontoxic gene Avr-Pi9 Separating clone and application.

Background technology

Oryza sativa L. (Oryza sativa L.) is China the first generalized grain crop, is also the most important cereal crops in the whole world. Rice blast fungus (Magnaporthe oryzae) are the pathogens causing rice blast, and the whole world is annual to be caused because of rice blast 10%～30% Rice Yield Loss Caused, even No kernels or seeds are gathered, as in a year of scarcity for serious field.The means of prevention and control rice blast are mainly and apply at present Pesticide and plantation resistant variety.Applying pesticides have pollution environment increases production cost, and impact rice quality etc. is a series of unfavorable The selection-breeding of factor, therefore disease-resistant variety and popularization are current prevention and control rice blast the most economically and efficiently means.However, natural ring In border, in the selection pressure of rice resistant variety, rice blast fungus flora replaces the new physiology that can break through rapidly resistant variety direction Microspecies simultaneously form dominant population, ultimately result in resistant variety resistance " forfeiture ".In production practices, resistant variety continuously plants 3-5 Nian Hou, resistance will substantially fail, disease-resistant cannot persistence.How to monitor physiological races of rice blast fungus occurrence dynamics, cultivation is held Kind disease-resistant long and the more efficiently resistant breeding strategy of searching, become an urgent demand on current production.

Coupling effects between Oryza sativa L. and Pyricularia oryzae meet " gene-for-gene " hypothesis (Flor, 1947；Silue et al.,2000；Bryan et al.,2000；Orbach et al.2000).Theoretical according to this, Oryza sativa L. passes through its resistant gene (R gene) specific recognition derives from the nontoxic gene (Avr gene) of Pyricularia oryzae, thus exciting a series of disease-resistant in Oryza sativa L. body Defense response.In the past twenty years, the research about rice blast resistant gene aspect achieves very big entering both at home and abroad Exhibition, at least it has been reported that 63 blast resisting sites totally 77 major gene resistances (national Oryza sativa L. data center. rice blast main effect resists Property list of genes [DB/OL] .http://www.ricedata.cn/gene.).These genes greatly accelerate paddy disease-resistant and educate The process of kind.However, the resistance of R gene mediated has strict kind specificity, different R genes is had sternly with its recognition factor The corresponding relation of lattice, the high variability of pathogen microspecies pathogenicity is easily caused the resistance disappearance of R gene mediated.Solve this to ask Topic, needs to further investigate the essence of interaction between resistant gene and nontoxic gene, then provides new broad spectrum antidisease gene Engineering strategy and method (Wit.1992；Bryan et al.,2000；Orbach et al.2000).At present, with respect to Oryza sativa L. The research of blast resistant gene, rice blast fungus nontoxic gene clone's aspect relatively lags behind, although about 40 nontoxic genes are It is reported, but only 9 nontoxic genes are cloned, wherein PWL1 (Kang et al., 1995) and PWL2 (Sweigard et Al., it is 1995) weeping love grass host specificity, remaining AvrPi-ta (Orbach et al., 2000), ACE1 (Bohnert et al.,2004)、Avr1-CO39(Farman and Leong,1998)、AvrPiz-t(Li et al., 2009), AvrPii, AvrPia and AvrPik/km/kp (Yoshida et al., 2009) this 7 is to control rice blast fungus to Oryza sativa L. Pathogenic.ACE1 is wherein only had to encode a non-secretory polyketide synthases gene, other 8 nontoxic genes all encode The secretory protein of prediction.Only Pi-ta and Avr-Pita, Pia between the resistant gene cloned at these and nontoxic gene It is paired with Avrpia, Piz-t and AvrPiz-t and Pikm and AvrPik | km | kp, and only have Pi-ta and AvrPi-ta Between have the molecular Evidence of direct interaction.

Content of the invention

It is an object of the invention to provide the cloning process of avirulence gene of rice blast and molecular marker.

In a first aspect of the present invention, provide a kind of polypeptide of avirulence gene of rice blast AvrPi9 coding, this polypeptide selects From the following group：

A () has SEQ ID NO:The polypeptide of 3 aminoacid sequences；

B () is by SEQ ID NO:3 aminoacid sequences through one or more (such as 1-50, preferably 1-30, more preferably 1-10, more preferably 1-5, more preferably 1-3) replacement of amino acid residue, disappearance or interpolation and formed, and have can be with Rice blast resistant gene Pi9 coding protein interaction function by polypeptide derived from (a).

In another aspect of this invention, provide avirulence gene of rice blast AvrPi9 it is characterised in that it comprises a nucleoside Acid sequence, this nucleotide sequence is selected from the group：

The polynucleotide of coding said polypeptide；The complementary polynucleotide with above-mentioned polynucleotide.

In another preference, described polynucleotide are：

There is SEQ ID NO:The polynucleotide of nucleotide sequence shown in 1；There is SEQ ID NO:Nucleotide sequence shown in 2 Polynucleotide；

In another aspect of this invention, provide a kind of carrier, it contains described polynucleotide.

In another aspect of this invention, provide a kind of genetically engineered host cell, it contains described carrier；Or its It is integrated with described polynucleotide in genome.

In another aspect of this invention, using described aminoacid sequence or nucleotide sequence or carrier or genetically engineered Host cell, for cultivating disease-resistant plant variety.

In another aspect of this invention, using described aminoacid sequence or nucleotide sequence, for identifying Pyricularia oryzae Infection ability, or the label for preparation detection plant Pyricularia oryzae toxicity population distribution.These labels include but do not limit In SNP (mononucleotide polymorphic), SSR (simple sequence repeats polypeptide), RFLP (restricted enzyme length polypeptide), CAP (cutting Amplified fragments polypeptide).

In another aspect of this invention, provide the purposes of described molecular marked compound, for the planting structure distribution of rice varieties.

The invention has the beneficial effects as follows：

The present invention obtains a kind of new avirulence gene of rice blast AvrPi9, the 26S Proteasome Structure and Function research based on described gene As a result, can be widely used for novel agrochemical exploitation, physiological races of rice blast fungus monitoring and rice blast Molecular interaction theory to grind Study carefully；Based on the sequence of described gene, the Markers for Detection system of monitoring Pyricularia oryzae natural population pathogenicity variation can be set up； Understand that change in the natural population of field for the described gene is dynamic, thus instructing rice varieties planting structure distribution and rotation, with effective Control the generation of disease；Assembled with corresponding disease-resistant gene and suitable promoter using described gene, built wide spectrum The engineering carrier of permanent disease-resistant, and and then Introduced into Rice or other plant cell, cultivate the disease-resistant Oryza sativa L. product of broad spectrum durable Kind.

Brief description

Fig. 1 passes through the prominent of R01-1 in specific 26 hybrid bacterial strains of molecular markers for identification of 192 R01-1 for the present invention Become F+strain, and the comparison according to PCR amplified band and result statistics, built using adjacent method (Neighbor-joining) Systematic evolution tree；

Fig. 2 is the natural bacterial strain scanning result of AvrPi9 candidate gene and gene structure, avirulent strains R88-002 and In Guy11,1434 all exist and consistent with No. 1435 genes；In virulent strain R01-1, but two genes all exist No. 1434 bases Because inside has transposon Mg-SINE to insert；

Fig. 3 is that the present invention identifies to the complementary function of AvrPi9 candidate gene, and candidate gene 1434 and 1435 is led respectively Enter in virulent strain R01-1 and 75-5, the conversion bacterial strain that each transformation event obtains utilizes Tp309 and imports disease-resistant gene The Tp309 Monogenic lines (Pi9-Tp309) of Pi9 carry out pathogenic identification.Fig. 3 A inoculates the result, figure for R01-1 transformant 3B inoculates the result for 75-5 transformant.Result shows, R1434 is exactly AvrPi9 gene.

Fig. 4 is PCR testing result and the Function Identification result figure that the present invention loses transformant to AvrPi9 gene function.Figure 4A is the PCR testing result that AvrPi9 gene function is lost with transformant, respectively knocks out transformant RJ1-43 of R1434, strikes Except transformant RJ2-7 of R1434 and RJ1435 and transformant RJ3-53 of RJ2-15, knockout RJ1435.Fig. 4 B converts to losing Subfunction qualification result, experimental result has further confirmed that the R1434 in R88-002 is exactly AvrPi9 gene.

Specific embodiment

Further illustrate present disclosure with specific embodiment below in conjunction with the accompanying drawings, but should not be construed as to the present invention's Limit.If not specializing, the conventional meanses that the technological means employed in embodiment are well known to those skilled in the art.

Embodiment 1：Carry the determination of nontoxic gene AvrPi9 wild type rice blast bacteria strain R88-002.

R01-1 is the avrPi9 mutant being screened by the method for 26 AvrPi9 strain mixture vaccinizations (Kim BR,et al.,2004).Therefore, it can reasonably speculate very much that R01-1 should derive from 26 hybrid bacterial strains Individual.It is prominent to identify R01-1's in 26 hybrid bacterial strains that the present invention passes through to develop the specific molecular marker (table 1) of 192 R01-1 Become F+strain, as shown in table 2, compared with R01-1, bacterial strain R88-002 is not observed polymorphic result, and bacterial strain 85-242 is then Polymorphism is the abundantest.Comparison according to PCR amplified band and result statistics, are built using adjacent method (Neighbor-joining) Systematic evolution tree (accompanying drawing 1), the final wild type determining that R88-002 is R01-1 bacterial strain.

Table 1 is used for identifying the PCR primer of R01-1 and 26 bacterial strain sibship

Table 2.192 polymorphic situation .NA in 26 bacterial strains to primer:No amplified production；AD:Primer size is different.

Embodiment 2：R88-002 and R01-1 genome sequence determination with compare.

The present invention carries out gene order-checking using the sequencing of Illumina platform to R88-002 and R01-1, for reaching equal matter The assembling genome of amount, the present invention carries out 100 times of coverage sequencings to them, and sequencing amount reaches about 4.5Gb.On the whole, The genome of R88-002 and R01-1 assembling in Genome Size, surveyed with other by the aspect such as predicted gene number and secretory protein number Sequence rice blast fungus genome no significant difference.Comparison and analysis for convenience, the present invention also introduces two other and has announced sequence AvrPi9 avirulent strains 70-15 and Ina168.R88-002 is AvrPi9 avirulent strains and R01-1 is AvrPi9 virulent strain.Root Courageously assume AvrPi9 encoding secreted proteins, and the machine that nontoxic gene function is lost according to the rice blast fungus nontoxic gene experience of clone System is that have, in gene elmination, gene or gene regulatory region (upstream 1kb), the afunction that big Indel leads to.So AvrPi9 candidate gene should be the secreted protein gene of R88-002, and upstream 1kb regulatory region and 70-15 and Ina168 base Cause is consistent, and there is larger difference with R01-1.

The present invention uses Repeatmask3.3 (http to rice blast fungus genomic data:// Www.repeatmasker.org, species=" magnaporthe grisea ") to filter, the repetitive sequence storehouse of use is derived from RepBasehttp://www.girinst.org/).Predictive genes employ the fgenesh module (species of Molquest： magnaporthe；Sequence length is more than 20aa).R88-002 has 12344 predicted genes, and secretory protein prediction uses SignalP4.1 detection contains signal peptide gene 1764, then uses the Protcomp-AN module prediction albumen of Molquest thin Born of the same parents position, and containing secretion signal gene 1015, finally filter out the albumen of two or more membrane spaning domain with TMHMM2.0, finally Remaining 1008 genes are prediction point albumen.Transfer this 1008 secreted protein genes and upstream 1Kb regional sequence as inquiry Storehouse, blastn compares R01-1/70-15 and In168 genome, transfers consistent with 70-15/In168 gene and exists with R01-1 As AvrPi9 candidate gene, detailed results are shown in Table 3 to 23 secreted protein genes of larger difference.For this 23 candidate's bases Cause, we exclude sequence assembly through careful human interpretation and compare the false positive gene leading to, design specific aim primer row Except the gene that there is variation in avirulent strains.Natural bacterial strain scanning (see Fig. 3 A and B) result avirulent strains R88-002 and In Guy11,1434 all exist and consistent with No. 1435 genes；In virulent strain R01-1, but two genes all exist No. 1434 bases Because inside has transposon Mg-SINE to insert；In virulent strain 75-5, two genes do not exist.Result above primarily determines that R88- 002 1434 and No. 1435 genes (gene structure is shown in Fig. 3 B) are AvrPi9 emphasis candidate genes, then by following gene work( Its function can be confirmed by complementation test.

Embodiment 3：The complementary identification of candidate gene.

The primer of candidate gene of the present invention complementary identification vector construction and vector background are as shown in table 3.Using KOD high-fidelity Enzyme, expands out from R88-002 by R1434 and R1435 using 12655CF/12655CR and 12654CF/12654CR respectively, And be cloned into from the T-easy carrier that Quan Shi King Company buys.Recycle Sac I and Asc I by genetic fragment recombinate to PCSN43 carrier, is respectively designated as pR55 and pR54.Carrier construction is imported with toadstool by the method using PEG protoplast transformation In strain R01-1 and 75-5 (Sweigard et al.1995).Wherein, in R01-1, R1434 gene internal has Mg-SINE to insert, And R1435, as R88-002, therefore, only carries out the complementation of pR55.And two genes do not exist in 75-5, therefore, respectively Carry out the complementation of pR55 and pR54.

The conversion bacterial strain that the present invention obtains to each transformation event utilizes the Tp309 of Tp309 and importing disease-resistant gene Pi9 mono- Gene line (Pi9-Tp309) carries out pathogenic identification, using R01-1 and R88-002 as comparison.Result shows, R01-1 is to Pi9- Tp309 have pathogenic, but import R88-002 R1434 gene after, transformant (Fig. 3 A, pR55-1/pR55-5) shows With F+strain R88-002 identical avirulence.Virulent strain 75-5 is importing R1434 gene (Fig. 3 B, the 127- of R88-002 After 5-1-15/127-5-1-22), equally obtain the avirulence to Pi9.But import R1435 gene (Fig. 3 B, the 127- of R88-002 5-3-9) then can not complementary Pi9 avirulence.Equally, convert the comparison transformant (Fig. 3 B, 127-5-4-5) of pCSN43 empty carrier Nor the avirulence of complementation Pi9, eliminate the impact to complementary result for the transformation experiment itself.These results indicate that R1434 is just It is AvrPi9 gene.

Embodiment 4：Knock out the function property the lost experiment of AvrPi9 gene.

In order to further confirm that the function of AvrPi9, applicant carried out the gene knockout based on homologous recombination gene substitution The methodological function property lost experiment.Recombinant dna fragment enters performing PCR amplification respectively using primer as shown in table 3, recycles RJ1LB/HPH/RJ1RB, RJ1LB/HPH/RJ2RB, RJ3LB/HPH/RJ2RB tri- fragment are connected, obtain by Overlapping PCR To RJ1, RJ2, RJ3PCR product, carry out purification with the PCR primer purification kit bought from Omega company afterwards.Using PEG The method of protoplast transformation, imports in avirulent strains R88-002, subsequently the transformant of gene substitution restructuring is carried out dual The Molecular of round pcr, result shows, RJ1-43, RJ2-7/RJ2-15, RJ3-53 are the transformant (figures of homologous recombination 4A).

Tp309 Monogenic lines (Pi9-Tp309) also with Tp309 and importing disease-resistant gene Pi9 carry out pathogenic mirror Fixed, artificial vaccination is carried out to above-mentioned knock-out bacterial strain.Result shows, knock out R1434 transformant RJ1-43 and knock out R1434 and The nontoxic function of transformant RJ2-7/RJ2-15 of RJ1435 completely loses, and the bacterial strain RJ3-53 avirulence only knocking out RJ1435 does not have There is change (Fig. 4 B).Experimental result has further confirmed that the R1434 in R88-002 is exactly AvrPi9 gene.

Carrier or PCR fragment and its primer that table 3. Function Identification of the present invention is used

Embodiment 5：The sequence of AvrPi9 gene and structure.

AvrPi9 full length cDNA sequence (SEQ ID NO:1) and genome sequence (SEQ ID NO:2) as follows.To AvrPi9 Gene order be analyzed showing, AvrPi9 comprises 2 exons and 1 intron.

AvrPi9 full length cDNA sequence (SEQ ID NO:1)：

atgcagttctctcagatcctcaccgtcttgttccttggcgtctccgtcagcgcccttcccgccggcggtctgcccgg cagccctggcagcgctgtccagaggtgccactgcccccctcgtggctcccacgcccacggctccctcgccgctcggg aggaagcgcccgaggccgaaggtgacgccaagatttccgcccgctacacctgccccaactgccacaagacgggcaaa ggctgcgatgatggctggtgccaagtcgaaaagacgcactgg

AvrPi9 genome sequence (SEQ ID NO:2, comprise intron and exon)：

aaaaaaaaaaaaaaagacaaacacgggctgacagtgacaaattctcccagtggtaccggagtcggtttggttcacga gtcgcagcccgccggcgagattgtcagagaagtccgatcagaagctctggctgttattgacaagcttaaagcgcagc tcacagtgccgtgagttttccatgtcctgaaacacgttgttggtggtgggctgcggctttagctacgttcagagtca tgacatcaacaccgcccccctcgcacataagcacatggtgaggtgggctcggttaacgcttaaagaccgcatcccaa aatcccatgtgtgcttatcgcgtgatttctttttgttcttgtttttttttccttttttttgttttctcctgtgctgc cgtctattccaagttacttccctcggcatggtcaagctatgaataggtctaggtagtcgccgcggcggttgtgggag tgcctcgctaccaccgaagatatccactccccacaactcgaagctgcgcggcgatcctaataaccgcatgactggtc tgggtggtattgttgcacgaaaagggtaaaacccaaaccatgcacttgggagcagaacaacataggtccactgctcc atcttgtttggccttctttgtggcctactgctgatcccccctttccagaatcgctacatttgttcttggacaggaca aaaatatgacctgtgtaagcataaagtaacattattgcatgatcataccagatctgatatagcagcccatgagtcct gtctgctttgcttaccccctcccaactccactatccagtcctatactcgacgtgaccaaaaggcgcttcggggcgtg gactatagtctgatgagtgcggttacgagtcggcaggtgggtttatcatcagtcacgcctcatgacggtctggccga ttggttcgttgggggctggagttggaacgcacttgttttgagtgtcgccatgaacatcattctcctacactggggct ctcctacactggggctgtcctacactgagaaaagatattgggcacctgttcgacgagtataaaggccgcctttcgct gccgaagaaatcacttcctctgctccttcctatccccatccacaggattccagctattcgacaaccccttctctctt ttctttatctcttggctttttttccttactgtcactctctttctctctctctctgtcttccttctagtcattccttt Ggcaaagttctttttttccttccagcccaac (promoter and 5-UTR sequence)

atgcagttctctcagatcctcaccgtcttgttccttggcgtctccgtcagcgcccttcccgccggcggtctgcccgg cagccctggcagcgctgtccagaggtgccactgcccccctcgtggctcccacgcccacggctccctcgccgctcggg aggaagcgcccgaggccgaaggtgacgccaagatttccgcccgctacacctgccccaactgccacaagacgggcaaa Ggctgcgatgatgg (exons 1)

Gtaaataagcaaccccccatccccttgaaatcctttttgcggtcatgttgctgacg tctttgatag is (interior Containing son 1)

Ctggtgccaagtcgaaaagacgcactggtag (exon 2)

aggaaaagtcaatcgagtaattatacaggttagtgcttgtactagtgctctttctaataacaatccatagtatatta tactaagtagtaagcacattcgcctttaattaattctggtatcagtaatgcgcagaagggtgttgatgctggggctt caaaagtcgggccactcgcattatcgcataattgcgaccaagttaaaagctggtcaattttccaacaatagtaagct aagatgttggttaatactagtatggccttggtaataaataactcttctgcaagcagagaacaactatgtcacaaagt ttcaacaaatatatagagtcgctgtctgctttggaaattataccccgtctcttctcccccttaccatcgccgcaacc Attcgatattcgccttcttcttcttttgtcttttttctttcttcttttct (3-UTR sequence and terminator)

Embodiment 6：The sequence of AvrPi9 nontoxic protein and structure.

The nontoxic protein aminoacid sequence of AvrPi9 gene code such as SEQ ID NO:Shown in 3.Nontoxic gene AvrPi9 compiles 1 polypeptide being made up of 92 amino acid residues of code.With SignalP4.1, structure elucidation is carried out to the albumen of this gene code, Result shows that this albumen has the feature of secretory protein, and 1-18 amino acid region of its N-terminal is signal peptide region.

AvrPi9 aminoacid sequence (SEQ ID NO:3)：

MQFSQILTVLFLGVSVSA(signal peptide sequence)

LPAGGLPGSPGSAVQRCHCPPRGSHAHGSLAAREEAPEAEGDAKISARYTCPNCHKTGKGCDDGWCQVE KTHW

Claims (3)

1. the polypeptide of a kind of avirulence gene of rice blast AvrPi9 and its coding and the polynucleotide being equal to it are resisted in cultivation Application in sick plant variety it is characterised in that：Described avirulence gene of rice blast AvrPi9 is SEQ ID NO:Nucleoside shown in 1 Polynucleotide of acid sequence or SEQ is ID NO:The polynucleotide of nucleotide sequence shown in 2；Described avirulence gene of rice blast The polypeptide of AvrPi9 coding is SEQ ID NO:The polypeptide of aminoacid sequence shown in 3；Described and avirulence gene of rice blast The polynucleotide that AvrPi9 is equal to are the polynucleotide of encoding such polypeptides or the polynucleotide being complementary to.

2. a kind of application in cultivating disease-resistant plant variety for the carrier that avirulence gene of rice blast AvrPi9 builds, its feature It is：Described carrier contains avirulence gene of rice blast AvrPi9 or avirulence gene of rice blast AvrPi9 in claim 1 and is equal to Polynucleotide described in polynucleotide sequence.

3. the polypeptide of a kind of avirulence gene of rice blast AvrPi9 and its coding and the polynucleotide being equal to it are planted in detection Application in thing Pyricularia oryzae toxicity population distribution and rice varieties planting structure distribution it is characterised in that：Described Pyricularia oryzae is nontoxic Gene A vrPi9 is SEQ ID NO:Polynucleotide of nucleotide sequence shown in 1 or SEQ is ID NO:Nucleotide sequence shown in 2 Polynucleotide；The polypeptide of described avirulence gene of rice blast AvrPi9 coding is SEQ ID NO:Aminoacid sequence shown in 3 Polypeptide；The described polynucleotide that are equal to avirulence gene of rice blast AvrPi9 be encoding such polypeptides polynucleotide or and its Complementary polynucleotide.