CN101386856A - Rice disease resistance relevant gene OsWRKY45-2 and application thereof in improving rice disease resistance - Google Patents
Rice disease resistance relevant gene OsWRKY45-2 and application thereof in improving rice disease resistance Download PDFInfo
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Abstract
The invention relates to the technical field of plant genetic engineering, in particular relates to isolating clone and functional verification of a DAN fragment containing a gene OsWRKY45-2 related to rice disease resistance. The gene OsWRKY45-2 codes WRKY proteinoids and can endow rice with resistance to the diseases caused by bacterial pathogenic bacteria, namely the leaf blight bacteria (Xanthomonas oryzae pv.oryzae) and fungal pathogenic bacteria, namely the rice blast bacteria (Magnaporthe grisea). The fragment and the exogenous regulatory sequence thereof are directly transferred into rice, thus the resistance capability of transgenic rice with over-expression OsWRKY45-2 against bacterial blight in and rice blast is improved remarkably.
Description
Technical field
The present invention relates to the plant gene engineering technology field.Be specifically related to separating clone, functional verification and the application of a paddy disease-resistant related gene OsWRKY45-2.The OsWRKY45-2 gene of separating clone of the present invention is the positive regulatory factor in the paddy disease-resistant reaction.The resisting bacterial leaf-blight and the blast resisting ability of the transfer-gen plant of overexpression OsWRKY45-2 gene significantly improve.
Background technology
Plant is subjected to the infringement of multiple pathogen in the process of growth.The phytopathy original of a great variety comprises virus, bacterium, fungi and nematode etc.The pathogen invaded plants causes two kinds of results: breeding in host plant of (1) pathogenic agent success causes relevant illness; (2) host plant produces disease resistance response, kills pathogen or stops its growth.Utilizing the disease resistance of resistant gene resource improvement plant, is the basic outlet that pre-disease prevention is protected environment simultaneously again.
The disease resistance response of plant is the complex process that polygene participates in regulation and control.The gene that participates in the plant disease-resistant reaction is divided into two classes: (1) disease-resistant gene claims R (resistance) gene and (2) disease-resistant related gene again.
According to the understanding of present people's enantiopathy gene function, the product of R gene mainly is as acceptor, directly or indirectly with the cause of disease protein-interacting, and the intravital disease-resistant signal conducting path of startup plant (Tang etc., 1996; Baker etc., 1997; Jia etc., 2000; Dangl and Jones, 2001; Nimchuk etc., 2001).The disease resistance response strong resistance of R gene mediated is good genetic resources.But owing to following reason, make and utilize disease-resistant gene improvement plant resistance to environment stress to be restricted: the resource-constrained of (1) R gene, disease-resistant gene as the important disease bacterial leaf-blight of opposing paddy rice known at present approximately has only 30, and the R gene of resisting the important disease-rice blast of another paddy rice also has only about 50; (2) the R gene has cause of disease kind and cause of disease physiological strain specificity, disease-resistant being limited in scope; (3) because the quick sudden change of cause of disease, the effect of a R gene has often just been lost after several years or more than ten years.
Disease-resistant related gene is meant that all participate in the gene of disease resistance response except that disease-resistant gene, and their coded product participates in disease-resistant signaling molecule in the synthetic plant materials, participates in the signal conduction, stops the signal conduction or participates in defense response etc.The common feature of this genoid is that their expression amount of pathogeny evoked back raises or reduces, so people can be according to the difference of pathogeny evoked front and back expression of gene amount plant identification disease-resistant related gene (Schenk etc., 2000 on a large scale; Zhou etc., 2002; Chu etc., 2004).At present, the understanding of people's enantiopathy genes involved is limited.According to existing report, most disease-resistant related genes are made the resistance capacity of time spent separately may be littler than R gene.But according to following reason, they are to be worth the genetic resources of exploitation energetically: (1) because the product of most disease-resistant related genes does not need directly and pathogen interacts, this genoid is the genetic resources with durable resistance; (2) disease resistance response that participates in of most of disease-resistant related genes does not have the cause of disease specificity, so they are the genetic resourceses with resistance of wide spectrum; (3) aboundresources of this genoid.But, though identified a lot of disease-resistant related genes (Zhou etc., 2002 in the paddy rice; Chu etc., 2004), whether the mechanism of action and the single disease-resistant related gene of these genes in the paddy disease-resistant reaction can cause that the change of paddy disease-resistant phenotype is all unclear.
Paddy rice is an important crops in the world, but the influence of disease usually causes the decline of its yield and quality.Bacterial blight of rice causes by bacterial leaf spot pathogenic bacteria (Xanthomonas oryzae pv.oryzae), is in the world to the bacterial disease (crossing Chong Jian, 1995) of rice hazard maximum.Rice blast causes by Pyricularia oryzae (Magnaporthe grisea), is in the world to one of the fungal disease of rice hazard maximum (Zeigler etc., 1994).Excavating and utilizing good resistant gene resource improvement paddy rice is the generation of control disease, the most economical effective measures of loss that reduce or avoid rice disease to bring.
Compare with the application of R gene, the application of disease-resistant related gene can provide plant more wide spectrum and long lasting resistance.Carry out the improvement of rice varieties by overexpression as the disease-resistant related gene of the positive regulatory factor of disease resistance response, will further strengthen the disease resistance of plant, widen the anti-spectrum of plant.These aspects are that employing conventional plant breeding and improving technology institute are inaccessiable.
Summary of the invention
A disease-resistant related gene global DNA fragment that the objective of the invention is to carry in the separating clone paddy rice, and utilize the overexpression technology to identify this gene role in the disease resistance response process by making target gene overexpression in transgene receptor, for the ability of utilizing this improvement of genes rice varieties or other plant to resist disease lays the foundation.The applicant is named as OsWRKY45-2 with this gene.
The present invention relates to separate a kind of dna fragmentation of the OsWRKY45-2 of comprising gene and identify its function, this fragment is given plant to producing disease resistance response by bacterial leaf spot pathogenic bacteria (Xanthomonas oryzae pv.oryzae) and the caused disease of Pyricularia oryzae (Magnaporthe grisea).Wherein, described fragment perhaps is equivalent to the dna sequence dna shown in the SEQ ID NO:1 basically shown in sequence table SEQ ID NO:1, and perhaps its function is equivalent to the subfragment of sequence shown in the SEQ ID NO:1.Its sequence is carried out analysis revealed, and the conserved domain that its encoded protein matter has the WRKY proteinoid shows that it belongs to WRKY proteinoid family.Sequence can strengthen the resistance of paddy rice to bacterial leaf-blight and rice blast shown in the overexpression sequence table SEQ ID NO:1.
Can adopt the OsWRKY45-2 gene of having cloned to make probe, from cDNA and genomic library, screen gene of the present invention or homologous gene.Equally, adopt PCR (polymerase chain reaction) technology, also can from genome, mRNA and cDNA, increase obtain OsWRKY45-2 gene of the present invention and any interested section of DNA or with its homologous section of DNA.Adopt above technology, can separate the sequence that obtains comprising the sequence of OsWRKY45-2 gene or comprise one section OsWRKY45-2 gene, this sequence is connected with suitable carriers, can changes vegetable cell overexpression OsWRKY45-2 gene over to, produce disease-resistant transgenic plant.Adopting this transgenic technology to create disease-resistant plants is that traditional breeding technology institute is inaccessiable.
The present invention provides a kind of new method for strengthening paddy rice to the resistance of bacterial leaf spot pathogenic bacteria and Pyricularia oryzae.This method comprises the genomic fragment of OsWRKY45-2 gene complete coding region is connected, changes over to susceptible paddy rice with carrier that can the overexpression target gene, by the resistance of overexpression OsWRKY45-2 improvement of genes paddy rice to bacterial leaf-blight and rice blast.
In the embodiments of the invention part, the applicant has set forth separation, functional verification and the application process of OsWRKY45-2 gene and the characteristics of this gene.
Description of drawings
Sequence table SEQ ID No:1.: be the nucleotide sequence of OsWRKY45-2 gene of separating clone of the present invention and the aminoacid sequence of its encoded protein matter.
Fig. 1: the schema that is evaluation of the present invention and separating clone paddy disease-resistant related gene OsWRKY45-2 and checking OsWRKY45-2 gene function.
Fig. 2: be to infect OsWRKY45-2 expression of gene variation in back disease resisting rice kind bright extensive 63 and the precious Shan 97 of susceptible rice varieties with quantitative RT-PCR technical Analysis bacterial leaf spot pathogenic bacteria PXO61.Contrast is the sample before the inoculation PXO61; Other are the samples behind the inoculation PXO61.OsWRKY45-2 expression of gene amount is with respect to OsWRKY45-2 expression of gene amount in precious Shan 97 samples before the inoculation in each sample.
Fig. 3: the structure of pcr amplification product and OsWRKY45-2 gene." ATG " and " TAG " is respectively translation initiation password and termination codon.Numeral is shown the Nucleotide number of each structure.
Fig. 4: the physical map that is the genetic transformation carrier pU1301 that adopts of the present invention.
Fig. 5: be part T among the present invention
0Inoculating the phenotype of bacterial leaf spot pathogenic bacteria PXO61 after two weeks for the genetic transformation plant." Mudanjiang 8 " is the acceptor (contrast) of genetic transformation, and " D114UM4 " and " D114UM6 " is positive transformed plant, and " D114UM15 " is negative transformed plant.
Fig. 6: be T of the present invention
0Relevant to resistance (A among Fig. 6) enhancing of bacterial leaf spot pathogenic bacteria strain PXO61 for the OsWRKY45-2 gene expression amount in the genetic transformation plant (D114UM) (B among Fig. 6) with plant.Contrast is paddy rice susceptible variety " Mudanjiang 8 " (acceptor of genetic transformation).OsWRKY45-2 expression of gene amount is with respect to OsWRKY45-2 expression of gene amount in the contrast " Mudanjiang 8 " in the genetic transformation plant.Asterisk (
*) represent that compared with the control the lesion area of transfer-gen plant significantly (P＜0.01) reduces.
Fig. 7: be T of the present invention
1For the OsWRKY45-2 expression of gene amount in the genetic transformation plant and phenotype be divided into from.Among Fig. 7: A is T
1For genetic transformation plant D114UM4; B is T
1For genetic transformation plant D114UM6; C is T
1For genetic transformation plant D114UM10 and D114UM11.Contrast is paddy rice susceptible variety " Mudanjiang 8 " (acceptor of genetic transformation).Lesion area is the data of inoculation bacterial leaf spot pathogenic bacteria PXO61 after two weeks.OsWRKY45-2 expression of gene amount is with respect to OsWRKY45-2 expression of gene amount in the contrast " Mudanjiang 8 " in the genetic transformation plant.
Fig. 8: the plant that is overexpression OsWRKY45-2 gene has resistance of wide spectrum to bacterial leaf spot pathogenic bacteria.
Fig. 9: the remarkable resistance that strengthens Pyricularia oryzae CHL358 of plant that is overexpression OsWRKY45-2 gene.
Embodiment
Previous research work result of the present invention shows that a member of paddy rice WRKY gene family may participate in disease resistance response.According to the name to paddy rice WRKY gene family member such as Xie (Xie etc., 2005), this member is OsWRKY45.Researchist of the present invention thinks that the main foundation that OsWRKY45 may participate in disease resistance response regulation and control is after activating disease-resistant related gene OsWRKY13, OsWRKY45 expression of gene change (Qiu etc., 2008).OsWRKY13 albumen is the incitant of paddy disease-resistant reaction; The transfer-gen plant of overexpression OsWRKY13 gene all significantly strengthens (Qiu etc., 2007) to the resistance of bacterial leaf-blight and rice blast.Previous research work results suggest OsWRKY45 of the present invention may be a disease-resistant related gene.In order to verify this analysis supposition, produced the present invention.
Further definition the present invention in following examples, Fig. 1 have described the flow process of evaluation and separating clone OsWRKY45-2 gene and checking OsWRKY45-2 gene function.According to following description and these embodiment, those skilled in the art can determine essential characteristic of the present invention, and under the situation that does not depart from spirit and scope of the invention, can make various changes and modification, so that its suitable various uses and condition to the present invention.
The expression pattern analysis of embodiment 1:OsWRKY45-2 gene in different rice varieties
With the sequence of the OsWRKY45 gene in the paddy rice whole genome sequence [TIGR (The Institute for GenomicResearch,
Http:// rice.tigr.org) database gene locus numbering: LOC_Os05g25770] retrieval long-grained nonglutinous rice expressed sequence tag (expressed sequence tag, EST) database REDB (Rice EST DataBase,
Http:// redb.ricefgchina.orgZhang etc., 2005), find that a cDNA sequence EI77K16 and an OsWRKY45 gene that derives from bright extensive 63 (the Oryza sativa ssp.indica) of rice variety, long 1437bp has 98% sequence homology, being the allelotrope of OsWRKY45, is OsWRKY45-2 so this is derived from bright extensive 63 unnamed gene.
In order to confirm whether the OsWRKY45-2 gene participates in the regulation and control of disease resistance response, the present invention adopts quantitative reverse transcription-PCR (quantitative reverse transcription-PCR, qRT-PCR) technology (Qiu etc., 2007) analyzed the expression pattern OsWRKY45-2 gene is inoculated bacterial leaf-blight bacterial strain PXO61 (Philippines physiological strain 1) (Sun etc., 2004) in different rice varieties after.The bacterial leaf spot pathogenic bacteria inoculation adopts leaf-cutting method that the paddy rice that becomes the strain phase is inoculated (Sun etc., 2004).Bacterial leaf-blight bacteria strain PXO61 is so kind as to give (Sun etc., 2004) by the Philippines International Rice Research Institute.The method of having published (Sun etc., 2004) is followed in the cultivation of bacterial leaf spot pathogenic bacteria.Inoculation back branch different time points is got inoculation blade extracted total RNA (Zhou etc., 2002).Get the total RNA of 1～5 μ g and handle 15 minutes to remove the genomic dna pollution, with reference to the method for (2002) such as Zhou, use oligo (dT) then with DNaseI (American I nvitrogen company)
15Oligomerization primer and M-MLV ThermoScript II (U.S. Promega company) are carried out reverse transcription.Adopt the real-time quantitative PCR assay kit
Green PCR Master Mix (precious biotechnology (Dalian) company limited, Tokara), and, on ABI 7500 Real-TimePCR system (U.S. Applied Biosystems company) instrument, carry out the real-time quantitative PCR reaction according to these test kit working instructions.Weigh and homogenization sample rna content (Qiu etc., 2007) with the endogenous Actin muscle of paddy rice (actin) expression of gene amount.Os WRKY45-2 gene specific PCR primer during qRT-PCR analyzes is w45F (5 ＇-TTCCTTGTTGATGTGTCGTCTCA-3 ＇) and w45R (5 ＇-CCCCCAGCTCATAATCAAGAAC-3 ＇), and actin gene PCR primer is actinF (5 ＇-TGCTATGTACGTCGCCATCCAG-3 ＇) and actinR (5 ＇-AATGAGTAACCACGCTCCGTCA-3 ＇).
Experimental result induces the OsWRKY45-2 gene to express (Fig. 2) in rice varieties bright extensive 63 and precious Shan 97 (Oryza sativa ssp.indica) after showing inoculation bacterial leaf spot pathogenic bacteria PXO61.But the OsWRKY45-2 gene is in bright extensive 63 (Chen etc., 2002 of the disease resisting rice kind of carrying resisting bacterial leaf-blight major gene Xa25 (t) and Xa3/Xa26; Sun etc., 2004; Xiang etc., 2006) expression amount in is significantly higher than its expression amount (Fig. 2) at susceptible rice varieties " precious Shan 97 ".This results suggest: the OsWRKY45-2 gene may participate in the regulation and control of resisting bacterial leaf-blight reaction, strengthen this genetic expression and may can improve the disease resistance of paddy rice, and disease-resistant major gene have an expression that helps strengthen it.
Embodiment 2: separating clone OsWRKY45-2 gene and gene structure analysis
1.OsWRKY45-2 the prediction of gene structure
CDNA sequence EI77K16 with the OsWRKY45-2 gene makes template, with BLAST method (Altschul etc., 1997) retrieval japonica rice full-length cDNA database KOME database (http://cdna01.dna.affrc.go.jp/cDNA/) finds in EI77K16 sequence and this database that (number of registration: AK066255) homology reaches 98% for the full length cDNA sequence of, a long 1453bp fine from japonica rice variety Japan.The AK066255 sequence is exactly the cDNA sequence of numbering LOC_Os05g25770 gene in the paddy rice whole genome sequence, i.e. the cDNA sequence of the allelotrope of OsWRKY45-2 (OsWRKY45).The arrangement comparative analysis of this two sequences confirms that the cDNA sequence EI77K16 of OsWRKY45-2 gene has comprised the proteic total length opening code-reading frame of coding OsWRKY45.
2. the full length DNA of separating clone OsWRKY45-2 gene from rice varieties bright extensive 63
The present invention is according to paddy rice whole genome sequence (TIGR database
Http:// rice.tigr.org) in OsWRKY45-2 allelotrope OsWRKY45 both sides sequences Design one couple of PCR primers: w45F4 (5 ＇-AT
GGTACCGCCTACGCATCATCTCCTTC-3 ＇) (the underscore representative is used for the KpnI digestion with restriction enzyme site of the joint of carrier connection) and w45R4 (5 ＇-CA
GGATCCTTATGGCACAACATTTAGCA-3 ＇) (the underscore representative is used for the BamHI digestion with restriction enzyme site of the joint of carrier connection).Utilize this that PCR primer is increased from rice varieties bright extensive 63 and comprise the dna fragmentation (Fig. 3) of OsWRKY45-2 gene.Utilize PCR primer w45F4, w45R4 and be positioned at the PCR primer w45F6 (5 ＇-ATCACAAAGCATAGCATCATCT-3 ＇) and the w45R6 (5 ＇-CTCAGCACCTCCTCCTGGTCGG-3 ＇) of gene inside, and the sequencing kit of U.S. Applied Biosystems company, check order from the pcr amplified fragment two ends respectively with the terminal cessation method of dideoxy nucleotide (U.S. Applied Biosystems company).Obtain the dna sequence dna of a long 1778bp behind the sequence assembly, it comprises the paddy DNA sequence (joint sequence that 16bp is arranged in addition) of 1762bp and complete OsWRKY45-2 gene order (Fig. 3).
3.OsWRKY45-2 gene structure analysis
The genome sequence of comparative analysis OsWRKY45-2 gene and cDNA sequence, determine that the OsWRKY45-2 gene is made up of 1629 Nucleotide, comprise three exons and two introns, 5 ' end non-translational region (untranslated region of gene, UTR) form (1-112bp place that is positioned at sequence table SEQ ID NO:1) by 112 Nucleotide, first exon is formed (113-421bp place that is positioned at sequence table SEQ ID NO:1) by 309 Nucleotide, first intron is formed (422-512bp place that is positioned at sequence table SEQ ID NO:1) by 91 Nucleotide, second exon formed (513-617bp place that is positioned at sequence table SEQ ID NO:1) by 105 Nucleotide, second intron formed (618-718bp place that is positioned at sequence table SEQ ID NO:1) by 101 Nucleotide, the 3rd exon formed (719-1270bp place that is positioned at sequence table SEQ ID NO:1) by 552 Nucleotide, and 3 ' holds UTR to be made up of 359 Nucleotide (is positioned at 1271-1629bp) of sequence table SEQ ID NO:1.
4.OsWRKY45-2 the analysis of gene encoding production
(113-1270bp place that is positioned at sequence table SEQ ID NO:1) formed by 969 Nucleotide in the coding region of OsWRKY45-2 gene, and the length of encoding is 322 amino acid whose protein.According to the classification of the WRKY protein families of (2000) such as Eulgem, OsWRKY45-2 albumen belongs to typical WRKY class transcription factor.It belongs to the 3rd subclass in the WRKY albumen, has a conservative WRKY structural domain, a C2HC zinc fingers.
Embodiment three: the functional verification of OsWRKY45-2 gene
1. the structure of genetic transformation carrier
Used carrier of the present invention is pU1301 (Fig. 4).PU1301 is rice genetic conversion carrier (Cao etc., 2007 of using always; Qiu etc., 2007; Ding etc., 2008).It is the agriculture bacillus mediated genetic transformation carrier that carries the corn ubiquitin promoter with composing type and overexpression feature.
With the described dna fragmentation (Fig. 3) that comprises the OsWRKY45 gene of embodiment 2 the 2nd joint with restriction enzyme KpnI and BamHI digestion, behind the inactivator as the external source fragment.Simultaneously, carry the genetic transformation carrier pU1301 of corn ubiquitin promoter with restriction enzyme KpnI and BamHI digestion; Enzyme cuts complete, uses chloroform: primary isoamyl alcohol (volume ratio is 24:1) extracting, purifying enzyme is cut product.Do ligation with endonuclease bamhi that comprises the OsWRKY45-2 gene and the good carrier of purifying.By the sequence verification positive colony, the recombinant plasmid vector of acquisition is named as D114U.
2. genetic transformation and T
0In generation, lost plant analysis
Adopt agriculture bacillus mediated genetic transforming method (Lin and Zhang, 2005) that D114U is imported paddy rice susceptible variety Mudanjiang 8 (Oryza sativa ssp.japonica).The genetic transformation plant that obtains is named as D114UM (wherein previous section is that D114U is the genetic transformation container name, and M represents rice varieties Mudanjiang 8).The present invention obtains independent transformed plant 17 strains altogether.Whole transformed plants becoming stage strain phase inoculation bacterial leaf-blight bacterial strain PXO61, are compared with wild-type contrast Mudanjiang 8 and the negative plant of genetic transformation, the resistance of all positive genetic transformation plant significantly strengthen (P＜0.01) (table 1, Fig. 5).
Table 1.T
0For the reaction of genetic transformation plant (D114UM) to bacterial leaf spot pathogenic bacteria strain PXO61
Rice material lesion area (%)
(1)The P value
Mudanjiang 8 (contrast) 54.8 ± 5.9
D114UM11 8.6±4.5 0.0000
D114UM2 22.0±15.1 0.0033
D114UM3 26.1±4.7 0.0001
D114UM4 27.6±3.1 0.0006
D114UM5 25.6±10.5 0.0010
D114UM6 25.1±7.4 0.0001
D114UM7 27.5±10.1 0.0007
D114UM8 20.6±3.8 0.0001
D114UM9 18.0±4.9 0.0000
D114UM10 25.7±9.7 0.0004
D114UM11 17.3±5.1 0.0000
D114UM12 5.0±2.8 0.0001
D114UM13 24.0±2.2 0.0003
D114UM14 22.5±4.3 0.0001
D114UM15
(2) 57.3±5.5 0.2696
D114UM16 21.2±6.1 0.0000
D114UM 1714.0±7.2 0.0000
(1)3-5 leaves of every strain genetic transformation gene plant inoculation, 14 days " Invest, Then Investigate " scabs and sick leaf length, each data comes from the mean value of a plurality of blades.
(2)Negative transformed plant, the positive transformed plant of other plant
Change the expression amount that causes over to the OsWRKY45-2 gene and raise relevantly for whether the further resistance against diseases of checking genetic transformation plant strengthens, the present invention adopts OsWRKY45-2 expression of gene amount in the qRT-PCR analytical procedure detection genetic transformation plant described in the embodiment 1.The result shows the expression amount of OsWRKY45-2 and the resistance closely related (Fig. 6 A and 6B) of plant.This results suggest genetic transformation plant is because OsWRKY45-2 expression of gene amount strengthens to the resistance enhancing of bacterial leaf spot pathogenic bacteria.
3. the coseparation analysis of genetic transformation plant
For the further above-mentioned supposition of checking, at T
0T for resistance enhanced 4 strain genetic transformation plant (D114UM4, D114UM6, D114UM10 and D114UM11)
1Carry out gene expression amount and resistance coseparation analysis for family.Paddy rice during boot stage in inoculation bacterial leaf spot pathogenic bacteria PXO61, inoculate 14 days " Invest, Then Investigate "s, the total RNA of the fresh blade of paddy rice (Zhou etc., 2002) is extracted in sampling simultaneously, adopts OsWRKY45-2 expression of gene amount in the above-mentioned qRT-PCR analytical procedure detection plant.The result shows that OsWRKY45-2 gene expression amount of genetic transformation plant and resistance are divided into from (Fig. 7 A, 7B and 7C); All strengthen with the OsWRKY45-2 expression of gene amount compared in the remarkable enhanced plant of all resistances of contrast Mudanjiang 8, and resistance does not have the expression amount of the plant target gene of noticeable change compared with the control and does not have noticeable change.Further the resistance enhancing of proof genetic transformation plant is because of overexpression OsWRKY45-2 gene institute extremely.These results of study also illustrate the effect of coded product positive regulatory factor of performance in the rice bacterial blight resistance reaction of OsWRKY45-2 gene.Overexpression OsWRKY45-2 gene can strengthen the resistance of paddy rice to bacterial leaf-blight.
Embodiment four: the application prospect analysis of overexpression OsWRKY45-2 gene
1. the plant of overexpression OsWRKY45-2 gene has resistance of wide spectrum to bacterial leaf-blight
To genetic transformation family D114UM4-8 (T
2Generation) and contrast Mudanjiang 8 inoculate different bacterial leaf spot pathogenic bacterias in boot stage, analyze the anti-spectrum of genetic transformation plant.Above-mentioned or conventional leaf-cutting method is adopted in inoculation.Bacterial leaf spot pathogenic bacteria comprises Philippines physiological strain 2 (PXO86), Philippines physiological strain 3 (PXO79), Philippines physiological strain 6 (PXO99), Philippines physiological strain 10 (PXO341), Chinese physiological strain Zhe174 and KS-1-21.All bacterial leaf-blight bacterial strains all are bacterial strain (Yang etc., 2003 of using always; Sun etc., 2004; Yang etc., 2007).Overexpression OsWRKY45-2 gene significantly (P＜0.01) strengthens the resistance (Fig. 8) of paddy rice to these bacterial leaf spot pathogenic bacterias.Compared with the control, the lesion area of overexpression OsWRKY45-2 gene plant has reduced 51%-94%.The paddy rice of these presentation of results overexpressions OsWRKY45-2 gene has resistance of wide spectrum to bacterial leaf spot pathogenic bacteria, and OsWRKY45-2 is a gene that application prospect is arranged in the resisting bacterial leaf-blight breeding.
2. the plant of overexpression OsWRKY45-2 gene significantly strengthens the resistance of rice blast
The present invention is to the T1 family (D114UM13 and D114UM16) of two overexpression OsWRKY45-2 genes, the acceptor material Mudanjiang 8 (contrast) and the susceptible paddy rice CO39 of rice blast (Jaing etc., 2002 commonly used in the world of genetic transformation; Wen etc., 2003) at 4-5 leaf phases of paddy rice inoculation Pyricularia oryzae CHL358.The CHL358 bacterial strain is so kind as to give by Chinese professor Pan Qinghua of Agricultural University Of South China, is the rice blast fungi isolates commonly used in professor's Pan Qinghua research.Conventional spray method is adopted in the Pyricularia oryzae inoculation, inoculates and detects incidence (Chen etc., 2003) after 5 days.Compare with susceptible contrast CO39, CHL358 (Fig. 9) is also felt in Mudanjiang 8, and this is consistent (Qiu etc., 2007) with Mudanjiang 8 to the also susceptible report of other rice blast fungi isolates.And compare with Mudanjiang 8, D114UM13 and the high anti-CHL358 of D114UM16 plant (Fig. 9) with same genetic background, illustrate that overexpression OsWRKY45-2 gene of the present invention also can strengthen the resistance of paddy rice to rice blast, this gene also is a gene that application prospect is arranged in the blast resisting breeding.
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＜110〉Hua Zhong Agriculture University
＜120〉paddy disease-resistant related gene OsWRKY45-2 and its application in the improvement paddy disease-resistant
<130>
<141>2008-10-12
<160>2
<170>PatentIn?version3.1
<210>1
<211>1629
<212>DNA
＜213〉paddy rice (Oryza sativa)
<220>
<221>gene
<222>(1)..(1629)
<223>
<220>
<221>5’UTR
<222>(1)..(112)
<223>
<220>
<221>CDS
<222>(113)..(421)
<223>
<220>
<221>CDS
<222>(513)..(617)
<223>
<220>
<222>(719)..(1270)
<220>
<221>Intron
<222>(422)..(512)
<223>
<220>
<221>Intron
<222>(618)..(718)
<223>
<220>
<221>3’UTR
<222>(1271)..(1629)
<223>
<400>1
tgctttgagc?tccatcacca?gctgagctgc?gaggaagaga?gagtgcgaga?gtgcgcggca 60
gcggcagtgt?agtgtcagtc?actgggtgtg?cgcttgcttg?cttggattga?gg?atg?acg 118
Met?Thr
1
tca?tcg?atg?tcg?ccg?gcg?ccg?gcg?ccg?gcg?tac?gcg?cag?gtg?atg?gag 166
Ser?Ser?Met?Ser?Pro?Ala?Pro?Ala?Pro?Ala?Tyr?Ala?Gln?Val?Met?Glu
5 10 15
gac?atg?gag?aag?ggg?aag?gag?ctg?gcg?gcg?cag?ctg?cag?ggg?ctc?ctc 214
Asp?Met?Glu?Lys?Gly?Lys?Glu?Leu?Ala?Ala?Gln?Leu?Gln?Gly?Leu?Leu
20 25 30
cgc?gac?tcg?ccg?gag?gcc?ggc?cgc?ttc?gtc?gac?cag?att?ctc?cac?acc 262
Arg?Asp?Ser?Pro?Glu?Ala?Gly?Arg?Phe?Val?Asp?Gln?Ile?Leu?His?Thr
35 40 45 50
ttc?tcc?cgg?gcg?atg?cgg?gcg?ctc?gac?aag?gcg?gcg?gtc?tcc?gcc?gcc 310
Phe?Ser?Arg?Ala?Met?Arg?Ala?Leu?Asp?Lys?Ala?Ala?Val?Ser?Ala?Ala
55 60 65
gga?gga?gaa?ggg?tcg?gag?gtg?cag?agc?gag?gtc?acc?tgc?ggg?ggc?ggg 358
Gly?Gly?Glu?Gly?Ser?Glu?Val?Gln?Ser?Glu?Val?Thr?Cys?Gly?Gly?Gly
70 75 80
gcc?agc?gcc?ggc?ggg?aag?agg?aaa?gcc?ccc?gcc?gcc?aac?cgg?aag?gcc 406
Ala?Ser?Ala?Gly?Gly?Lys?Arg?Lys?Ala?Pro?Ala?Ala?Asn?Arg?Lys?Ala
85 90 95
aac?tgc?cgc?agg?agg?tgagaacgaa?ggccagagca?tagctcatca?caaagcatag 461
Asn?Cys?Arg?Arg?Arg
100
catcatctgt?gtgtaattaa?tgtactaaga?tttgtgtgta?cgtattgcag?g?acg?cag 518
Thr?Gln
105
caa?tcg?tcc?ggg?aat?acg?gtg?gtc?gtc?aag?aac?ctc?gac?gac?ggc?cag 566
Gln?Ser?Ser?Gly?Asn?Thr?Val?Val?Val?Lys?Asn?Leu?Asp?Asp?Gly?Gln
110 115 120
gca?tgg?cgc?aag?tac?ggg?cag?aag?gag?atc?caa?aac?tcc?aag?cac?cca 614
Ala?Trp?Arg?Lys?Tyr?Gly?Gln?Lys?Glu?Ile?Gln?Asn?Ser?Lys?His?Pro
125 130 135
aag?tgagtagact?tgtcccgaca?aaaaacaatg?tgttcgagac?tgtacagttg 667
Lys
gatgcgttgc?gcgctgacga?ggagttgttt?ggggtatgct?acgtgtacag?g?gcc?tac 724
Ala?Tyr
140
ttc?cgg?tgc?acg?cac?aag?tac?gac?cag?atg?tgc?acg?gcg?cag?cgg?cag 772
Phe?Arg?Cys?Thr?His?Lys?Tyr?Asp?Gln?Met?Cys?Thr?Ala?Gln?Arg?Gln
145 150 155
gtg?cag?cgc?tgc?gac?gac?gac?ccg?gcg?agc?tac?agg?gtc?acc?tac?atc 820
Val?Gln?Arg?Cys?Asp?Asp?Asp?Pro?Ala?Ser?Tyr?Arg?Val?Thr?Tyr?Ile
160 165 170
ggc?gag?cac?acc?tgc?cgg?gac?ccg?gcc?acc?gcc?ccc?atc?atc?gcg?gcg 868
Gly?Glu?His?Thr?Cys?Arg?Asp?Pro?Ala?Thr?Ala?Pro?Ile?Ile?Ala?Ala
175 180 185
cac?gtc?atc?cac?cag?gtc?gcc?gcc?ggc?gac?gac?gac?gac?ggc?tgc?ggc 916
His?Val?Ile?His?Gln?Val?Ala?Ala?Gly?Asp?Asp?Asp?Asp?Gly?Cys?Gly
190 195 200
ggc?ctc?cac?gcg?ggg?tcc?cgc?ctc?atc?agc?ttc?gtc?gcc?gcg?ccg?gcg 964
Gly?Leu?His?Ala?Gly?Ser?Arg?Leu?Ile?Ser?Phe?Val?Ala?Ala?Pro?Ala
205 210 215 220
gcg?cca?gta?gac?gct?gcc?gcg?gcg?ccg?acg?acc?agc?acg?atc?acc?acg 1012
Ala?Pro?Val?Asp?Ala?Ala?Ala?Ala?Pro?Thr?Thr?Ser?Thr?Ile?Thr?Thr
225 230 235
gtc?acc?gcg?ccg?ggc?ccg?ctg?ctg?cag?ccg?ctc?aag?gtg?gag?ggc?ggc 1060
Val?Thr?Ala?Pro?Gly?Pro?Leu?Leu?Gln?Pro?Leu?Lys?Val?Glu?Gly?Gly
240 245 250
atc?ggc?tcg?tcc?gac?cag?gag?gag?gtg?ctg?agc?agc?ctc?acg?ccc?ggc 1108
Ile?Gly?Ser?Ser?Asp?Gln?Glu?Glu?Val?Leu?Ser?Ser?Leu?Thr?Pro?Gly
255 260 265
agc?tcc?gcg?gcg?cgc?ggc?ggc?gga?gtc?gcg?ggt?ccc?ttc?ggg?ccg?gac 1156
Ser?Ser?Ala?Ala?Arg?Gly?Gly?Gly?Val?Ala?Gly?Pro?Phe?Gly?Pro?Asp
270 275 280
cag?ggc?gat?gtc?acg?tcc?tcc?ctg?cac?tgg?agc?tac?gac?gcc?gtc?gcc 1204
Gln?Gly?Asp?Val?Thr?Ser?Ser?Leu?His?Trp?Ser?Tyr?Asp?Ala?Val?Ala
285 290 295 300
ggc?atg?gag?ttc?ttc?aag?aac?gac?gag?gtt?gtc?ttc?gat?ctg?gac?gac 1252
Gly?Met?Glu?Phe?Phe?Lys?Asn?Asp?Glu?Val?Val?Phe?Asp?Leu?Asp?Asp
305 310 315
att?atg?ggt?ttg?agc?ttt?tgatcaccga?agaatcatgg?atggacacgg 1300
Ile?Met?Gly?Leu?Ser?Phe
320
gccgggtaaa?acgatcgaaa?gaagatggat?tccacgcgtg?tgtacagaaa?taattagcgg 1360
cagcgcggat?cttaatttgg?aacttgcaaa?gatactccta?attagcctgg?ctagattagt 1420
ttgtaaattc?cttgttgatg?tgtcgtctca?gctttaagct?gcagacatgc?tagcaagtaa 1480
caacacgatt?agtacgtagt?aatgtggttc?ttgattatga?gctgggggtc?ttaacctttt 1540
ttgtgtgaca?agcaagagaa?gaggatttgg?gtacaatgta?atcctgttct?tccgctttcg 1600
aaaaaaaaaa?catatagctt?cacgtgcct 1629
<210>2
<211>322
<212>PRT
＜213〉paddy rice (Oryza sativa)
<400>2
Met?Thr?Ser?Ser?Met?Ser?Pro?Ala?Pro?Ala?Pro?Ala?Tyr?Ala?Gln?Val
1 5 10 15
Met?Glu?Asp?Met?Glu?Lys?Gly?Lys?Glu?Leu?Ala?Ala?Gln?Leu?Gln?Gly
20 25 30
Leu?Leu?Arg?Asp?Ser?Pro?Glu?Ala?Gly?Arg?Phe?Val?Asp?Gln?Ile?Leu
35 40 45
His?Thr?Phe?Ser?Arg?Ala?Met?Arg?Ala?Leu?Asp?Lys?Ala?Ala?Val?Ser
50 55 60
Ala?Ala?Gly?Gly?Glu?Gly?Ser?Glu?Val?Gln?Ser?Glu?Val?Thr?Cys?Gly
65 70 75 80
Gly?Gly?Ala?Ser?Ala?Gly?Gly?Lys?Arg?Lys?Ala?Pro?Ala?Ala?Asn?Arg
85 90 95
Lys?Ala?Asn?Cys?Arg?Arg?Arg?Thr?Gln?Gln?Ser?Ser?Gly?Asn?Thr?Val
100 105 110
Val?Val?Lys?Asn?Leu?Asp?Asp?Gly?Gln?Ala?Trp?Arg?Lys?Tyr?Gly?Gln
115 120 125
Lys?Glu?Ile?Gln?Asn?Ser?Lys?His?Pro?Lys?Ala?Tyr?Phe?Arg?Cys?Thr
130 135 140
His?Lys?Tyr?Asp?Gln?Met?Cys?Thr?Ala?Gln?Arg?Gln?Val?Gln?Arg?Cys
145 150 155 160
Asp?Asp?Asp?Pro?Ala?Ser?Tyr?Arg?Val?Thr?Tyr?Ile?Gly?Glu?His?Thr
165 170 175
Cys?Arg?Asp?Pro?Ala?Thr?Ala?Pro?Ile?Ile?Ala?Ala?His?Val?Ile?His
180 185 190
Gln?Val?Ala?Ala?Gly?Asp?Asp?Asp?Asp?Gly?Cys?Gly?Gly?Leu?His?Ala
195 200 205
Gly?Ser?Arg?Leu?Ile?Ser?Phe?Val?Ala?Ala?Pro?Ala?Ala?Pro?Val?Asp
210 215 220
Ala?Ala?Ala?Ala?Pro?Thr?Thr?Ser?Thr?Ile?Thr?Thr?Val?Thr?Ala?Pro
225 230 235 240
Gly?Pro?Leu?Leu?Gln?Pro?Leu?Lys?Val?Glu?Gly?Gly?Ile?Gly?Ser?Ser
245 250 255
Asp?Gln?Glu?Glu?Val?Leu?Ser?Ser?Leu?Thr?Pro?Gly?Ser?Ser?Ala?Ala
260 265 270
Arg?Gly?Gly?Gly?Val?Ala?Gly?Pro?Phe?Gly?Pro?Asp?Gln?Gly?Asp?Val
275 280 285
Thr?Ser?Ser?Leu?His?Trp?Ser?Tyr?Asp?Ala?Val?Ala?Gly?Met?Glu?Phe
290 295 300
Phe?Lys?Asn?Asp?Glu?Val?Val?Phe?Asp?Leu?Asp?Asp?Ile?Met?Gly?Leu
305 310 315 320
Ser?Phe
Claims (2)
1, the paddy rice that influences of a separating clone produces the nucleotide sequence of resistance to bacterial leaf-blight and rice blast, and it is the nucleotide sequence shown in the sequence table SEQ ID NO:1, and it comprises complete OsWRKY45-2 gene.
2, the coding region of OsWRKY45-2 gene, it is sequence table SEQ ID NO; Nucleotide sequence in 1 shown in the 113-1270 or coding and the identical protein DNA sequence of SEQ ID NO:1 encoded protein matter.
3, each described nucleotides sequence of claim 1-2 is listed in increases paddy rice to the application in bacterial leaf spot pathogenic bacteria and the Pyricularia oryzae resistance.
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Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101921775A (en) * | 2010-06-04 | 2010-12-22 | 华中农业大学 | Application of rice OsWRKY45-1 gene in improving abiotic stress resistance of plants |
| CN101948847A (en) * | 2010-06-01 | 2011-01-19 | 华中农业大学 | Application of rice OsWRKY45-2 gene in improving plants against abiotic adverse environmental stress |
| CN102477431A (en) * | 2010-11-23 | 2012-05-30 | 中国科学院植物研究所 | Application of R protein Glyma11g00320 to promotion of biosynthesis of salicylic acid and enhancement of disease resistance of plants |
| CN102477430A (en) * | 2010-11-23 | 2012-05-30 | 中国科学院植物研究所 | Application of WRKY polypeptide Glyma07g02630 to promotion of biosynthesis of salicylic acid and enhancement of disease resistance of plants |
| CN109182348A (en) * | 2018-09-12 | 2019-01-11 | 华南农业大学 | The application of bacterial leaf spot resistance related gene OsPRX30 |
| CN111187779A (en) * | 2020-02-14 | 2020-05-22 | 西南大学 | Disease-resistant gene OsRLR1, transcription factor OsWRKY19 and application in breeding of rice resistant to bacterial blight |
| CN112195184A (en) * | 2020-10-04 | 2021-01-08 | 华中农业大学 | Application of OsMAPK6 gene in improving disease resistance of rice |
| CN112313338A (en) * | 2018-04-18 | 2021-02-02 | 金农有限公司 | Recombinant gene |
| CN112646010A (en) * | 2020-12-31 | 2021-04-13 | 浙江大学 | OsWRKY12 and application thereof in efficient phosphorus breeding of rice |
| CN112831505A (en) * | 2021-03-16 | 2021-05-25 | 昆明理工大学 | Pseudo-ginseng WRKY transcription factor genePnWRKY15And applications |
| CN114456248A (en) * | 2022-03-15 | 2022-05-10 | 湖北大学 | Abscisic acid receptor OsPYL2 gene for synergistically regulating resistance of rice to drought and rice blast and application thereof |
| CN114621975A (en) * | 2020-12-11 | 2022-06-14 | 华南农业大学 | Application of rice blast resistance related gene OsWRKY5 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101948847A (en) * | 2010-06-01 | 2011-01-19 | 华中农业大学 | Application of rice OsWRKY45-2 gene in improving plants against abiotic adverse environmental stress |
| CN101921775A (en) * | 2010-06-04 | 2010-12-22 | 华中农业大学 | Application of rice OsWRKY45-1 gene in improving abiotic stress resistance of plants |
| CN101921775B (en) * | 2010-06-04 | 2014-03-26 | 华中农业大学 | Application of rice OsWRKY45-1 gene in improving abiotic stress resistance of plants |
| CN102477431A (en) * | 2010-11-23 | 2012-05-30 | 中国科学院植物研究所 | Application of R protein Glyma11g00320 to promotion of biosynthesis of salicylic acid and enhancement of disease resistance of plants |
| CN102477430A (en) * | 2010-11-23 | 2012-05-30 | 中国科学院植物研究所 | Application of WRKY polypeptide Glyma07g02630 to promotion of biosynthesis of salicylic acid and enhancement of disease resistance of plants |
| CN102477430B (en) * | 2010-11-23 | 2014-08-27 | 中国科学院植物研究所 | Application of WRKY polypeptide Glyma07g02630 to promotion of biosynthesis of salicylic acid and enhancement of disease resistance of plants |
| CN112313338A (en) * | 2018-04-18 | 2021-02-02 | 金农有限公司 | Recombinant gene |
| CN109182348B (en) * | 2018-09-12 | 2020-07-31 | 华南农业大学 | Application of bacterial leaf blight resistance related gene OsPRX30 |
| CN109182348A (en) * | 2018-09-12 | 2019-01-11 | 华南农业大学 | The application of bacterial leaf spot resistance related gene OsPRX30 |
| CN111187779A (en) * | 2020-02-14 | 2020-05-22 | 西南大学 | Disease-resistant gene OsRLR1, transcription factor OsWRKY19 and application in breeding of rice resistant to bacterial blight |
| CN111187779B (en) * | 2020-02-14 | 2022-06-10 | 西南大学 | Disease-resistant gene OsRLR1, transcription factor OsWRKY19 and application in breeding of rice resistant to bacterial blight |
| CN112195184A (en) * | 2020-10-04 | 2021-01-08 | 华中农业大学 | Application of OsMAPK6 gene in improving disease resistance of rice |
| CN114621975A (en) * | 2020-12-11 | 2022-06-14 | 华南农业大学 | Application of rice blast resistance related gene OsWRKY5 |
| CN114621975B (en) * | 2020-12-11 | 2023-05-02 | 华南农业大学 | Application of rice blast resistance related gene OsWRKY5 |
| CN112646010A (en) * | 2020-12-31 | 2021-04-13 | 浙江大学 | OsWRKY12 and application thereof in efficient phosphorus breeding of rice |
| CN112831505A (en) * | 2021-03-16 | 2021-05-25 | 昆明理工大学 | Pseudo-ginseng WRKY transcription factor genePnWRKY15And applications |
| CN112831505B (en) * | 2021-03-16 | 2023-04-11 | 昆明理工大学 | Pseudo-ginseng WRKY transcription factor gene PnWRKY15 and application thereof |
| CN114456248A (en) * | 2022-03-15 | 2022-05-10 | 湖北大学 | Abscisic acid receptor OsPYL2 gene for synergistically regulating resistance of rice to drought and rice blast and application thereof |
| CN114456248B (en) * | 2022-03-15 | 2023-05-26 | 湖北大学 | Abscisic acid receptor OsPYL2 gene for synergistically regulating and controlling drought resistance and rice blast resistance of rice and application thereof |
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Granted publication date: 20110209 Termination date: 20211021 |