CN104059930A - Paddy rice disease resistance-related gene PAL4 - Google Patents

Abstract

The invention belongs to the technical field of plant gene engineering, and particularly relates to function verification and application of a paddy rice disease resistance-related gene PAL4. The PAL4 gene verified in the invention is a positive regulatory factor in paddy rice disease resistance reaction, and participates the regulation of paddy rice resistance to rice blast pathogens infected from roots. The nucleotide sequence of the PAL4 gene is as shown in sequence table SEQ ID NO:1, which codes pH and temperature-sensitive phenylalanine ammonialyase, and the amino acid sequence of the protein of the phenylalanine ammonialyase is as shown in sequence table SEQ ID NO:2. The invention also discloses an application of the gene in paddy rice genetic improvement.

Description

Paddy disease-resistant related gene PAL4

Technical field

The present invention relates to plant gene engineering technology field.Be specifically related to the functional verification of a paddy disease-resistant related gene PAL4.The PAL4 gene of the present invention's checking is the positive regulatory factor in paddy disease-resistant reaction, and it participates in adjusting and controlling rice and resists the Pyricularia oryzae infecting from root.

Background technology

Plant, in the process of growth, is subject to the infringement of multiple pathogen.The phytopathy original of a great variety, comprises virus, bacterium, fungi and nematode etc.Pathogen invaded plants causes two kinds of results: the pathogenic bacteria successful reproduction in (1) host plant causes relevant illness; (2) host plant produces disease resistance response, kills pathogen or stops its growth.By utilizing resistant gene resource to improve not only disease prevention but also can reduce the environmental hazard that spraying pesticide brings in advance of disease resistance of plant.

Paddy rice is important in the world food crop, but the impact of disease usually causes the decline of its yield and quality.Rice blast causes by Pyricularia oryzae (Magnaporthe oryzae), is one of fungal disease to rice hazard maximum in the world.Generally believe that at present rice blast fungus is the pathogenic bacteria of half biotroph that infects of a kind of typical tissue on the ground (such as blade etc.), its infection way is: conidium survives the winter in sick paddy and the residual straw of catching an illness, when pathogenic bacteria under temperature and the suitable condition of humidity produces a large amount of conidiums, and these spores arrive the ground tissue of paddy rice by air diffuser, complete the process that infects rice cell and cause paddy rice to occur illness.Be discharged into and in environment, be again diffused into other paddy rice and complete superinfection and these cells that infected produce new mycelia conidium, thereby cause the rapid spread of disease.But along with the continuous understanding of people to Pyricularia oryzae, investigator finds that Pyricularia oryzae can infect paddy rice by root, produce scab at root and over-ground part, and rice blast fungus root infects also and infects the same gene pairs gene hypothesis (Sesma and Osbourn, 2004) that is applicable to leaf portion.Further research shows, rice blast fungus infects paddy rice from root and belongs to active nutritional type, can on whole plant, produce systematicness and infect, and has feature and the characteristic (Marcel etc., 2010) of typical soil-borne pathogen.Classification of fungi scholar has been divided into the newly-established Magnaporthaceae family (Rossman etc., 2007) that uploads pathogenic bacteria that comprises Pyricularia oryzae again in recent years, and having changed people can only be from the traditional view that tissue infects on the ground to Pyricularia oryzae.And this discovery also the viewpoint of the epidemiology aspect to Pyricularia oryzae produced important meaning, infecting from root may be one of primary source of infection of Pyricularia oryzae, be Pyricularia oryzae occur and a popular important factor in order.Also the circulation that has research to point out Pyricularia oryzae be by rice paddy seed in spite of illness in the process of sprouting because the continuous accumulation of Pyricularia oryzae causes these seeds in spite of illness very little just dead, and the rice seedlings of catching an illness of these death just infects root and the blade of near healthy plant as the initial source of infection, infect new sprout rice shoot root with leaf become mycelia, then these plant that catch an illness are developed illness again and complete circulation (Faivre-Rampant etc., 2012) until again infect seed.For paddy rice, different infection ways can cause the difference of paddy rice defensive raction related gene expression intensity and space-time.Therefore, the pathogenesis related gene that the anti-rice blast root of Study On Rice infects contributes to understand the resistance mechanism of rice anti-rice blast, also provides new strategy for the seed selection of anti-rice blast kind, academicly and have profound significance in production application.

The disease resistance response of plant is the complex process that polygene participates in regulation and control.The gene that participates in Plant defense responses is divided into two classes: (1) main effect disease-resistant gene, claims again MR (major resistance) gene and (2) disease-resistant related gene.People generally believe that the major function of main effect disease-resistant gene proteins encoded is direct or indirect as acceptor and cause of disease matter interaction starts defense signaling path (Kou and Wang, 2010 in plant materials at present; Zhang and Wang, 2013).The disease resistance response strong resistance of disease-resistant gene mediation is good genetic resources.But in the process of using disease-resistant gene improvement plant resistance to environment stress, be subject to following some restriction: the resource-constrained of (1) main effect disease-resistant gene, as the main effect disease-resistant gene of the important disease bacterial leaf-blight of opposing paddy rice of knowing at present approximately only has 37; (2) main effect disease-resistant gene has cause of disease kind and cause of disease physiological strain specificity, causes disease-resistant being limited in scope; (3) because of the quick sudden change of cause of disease, the effect of a main effect disease-resistant gene often can not be lasting.Disease-resistant related gene refers to all genes that participate in disease resistance response except main effect disease-resistant gene, it has been generally acknowledged that their coded product participates in disease-resistant signaling molecule in synthetic plant materials, participates in signal conduction, stops signal conduction or participate in defense response etc.The common feature of this genoid be pathogeny evoked after they expression amount raise or reduce, therefore people can be according to the difference of the expression amount of pathogeny evoked front and back gene plant identification disease-resistant related gene (Maleck etc., 2000 on a large scale; Schenk etc., 2000; Zhou etc., 2002).At present, the understanding of people's enantiopathy genes involved is limited.According to having been reported, resistance capacity when most disease-resistant related gene independent role may be less than disease-resistant gene.But based on following reason, they are the genetic resourceses that are worth Devoting Major Efforts To Developing: (1), because the product of most disease-resistant related genes does not need directly and pathogen interaction, this genoid is the genetic resources with durable resistance; (2) disease resistance response that most of disease-resistant related genes participate in does not have cause of disease specificity, and therefore they are the genetic resourceses with resistance of wide spectrum; (3) aboundresources of this genoid.But, although identified a lot of disease-resistant related genes (Zhou etc., 2002 in paddy rice; Chu etc., 2004), whether the mechanism of action and the single disease-resistant related gene of these genes in paddy disease-resistant reaction can cause that the change of paddy disease-resistant phenotype is all unclear.Separating clone disease-resistant related gene is of great importance to the research of paddy disease-resistant mechanism, and disease-resistant related gene provides more wide spectrum and long-acting resistance than disease-resistant gene in application simultaneously.Understand the pathogenesis of disease, contribute to utilize the resistance of high effective way improvement rice varieties, control the generation of disease.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.

Phenylalanine ammonia lyase (phenylalanine ammonia-lyase, PAL) gene described in the invention belongs to a gene family, is extensively present in plant, is the key enzyme of multiple secondary metabolism approach in plant materials.The phenylalanine that shikimic acid pathway produces generates styracin after PAL separates ammonia effect, enter phenylpropyl alcohol alkanes pathways metabolism, can generate the multiple secondary metabolite such as flavonoid, xylogen, all all directly or are indirectly generated by this approach containing materials of phenylpropyl alcohol alkane skeleton, and the secondary metabolite producing all plays an important role in the growing of plant, disease-resistant, degeneration-resistant reaction.In the last few years, along with deepening continuously of research, found that many Flavonoid substances had Green Tea Extract, regulating blood lipoid, anticancer, the effect of antimicrobial antiphlogistic, has started the upsurge that Flavonoid substances research and development utilize.The pathways metabolism of phenylpropyl alcohol alkanes and flavonoid compound is one of important secondary metabolism approach of plant, and the biosynthesizing of flavonoid compound is all to come by phenylpropyl alcohol alkanes biosynthetic pathway, and PAL is wherein very crucial enzyme.Consequent flavonoid compound naringenin has multiple biological activity, can be carried out structural modification by multiple enzyme and generate derivative, it is the precursor of many meta-bolitess, there is antibacterial, anti-inflammatory, removing free radical, anti-oxidant, cough-relieving apophlegmatic, reducing blood-fat, the anticancer effect such as antitumor, can be widely used in the field such as medicine, food.Naringenin proceeds to a methyl through methylferase can form sakuranetin, and sakuranetin is a kind of well flavanone phytoalexin, is the important component part of plant defense system.

In sum, excavating and utilize good resistant gene resource improvement paddy rice is the generation of controlling rice blast disease, the most economical effective measures of loss that reduce or avoid rice disease to bring.On the other hand, PAL is very important enzyme in phenylpropyl alcohol alkanes and flavonoid compound pathways metabolism, by reaction has very large utility value for research secondary metabolism to its excavation, also flavonoid meta-bolites can better be applied to medicine, field of food simultaneously.

Summary of the invention

The object of the invention is to identify the function of a paddy rice PAL gene in paddy rice-cause of disease is done mutually, for the ability of utilizing this improvement of genes rice varieties or other plant to resist disease lays the foundation.Applicant is PAL4 by this unnamed gene.

The PAL4 gene the present invention relates to is given paddy rice the caused disease of the Pyricularia oryzae being infected by acting in accordance with YIN YANG changes in four seasons portion is produced to disease resistance response.The DNA sequence dna of this gene, as shown in sequence table SEQ ID NO:1, or is equivalent to the DNA sequence dna shown in SEQ ID NO:1 substantially, or its function is equivalent to the subfragment of sequence shown in SEQ ID NO:1.Its sequence analysis is shown to its a kind of phenylalanine ammonia lyase of encoding.Stop the expression of this kind of enzyme, weaken the resistance of paddy rice to rice blast, reduce in its root flavonoid secondary metabolite as the content of phytoalexin simultaneously.

The present invention relates to separate a kind of DNA fragmentation of the OsPAL4 of comprising gene and identify its function, nucleotide sequence and the aminoacid sequence of this gene is provided.Wherein, the nucleotide sequence of described fragment is as shown in sequence table SEQ ID NO:1, or be substantially equivalent to the nucleotide sequence shown in SEQ ID NO:1, be also included within the mutant allele or the derivative that add, replace, insert or delete one or more Nucleotide and produce, or its function is equivalent to the subfragment of sequence shown in SEQ ID NO:1.Its sequence analysis is shown to its a kind of phenylalanine ammonia lyase (phenylalanine ammonia-lyase) of encoding, by the amino phenylalanine solution styracin that forms.

In embodiments of the invention part, applicant set forth to the functional verification process of PAL4 gene with and the feature of phenylalanine ammonia lyase of coding.

Brief description of the drawings

Sequence table SEQ ID NO:1: the nucleotide sequence that is PAL4 gene.Sequence total length is 2261bp.

Sequence table SEQ ID NO:2: the aminoacid sequence that is the protein of PAL4 genes encoding.713 amino acid of encoding.

Fig. 1: qualification of the present invention and separating clone paddy disease-resistant related gene PAL4 and verify the schema of this gene function.

Fig. 2: the expression pattern that connects PAL4 gene after Pyricularia oryzae bacterial strain CHL358 with quantitative reverse transcription-PCR susceptible rice varieties CO39 of (quantitative reverse transcription-PCR, qRT-PCR) technology for detection and disease resisting rice family C101A51.Paddy rice sample source is the material after 0.5,1,2,24,72,120,168 hour after inoculation CHL358.

Fig. 3: the check analysis (B) of T-DNA insertion point (A) and mutant gene type in PAL4 gene structure and T-DNA insertion mutation body 05Z11DD35.In figure, ledgement represents the intron of PAL4 gene, and black spreader bar represents the encoding sequence of exon.Nucleotide number before the Nucleotide number of each structure of numeral and T-DNA insertion point (by sequence numbering shown in sequence table SEQ ID NO:1.)." ATG " and " TAG " is respectively translation initiation password and termination codon.Arrow P AL-1F, PAL1R-2, NTLB5 represent respectively the PCR primer for verifying that whether mutant is correct; Wherein PAL-1F and PAL1R-2 primer are according to the design of PAL4 gene order, lay respectively at T-DNA insertion point both sides; NTLB5 is according to the primer of T-DNA internal sequence design.Fig. 3, the WT (contrast) in B represents to spend 11 in wild-type (non-transgenic) rice varieties.

Fig. 4: be the prokaryotic expression carrier pMAL-c2 schematic diagram the present invention relates to.

Fig. 5: PAL4 albumen has the activity of phenylalanine solution amino.PAL4 albumen catalytic substrate L-Phe deaminizating forms product trans-cinnamic acid.Ck: prokaryotic expression label protein.

Fig. 6: temperature and pH value affect the enzymic activity of PAL4 albumen.

Fig. 7: PAL4 gene T-DNA insertion mutation body plant (05Z11DD35) weakens rice blast disease resistance.CO39 is rice blast susceptible variety.Contrast, for spending 11 (wild-types) in rice varieties, is the acceptor of T-DNA insertion mutation body genetic transformation.Contrast 1 is isolated negative plant from 05Z11DD35 strain offspring.The lesion area of mutant contrasts remarkable increase.Infection rate refers to the ratio of the Pyricularia oryzae cell containing in each rice cell.Infection rate in mutant plant higher than in spend 11 wild-types contrasts and negative controls.

Fig. 8: the content of the work of PAL enzyme, Whitfield's ointment and flavonoid plant protecting chemical in PAL4 gene T-DNA insertion mutation body plant (05Z11DD35) plant root reduces.Contrast, for spending 11 (wild-types) in rice varieties, is the acceptor of T-DNA insertion mutation body genetic transformation, and contrast 1 is isolated negative plant from 05Z11DD35 strain offspring.It is original 1/3rd that the decline that enzyme is lived causes Whitfield's ointment to drop to, and causes the content of sakuranetin and naringenin to reduce more than 95%.

Embodiment

Previous research work result of the present invention discloses, in paddy rice-bacterial pathogen is done mutually, the expression amount of PAL4 (naming PAL in former document) gene changes, and points out this gene may participate in paddy rice opposing pathogenic bacteria reaction (Qiu etc., 2007).In order to verify this analysis supposition, produce the present invention.

Further definition the present invention in following examples, Fig. 1 has described the flow process of qualification PAL4 gene function.According to above description and these embodiment, those skilled in the art can determine essential characteristic of the present invention, and in the situation that not departing from spirit and scope of the invention, can make various changes and amendment to the present invention, so that its applicable various uses and condition.

The expression pattern analysis of embodiment 1:PAL4 gene in different rice varieties

In paddy rice whole genome sequence, carry out Blastp retrieval with PAL structural domain PF00221 (Pfam database, http://pfam.sanger.ac.uk/), obtain 9 PAL family members.The gene that the present invention studies is numbered LOC_Os02g41680 in MSU Rice Genome Annotation Project (http://rice.plantbiology.msu.edu/), according to each PAL family member, in chromosomal positional alignment, applicant is PAL4 by this unnamed gene.

In order to confirm whether PAL4 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) has been analyzed PAL4 gene and inoculated the expression pattern after Pyricularia oryzae CHL358 in susceptible rice varieties CO39 and disease resisting rice family C101A51.CHL358 is conventional rice blast fungi isolates (Fu etc., 2011).CO39 is the susceptible rice varieties of rice blast of commonly using in the world, and C101A51 is the near isogenic line that contains blast resisting major gene Pi-2 (Jiang and Wang, 2002) taking CO39 as background.Pyricularia oryzae inoculation adopts spray method to inoculate (Fu etc., 2011) to the paddy rice of 3-4 leaf phase.After inoculation, a point different time points is got inoculation blade extracted total RNA (Zhou etc., 2002).Get 1～5 μ g total for RNA DNaseI (American I nvitrogen company) process and pollute to remove genomic dna for 15 minutes, then with reference to the method for (2002) such as Zhou, use oligo (dT) ₁₅oligomerization primer and M-MLV ThermoScript II (purchased from Promega company of the U.S.) are carried out reverse transcription.Adopt Real-time PCR Analysis test kit (precious biotechnology (Dalian) company limited), and according to test kit working instructions, on ABI 7500Real-Time PCR system (Applied Biosystems company of the U.S.) instrument, carry out real-time quantitative PCR reaction.With expression amount measurement the homogenization sample rna content (Qiu etc., 2007) of the endogenous Actin muscle of paddy rice (actin) gene.PAL4 gene specific PCR primer during qRT-PCR analyzes is DL-OsPAL4F (5 ＇-GGGCAACCCAGTGACCAA-3 ＇) and DL-OsPAL4R (5 ＇-CGATTGCCTCGTCGGTCTT-3 ＇), and actin gene PCR primer is actinF (5 ＇-TGCTATGTACGTCGCCATCCAG-3 ＇) and actinR (5 ＇-AATGAGTAACCACGCTCCGTCA-3 ＇).

Experimental result shows after inoculation Pyricularia oryzae CHL358, PAL4 gene is induced rapidly until within 72 hours, there is peak expression in susceptible variety CO39, and first the expression of PAL4 has individual process of inhibition in disease-resistant variety, until within 72 hours, be just induced rise (Fig. 2).C101A51 carries blast resisting major gene Pi-2, and in the time not having Pyricularia oryzae to infect, the expression amount of PAL4 gene in disease resisting rice C101A51 is significantly higher than the expression amount (Fig. 2) in susceptible paddy rice CO39.This results suggest: PAL4 gene may participate in the regulation and control of blast resisting reaction.

Embodiment 2: determine sequence and the structure of spending the PAL4 gene in 11 in rice varieties

1. in separating rice kind, spend the sequence of the PAL4 gene in 11

In the LOC_Os02g41680 site information of paddy rice whole genome sequence database, include the cDNA sequence [GenBank (http://www.ncbi.nlm.nih.gov) number of registration: AK068993] of PAL4 gene, 713 amino acid of this cDNA sequence encoding.Paddy rice sequence in paddy rice whole genome sequence database comes from japonica rice variety Japan fine (Oryza sativa ssp.Japonica), and it is fine that cDNA sequence A K068993 also comes from Japan.

The sequences Design of the both sides, PAL4 gene coding region during we are fine according to rice varieties Japan one couple of PCR primers: ECOR1-OSPAL4F (5 ＇-AT gAATTCaTGGAGTGCGAGAACGGCCGC-3 ＇) (the joint EcoRI digestion with restriction enzyme site that underscore representative connects for carrier) and XBA1-OSPAL4R (5 ＇-AT tCTAGAtCAGCAGATTGGCAGGGGCTC-3 ＇) (the XbaI digestion with restriction enzyme site of the joint that underscore representative connects for carrier).Utilize this that PCR primer is spent the cDNA fragment that in 11, amplification comprises PAL4 gene and this fragment is cloned on carrier from japonica rice variety.In spend 11 to be conventional rice varieties (Wu etc., 2003).Utilize the sequencing kit of Applied Biosystems company of the U.S., obtain the coding region sequence of PAL4 with dideoxy nucleotide end cessation method (Applied Biosystems company of the U.S.) from the order-checking of carrier two ends.Utilize the sequence of PCR primer PAL3R (5 ＇-TGAGTCAGGTGGTCGGTGTA-3 ＇) the measurement intron that is positioned at gene inside simultaneously, after sequence assembly, obtain the DNA sequence dna (seeing sequence table SEQ ID NO:1) of a long 2261bp, the encoding sequence (Fig. 3, A) that it comprises complete PAL4 gene.

2. in rice varieties, spend the PAL4 gene structure analysis in 11

Applicant by the middle PAL4 gene order-checking of spending in 11 after, the PAL4 gene order in fine with rice varieties Japan compares, the sequence of finding coding region and intron with Japanese fine in identical.Relatively, spend genome sequence and the cDNA sequence of the PAL4 gene in 11, in determining, spend the PAL4 gene coding region in 11 to comprise two exons and an intron (Fig. 3, A).First exon forms (the 1-395bp place that is positioned at sequence table SEQ ID NO:1) by 395 Nucleotide, First Intron forms (the 396-514bp place that is positioned at sequence table SEQ ID NO:1) by 119 Nucleotide, second exon forms (the 515-2258bp place that is positioned at sequence table SEQ ID NO:1) (Fig. 3, A) by 1747 Nucleotide.The PAL4 gene hereinafter relating to is all from spending 11 in rice varieties.

The analysis of 3.PAL4 gene encoding production

Protein being made up of 713 amino acid of PAL4 genes encoding (sequence table SEQ ID NO:2), has phenylalanine/Histidine ammonialyase conserved domain (MacDonald and D ＇ Cunha, 2007).Known phenylalanine ammonia lyase can form trans-cinnamic acid (Koukol and Conn, 1961) by catalysis L-Phe.In order to verify that PAL4 albumen has PAL activity, the present invention is at expression in escherichia coli PAL4 albumen, and this albumen of separation and purification is for enzyme activity assay.Concrete operations are as follows: from rice varieties, spend and in 11, extract total RNA and obtain cDNA by reverse transcription, adopt the cDNA fragment of the coding region of round pcr amplification PAL4 gene as template.Be ECOR1-OSPAL4F (5 ＇-AT for the PCR primer increasing gAATTCaTGGAGTGCGAGAACGGCCGC-3 ＇) (the joint EcoRI digestion with restriction enzyme site that underscore representative connects for carrier) and XBA1-OSPAL4R (5 ＇-AT tCTAGAtCAGCAGATTGGCAGGGGCTC-3 ＇) (the XbaI digestion with restriction enzyme site of the joint that underscore representative connects for carrier).PCR product is connected with TA cloning vector pMD18-T (purchased from precious biotechnology (Dalian) company limited), cuts screening positive clone by enzyme, then through sequence verification without coding mutation.Digest and [see Fig. 4 with positive colony and the pMAL-c2 carrier of PAL4 genes encoding section with restriction enzyme EcoRI and XbaI; Purchased from Niu Yinglun biotechnology (Beijing) company limited]; Enzyme cuts to be finished at 10 minutes deactivation restriction enzymes of 65 DEG C of water-baths.Endonuclease bamhi is cut after 0.8% agarose gel electrophoresis to glue and reclaimed object band.Carrier after cutting with the endonuclease bamhi that comprises PAL4 gene and enzyme does ligation.Cut and sequence verification positive colony by enzyme, by the recombinant plasmid electricity conversion obtaining, (electroporation is eppendorf company product, the present embodiment applied voltage is 1800v, and concrete operations are with reference to the working instructions of this instrument) enter escherichia coli expression host strain BL21 (purchased from German Novagen company) and carry out prokaryotic expression.PMAL-c2 empty carrier is also imported to BL21 in contrast.PMAL-c2 carrier is with maltose binding protein (maltose binding protein, MBP) label.Picking mono-clonal overnight incubation, with 1: 100 (volume ratio) enlarged culturing 2-3 hour, treat that its OD value reaches 0.5, adding isopropylthio-β-D-galactoside (IPTG) to final concentration is 1mM/L, induce 3 hours, collect thalline, (empty carrier contrasts with MBP label expressing protein, PAL4 recombinant protein aminoterminal is with MBP label) with the affine Resin A mylose Resin[of maltose purchased from Niu Yinglun biotechnology (Beijing) company limited] carry out purifying, operation is with reference to specification sheets.PAL4 mmp reaction program is with reference to forefathers' method slightly modified (Hu etc., 2011).Concrete operations are as follows: the PAL4 albumen that adds 500 μ L 20mM substrate L-Phes and 3 μ L purifying in 1mL Tris-HCl damping fluid, the label protein giving expression to empty carrier in contrast, after 37 DEG C of reaction 1h, add the hydrochloric acid termination reaction of 0.25mL 4M.With methyl alcohol: reaction product dilution 10 times of laggard supper-fast liquid chromatography-mass spectrography/GC-MS (UFLC-MS/MS) (SHIMADZU, Japan) are done qualitative analysis by the solvent that water (volume ratio) is 1: 1.Measure the light absorption value calculation sample enzyme work of 290nm by Tecan grating type microplate reader (INFINITE 200, Austria) and do quantitative analysis.In sample, the mensuration of protein content adopts the method for Bradford (1976) report.The standard model of L-Phe and trans-cinnamic acid is buied from Duchefa and Sigma company respectively.

Result shows, can generate product trans-cinnamic acid after adding MBP-PAL4 recombinant protein to react, and the contrast of MBP label protein can not generate trans-cinnamic acid, proves that PAL4 albumen is phenylalanine ammonia lyase (Fig. 5).MBP-PAL4 albumen enzymic activity under alkaline environment is higher, and enzyme work is along with the rising of temperature is risen gradually, until 55 DEG C reach the rising fast-descending (Fig. 6) with temperature after peak value.These presentation of results PAL4 is to pH value and temperature sensitive phenylalanine ammonia lyase.

The functional verification of embodiment 3:PAL4 gene in paddy rice-cause of disease is done mutually

1. Screening of Rice mutant library and mutant plant insertion point coseparation analysis

In order to verify the function of PAL4 gene in paddy rice-cause of disease is done mutually, applicant utilizes the sequence retrieval paddy rice T-DNA insertion mutation volume data storehouse RMD (Zhang etc. of PAL4 gene, 2006), find that the mutant 05Z11DD35 in this database has the flanking sequence of 375bp, one section of sequence (483-857bp in sequence table SEQ ID NO:1) homology of this flanking sequence and PAL4 gene.Prompting T-DNA is inserted in PAL4 gene inside.Sequence comparing analysis prompting T-DNA is inserted in after the 857bp of sequence shown in sequence table SEQ ID NO:1, and T-DNA is inserted in the 2nd exon interior (Fig. 3, A) of PAL4 gene.PAL4 gene T-DNA insertion mutation body (05Z11DD35) has the genetic background (Wu etc., 2003) of spending 11 in japonica rice.

In order to verify the exactness of obtained mutant, applicant according to the sequences Design of PAL4 gene a pair of PCR primer (Fig. 3, A) that is positioned at T-DNA insertion point both sides: PAL1F (5 ＇-TCACGGAGTTGATCTCACGC-3 ＇) and PAL1R-2 (5 ＇-CACAGGAGGACCAAGGAGGG-3 ＇).In addition, according to T-DNA sequences Design PCR primer NTLB5 (5 ＇-AATCCAGATCCCCCGAATTA-3 ＇) (Wu etc., 2003).Utilize the T-DNA of these 3 PCR primer mutant that analysis obtains whether to be inserted in the inside of PAL4 gene.The ultimate principle of this analysis is: in 05Z11DD35 homozygous mutation body (having the insertion of T-DNA in pair of homologous karyomit(e)) plant, due to the about 14kb (Wu etc. of T-DNA fragment that insert, 2003), utilize the conventional PCR method of primer PAL1F and PAL1R-2, employing so large DNA fragmentation that cannot increase, and by primer PAL1R-2 and NTLB5 can the increase known rice genome of one section of size and the hybrid DNA fragment of T-DNA; In heterozygous mutant body (having T-DNA to insert in only having item chromosome in the pair of homologous karyomit(e)) plant of 05Z11DD35, with can the increase DNA fragmentation of the known rice genome of one section of size of primer PAL1F and PAL1R-2, by primer PAL1R-2 and NTLB5 also can the increase known rice genome of one section of size and the hybrid DNA fragment of T-DNA; In the feminine gender of 05Z11DD35 (isolated in family do not have T-DNA to insert) plant, by primer PAL1F and the PAL1R-2 one section of oryza sativa genomic dna fragment that size is known that can increase, but can not amplify DNA fragmentation with primer PAL1R-2 and NTLB5.The T of applicant to the 10 strain 05Z11DD35 familys of choosing at random ₁plant has carried out above-mentioned pcr analysis.Result shows that wherein 6 strains (1,2,3,4,6,7) are homozygous mutation bodies, and 4 strains (5,8,9,10) are heterozygous mutant body (figure, 3B).The rice material 05Z11DD35 that these presentation of results applicant obtains is PAL4 gene mutation body.

2. the Analysis of Resistance of mutant plant to rice blast

Applicant is to the PAL4 gene T-DNA insertion mutation body (05Z11DD35) isozygotying and negative control (05Z11DD35 feminine gender) plant of separating and have in the wild-type contrast rice varieties of same genetic background and spend 11 to carry out root inoculation Pyricularia oryzae RB11 (Fu etc., 2011).Concrete grammar is as follows: the vermiculite (vermiculite drains after 1 hour with distilled water immersion) of 3 cm thicks is filled in culture tube bottom, put into the growth of a size of culture tube aperture evenly and be paved with the mycelia piece of Pyricularia oryzae, then spread the vermiculite of 1 cm thick, put into the rice paddy seed after normal germination, then spread a little vermiculite on top layer to fix seed.Sealing culture tube, to stop the loss of moisture, is put into biochemical cultivation case growth.Light intensity is 12000-15000 Lux, 28 DEG C of set temperatures, illumination in 16 hours dark cycle processing in 8 hours.Until paddy rice grows to the 3 leaf phases (about about 15 days), the scab occurring on investigation blade.The rice leaf DNA of simultaneously fetching water sample carries out Pyricularia oryzae infection rate analysis (Kawano etc., 2010).The method for extracting of DNA is: sample is at extraction buffer [50mM Tris-HCl (pH8.0), 150mM NaCl, 100mM EDTA (pH8.0)] in fully grind, add 50 μ L 10%SDS, process 1-3 hour for 37 DEG C, after processing, add 75 μ L5M NaCl, after mixing, add 65 μ L CTAB/NaCl solution (10% CTAB is dissolved in 0.7M NaCl) to process 30-60 minute with 65 DEG C, add equal-volume chloroform: primary isoamyl alcohol (volume ratio 24: 1) mixes 15 minutes, centrifugal 10 minutes of 10000g, carefully draws supernatant.Centrifugal 10 minutes of 10000g after adding 0.6 times of volume Virahol to precipitate 1 hour with-20 DEG C.Abandon supernatant, precipitation adds 75% ethanol, and centrifugal 5 minutes of 10000g, dries up rear molten water.The DNA obtaining adopts Real-time PCR Analysis test kit SYBR green PCR Master Mix (precious biotechnology (Dalian) company limited), and according to test kit working instructions, on ABI7500 Real-Time PCR system (Applied Biosystems company of the U.S.) instrument, carry out real-time quantitative PCR reaction.Use respectively the rice cell amount containing in the quantitative DNA sample of samples contg of the PCR primer ubiquitinF (5 '-AACCAGCTGAGGCCCAAGA-3 ') of the endogenous ubiquitin of paddy rice (ubiquitin) gene and ubiquitinR (5 '-ACGATTGATTTAACCAGTCCATGA-3 ') amplification, the Pyricularia oryzae cell concentration containing in the quantitative DNA sample of samples contg increasing with the PCR primer Pot2F of the endogenous gene Pot2 of Pyricularia oryzae (5 '-ACGACCCGTCTTTACTTATTTGG-3 ') and Pot2R (5 '-AAGTAGCGTTGGTTTTGTTGGAT-3 ').Because ubiquitin gene only has 1 copy in paddy rice, Pot2 gene has 100 copies in Pyricularia oryzae, so the formula that infection rate calculates is I=N _pot2/ (N _ubiquitin× 100) (Kawano etc., 2010).

Experimental result shows after inoculation Pyricularia oryzae, susceptible contrast CO39 shows as susceptible to rice blast fungi isolates RB11, in wild-type, spend the disease index of the negative plant of 11 (contrasts) and 05Z11DD35 (contrast) to be respectively 15.2 and 11.5, and the disease index of the PAL4 gene T-DNA insertion mutation body that isozygotys is 23 (tables 1); Illustrate compared with the control, suppress PAL4 gene function and cause resistance to weaken (the left figure of Fig. 7).Real-time quantitative PCR result based on DNA cloning also shows simultaneously, and the infection rate of the PAL4 gene T-DNA insertion mutation body that isozygotys will be higher than adjoining tree (the right figure of Fig. 7).This presentation of results PAL4 gene is a gene that has application prospect in blast resisting breeding.

The reaction of table 1.PAL4 gene T-DNA insertion mutation body (05Z11DD35) to rice blast fungi isolates RB11

⁽¹⁾the statistics of disease index is the blade of investigation 15 strain plant.Disease index=∑ (the sick level of morbidity strain number × corresponding blade) × 100/ (always investigating the sick level of the heaviest blade of strain number × morbidity)

⁽²⁾negative transformed plant, isolated negative plant from 05Z11DD35.

The content analysis of PAL enzymic activity and Whitfield's ointment and flavonoid plant protecting chemical-naringenin and sakuranetin in embodiment 4:PAL4 gene mutation body

Applicant is to the PAL4 gene T-DNA insertion mutation body (05Z11DD35) isozygotying and negative control (05Z11DD35 feminine gender) plant of separating and have in the wild-type contrast rice varieties of same genetic background and spend the PAL enzymic activity in 11 to carry out analyzing (Hsieh etc., 2011).Concrete method is as follows, gets the fresh rice root in 2 tillering phase and is organized in liquid nitrogen and grinds, and adds in the borate buffer of 5mL pH8.8 (containing 5mM beta-mercaptoethanol and 1mM EDTA) and hatches on ice.Mixture, in centrifugal 15 minutes of 4 DEG C of 9000g, is collected supernatant and is enzyme crude extract.Get 0.2mL enzyme crude extract and add in the borate buffer of 2mL 50mM, then add the substrate L-Phe of 1mL 20mM in 37 DEG C of reactions 30 minutes, add 0.25mL 5M hydrochloric acid termination reaction.Measure the light absorption value calculation sample content of 290nm by Tecan grating type microplate reader (INFINITE 200, Austria).In sample, the mensuration of protein content adopts the method for Bradford (1976) report.Meanwhile, also the content of the salicylic content of plant hormone and flavonoid plant protecting chemical-naringenin and sakuranetin in above-mentioned rice material root has been carried out to quantitative analysis.Specific analytical method is shown in the working method (Liu etc., 2012) of having delivered.

Experimental result shows, the PAL enzyme that PAL4 gene T-DNA inserts in the root that causes mutant plant is lived, and salicylic acid content significantly declines.It is more huge that simultaneously T-DNA inserts the impact that the PAL4 inactivation that causes causes flavonoid plant protecting chemical, causes the content minimizing more than 95% (Fig. 8) of naringenin and sakuranetin.

Whitfield's ointment is in Plant defense responses and important endogenous signal molecule, it is not only, and paddy rice produces anaphylaxis and systemic resistance institute is essential, also be to infect with the important component in the signal transduction process of postactivated a series of defense responses (An and Mou, 2011) when paddy rice is subject to rice blast.The run-up after being subject to Infected with Pathogenic Fungi of naringenin and sakuranetin, is important phytoalexin in paddy rice body, there are some researches prove that these two kinds of toxinicides all can suppress growth (Kodama etc., 1992 of rice blast fungus in vitro; Padmavati etc., 1997).These presentation of results PAL4 gene T-DNA insertion mutation body (05Z11DD35) weakens and reduces closely related with the content of Whitfield's ointment and plant protecting chemical (naringenin and sakuranetin) the resistance of rice blast.According to the Gene Expression Profile Analysis of Fig. 2, after inoculation Pyricularia oryzae, the expression amount of PAL4 gene is all induced to rise and has been expressed in disease-resistant variety C101A51 and susceptible variety CO39, but the disease-resistant variety content of relatively can seeing clearly of the basic content of PAL4 gene in the time not inoculating Pyricularia oryzae exceeds 4 times than susceptible variety, this absolutely proves that the basal expression level that improves PAL4 gene by overexpression or the allelotrope of excavating high expression level amount can strengthen paddy rice to infect the resistance of Pyricularia oryzae from root.

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Claims (3)

1. strengthen paddy rice produces a resistance PAL4 gene to the Pyricularia oryzae infecting from root, its nucleotide sequence is as shown in sequence table SEQ ID NO:1.

2. gene claimed in claim 1, is characterized in that, described genes encoding is to pH value and temperature sensitive phenylalanine ammonia lyase, and the aminoacid sequence of this phenylalanine ammonia lyase is as shown in sequence table SEQ ID NO:2.

Gene described in claim 1 or 2 strengthen paddy rice to the application the Pyricularia oryzae resistance infecting from root.