CN105859860A - Application of disease resistance-related protein to regulation and control of plant disease resistance - Google Patents
Application of disease resistance-related protein to regulation and control of plant disease resistance Download PDFInfo
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Abstract
The invention discloses application of disease resistance-related protein to regulation and control of plant disease resistance. In the application disclosed by the invention, the disease resistance-related protein is A1), A2) or A3), wherein A1) is protein with an amino acid sequence shown in the sequence 2; A2) is protein which is obtained by performing the replacement and/or missing and/or addition of one or more of amino acid residues on the amino acid sequence in the sequence 2 and is related to the plant disease resistance; A3) is fusion protein obtained by connecting a tag at the N end or/and the C end of the A1) or A2). An experiment proves that the disease resistance-related protein disclosed by the invention and a coding gene thereof can regulate and control the plant disease resistance; the plant disease resistance can be improved through improving the expression of the disease resistance-related protein, and the plant disease resistance can be reduced through reducing the expression of the disease resistance-related protein, therefore, the plant disease resistance can be regulated and controlled by utilizing the disease resistance-related protein and the coding gene thereof.
Description
Technical field
The present invention relates to disease-resistance-related protein application in regulation and control disease resistance of plant in biological technical field.
Background technology
Oryza sativa L. is not only the most of paramount importance cereal crops, there are about global population more than half as food, and it is also
It it is the model plant of monocotyledon research.But, constantly reducing at arable area, the size of population constantly expands
Under situation, rice yield has become one of key factor affecting China's grain security.Although experienced by the Green revolution and
The extensive application of hybridization technique, but pest and disease damage, the factor such as severe natural environment the most seriously constrains rice yield
Growth.Bacterial leaf-blight is the most serious disease run in Rice Production, after Oryza sativa L. infects bacterial leaf-blight,
General underproduction 20-30%, even has no harvest.It is identified and isolated from Gene For Resistance To Rice Bacterial Blight, extensively should in particularly producing
Broad spectrum antidisease gene become the important step of Rice molecular breeding.
Summary of the invention
The technical problem to be solved is how to improve the disease resistance of plant.
For solving above-mentioned technical problem, present invention firstly provides disease-resistance-related protein answering in regulation and control disease resistance of plant
With；The entitled OsAPX8 of described disease-resistance-related protein, is following A1), A2) or A3):
A1) aminoacid sequence is the protein of sequence 2；
A2) by shown in sequence in sequence table 2 aminoacid sequence through one or several amino acid residue replacement and/
Or lack and/or add and the protein relevant to plant disease-resistant；
A3) at A1) or N end A2) or/and C end connects the fused protein that label obtains.
In order to make A1) in protein be easy to purification, can in by sequence table the aminoacid sequence shown in sequence 2
The amino terminal of the protein of row composition or carboxyl terminal connect upper label as shown in table 1.
Table 1, the sequence of label
| Label | Residue | Sequence |
| Poly-Arg | 5-6 (usually 5) | RRRRR |
| Poly-His | 2-10 (usually 6) | HHHHHH |
| FLAG | 8 | DYKDDDDK |
| Strep-tag II | 8 | WSHPQFEK |
| c-myc | 10 | EQKLISEEDL |
Above-mentioned A2) in, one or the replacement of several amino acid residue and/or disappearance and/or be added to less than 10
The replacement of individual amino acid residue and/or disappearance and/or interpolation.
Above-mentioned A2) in OsAPX8 can synthetic, it is possible to first synthesize its encoding gene, then carry out biological expression and obtain.
Above-mentioned A2) in OsAPX8 encoding gene can by will in the DNA sequence shown in sequence 1 or sequence 4 lack
The codon of one or several amino acid residue, and/or carry out the missense mutation of one or several base pair, and/or
Its 5 ' end and/or 3 ' ends connect the coded sequence of the label shown in table 1 and obtain.
For solving above-mentioned technical problem, present invention also offers the biomaterial relevant to OsAPX8 at regulation and control plant disease-resistant
Application in property；Described biomaterial is following B1) to B16) in any one:
B1) nucleic acid molecules of OsAPX8 is encoded；
B2) containing B1) expression cassette of described nucleic acid molecules；
B3) containing B1) recombinant vector of described nucleic acid molecules；
B4) containing B2) recombinant vector of described expression cassette；
B5) containing B1) recombinant microorganism of described nucleic acid molecules；
B6) containing B2) recombinant microorganism of described expression cassette；
B7) containing B3) recombinant microorganism of described recombinant vector；
B8) containing B4) recombinant microorganism of described recombinant vector；
B9) containing B1) the transgenic plant cells system of described nucleic acid molecules；
B10) containing B2) the transgenic plant cells system of described expression cassette；
B11) containing B1) Transgenic plant tissue of described nucleic acid molecules；
B12) containing B2) Transgenic plant tissue of described expression cassette；
B13) containing B1) the transgenic plant organ of described nucleic acid molecules；
B14) containing B2) the transgenic plant organ of described expression cassette；
B15) nucleic acid molecules that OsAPX8 expresses is reduced；
B16) containing B15) expression cassette of described nucleic acid molecules, recombinant vector, recombinant microorganism or transgenic plant thin
Born of the same parents are.
In above-mentioned application, B1) described nucleic acid molecules can be following b1)-b5) and in any one:
B1) the cDNA molecule of sequence 4 or DNA molecular during coded sequence is sequence table；
B2) the cDNA molecule of sequence 1 or DNA molecular during nucleotide sequence is sequence table；
B3) DNA molecular of sequence 3 during nucleotide sequence is sequence table；
B4) and b1) or b2) or b3) nucleotide sequence that limits there is 75% or more than 75% homogeneity, and coding
The cDNA molecule of OsAPX8 or genomic DNA molecule；
B5) under strict conditions with b1) b2) or b3) nucleotide sequence hybridization that limits, and coding OsAPX8
CDNA molecule or genomic DNA molecule；
B15) described nucleic acid molecules is and the DNA molecular of arbitrary fragment reverse complemental in the DNA molecular shown in sequence 4.
Wherein, described nucleic acid molecules can be DNA, such as cDNA, genomic DNA or recombinant DNA；Described nucleic acid divides
Son can also be RNA, such as mRNA or hnRNA etc..
Wherein, the protein shown in DNA molecular coded sequence 2 shown in sequence 1.Sequence 3 derives from Oryza sativa L., for
The genome sequence of OsAPX8 gene, sequence 4 is the cDNA sequence of OsAPX8 gene.
Those of ordinary skill in the art can use the side of known method, such as orthogenesis and point mutation easily
Method, the nucleotide sequence that the present invention encodes OsAPX8 suddenlys change.Those are through manually modified, have and this
The nucleotide sequence 75% of the OsAPX8 of invention isolated or the nucleotide of higher homogeneity, if coding OsAPX8
And there is OsAPX8 function, all it is derived from the nucleotide sequence of the present invention and is equal to the sequence of the present invention.
Term used herein " homogeneity " refers to the sequence similarity with native sequence nucleic acid." homogeneity " includes and this
The nucleotide sequence of protein of the composition of the aminoacid sequence shown in coded sequence 2 of invention has 75% or higher, or
85% or higher, or 90% or higher, or the nucleotide sequence of 95% or higher homogeneity.Homogeneity can with the naked eye or
Computer software is evaluated.Using computer software, the homogeneity between two or more sequences can use percentage ratio
(%) representing, it can be used to the homogeneity evaluating between correlated series.
In above-mentioned application, described stringent condition is at 2 × SSC, in the solution of 0.1%SDS, hybridizes also at 68 DEG C
Wash film 2 times, each 5min, again in 0.5 × SSC, in the solution of 0.1%SDS, hybridize at 68 DEG C and wash film 2
Secondary, each 15min；Or, 0.1 × SSPE (or 0.1 × SSC), 0.1%SDS solution in, under the conditions of 65 DEG C
Hybridize and wash film.
Above-mentioned 75% or more than 75% homogeneity, can be the homogeneity of 80%, 85%, 90% or more than 95%.
In above-mentioned application, B2) described in expression cassette (the OsAPX8 gene table of the nucleic acid molecules containing coding OsAPX8
Reach box), refer to express the DNA of OsAPX8, this DNA in host cell and not only can include starting OsAPX8 base
Because of the promoter transcribed, may also include the terminator terminating OsAPX8 genetic transcription.Further, described expression cassette also may be used
Including enhancer sequence.The promoter that can be used for the present invention includes but not limited to: constitutive promoter, tissue, organ
The promoter special with growth, and inducible promoter.The example of promoter includes but not limited to: cauliflower mosaic
The constitutive promoter 35S of poison: from the wound-inducible promoter of Fructus Lycopersici esculenti, leucine aminopeptidase (" LAP ",
Chao et al. (1999) Plant Physiol 120:979-992)；From chemical inducible promoter of Nicotiana tabacum L.,
Pathogeny is correlated with 1 (PR1) (being induced by salicylic acid and BTH (diazosulfide-7-carbothioic acid S-methyl ester))；
Fructus Lycopersici esculenti protease inhibitor II promoter (PIN2) or LAP promoter (all can use methyl jasmonate to induce)；
Heat-shock promoters (United States Patent (USP) 5,187,267)；Tetracycline inducible promoter (United States Patent (USP) 5,057,422)；
Seed specific promoters, such as Millet Seed specificity promoter pF128 (CN101063139B (Chinese patent
200710099169.7)), and the special promoter of seed storage protein matter (such as, phaseollin., napin, oleosin
Promoter (Beachy et al. (1985) EMBO is J.4:3047-3053) with Semen sojae atricolor beta conglycin).
They can be used alone or be used in combination with other plant promoter.All references cited herein is drawn all in full
With.Suitably transcription terminator includes but not limited to: Agrobacterium nopaline syntase terminator (NOS terminator), flower
Cauliflower mosaic virus CaMV 35S terminator, tml terminator, Semen Pisi sativi rbcS E9 terminator and nopaline and Octopus
Propylhomoserin synthase terminator (see, e.g.: Odell et al. (I985)Nature 313:810；Rosenberg
Et al. (1987) Gene, 56:125；Guerineau et al. (1991) Mol.Gen.Genet, 262:141；
Proudfoot(1991)Cell,64:671；Sanfacon et al. Genes Dev., 5:141；Mogen etc.
People (1990) Plant Cell, 2:1261；Munroe et al. (1990) Gene, 91:151；Ballad
Et al. (1989) Nucleic Acids Res.17:7891；Joshi et al. (1987) Nucleic Acid
Res.,15:9627)。
Available existing expression vector establishment contains the recombinant vector of OsAPX8 expression casette.Described plant expression vector
Including double base agrobacterium vector and the carrier etc. that can be used for plant micropellet bombardment.As pAHC25, pBin438,
PCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or
PCAMBIA1391-Xb (CAMBIA company) etc..Described plant expression vector also can comprise the 3 ' of exogenous gene and hold non-turning over
Translate region, i.e. comprise polyadenylation signals and any other participates in mRNA processing or the DNA fragmentation of gene expression.Described
The bootable polyadenylic acid of polyadenylation signals joins 3 ' ends of mRNA precursor, as Agrobacterium crown gall nodule induces (Ti)
Plasmid gene (such as rouge alkali synthetase gene Nos), plant gene (such as soybean storage protein genes) 3 ' end is transcribed
Untranslated region is respectively provided with similar functions.When using the gene constructed plant expression vector of the present invention, it be also possible to use enhancer,
Including translational enhancer or transcriptional enhancer, these enhancer regions can be ATG initiation codon or neighboring region rises
Beginning codon etc., but must be identical with the reading frame of coded sequence, to ensure the correct translation of whole sequence.Described turn over
The source translating control signal and start codon is widely, can be natural, it is also possible to be synthesis.Translate
Beginning region can come from transcription initiation region or structural gene.For the ease of transgenic plant cells or plant are reflected
Determine and screening, plant expression vector used can be processed, face can be produced as added the coding can expressed in plant
The enzyme of complexion changed or the gene (gus gene, luciferase genes etc.) of luminophor, antibiotic marker gene (as
Give the nptII gene to kanamycin and associated antibiotic resistance, give herbicide phosphinothricin resistance
Bar gene, gives the hph gene to antibiotic hygromycin resistance, and gives the dhfr to methotrexate resistance
Gene, gives EPSPS gene to glyphosate) or anti-chemical reagent marker gene etc. (such as anti-herbicide
Gene), provide metabolism mannose ability mannose-6-phosphate isomerase gene.From the safety of transgenic plant
Consider, any selected marker can be not added with, directly screen transformed plant with adverse circumstance.
In above-mentioned application, described carrier can be plasmid, glutinous grain, phage or viral vector.Described plasmid is concretely
PCAMBIA1300 or pANDA, it is possible to for will pCAMBIA1300 transform after the carrier that obtains, as
pCAMBIA1300-35S。
B3) described recombinant vector can contain sequence 1, sequence 3 or the DNA for encoding OsAPX8 shown in sequence 4
Sequence；B3 further) described recombinant vector concretely pCAMBIA1300-35S-OsAPX8.Described
PCAMBIA1300-35S-OsAPX8 be by EcoRI and the XbaI recognition site of pCAMBIA1300-35S between DNA
Sequence replaces with the recombinant vector of the OsAPX8 shown in expressed sequence 2 that the DNA sequence shown in sequence 4 obtains.
B16) described recombinant vector can be containing reducing the nucleic acid molecules that OsAPX8 expresses.In one embodiment of the present of invention
In, described recombinant vector is that the structure between LB and RB of pANDA-OsAPX8i, pANDA-OsAPX8i is as follows:
Ubiquitin promoter-reverse APX492-gus linker-forward APX492-NOS terminator, pANDA-OsAPX8i's
Other sequences are identical with pANDA.
In above-mentioned application, described microorganism can be yeast, antibacterial, algae or fungus.Described antibacterial concretely Agrobacterium,
Such as Agrobacterium EHA105.
In above-mentioned application, described transgenic plant cells system, Transgenic plant tissue and transgenic plant organ do not wrap
Include propagating materials.
In above-mentioned application, B15) described nucleic acid molecules concretely with the 1108-1599 position core of sequence in sequence table 4
The DNA molecular of the DNA fragmentation reverse complemental shown in thuja acid.
For solving above-mentioned technical problem, present invention also offers following arbitrary application of OsAPX8 or described biomaterial:
H1, the application being increased or decreased in plant in cultivation disease resistance；
H2, improve in preparation or reduce the application in disease resistance of plant product.
For solving above-mentioned technical problem, present invention also offers following G1 or G2:
G1, OsAPX8 or described biomaterial；
G2, regulation and control disease resistance of plant product, described product contains OsAPX8 or described biomaterial.
In the said goods, described regulation and control disease resistance of plant product can become using OsAPX8 or described biomaterial as activity
Point, it is also possible to the compositions being combined obtaining using OsAPX8 or described biomaterial and other disease resistance material is as work
Property composition.
For solving above-mentioned technical problem, present invention also offers following M1) or M2):
M1) method cultivating disease resistant transgenic plants, including importing following 1 in recipient plant) or 2) turned
Gene plant；
1) encoding gene of OsAPX8；
2) expression cassette of the encoding gene containing OsAPX8；
Described transgenic plant disease resistance compared with described recipient plant increases；
M2) method cultivating the transgenic plant that disease resistance reduces, including reducing the coding base of OsAPX8 in purpose plant
The expression of cause, obtains the disease resistance transgenic plant less than described purpose plant.
In said method, in reduction purpose plant, the expression of the encoding gene of OsAPX8 can be by by B15) described nucleic acid
Molecule imports described purpose plant and realizes.
In said method, the encoding gene of OsAPX8 can be B1) described nucleic acid molecules；Described expression cassette is B2) institute
State expression cassette.
In an embodiment of the present invention, the encoding gene (i.e. DNA molecular shown in sequence 1) of described OsAPX8 passes through
OsAPX8 gene recombinant vectors containing OsAPX8 expression casette imports in purpose plant.Described OsAPX8
In expression casette, the promoter starting OsAPX8 genetic transcription is 35S.
In said method, wherein said OsAPX8 gene can be modified the most as follows, then imports in receptor seed plant,
To reach more preferable expression effect:
1) modify according to actual needs and optimize, so that gene efficient expression；Such as, can plant according to receptor
The codon that thing is had a preference for, changes its password while keeping the aminoacid sequence of OsAPX8 gene of the present invention
Son is to meet plant-preference；During optimization, it is desirable that the coded sequence after optimization keeps certain GC
Content, to be best implemented with the high level expression of quiding gene in plant, wherein G/C content can be 35%, be more than
45%, more than 50% or more than about 60%；
2) gene order of neighbouring initial methionine is modified, so that translation is the most initial；Such as, utilization is being planted
In thing, known effective sequence is modified；
3) promoter expressed with various plants is connected, and is beneficial to its expression in plant；Described promoter can
Including composing type, induction type, sequential regulation, Growth adjustment, Chemical Regulation, tissue preferably and tissue specificity
Promoter；The selection of promoter will change along with expression time and space requirement, and also depend on target kind；
Such as tissue or the specific expressing promoter of organ, receptor is depending on what period grown as required；To the greatest extent
Pipe demonstrates and derives from many promoteres of dicotyledon is operational in monocotyledon, otherwise also
So, but it is desirable to select dicot promoters expression in the dicotyledon, monocotyledon
Promoter expression in the monocotyledon；
4) it is connected with the transcription terminator being suitable for, it is also possible to improve the expression efficiency of gene of the present invention；Such as originate
In the tml of CaMV, derive from the E9 of rbcS；Any known available terminator worked in plant
Can be attached with gene of the present invention；
5) enhancer sequence is introduced, before intron sequences (such as deriving from Adhl and bronzel) and virus
Lead sequence (such as deriving from TMV, MCMV and AMV).
Described OsAPX8 expression vector can be by using Ti-plasmids, Ri plasmid, plant viral vector, direct DNA
The conventional biology methods such as conversion, microinjection, conductance, agriculture bacillus mediated, particle gun convert plant cell or tissue,
And the plant tissue of conversion is cultivated into plant.
Described method also includes the plant from the encoding gene importing sequence 1, sequence 3 or the OsAPX8 shown in sequence 4
The plant of described encoding gene is expressed in middle screening, obtains transgenic plant.
In one embodiment of the invention, with the DNA shown in the 1108-1599 position nucleotide of sequence in sequence table 4
The DNA molecular of fragment reverse complemental is by purpose plant described in pANDA vector introduction.
In the present invention, described transgenic plant is interpreted as not only comprising described OsAPX8 gene transformation purpose plant
The transgenic plant that the first generation transgenic plant obtained and reduction OsAPX8 express, also includes its filial generation.For
Transgenic plant, can breed this gene, it is also possible to this gene is transferred into by traditional breeding method in these species
Enter other kind of same species, in commercial variety.Described transgenic plant includes seed, wound healing group
Knit, whole plant and cell.
In the present invention, described plant can be monocotyledon or dicotyledon；Described purpose plant and described receptor are planted
Thing can be all monocotyledon or dicotyledon.Described monocotyledon can be grass.Described grass family is planted
Thing can be Oryza sativa L..
In the present invention, described disease resistance can be bacterial blight-resisting.Described bacterial leaf-blight is caused by leaf spot bacteria, as in vain
The bacterial leaf-blight that leaf spoting bacteria Philippiiies race PXO99 causes.
It is demonstrated experimentally that the OsAPX8 of the present invention and encoding gene thereof can regulate and control the disease resistance of plant, improve OsAPX8
Expression can improve the disease resistance of plant, the expression reducing OsAPX8 can reduce the disease resistance of plant: utilizes white leaf
Rot infects the plant of plant (transfer-gen plant first) and OsAPX8 expression reduction that OsAPX8 expresses rising and (turns base
Because of plant second) find afterwards, inoculation pathogenic bacteria a length of 0.5 ± 0.3cm of scab of transfer-gen plant first after 5 days, the shortest
In the scab length (1.5 ± 0.3cm) of WT lines, a length of 2.3 ± 0.2cm of scab of transfer-gen plant second,
It is considerably longer than the scab length (1.5 ± 0.3cm) of WT lines.Explanation, it is possible to use OsAPX8 and coding base thereof
Because regulating and controlling the disease resistance of plant.
Accompanying drawing explanation
Fig. 1 is the Semiquatitative RT-PCR assay result of transfer-gen plant.Wherein, swimming lane 1-4 is respectively transfer-gen plant first
Four strains, swimming lane 5-8 is respectively four strains of transfer-gen plant fourth, and Marker represents DNA molecular amount standard.
Fig. 2 is the disease resistance testing result of each transgenic paddy rice.Wherein, tAPX8OX represents transgenic paddy rice first,
TAPX8Ri represents transgenic paddy rice second, and TP309-CK represents rice varieties " TP309 ".
Detailed description of the invention
Below in conjunction with detailed description of the invention, the present invention is further described in detail, the embodiment be given only for
Illustrate the present invention rather than in order to limit the scope of the present invention.
Experimental technique in following embodiment, if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, the most commercially obtain.
Leaf spot bacteria Philippiiies race PXO99 in following embodiment, for document, (bacterial blight of rice quantity resists
Property location, seat and Race specificity thereof. Acta Agronomica Sinica, 2006,32 (11): 1611-1617)) in Philippine
Microspecies P6, after the Chinese Academy of Agricultural Sciences crop science institute Zhou Yongli agrees to, the public can obtain at applicant, should
Biomaterial only attach most importance to duplicate invention related experiment used by, can not use as other purposes.
Embodiment 1, OsAPX8 can be with the disease resistances of adjusting and controlling rice
The invention provides the disease-resistance-related protein deriving from rice varieties " TP309 ", its entitled OsAPX8.
The aminoacid sequence of OsAPX8 as shown in sequence 2 in sequence table, the genome sequence of OsAPX8 encoding gene such as sequence 3
Shown in, the coded sequence of OsAPX8 such as sequence 1 institute, OsAPX8 gene cDNA sequence as shown in sequence 4 in sequence table,
The 31-1467 position of sequence 4 is identical with sequence 1.
One, the structure of transgenic paddy rice
1, the structure of recombinant vector
DNA fragmentation between EcoRI and the XbaI recognition sequence of pCAMBIA1300-35S is replaced with sequence 4 institute
The DNA molecular shown, other sequences keeping carrier are constant, obtain recombinant vector, by named for this recombinant vector
pCAMBIA1300-35S-OsAPX8.PCAMBIA1300-35S-OsAPX8 can disease-resistant relevant shown in expressed sequence 2
Albumen OsAPX8.
Wherein, pCAMBIA1300-35S is prepared as follows: utilize BstXI and KpnI restriction endonuclease to be carried by double base
Body pEZR-LN (being given by Glasgow university of Britain John professor Christie) (The Plant Cell, 2003,
Vol.15,1781 1794) on the 1692bp fragment containing 35S promoter replace the BstXI of pCAMBIA1300
And the 268bp fragment between KpnI, it is built into containing EcoRI-KpnI-SmaI-BamHI-XbaI multiple clone site
PCAMBIA1300-35S carrier.Wherein, pCAMBIA1300 is cambia product.
Total serum IgE the reverse transcription of extracting rice varieties " TP309 " are cDNA, with cDNA as template, form with F2 and R2
Primer to carry out PCR amplification (F2:5 '-CCAGCCGTCGAAGAGAAGGATCC-3 '；R2:5 '-
CACATGCCCATCCTTTTACCTC-3 '), the pcr amplification product APX of 492bp that will obtain492, it is cloned into
pENTRTM/SD/Carrier (Thermo Fischer Scient Inc. (Thermo Fisher Scientific) product),
By the named pENTR-OsAPX8i of recombinant vector correct for sequence；With LR Clonase II Plus Enzyme Mix,
By pENTR-OsAPX8i plasmid and pANDA carrier (Proc Natl Acad Sci U S A 103,10503-10508
(2006)) restructuring, by the named pANDA-OsAPX8i of recombinant vector correct for sequence.The LB of pANDA-OsAPX8i with
Structure between RB is as follows: Ubiquitin promoter-reverse APX492-gus linker-forward APX492-NOS terminator,
Other sequences of pANDA-OsAPX8i are identical with pANDA.
PCAMBIA1300-35S-OsAPX8 is imported agrobacterium strains EHA105 (Plant Molecular
Biology, 2003,52:957-966) in, the named E-pCAMBIA1300-35S-OsAPX8 of recombinant bacterium that will obtain,
PCAMBIA1300-35S is imported in agrobacterium strains EHA105, by named for the recombinant bacterium obtained
E-pCAMBIA1300-35S；PANDA-OsAPX8i is imported in agrobacterium strains EHA105, the recombinant bacterium life that will obtain
Entitled E-pANDA-OsAPX8i, imports in agrobacterium strains EHA105 by pANDA, by named for the recombinant bacterium obtained
E-pANDA。
2, the structure of transgenic paddy rice
Step 1 is utilized by agriculture bacillus mediated transgenic method (Kumar et al., 2005)
The mature embryo callus of E-pCAMBIA1300-35S-OsAPX8 rice transformation kind " TP309 ", uses 50mg/L
Hygromycin selection resistant calli, be then passed through pre-differentiation, break up, take root after obtain T0In generation, turns OsAPX8 gene
Plant, by its named T0For transgenic paddy rice first.Utilize E-pCAMBIA1300-35S rice transformation kind " TP309 ",
Obtain T0In generation, turns empty carrier Oryza sativa L., by its named T0For transgenic paddy rice third, as comparison.
Step 1 is utilized by agriculture bacillus mediated transgenic method (Kumar et al., 2005)
The mature embryo callus of E-pANDA-OsAPX8i rice transformation kind " TP309 ", uses 50mg/L hygromycin sieve
Select resistant calli, be then passed through pre-differentiation, break up, take root after obtain T0For OsAPX8 gene RNAi plant,
By its named T0For transgenic paddy rice second.Utilize pANDA rice transformation kind " TP309 ", obtain T0In generation, turns zero load
Body Oryza sativa L., by its named T0For transgenic paddy rice fourth, as comparison.
Utilize primer in hygromycin gene to (Hyg_F:5 '-GACGGTGTCGTCCATCACAGTTT-3 ' with
Hyg_R:5 '-ACTCACCGCGACGTCTGTCGAGAA-3 ') to T0For transgenic paddy rice first, T0For transgenic paddy rice
Second, T0For transgenic paddy rice third and T0The qualification in genomic level is carried out, by rice varieties for transgenic paddy rice fourth
" TP309 " shows as negative control, result, T0For transgenic paddy rice first, T0For transgenic paddy rice second, T0In generation, turns
Trans-genetic hybrid rice third and T0For all containing hygromycin gene in transgenic paddy rice fourth, rice varieties " TP309 " is without containing
The carrier segments of target DNA.
Extract T0For transfer-gen plant first, T0For transfer-gen plant second, T0For transfer-gen plant third, T0Plant for transgenic
The blade total serum IgE of strain fourth and rice varieties " TP309 " carries out semi-quantitative RT-PCR analysis, and primer is
F:5 '-gctgcgaaatactcctacg-3 ' and R:5 '-AGAGGAGGTCATCAGACCATCG-3 ', uses Actin
As internal reference.Result shows, the OsAPX8 in transfer-gen plant third, transfer-gen plant fourth and rice varieties " TP309 "
The expression of gene is significantly higher than rice varieties without significant difference, the expression in transfer-gen plant first of the OsAPX8 gene
" TP309 " (Fig. 1), is nearly no detectable the expression (Fig. 1) of OsAPX8 gene, turns base in transfer-gen plant second
Because in plant second, the expression of OsAPX8 gene is suppressed.
Two, transgenic paddy rice disease resistance detection
Infect with leaf spot bacteria Philippiiies race PXO99 and normally cultivate the T to tillering stage0For transfer-gen plant first, T0Generation
Transfer-gen plant second, T0For transfer-gen plant third, T0For transfer-gen plant fourth and rice varieties " TP309 ", detect each water
The disease resistance of rice, in triplicate, concrete grammar is as follows in experiment:
By preserve-70 DEG C bacterial leaf spot bacterium Philippiiies race PXO99 strain in PSA culture medium (Rhizoma Solani tuber osi 300g/L,
Ca(NO3)2·4H2O 0.5g/L, Na2HPO4·12H2O 2.0g/L, sucrose 15g/L, agar powder 15g/L) on
Rejuvenation, standby in being stored in 4 DEG C of refrigerators.First 2 days of inoculation on PSA plating medium streak culture, 28 DEG C of cultivations
72h, treats the dense and uniform of antibacterial length, and with pure water eluting thalline, regulation concentration is to 109Individual cell/ml inoculates.
Concrete inoculation method is with reference to the bacterium method that the connects artificial leaf-cutting inoculation of (1973) such as Kauffman, every kind of Oryza sativa L. inoculation 3-5
Individual blade, each blade deducts top 3-5cm, investigates when scab length is obvious and stablizes, often after inoculating two weeks
One plant measures 3-5 blade.
Result (Fig. 2) shows, T0For transfer-gen plant third, T0For transfer-gen plant fourth and rice varieties " TP309 "
Scab length without significant difference, a length of 1.5 ± 0.3cm of scab of rice varieties " TP309 "；T0For transgenic
A length of 0.5 ± the 0.3cm of scab of plant first, considerably shorter than the scab length of rice varieties " TP309 "；T0In generation, turns
A length of 2.3 ± the 0.2cm of scab of gene plant second, considerably longer than the scab length of rice varieties " TP309 ".Say
Bright, the expression improving OsAPX8 gene can improve the resistance of the bacterial leaf-blight that leaf spot bacteria is caused by Oryza sativa L.,
The expression reducing OsAPX8 gene can reduce the resistance of the bacterial leaf-blight that leaf spot bacteria is caused by Oryza sativa L., table
Bright, OsAPX8 gene can be with the adjusting and controlling rice resistance to bacterial leaf-blight.
Claims (10)
1. disease-resistance-related protein application in regulation and control disease resistance of plant；Described disease-resistance-related protein is following A1), A2)
Or A3):
A1) aminoacid sequence is the protein of sequence 2；
A2) by shown in sequence in sequence table 2 aminoacid sequence through one or several amino acid residue replacement and/
Or lack and/or add and the protein relevant to plant disease-resistant；
A3) at A1) or N end A2) or/and C end connects the fused protein that label obtains.
2. the biomaterial relevant to disease-resistance-related protein described in claim 1 answering in regulation and control disease resistance of plant
With；Described biomaterial is following B1) to B16) in any one:
B1) nucleic acid molecules of disease-resistance-related protein described in coding claim 1；
B2) containing B1) expression cassette of described nucleic acid molecules；
B3) containing B1) recombinant vector of described nucleic acid molecules；
B4) containing B2) recombinant vector of described expression cassette；
B5) containing B1) recombinant microorganism of described nucleic acid molecules；
B6) containing B2) recombinant microorganism of described expression cassette；
B7) containing B3) recombinant microorganism of described recombinant vector；
B8) containing B4) recombinant microorganism of described recombinant vector；
B9) containing B1) the transgenic plant cells system of described nucleic acid molecules；
B10) containing B2) the transgenic plant cells system of described expression cassette；
B11) containing B1) Transgenic plant tissue of described nucleic acid molecules；
B12) containing B2) Transgenic plant tissue of described expression cassette；
B13) containing B1) the transgenic plant organ of described nucleic acid molecules；
B14) containing B2) the transgenic plant organ of described expression cassette；
B15) nucleic acid molecules that described in claim 1, disease-resistance-related protein is expressed is reduced；
B16) containing B15) expression cassette of described nucleic acid molecules, recombinant vector, recombinant microorganism or transgenic plant thin
Born of the same parents are.
Apply the most according to claim 2, it is characterised in that: B1) described nucleic acid molecules is following b1)-b5)
In any one:
B1) the cDNA molecule of sequence 4 or DNA molecular during coded sequence is sequence table；
B2) the cDNA molecule of sequence 1 or DNA molecular during nucleotide sequence is sequence table；
B3) DNA molecular of sequence 3 during nucleotide sequence is sequence table；
B4) and b1) or b2) or b3) nucleotide sequence that limits there is 75% or more than 75% homogeneity, and coding
The cDNA molecule of disease-resistance-related protein described in claim 1 or genomic DNA molecule；
B5) under strict conditions with b1) b2) or b3) nucleotide sequence hybridization that limits, and coding right wants
Ask cDNA molecule or the genomic DNA molecule of disease-resistance-related protein described in 1；
B15) described nucleic acid molecules is and the DNA molecular of arbitrary fragment reverse complemental in the DNA molecular shown in sequence 4.
4. biomaterial described in disease-resistance-related protein described in claim 1 or Claims 2 or 3 is following arbitrary
Application:
H1, the application being increased or decreased in plant in cultivation disease resistance；
H2, improve in preparation or reduce the application in disease resistance of plant product.
The most following G1 or G2:
Biomaterial described in disease-resistance-related protein described in G1, claim 1 or Claims 2 or 3；
G2, regulation and control disease resistance of plant product, containing disease-resistance-related protein described in claim 1 or claim 2 or
Biomaterial described in 3.
The most following M1) or M2):
M1) method cultivating disease resistant transgenic plants, including importing following 1 in recipient plant) or 2) turned
Gene plant；
1) encoding gene of disease-resistance-related protein described in claim 1；
2) expression cassette of the encoding gene containing disease-resistance-related protein described in claim 1；
Described transgenic plant disease resistance compared with described recipient plant increases；
M2) method cultivating the transgenic plant that disease resistance reduces, including institute in claim 1 in reduction purpose plant
State the expression of the encoding gene of disease-resistance-related protein, obtain the disease resistance transgenic plant less than described purpose plant.
Method the most according to claim 6, it is characterised in that: in reduction purpose plant described in claim 1
The expression of the encoding gene of disease-resistance-related protein is by by B15 in Claims 2 or 3) importing of described nucleic acid molecules
Described purpose plant realizes；
The encoding gene of disease-resistance-related protein described in claim 1 is B1 in Claims 2 or 3) described nucleic acid divides
Son；Described expression cassette is B2 in Claims 2 or 3) described expression cassette.
8. according to product or claim 6 or 7 described in described application arbitrary in claim 1-4, claim 5
Described method, it is characterised in that: in claim 1-5, arbitrary described plant is monocotyledon or dicotyledon；
Purpose plant described in claim 6 or 7 and described recipient plant are monocotyledon or dicotyledon.
Application, described product or described method the most according to claim 8, it is characterised in that: described unifacial leaf is planted
Thing is grass.
10. according to described application arbitrary in claim 1-4, product described in claim 5, claim 6 or 7
Described method, or application, described product or described method described in claim 8 or 9, it is characterised in that:
Described disease resistance is bacterial blight-resisting, and/or, described bacterial leaf-blight is caused by leaf spot bacteria.
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| CN106636183A (en) * | 2016-11-07 | 2017-05-10 | 中国科学院遗传与发育生物学研究所 | Application of TBL1 protein in regulation and control of plant acetylation level and prevention and treatment of bacterial leaf blight of rice |
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| CN111560056B (en) * | 2020-05-06 | 2022-04-19 | 中国农业科学院植物保护研究所 | Wheat stripe rust resistance related protein TaERF8 and coding gene and application thereof |
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