CN109206493A - Application of the Zm-Remorin gene in the prevention and treatment of corn southern rust - Google Patents

Application of the Zm-Remorin gene in the prevention and treatment of corn southern rust Download PDF

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CN109206493A
CN109206493A CN201811124032.7A CN201811124032A CN109206493A CN 109206493 A CN109206493 A CN 109206493A CN 201811124032 A CN201811124032 A CN 201811124032A CN 109206493 A CN109206493 A CN 109206493A
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remorin
plant
disease
sequence
albumen
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吴刘记
陈赞
王顺喜
张君
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Henan Agricultural University
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    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8282—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for fungal resistance

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Abstract

The invention discloses application of the Zm-Remorin gene in the prevention and treatment of corn southern rust.The present invention provides following 1) -3) in application of any substance in regulation disease resistance of plant；1) albumen Zm-Remorin；2) DNA molecular of albumen Zm-Remorin is encoded；3) recombinant vector, expression cassette, transgenic cell line or the recombinant bacterium of the DNA molecular containing coding albumen Zm-Remorin；The experiment proves that expression of the Zm-Remorin in plant can significantly improve the resistance to southern rust.

Description

Application of the Zm-Remorin gene in the prevention and treatment of corn southern rust
Technical field
The invention belongs to field of biotechnology more particularly to a kind of Zm-Remorin gene in the prevention and treatment of corn southern rust Application.
Background technique
Corn is one of the cereal crops cultivated the most extensively in the world.On the one hand, corn provides for human and animal A large amount of food source, but then, maize diseases can lead to huge economic loss.Especially corn in recent years Disease generation is increasingly frequent, and disease species are increasing, seriously affected the development of Maize Industry.The use of chemical pesticide is one Determine to improve the control action to maize diseases in degree, but the use of pesticide not only polluted environment, long-time service can also Make corn generate drug resistance, meanwhile, caused by medicament residue also have potential threat to the life security of the mankind.Therefore, right In the disease-resistant research of corn, people, which are dedicated to finding neither, influences the method that environment again can effectively prevent maize diseases.
Corn southern rust is a kind of gas transmissibility as caused by Puccinia polysora (Puccinia polysora Underw.) Disease.Main harm blade and leaf sheath (can also infect bract), aggrieved blade two sides generate a large amount of sorus, influence blade light Cooperation is used and causes blade withered, and then reduces corn yield.For the current biotic for threatening maize production, using suitable Molecular biology method, carry out corn anti-disease mechanism research, excavate and identify the important gene in corn disease resistance response, it is right The disease-resistant research of corn and genetic breeding have important theory significance and more practical value.
Summary of the invention
A purpose of the invention is to provide following 1) -3) in any substance purposes.
Application of any substance in regulation disease resistance of plant in following 1) -3) provided by the invention；
1) albumen Zm-Remorin；
2) DNA molecular of albumen Zm-Remorin is encoded；
3) recombinant vector, expression cassette, transgenic cell line or the recombination of the DNA molecular containing coding albumen Zm-Remorin Bacterium；
The albumen Zm-Remorin is following (1) or (2):
(1) protein that the amino acid sequence shown in sequence 2 in sequence table forms；
(2) by amino acid sequence shown in sequence 2 in sequence table by one or several amino acid residues substitution and/or Deletion and/or addition and the protein with the same function as derived from (1).
In above-mentioned application, the DNA molecular is following 1) -4) in any DNA molecular:
1) code area is DNA molecular shown in sequence 1 in sequence table；
2) code area is DNA molecular shown in sequence 3 in sequence table；
1) or 2) 3) hybridize under strict conditions with the DNA sequence dna limited and encode the DNA with identical function protein Molecule；
1) or 2) 4) at least have 70% with the DNA sequence dna limited, at least have 75%, at least having with 80%, at least Have 85%, at least have with 90%, at least with 95%, at least with 96%, at least with 97%, at least 98% or at least With 99% homology and coding has the DNA molecular of identical function protein.
In above-mentioned application, the regulation disease resistance of plant is to improve disease resistance of plant.
In above-mentioned application, the disease is southern rust；
Or the pathogen of the disease is Puccinia polysora (Puccinia polysora Underw.)；
Or the plant is dicotyledon or monocotyledon；
Or the plant is monocotyledon, the monocotyledon is specially corn；
Or the plant is dicotyledon, the monocotyledon is specially arabidopsis.
Above-mentioned 1) -3) application of any substance in controlling plant diseases is also the scope of protection of the invention in；
Or, above-mentioned 1) -3) in any substance cultivating the application in disease-resistant plants be also the scope of protection of the invention；
Or, above-mentioned 1) -3) in any substance cultivating the application in high disease-resistant plants be also the model that the present invention protects It encloses.
In above-mentioned application, the disease is southern rust；
Or the pathogen of the disease is Puccinia polysora (Puccinia polysora Underw.)；
Or the plant is dicotyledon or monocotyledon.
Or the plant is monocotyledon, the monocotyledon is specially corn；
Or the plant is dicotyledon, the monocotyledon is specially arabidopsis.
Another object of the present invention is to provide a kind of method for cultivating high disease resistant transgenic plants.
Method provided by the invention, includes the following steps:
The expression quantity and/or activity for encoding the DNA molecular of albumen Zm-Remorin in purpose plant are improved, transgenosis is obtained Plant,
Or, improving albumen Zm-Remorin activity in purpose plant, genetically modified plants are obtained,
The disease resistance of the genetically modified plants is higher than the purpose plant；
The albumen Zm-Remorin is following (1) or (2):
(1) protein that the amino acid sequence shown in sequence 2 in sequence table forms；
(2) by amino acid sequence shown in sequence 2 in sequence table by one or several amino acid residues substitution and/or Deletion and/or addition and the protein with the same function as derived from (1).
In the above method, it is described improve purpose plant in encode albumen Zm-Remorin DNA molecular expression quantity and/or Activity is that the DNA molecular of the coding albumen Zm-Remorin is imported purpose plant.
In the above method, the disease is southern rust；
Or the pathogen of the disease is Puccinia polysora (Puccinia polysora Underw.).
In the above method, the plant is dicotyledon or monocotyledon.
Or the plant is monocotyledon, the monocotyledon is specially corn；
Or the plant is dicotyledon, the monocotyledon is specially arabidopsis.
The experiment proves that the present invention is divided by the Disease Resistance Identification to Zm-Remorin transgenic line with expression Analysis, the study found that the Lesion number of Zm-Remorin transgenic positive strain, size are all significantly less than yin after being inoculated with rust Property strain, analyzed in conjunction with real-time fluorescence quantitative PCR, Zm-Remorin can stablize great expression in transgenic positive strain, It is significantly higher than negative strain, it is found that expression of the Zm-Remorin in plant can be significantly improved to southern rust Resistance.
Detailed description of the invention
Fig. 1 is subcellular localization of the Zm-Remorin in arabidopsis as a result, each icon ruler is 50 μm.
Fig. 2 is Bar gene PCR testing result.
Fig. 3 is transgenic positive strain 231,233 phenotypic evaluations.
Fig. 4 is to turn area under Zm-Remorin corn strain 231,233 disease progress curves.
Fig. 5 is Zm-Remorin in transgenic positive strain 231,233 expression analysis.
Specific embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Corn inbred line B73 is the preservation of this laboratory in following embodiments, and bacillus coli DH 5 alpha is the preservation of this laboratory, RNA extraction reagent TRIZOL Reagent, reverse transcription reagent box, Ago-Gel DNA QIAquick Gel Extraction Kit, carrier T PMD18-T, Taq enzyme, plasmid extraction kit, restriction enzyme, T4 ligase, DEPC water are purchased from precious bioengineering Co., Ltd, survey Sequence and primer synthesis are carried out in the raw work of Hua Da and Shanghai respectively.
The clone of embodiment 1, Zm-Remorin gene
1, the extraction of plant total serum IgE
The RNA of blade when extracting tetra- leaf of corn inbred line B73 wholeheartedly.
2, DNA of plants extracts
The genomic DNA of blade when extracting tetra- leaf of corn inbred line B73 wholeheartedly.
3, the synthesis of the first chain of cDNA
The RNA that above-mentioned 1 obtains is illustrated that configuration reverse transcription system carries out the conjunction of the first chain of cDNA according to reverse transcription reagent box At obtaining cDNA.
Reverse transcription is as follows:
RNA 6μl
Oligo(DT)1μl
4 μ l of DEPC water
65 DEG C warm bath 5 minutes, then on ice place 2 minutes, add following reagent:
RNase inhibitor 1μl
M-mlv buffer 4μl
DNTP(25nml)3μl
1 μ l of M-mlv reverse transcriptase
It is as follows that reverse transcription PCR program is set,
42℃ 1h
72℃ 10min
4℃ forever
After be stored in -20 DEG C.
4, the clone of Zm-Remorin
1) clone of cDNA
Design primer is as follows:
Zm-Remorin F1:5'AAAACCGGTTGCTATTAGTAC 3'；
Zm-Remorin R1:5'GAAGAAAACAAAATGTCTGC 3'；Target fragment length 971bp.
The cDNA obtained with above-mentioned 3 is template, carries out PCR expansion with Zm-Remorin F1 and Zm-Remorin F1 primer Increase, obtains pcr amplification product.
Above-mentioned PCR amplification system is as follows:
Above-mentioned PCR response procedures are as follows:
Above-mentioned pcr amplification product is connected on PMD 18-T and is sequenced, by sequencing, which has sequence 1 Shown in gene, which is Zm-Remorin, and the ORF sequence of the gene is sequence 1, and the albumen of gene coding is sequence Sequence 2 in list, the albumen are named as Zm-Remorin.
2) clone of DNA
It is as follows to design Zm-Remorin DNA cloning primer:
Zm-Remorin R2:5'GTGTTGAACGAAACGAACGTTGC 3'；
Zm-Remorin F2:5'GTCATCAAATAACTGCTAGCAT 3', target fragment 1586bp.
Zm-Remorin R3:5'ACGACGTGGCCATCGTAGGTT 3'；
Zm-Remorin F3:5'CTTTGGTTGTGCATAAGCAGGCT 3'.Target fragment 1443bp.
Zm-Remorin R4:5'TGCTGTTATCTGGTTTATCT 3'；
Zm-Remorin F4:5'GATAGAGCGAGAGGACCAAGCA 3'.Target fragment 2054bp.
The DNA obtained respectively using above-mentioned 2 carries out PCR amplification as template, with above-mentioned 3 pairs of primers, obtains 3 kinds of PCR amplifications and produces Object.By above-mentioned 3 kinds of pcr amplification product amplified productions through connection, conversion, sequencing, splicing, the Zm-Remorin of 4140bp is obtained DNA sequence dna (sequence 3), by with its cDNA sequence compare, discovery Zm-Remorin gene DNA sequence contain 5 exons With 4 intrones.
Two, the subcellular localization of Zm-Remorin
B73 corn seed, arabidopsis seed, vector plasmid pCAMBIA 1304 are the preservation of this laboratory.Plasmid extracts examination Agent box, restriction enzyme NcoI, SpeI, T4DNA ligase, PCR related reagent etc..Laser Scanning Confocal Microscope, centrifuge, liquid relief Device, shaking table, gel imaging system, culture dish, glass slide, coverslip, tweezers etc..
1, the building of GFP fusion expression vector
GFP fusion expression vector pCAMBIA 1304-Zm-Remorin-GFP is by Zm- shown in sequence 1 in sequence table Remorin gene replacement carrier pCAMBIA-1304 (is recorded in the following literature: " Ruchi Pandey, Avinash Mishra,G.K.Garg.Plant promoter driven heterologous expression of HMW glutenin gene(s)subunit in E.coli.Mol Biol Rep(2008)35:153–162."；The public can be from Agricultural University Of He'nan It obtains；Have GFP on the carrier) NcoI and SpeI double enzyme site between carrier
Above-mentioned pCAMBIA 1304-Zm-Remorin-GFP is transferred in Agrobacterium GV3101, recombinational agrobacterium is obtained GV3101/pCAMBIA 1304-Zm-Remorin-GFP。
Empty carrier pCAMBIA 1304 is transferred in Agrobacterium GV3101, recombinational agrobacterium GV3101/ is obtained pCAMBIA1304。
2, the conversion of arabidopsis
1) respectively by GV3101/pCAMBIA 1304-Zm-Remorin-GFP and recombinational agrobacterium GV3101/pCAMBIA In the 1304 YEB fluid nutrient mediums (kanamycins concentration is 50 μ g/ml) for being inoculated in that resistance of 500ml card (while being inoculated with several Difference clone), 28 DEG C shaken cultivation 6-12 hours (OD600=0.8-1.0).
2) 4000rpm/min, room temperature are centrifuged 5 minutes, collect thallus.
3) 200ml MS salting liquid suspension thalline is used.
4) it is watered with water on the day before mentioning the wildtype Arabidopsis thaliana of full-bloom stage (col-0), arabidopsis floral is immersed in Agrobacterium Suspension in one minute or so, put on moisturizing one day with freshness protection package.Plant was taken out from freshness protection package in second day, dark is placed It is put back to after one day on illumination cultivation frame, normal growth to harvest 0 generation of mature T turns Zm-Remorin arabidopsis seed and T0 generation turns sky Carrier arabidopsis seed.
3, the screening of transgenic arabidopsis positive plant
1) resistance screening of transgenic arabidopsis
According to the experiment that laboratory is previous, 30mg/ml be hygromycin arabidopsis screening most suitable concentration, therefore just with Concentration of the hygromycin of 30mg/ml as arabidopsis resistance screening.
In T0 generation after drying, is turned into Zm-Remorin arabidopsis seed, it is flat to be seeded in the 1/2MS containing 30mg/ml hygromycin In plate culture medium, until arabidopsis it is long to 6 leaves or so when, can normal growth be to turn Zm- in resistance screening positive T0 generation Remorin arabidopsis seed will grow normal arabidopsis and move to normal growth in compost.
2) the PCR detection of transgenic arabidopsis
In resistance screening positive T0 generation, turns Zm-Remorin arabidopsis and moves in Nutrition Soil when growing to 14 leaves or so, extracts Arabidopsis thaliana genomic dna carries out PCR detection.
Above-mentioned PCR detection the primer is as follows:
18S R:5'CCTGCGGCTTAATTGACTC 3'；
18S F:5'GTTAGCAGGCTGAGGTCTCG 3'
Pcr amplification product is detected, purpose product segment 174bp is to turn Zm-Remorin arabidopsis in positive T0 generation.
In above-mentioned transformant per generation, will be subjected to resistance screening and PCR detection, positive strain plant division harvest turns until obtaining T3 Zm-Remorin arabidopsis seed carries out follow-up test.
4, subcellular localization of the Zm-Remorin in arabidopsis
In T3 generation, is turned into Zm-Remorin arabidopsis referring to above-mentioned arabidopsis implantation methods and T3 turns the plantation of empty carrier arabidopsis In the 1/2MS plating medium containing hygromycin resistance, after being protected from light 4 DEG C of vernalization 3 days, are moved under light (21 DEG C) one weeks of growth When about 6mm or so (root growth to), the tip of a root of clip transformant is placed on the centre of glass slide, drips clear water, covers Coverslip after being pressed lightly on thumb, moves under Laser Scanning Confocal Microscope, observes position of the green fluorescent protein in arabidopsis cell It sets.
As a result as shown in Figure 1, A, B, C are the fluorescence results for turning Zm-Remorin arabidopsis root in T3 generation；D, E, F are that T3 turns sky The fluorescence results of carrier arabidopsis root；A, D is the arabidopsis root cells picture under fluorescence signal；B, E is quasi- under light field signal Southern mustard root cells picture；C, F is that A, B close the superimposed photo of D, E；It can be seen from the figure that T3 turns the empty carrier arabidopsis tip of a root There is fluorescence signal in nucleus, cell membrane and cytoplasm, and in T3 generation, turns the arabidopsis tip of a root of Zm-Remorin arabidopsis only Fluorescence signal is detected in cell membrane, illustrates that Zm-Remorin albumen is a film positioning protein matter.
The application of embodiment 2, Zm-Remorin in the prevention and treatment of corn southern rust
1, the building of Zm-Remorin over-express vector
Zm-Remorin over-express vector pCAMBIA 3301-Zm-Remorin is by Zm-Remorin shown in sequence 1 3301 carrier of gene replacement pCAMBIA (is recorded in the following literature: " Production of purple-colored creeping bentgrass using maize transcription factor genes Pl and Lcthrough It is disclosed in Agrobacterium-mediated transformation.Plant Cell Rep (2009) 28:397-406 " Cross, the public can obtain from Agricultural University Of He'nan) in II restriction enzyme site of NcoI restriction enzyme site and Bgl between DNA molecular, obtain Carrier.
Above-mentioned pCAMBIA 3301-Zm-Remorin is transferred in Agrobacterium GV3101, recombinational agrobacterium GV3101/ is obtained pCAMBIA 3301-Zm-Remorin。
Empty carrier pCAMBIA 3301 is transferred in Agrobacterium GV3101, recombinational agrobacterium GV3101/ is obtained pCAMBIA3301。
2, turn the acquisition of Zm-Remorin corn
Above-mentioned GV3101/pCAMBIA 3301-Zm-Remorin is conducted into corn with the method that Agrobacterium is infected II (hereinafter also referred to wild-type corn of kind Hi；Corn variety Hi II is in document " Biolistic gun-mediated maize genetic transformation.Methods Mol Biol.(2009)；It is disclosed in 526:29-45 ", the public Can be obtained from Agricultural University Of He'nan) callus of IMMATURE EMBRYOS CULTURE, it is subsequent to carry out screening and regeneration induction (the method bibliography " the foundation of the good inbred lines genetic transformation body of mediated by agriculture bacillus.Agricultural University Of Shenyang's journal, 2002-06,33 (3): 195-199 "), in acquisition T0 generation, turns Zm-Remorin corn.
Recombinational agrobacterium GV3101/pCAMBIA 3301 is transferred in wild-type corn using same method, obtains T0 In generation, turns empty carrier corn.
3, turn the identification of Zm-Remorin corn
1) herbicide screening (PPT:200mg/L)
Non-transgenic corn seedling is smeared with the PPT of various concentration first, determines suitable smearing concentration；Choose second newly Raw maize leaf, and smear in blade tip the PPT of various concentration simultaneously in tow sides, concentration used be followed successively by 50mg/L, 100mg/L,200mg/L,300mg/L,400mg/L.After a week, blade tip phenotype is observed.By repeating three times, discovery is dense when smearing When degree is 200mg/L, blade tip starts obviously withered, and when 400mg/L, blade tip is substantially all withered.So final choice 200mg/L PPT smear identification transgenic seedling.Then, Zm-Remorin maize seedling is turned to T0 generation and carries out smearing identification, carried out after a week Phenotypic Observation, blade tip turn Zm-Remorin without obviously withered as Herbicid resistant seedling, as herbicide screening positive T0 generation Corn.
2) reporter gene Bar gene PCR
When herbicide screening positive T0 generation turning Zm-Remorin plant and growing to 4 leaf, blade genome is extracted DNA is expanded with following primer:
BarF8:TGACGCACAATCCCACTATCCTTC
BarR8:CCAGAAACCCACGTCATGCCAGT
As a result as shown in Fig. 2, swimming lane is the not homophyletic for turning Zm-Remorin plant in herbicide screening positive T0 generation System, the first two swimming lane do not detect that Bar gene, rear ten swimming lanes detected Bar gene, it can be seen that in addition to 243,241 Outside strain, other strains are to turn Zm-Remorin plant in positive T0 generation.
Sowing, per generation carries out herbicide screening and Bar gene PCR screening, select two kinds screening be the positive as Next time, selfing was maternal, obtained that T2 generation turns Zm-Remorin plant and T2 generation turns empty carrier corn.
4, Resistance Identification of the transgenic corns to southern rust
For trying southern rust pathogen: Puccinia polysora (Puccinia polysora Underw.)
1) preparation of spore suspension
Puccinia polysora (Puccinia polysora Underw.) spore is collected from the maize leaf with pathogen Son, being configured to concentration with distilled water is 1 × 105The spore suspension (3-4 drop Tween-20 is added in every 100ml) of a/ml, for use.
2) plantation of transgenic corn plant
In T2 generation, is turned into Zm-Remorin corn strain 231, T2 generation turns Zm-Remorin corn strain 233, T2 generation turns zero load In body corn 231sib, T2 generation, turns empty carrier corn 233sib and wild-type corn plantation in greenhouse, when the corn 4-5 leaf phase, Prepare inoculation.40 plants of each strain.
3) it is inoculated with
Firstly, 2) the T2 generation of 4-5 leaf phase in, is turned Zm-Remorin corn strain 231, T2 generation turns Zm-Remorin corn Strain 233, T2 generation turn empty carrier corn 231sib, T2 generation and turn empty carrier corn 233sib and wild-type corn blade with dissolved with spitting Distilled water (3-4 drop Tween-20 is added in every 100ml) wetting of temperature -20, then by 1) prepared spore suspension pressure Sprayer is uniformly sprayed on maize leaf, last bagging dark moisturizing for 24 hours, to plant after the onset of, be sampled, survey nature Shape.
Inoculation 7 days after, carry out phenotypic evaluation, as a result fig. 3, it is shown that T2 generation turn empty carrier corn 231sib, In T2 generation, turns occur a large amount of scab on empty carrier corn 233sib integrated plate blade, and produces sorus on scab surface, And in T2 generation, turns Zm-Remorin corn strain 231,233 and produces a small amount of lesser sorus at blade tip position, shows Zm- The anti-southern rust of Remorin.
In wild-type corn and T2 generation, turn empty carrier corn result without significant difference.
In order to preferably react the resistance difference of transgenic positive strain and wild-type corn to corn to southern rust, count It lets it pass area (AUDPC) under the 8th day to the 14th day disease progress curve of inoculation, calculation formula isTo calculate disease severity, yiIndicate the disease severity in time i, ti+1-tiIndicate that the number of days being spaced between evaluation twice, n refer to the number of evaluation.
The infection rank of southern rust is divided into seven ranks according to disease occurrence degree, specific division is as follows:
1 grade: highly resistance: the area infected on blade by sorus is less than 1%
2-3 grades: disease-resistant: the area infected on blade by sorus is less than 2%~20%
4 grades: in resist: on blade by sorus infect area be less than 21%~35%
5 grades: osculant: the area infected on blade by sorus is less than 36%~50%
6 grades: micro- sense: the area infected on blade by sorus is less than 51%~65%
7-8 grades: susceptible: the area infected on blade by sorus is less than 66%~80%
9 grades: height sense: the area infected on blade by sorus is 80% or more
As a result as shown in Figure 4, it can be seen that T2 generation turns under Zm-Remorin corn strain 231,233 disease progress curves Area be far smaller than T2 generation turn empty carrier corn 231sib, 233sib；Show the anti-southern rust of Zm-Remorin.
5, expression analysis of the Zm-Remorin in transgenic corns
Experimental material: RNA extraction reagent TRIZOL Reagent, reverse transcription reagent box, I fluorescent dye of SYBR GREEN, Quantitative fluorescent PCR pipe, LightCycler 480II fluorescence quantitative PCR instrument, design of primers use 5.0 software of Primer, data Processing and mapping use Excel software etc..
Sample: in T2 generation of the inoculation southern rust pathogen spore after 7 days, turns Zm-Remorin corn strain 231 in above-mentioned 4 And in 233 and T2 generation, turns empty carrier corn 231sib, T2 for the blade for turning empty carrier corn 233sib；
Experimental method:
In T2 generation of the extraction inoculation southern rust pathogen spore after 7 days, turns Zm-Remorin corn strain 231 and 233 and T2 In generation, turns the RNA for turning the blade of empty carrier corn 233sib in empty carrier corn 231sib, T2 generation, and reverse transcription obtains cDNA as mould Plate carries out fluorescent quantitative PCR with following primer Zm-Remorin R9 and Zm-Remorin F9.
It is as follows according to the cDNA sequence of Zm-Remorin and across the introne design fluorescence quantification PCR primer of DNA sequence dna:
Zm-Remorin R9 5'GATGAACCTGCTCCCG 3'；
Zm-Remorin F9 5'CTTCTATGTTTGCTTTCTTTGT 3'；Expanding fragment length 209bp.
18S is reference gene, primer 18S R and 18S F.
Real-time PCR system is as follows:
Fluorescent quantitation reaction is expanded on LightCycler 480II, and program is as follows:
Using the analysis method of relative quantification, the quantitative result of Zm-Remorin gene is analyzed.Relative quantification is For determining the differential expression between the sample object transcript Jing Guo different disposal, calculation method is as follows:
(1) △ CT calibration sample=sample-internal reference.
(2) △ CT sample to be tested=sample-internal reference.
(3) △ △ CT=△ CT sample to be tested-△ CT calibration sample.
(4) 2 are calculated-△△CTValue.
Detection takes the expression quantity of Zm-Remorin and 18S gene in every part of sample, and carries out 3 PCR and repeat, and takes 3 times The average value of CT value calculates 2 by calibration object-△△CTValue.
As a result such as Fig. 5, it can be seen that after rubiginose, it is beautiful for empty carrier is turned to turn empty carrier corn 231sib, T2 with T2 generation Rice 233sib is compared, and the expression quantity that T2 generation turns Zm-Remorin in Zm-Remorin corn strain 231 and 233 dramatically increases, about Be 650 times, this illustrate plant after rubiginose, Zm-Remorin in transgenic plant stablize, great expression.
Sequence table
<110>Agricultural University Of He'nan
<120>application of the Zm-Remorin gene in the prevention and treatment of corn southern rust
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<170> PatentIn version 3.5
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atggctgagg aggaggccaa gaaggtggag gtggaggtca ccaaggagcc cgaggcggcg 60
gcgaaggagg acgtagccga tgacaaggcc gtcatccccg cgaccgaccc gccgccgccg 120
ccgccgccgg ccgacgactc caaggccctg gccatcgtcg agaaagttgc agatgaacct 180
gctcccgcga agcctgcccc tgcgaagcaa gggggctcca atgacaggga tctcgctctt 240
gcaagggtgg aaacagagaa gaggaactct ttgatcaaag cttgggaaga gaatgagaag 300
acaaaagctg agaacaaggc tgctaagaaa gtatccgcta ttctttcatg ggaaaacaca 360
aagaaagcaa acatagaagc tgaactgaag aagattgagg aacaactgga aaagaagaag 420
gctgaatatg cagagaagat gaagaacaag gttgcaatga tacacaagga agccgaagag 480
aagcgagcga tggtggaggc aaaacgcggt gaggaggtcc tgaaggccga ggagatggct 540
gccaagtacc gggccaccgg ccacgctcct aagaagctca tcggttgctt cggagcc 597
<210> 2
<211> 199
<212> PRT
<213> 2
<400> 2
Met Ala Glu Glu Glu Ala Lys Lys Val Glu Val Glu Val Thr Lys Glu
1 5 10 15
Pro Glu Ala Ala Ala Lys Glu Asp Val Ala Asp Asp Lys Ala Val Ile
20 25 30
Pro Ala Thr Asp Pro Pro Pro Pro Pro Pro Pro Ala Asp Asp Ser Lys
35 40 45
Ala Leu Ala Ile Val Glu Lys Val Ala Asp Glu Pro Ala Pro Glu Lys
50 55 60
Pro Ala Pro Ala Lys Gln Gly Gly Ser Asn Asp Arg Asp Leu Ala Leu
65 70 75 80
Ala Arg Val Glu Thr Glu Lys Arg Asn Ser Leu Ile Lys Ala Trp Glu
85 90 95
Glu Asn Glu Lys Thr Lys Ala Glu Asn Lys Ala Ala Lys Lys Val Ser
100 105 110
Ala Ile Leu Ser Trp Glu Asn Thr Lys Lys Ala Asn Thr Glu Ala Glu
115 120 125
Leu Lys Lys Ile Glu Glu Gln Leu Glu Lys Lys Lys Ala Glu Tyr Ala
130 135 140
Glu Lys Met Lys Asn Lys Val Ala Met Ile His Lys Glu Ala Glu Glu
145 150 155 160
Lys Arg Ala Met Val Glu Ala Lys Arg Gly Glu Glu Val Leu Lys Ala
165 170 175
Glu Glu Met Ala Ala Lys Tyr Arg Ala Thr Gly His Ala Pro Lys Lys
180 185 190
Leu Ile Gly Cys Phe Gly Ala
195
<210> 3
<211> 4140
<212> DNA
<213> 3
<400> 3
atggctgagg aggaggccaa gaaggtggag gtggaggtca ccaaggagcc cgaggcggcg 60
gcgaaggagg acgtagccga tgacaaggcc gtcatccccg cgaccgaccc gccgccgccg 120
ccgccgccgg ccgacgactc caaggccctg gccatcgtcg agagtgagta cagcctgctc 180
tctgctctgc tttattaatt aattctgccc ccctctatgt agtccaaggc ttgcgatgga 240
aacatggcgc tggcggactg gtgtgtgctt tagattcatg tgccgcgaat tgggagaaac 300
tttcctcctc tctctaccct atgttttggt tcgtcgctct gtactttctg gtagctgtac 360
tgtggctggc agtactaggt acttgtgact tgtaatcctt ggcagtcggt ttgcttctcc 420
ttttcatggg ttccctgatt tgttgccggc gtgatccttt ggtgttggtt gtacttgtac 480
acccgaacgt atttggtccg attacttctc ttagacaaca aaattttctt gttagggggt 540
tgtgaggtgc aaatcacatt ggaaccttcg accaagttct aaagtactct agttctgtcg 600
taaaaaaaaa cgtactgaag taaatctgcg agtcatcgat tttcaggatt atttaccagc 660
ttcctaactg gtttccccct ttttttagag tggtagccac gcctgctaac ttgccaacta 720
ctacggacga attgcttgtc aagttccgaa agggcgcacg gggtaaaaag gttttaacat 780
ttttgggata gtcgcctcac ttcgaaaatt ggttttagga cgacgtggcc atcgtaggtt 840
tttggtttga gggttctaaa agtatgctag cagttatttg atgactaggc atgtagtatt 900
tcctttgctt gcgatttatt ttcagcgaaa atttcagatt ttgattcaga gaaaaagatg 960
gctctggaac tttcctgaat tctccacttt ctctccgcct tttgggcgtg ttcggctggc 1020
tacaagccga cactgttgca gctgtttgga ttgctgcagc tgcaatccat agagagaaaa 1080
atactgtaga agccgcagcc gcagccggat tgcagccgca gcaagccgca gcgaacaagc 1140
tgtttgtccc ttgttgcctg ctcttatcgt tcttcccttt attcaatccc tgttccctct 1200
aaaataacaa aaaaaaggcc ttagggcctg tttgtttcgg cttctggcag cttctggcca 1260
ccaaaatctg ctgcggactg ccaaacgctc agcttttcag ccagcctcta taaaattcgt 1320
tgggggcaaa aaccatccaa aatcaacata aacacataac cggttgagtc gttgtaatag 1380
taggaatccg tcactttcta gatcctaagt cctatgaata actttatctt cctccacacg 1440
taatcgtaat gatactcaga ttcttcccat agccagattc ttcctacagt cagattttca 1500
gaaaaactgg tcagaaaaaa ctgaaccaaa catgccctta gaatctgcat gtatgccgac 1560
cgtttttcta tctcagtata atgacgtgca ggcgcagctt tcgcgttcaa aaaagaataa 1620
atcctaaact ctatagaaag attcgaagtt tcatgctaag aggaaagtaa tcagacagaa 1680
acaaaggagt atgaagaaca attttgcata ctgttaagaa agttagaatt taccataaac 1740
cttattatga ttcatctgtc atttgaaatc tgtactcact aatctaaatc gaacaacaaa 1800
gcaggaaaaa aacagaattt gtcttttcta acacccaaat aattcctcca tgatttgaag 1860
aaataggaag aactgtctga tacttcacaa aaaatagaca tgccccatca cagcttgatg 1920
acaattgctt tctgttgctg ctaagtcttc aaagatgtac atcagttcct tgcataggcg 1980
ggcaaatgat gaaaccatgc gcttttgaag cattcagttt cactaaggtt atgcagatat 2040
acaaaataag ctcctgaaaa ttcaacttat ttctattctt taaatagcca aaaacatgag 2100
catataatca accgaacaga gtgggtggca aatttgagct ggcgttccag ttccaggcaa 2160
cttgctgtta tctggtttat ctattttatc atgttgatcc atcatccatc tttagttata 2220
tgcctatatt aatcctccaa agcctgctta tgcacaacca aagccgtctt gcctggaact 2280
atatattttt ctcgaggcac aaaaatttag caaggtgcca acatacttgt atacaagttg 2340
aacattatgt ctcacacata cagctaatta atcaaccaag acaccatatt ctaaaaattt 2400
gttaaacaca gcacaaatac atagaagaat attcagtttt gtagtcacta gtttttttta 2460
attattttgc attaactatg ctggtgacct ggtcttggat cttcagaaga ccaaccactt 2520
tcgttttctg aacaaaaaga caagagctct gccactcttt gagaagaaga gcagaagact 2580
gaccattgaa tacttagaag ttggaacact atgtcatcat ttggtaattc cttcctgaaa 2640
gatctagaga ggtttctatg gttactatca atactatcta ttgctttgta gttcgtgtag 2700
tttagaataa gattaatcct acgcatgcaa gagtgtacta gtcaaataag agaaaggaaa 2760
tctacaatct ttctcactga acaagctatg taagcagcag ttacgcagga cctggatatg 2820
gacacaaata agttgagcat tgcaaactca agtgcatgct cctgcaaaga taatagtata 2880
gcgacataaa ttacattttg ctctgtgatg ctaatgggtt ggattggatc attgatcagc 2940
atacaaccac ttgctagctt ctttcttcca tataaaattg ttcagcaaaa actgtttatg 3000
aatctgaatt tagtttttgc gccaaagcag gcataaattc tctatttttt tctgtctttc 3060
cagaagttgc agatgaacct gctcccgcga agcctgcccc tgcgaagcaa gggggctcca 3120
atgacagggg taattattga ataattgttg tgtcatcgag attgagtaca agttgccaat 3180
taaaacctga aacatggtct gcagatctcg ctcttgcaag ggtggaaaca gagaagagga 3240
actctttgat caaagcttgg gaagagaatg agaagacaaa agctgagaac aagtatgttt 3300
ctcaccttat ccattcccct gaaatcgtct tcagtggaac cattatttgt ctgttgcagt 3360
agatgcaatt tttgcaatgc tttcttcagt catctataat tctttcatca aagcaaattg 3420
gaagtttaat actggtttct gtacagccag ggcagtgtgg atataaaatt ctgctcagtt 3480
caaactaaaa caagaaaaag ccataagggc aaacaggata acatagtatg tcaattggaa 3540
gatatcatat ttagattctc aatatccaat agaagttgat ataaaccata ggcaggagat 3600
gtggaaattc ctaacttgaa ttaatacgca taactgtgac agggctgcta agaaagtatc 3660
cgctattctt tcatgggaaa acacaaagaa agcaaacata gaagctgaac tgaagaagat 3720
tgaggtatgt cattgaagga atactctttt ttatcgtctg tgcacttact agtttaagtc 3780
tgtcccatat tttaatagag acttgagcca ccgataacaa ttaatcaatt atagaggaat 3840
ttggaaaaga atagatccag tggaactaat atgccacccc ccctcccccc ctaaaaggtg 3900
atatcctctt agctacagct tatatggttt attcatgttc aggaacaact ggaaaagaag 3960
aaggctgaat atgcagagaa gatgaagaac aaggttgcaa tgatacacaa ggaagccgaa 4020
gagaagcgag cgatggtgga ggcaaaacgc ggtgaggagg tcctgaaggc cgaggagatg 4080
gctgccaagt accgggccac cggccacgct cctaagaagc tcatcggttg cttcggagcc 4140

Claims (10)

1. following 1) -3) application of any substance in regulation disease resistance of plant in；
1) albumen Zm-Remorin；
2) DNA molecular of albumen Zm-Remorin is encoded；
3) recombinant vector, expression cassette, transgenic cell line or the recombinant bacterium of the DNA molecular containing coding albumen Zm-Remorin；
The albumen Zm-Remorin is following (1) or (2):
(1) protein that the amino acid sequence shown in sequence 2 in sequence table forms；
(2) amino acid sequence shown in sequence 2 in sequence table is passed through to the substitution and/or missing of one or several amino acid residues And/or addition and the protein with the same function as derived from (1).
2. application according to claim 1, it is characterised in that:
The DNA molecular is following 1) -4) in any DNA molecular:
1) code area is DNA molecular shown in sequence 1 in sequence table；
2) code area is DNA molecular shown in sequence 3 in sequence table；
1) or 2) 3) hybridize under strict conditions with the DNA sequence dna limited and encode the DNA molecular with identical function protein；
1) or 2) 4) at least have 70% with the DNA sequence dna limited, at least have 75%, at least having with 80%, at least 85%, at least with 90%, at least with 95%, at least with 96%, at least with 97%, at least have 98% or at least have There is 99% homology and coding has the DNA molecular of identical function protein.
3. application according to claim 1 or 2, it is characterised in that:
The regulation disease resistance of plant is to improve disease resistance of plant.
4. application according to claim 1 to 3, it is characterised in that:
The disease is southern rust；
Or the pathogen of the disease is Puccinia polysora (Puccinia polysora Underw.)；
Or the plant is dicotyledon or monocotyledon；
Or the plant is monocotyledon, the monocotyledon is specially corn；
Or the plant is dicotyledon, the monocotyledon is specially arabidopsis.
Following 1) -3 during 5. claim 1-4 is any) application of any substance in controlling plant diseases in；
Or, following 1) -3 during claim 1-4 is any) in any substance cultivating the application in disease-resistant plants；
Or, following 1) -3 during claim 1-4 is any) in any substance cultivating the application in high disease-resistant plants.
6. application according to claim 5, it is characterised in that:
The disease is southern rust；
Or the pathogen of the disease is Puccinia polysora (Puccinia polysora Underw.)；
Or the plant is dicotyledon or monocotyledon.
7. a kind of method for cultivating high disease resistant transgenic plants, includes the following steps:
The expression quantity and/or activity for encoding the DNA molecular of albumen Zm-Remorin in purpose plant are improved, transgenosis plant is obtained Object,
Or, improving albumen Zm-Remorin activity in purpose plant, genetically modified plants are obtained,
The disease resistance of the genetically modified plants is higher than the purpose plant；
The albumen Zm-Remorin is following (1) or (2):
(1) protein that the amino acid sequence shown in sequence 2 in sequence table forms；
(2) amino acid sequence shown in sequence 2 in sequence table is passed through to the substitution and/or missing of one or several amino acid residues And/or addition and the protein with the same function as derived from (1).
8. according to the method described in claim 7, it is characterized by:
The expression quantity and/or activity that the DNA molecular of albumen Zm-Remorin is encoded in the raising purpose plant are by the volume The DNA molecular of code albumen Zm-Remorin imports purpose plant.
9. method according to claim 7 or 8, it is characterised in that:
The disease is southern rust；
Or the pathogen of the disease is Puccinia polysora (Puccinia polysora Underw.).
10. according to the method described in claim 9, it is characterized by:
The plant is dicotyledon or monocotyledon.
CN201811124032.7A 2018-09-26 2018-09-26 Application of the Zm-Remorin gene in the prevention and treatment of corn southern rust Pending CN109206493A (en)

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CN113372424A (en) * 2020-06-22 2021-09-10 北京市农林科学院 Corn southern rust resistance gene and application thereof
CN114349835A (en) * 2022-01-07 2022-04-15 河北农业大学 Application of GhREM protein and coding gene thereof in regulating and controlling aphid resistance of cotton
CN114349835B (en) * 2022-01-07 2023-10-10 河北农业大学 GhREM protein and application of encoding gene thereof in regulating and controlling cotton aphid resistance

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Application publication date: 20190115