CN105861540A - Application of magnaporthe grisea protein elicitors in enhancement and improvement of drought-resisting capacity of plants - Google Patents
Application of magnaporthe grisea protein elicitors in enhancement and improvement of drought-resisting capacity of plants Download PDFInfo
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Abstract
The invention discloses application of protein elicitors MoHrip1 and N2 in enhancement and improvement of the drought-resisting capacity of plants. The amino acid sequence of the protein elicitor MoHrip1 is shown as SEQ ID NO:1 in a sequence table, and the amino acid sequence of the protein elicitor N2 is shown as SEQ ID NO:2 in the sequence table. According to the application, molecular detection is conducted on the T1 generation and the T2 generation of MoHrip1 transgenic rice and N2 transgenic rice separately, and it is proved that an MoHrip1 gene and an N2 gene can be normally expressed and transcribed in the transgenic rice; the resistance of the transgenic rice on drought stress is significantly enhanced. Test results show that the MoHrip1 and the N2 have the function of enhancing the drought-resisting capacity of the plants by being expressed in the rice.
Description
Technical field
The present invention relates to biology field, particularly relate to two kinds of rice blast fungus albumen excitons and improving and improving the anti-of plant
Application in drought ability.
Background technology
Oryza sativa L. is one of Chinese and the most topmost cereal crops, the rice blast that rice blast fungus (Magnaporthe oryzae) causes
Disease affects Oryza sativa L. normal development, typically results in Oryza sativa L. underproduction 10%-30%.Exogenous gene is imported plant by modern transgenic technology
In, have been considered as cultivating degeneration-resistant, the effective way of high yield.The research of transgenic paddy rice can be divided three classes the most in the world, and one
It is that there is the resistance transgenic paddy rice such as (antiweed, pest-resistant, degeneration-resistant)；Two is to have high nutritive value (projectile vomiting of milk ferritin base
Because of, turn high-lysine gene etc.) transgenic paddy rice；Three is to have medical value (to turn Hepatitis B virus vaccine gene, turn cholera vaccine base
Because of etc.) transgenic paddy rice.Albumen exciton is when plant is with pathogen interaction, the pathogen such as oomycetes, antibacterial, fungus divide
Secrete the class signaling molecule, it is possible to excite plant defense signal reaction, induction plant to obtain systemic resistance, cause allergy anti-
Should, coordinate plant growth metabolism etc..Research shows, by transgenic approach, proceeds to albumen exciton can improve in Oryza sativa L.
Crop disease-resistant ability.
MoHrip1 and N2 be inventor from rice blast fungus secretory protein isolated and purified go out two kinds of albumen excitons, early-stage Study
Proving, MoHrip1 and N2 can cause Nicotiana tabacum L. anaphylaxis；Evoking tobacco plant produces early reaction, as active oxygen breaks out
Accumulate with NO；Activate immunity signal pathway, improve the resistance to rice blast.But do not mention albumen exciton MoHrip1 and
The effect in drought stress of the N2 encoding gene.
Summary of the invention
The technical problem to be solved in the present invention there is provided a kind of protein elicitor MoHrip1 and N2 and improving and improving plant
Drought-resistant ability in application, SEQ ID NO:1 institute in the aminoacid sequence of described protein elicitor MoHrip1 such as sequence table
Showing, the aminoacid sequence of described protein elicitor N2 is as shown in SEQ ID NO:2 in sequence table.
Application of the present invention, wherein, described plant is Oryza sativa L..
The present invention be number of patent application be ZL201210035653.4 and 201310284892.8 Chinese patent invention serial Shen
Please, the specification and claims of described patent application is in full cited in the present invention.
In above-mentioned patent application, inventor discloses two kinds of albumen excitons both from rice blast fungus secretory protein, names respectively
For MoHrip1 and N2, the described protein of this patent application primary study can significantly improve the resistance of plant, it is possible to high
The degree of effect ground suppression rice blast fungus morbidity.
The rice blast fungus albumen exciton of the present invention application difference from prior art in the drought-resistant ability improving and improving plant exists
In: albumen exciton MoHrip1 and N2 encoding gene are proceeded in Oryza sativa L. by the present invention respectively, it is thus achieved that transgenic paddy rice, pass through
The expression of Study On Rice phenotype, hormone-content and related gene under drought condition, discloses albumen exciton and coerces in arid
Effect in Poing.
The present invention is on the basis of foregoing invention, continues can show after research MoHrip1 and N2 gene is expressed in Oryza sativa L.
Write the drought-resistant ability improving and improving plant.The resistance of drought stress is obviously enhanced by transgenic paddy rice.
Present invention T to MoHrip1 and N2 transgenic paddy rice respectively1Generation, T2In generation, carries out Molecular Detection, it was demonstrated that MoHrip1
Can normal expression and transcribing in transgenic paddy rice with N2 gene.The resistance of drought stress is obviously enhanced by transgenic paddy rice.
Under same drought condition, the content during ABA content is significantly higher than wild rice blade in transgenic paddy rice blade, and GA
Content is substantially less than the content in wild rice blade；Four kinds of gene related to drought tolerance OsNCED2, OsNCED3 in rice leaf,
The relative expression levels of OsZEP and OsbZIP23, is 17,6,28 Hes of wild type in MoHrip1 transgenic paddy rice respectively
2.9 times, be 35,3,52 and 1.6 times of wild type in N2 transgenic paddy rice respectively.Result above shows, MoHrip1 and
N2 expresses in Oryza sativa L. has the function improving plant drought ability.
Below in conjunction with the accompanying drawings to the rice blast fungus albumen exciton of the present invention in the drought-resistant ability improving and improving plant should be used as into
One step explanation.
Accompanying drawing explanation
Fig. 1 is plant expression vector pCXUN-P-Ubi-MoHrip1/N2 structure in the present invention；
Fig. 2 is the Southern (A) of MoHrip1 and N2 transgenic paddy rice in the present invention, Northern (B) and Western blot (C)
Analyze；
Fig. 3 is arid front in the present invention and transgenic paddy rice and wild rice upgrowth situation after arid；
Fig. 4 is drought stress after 14 days in the present invention, ABA and GA content in transgenic paddy rice and wild rice blade；
Fig. 5 is drought stress after 14 days in the present invention, transgenic paddy rice and wild rice blade gene related to drought tolerance OsNCED2,
The relative expression levels of OsNCED3, OsZEP, and OsbZIP23.
In accompanying drawing, the translator of Chinese of all English that (Fig. 3, Fig. 4, Fig. 5) occurs is as follows:
MoHrip1: turn exciton MoHrip1 trans-genetic hybrid rice；
N2: turn exciton N2 trans-genetic hybrid rice；
PCXUN: empty vector control Oryza sativa L.；
Wt: wild rice；
ABA content: ABA content；
GA content: GA content；
Relative expression level: gene relative expression levels；
OsNCED2, OsNCED3, OsZEP:ABA synthesis related gene, the epoxy Semen Maydis in coding ABA synthesis path
Xanthin；Cyclase and 9-epoxy are along carotenoid dioxygenase；
OsbZIP23: transcription factor, belongs to a member in bZIP family.
Detailed description of the invention
Embodiment 1
1 materials and methods
1.1 test material
Fine (O.Sativa L.spp.japonica, var nipponbare, the AAgenome) wild rice of Japan and Agrobacterium tumefaciems
(Agrobacterium tumefaciens) bacterial strain is LBA4404, this laboratory preserve.Plant expression vector pCXUN by
The beam researcher's laboratory present of kingdom of Institute of Plant Protection of the Chinese Academy of Agricultural Sciences, this carrier contains Ubiqutin promoter, ccd 1 B gene and NOS
Terminator sequence.Goat-anti rabbit two used by Western hybridization is anti-purchased from Beijing Quanshijin Biotechnology Co., Ltd.
1.2 expression vector establishment
Design protein elicitor gene M oHrip1 and the specific primer of N2, with primer
Signal peptide is removed in 5 '-atggcccctgccccgcaggcgcaggccacctcg-3 ' and 5 '-taagcggagccgtcaatggcaatagcggc-3 ' amplification
MoHrip1 gene；With primer 5 '-atgcagcaggctatcgtccacaacaactgccag-3 ' and
The N2 gene of signal peptide is removed in 5 '-ctaggcgcagagggtagcctcgacgttgatgg-3 ' amplification.With Xcm I enzyme, pCXUN is carried
Body carries out cloning fragment with MoHrip1 and N2 mesh respectively by TA after single endonuclease digestion and dephosphorylation process and is connected, structure
Plant expression vector: pCXUN::MoHrip1, pCXUN::N2 and empty vector control pCXUN (Fig. 1).
1.3MoHrip1 the acquisition with N2 transgenic paddy rice strain and screening
Agrobacterium-mediated Transformation method is used to obtain MoHrip1 and N2 transgenic paddy rice.Choose the T of mature and plump0For transgenic paddy rice kind
Son, processes 30min with 50%Na Cl O or 84 disinfectant solution after removing grain husk shell on 37 DEG C of shaking tables.Afterwards in aseptic operating platform
Middle sterilizing dd H2O rinse water rice 6-10 is all over so that by disinfectant solution wash clean.By rice paddy seed kind to MS solid culture
In base (100mg L-1Hyb), close, under illumination/dark=14h/10h, within 4-5 days, grow transgenic paddy rice Seedling.Treat Oryza sativa L.
Seedling grows to be transplanted in the Nutrition Soil in flowerpot during about 10cm, cultivates under appropriate light is according to humidity.Results T1For transgenic water
After rice, the T that will obtain by identical method1T is obtained for transgenic paddy rice seed plantation2For transgenic paddy rice seed.Kind
In 4 DEG C of preservations after son drying, for subsequent experimental.
By T1For the seed soaking 24 in 1/2MS fluid medium (100mg L-1kan) of MoHrip1 and N2 transgenic seed
Accelerating germination after h.15d " Invest, Then Investigate " number of seedling, in the meantime regular replenishment sieve is cultivated in light dark is 14h/10h illumination box
Select liquid, to gather in the crops the seed of positive plant.
The Molecular Detection of 1.4 transgenic paddy rices
Extract the DNA (CTAB method) and RNA (TRIzol method) of MoHrip1 and N2 transgenic paddy rice respectively, with
DNA and cDNA is template, with primers F: 5 '-atggcccctgccccgcaggcgcaggccacctcg-3 ' and
R:5 '-taagcggagccgtcaatggcaatagcggc-3 ' PCR expands MoHrip1, uses primer
F:5 '-atgcagcaggctatcgtccacaacaactgccag-3 ' and R:5 '-Ctaggcgcagagggtagcctcgacgttgatgg-3 ' PCR expands
Increase N2.
By the genomic DNA of the transgenic paddy rice individual plant of acquisition, with 37 DEG C of double digestions of EcoRI and Bam HI overnight, through 1.2%
Agarose gel, goes to nylon membrane, for Southern blot.Total serum IgE is used for Northern hybridization, and method is same
Southern hybridizes.Use digoxin test kit, with the primer mark probe of above-mentioned amplifying target genes.Transgenic paddy rice blade
Add and after liquid nitrogen is fully ground, be immediately transferred to phosphate buffer (0.025mol L-1K2HPO4,0.025mol L-1KH2PO4,2
Mmol L-1EDTA, pH 8.0), fully 12000 × g, 4 DEG C of centrifugal 10min after mixing.After supernatant ice bath 1h
12000 × g, 4 DEG C are centrifuged 10min again.Pvdf membrane is transferred to after being separated by the albumen 12%SDS-PAGE glue of extraction,
Carry out immuning hybridization.Purification rabbit multi-resistance resists as one, and goat-anti rabbit resists as two.
1.5 transgenic paddy rice drought resistances measure
T2For MoHrip1 and N2 transgenic paddy rice, empty vector control pCXUN and wild rice, respectively take 20 seeds in
On filter paper in culture dish, add PEG (6000) solution of 1ml 20%, change solution every day, measure seed after five days and send out
Bud rate and seedling root length.In triplicate.
T2For MoHrip1 and N2 transgenic paddy rice, empty vector control pCXUN and wild rice, normal irrigation two weeks.
Stop afterwards pouring water, carry out drought stress process.Treating that drought stress processes 14 days, taking leaf rolling degree respectively is unanimously 50%
Blade, be placed in the centrifuge tube of 1.5ml, each kind take three pipe blades (weigh, it is ensured that the sample quality that often pipe takes be about
30mg) and be immediately positioned in liquid nitrogen, continue subsequent detection test, temporarily need not, preserve in-80 DEG C of refrigerators.
1.6 transgenic paddy rice ABA and GA assay
The content of ABA content and GA uses ABA and GA ELISA detection kit respectively.Often pipe rice leaf (30mg)
Add a formaldehyde (30ul), after being fully ground, add nine parts of PBS (pH value is 7.4,0.1M), then enter with refiner
Row homogenate (3-5 minute) makes tissue sample thoroughly pulverize, and obtains the homogenate of 10%.10% homogenate is positioned over centrifuge
In centrifugal about 20 minutes (2000-3000 rev/min), collect supernatant, a to be measured after subpackage, remaining freezing is standby.Directly
Connect and use the sample supernatant collected to carry out ELISA experiment.
1.7 transgenic paddy rice gene related to drought tolerance fluorescence quantitative RT-RCRs are analyzed
Taking required sample liquid nitrogen to pulverize, add Trizol by 50-100mg/mL, room temperature 5min stands, 12000rpm
Centrifugal 5min, abandons precipitation；Placing by 200 μ L chloroforms/mL Trizol, concussion mixing 15min, 4 DEG C, 12000rpm is centrifuged 15min,
Drawing upper strata, add 0.5mL isopropanol/mL Trizol, mixing ,-20 DEG C stand 20min；Centrifugal, 75% ethanol rinse,
Centrifugal segregation ethanol, 5-10min dries.Add diethypyrocarbonate (being abbreviated as DEPC) and process water, survey RNA concentration,
And record.
The synthesis of the first chain cDNA, uses the FastQuant RT kit (with gDNase) of TianGen company.Take 1ug total
RNA, synthesizes the first chain cDNA according to test kit description.Take 1ul reverse transcription product and be quantitative fluorescent PCR (SYBR
Green.Tiangen), the PCR instrument type number of use is CFX96 (Bio-Rad Laboratories, Hercules, CA, USA).
PCR step is 95 DEG C, 15s；95 DEG C, 10s and 60 DEG C, 30s carries out 40 circulations.Osactin is as internal standard gene
(F:5′-GAGTATGATGAGTCGGGTCCAG-3′R:5′-GAGTATGATGAGTCGGGTCCAG-3′)。
With relevant drought related gene special primer be PCR (OsNCED2 gene F:5 '-GGAGAGAGTTGGTTTGTG-3 ',
R:5′-ATTGTTGTGCGAGAAGTT-3′；OsNCED3 gene F:5 '-CAGGATATGCTCACATACAG-3 ',
R:5′-GGAGAATCTCACCGAATTG-3′；OsZEP gene F:5 '-GAAGTCTAATGATACGGAATCT
-3 ', R:5 '-TGGTTCTCAAGTGTCTCA-3 '；OsbZIP23 gene F:5 '-CCAGAGGAAACAGGCATAT
-3 ', R:5 '-ACTTGTCGGCTCATTCTC-3 '；).Observe drought related gene in transgenic paddy rice and wild rice
Expression.
2 results and analysis
The acquisition of 2.1 transgenic paddy rices and Molecular Detection
T is gathered in the crops after the plant expression vector arabidopsis thaliana transformation that will build0For seed, breed afterwards to T2Generation.
T to PCR tests positive1Southern hybridization analysis has been carried out for transgenic paddy rice.Swimming lane 1-7 is albumen
The Southern bolt of exciton 7 strains of MoHrip1 transgenic paddy rice analyzes, and wherein 3,4 is negative strain, the most miscellaneous
Hand over band；Swimming lane 8-13 is that the southern blot of albumen exciton 6 strains of N2 transgenic paddy rice analyzes, and wherein 12 show
It is shown as double copy strain, two hybrid belts；M:DL15000Marker.Result shows that MoHrip1 and N2 gene is integrated
In rice genome (Fig. 2-A).
T2Northern hybridization check is carried out for transgenic paddy rice strain.Swimming lane 1-3 is 3 strains of MoHrip1 transgenic paddy rice
The Northern blot result of system, 2 represent negative strain；4-7 represents the Northern blot of 4 strains of N2 transgenic paddy rice
As a result, 5 is negative strain.Result shows, MoHrip1 and N2 gene has been transcribed into mRNA (figure in transfer-gen plant
2-B)。
T2Western blot detection has been carried out for transgenic paddy rice.1 and 2 represent 1 strain of MoHrip1 transgenic paddy rice
The Western blot analysis result being repeated twice, 3 and 4 represent what 1 strain of MoHrip1 transgenic paddy rice was repeated twice
Western blot analysis result.Show that MoHrip1 and N2 gene correctly expresses (Fig. 2-C) in transgenic paddy rice.
2.2MoHrip1 and N2 transgenic paddy rice phenotype under drought stress and percentage of seedgermination
The patience of drought stress is significantly improved by MoHrip1 and N2 transgenic paddy rice, after the rice seedlings of normal irrigation is cut off the water supply 7 days,
Wild rice and empty vector control Oryza sativa L. all present lodging and wilt, and transgenic paddy rice still keeps green and erectility (Fig. 3).
Drought stress patience is significantly improved by MoHrip1 and N2 transgenic paddy rice seed, and result shows (table 1), does at 20%PEG
Drought is coerced down, MoHrip1 and N2 transgenic paddy rice seed germination percentage is above wild type (P < 0.05).
The germination percentage of rice paddy seed under table 1 drought stress
| Process | MoHrip1 transgenic paddy rice | N2 transgenic paddy rice | PCXUN empty vector control Oryza sativa L. | WT |
| Percentage of seedgermination/% | 79.78a | 80.90a | 57.14b | 58.24b |
Transgenic paddy rice ABA and GA content under 2.3 drought stress conditions
The abscisic acid (ABA) of MoHrip1, N2 transgenic paddy rice and wild rice blade and gibberellins (GA) content knot
Fruit display, after Osmotic treatment 14 days, the crop leaf measuring of identical amount of crimp.Transgenic paddy rice blade ABA content is significantly high
In wild rice (P < 0.05), the ABA content of MoHrip1, N2 transgenic paddy rice is 1.44 times of wild rice respectively
With 1.36 times.Transgenic paddy rice blade GA content is substantially less than wild rice (P < 0.05), the GA of wild rice blade
Content is 1.29 times and 1.34 times (Fig. 4) of MoHrip1, N2 transgenic paddy rice respectively.
The expression of drought related gene under 2.4 drought stresses
OsNCED2, OsNCED3 and OsZEP are ABA synthesis related genes, and OsbZIP23 is that ABA signal path is correlated with
Gene, after Osmotic treatment 14 days, detects MoHrip1, N2 transgenic paddy rice, empty vector control pCXUN and open country respectively
The relative expression levels of four kinds of genes in raw type rice leaf.The drought related gene of transgenic paddy rice apparently higher than wild type and
Empty vector control.The relative expression levels of four kinds of genes of MoHrip1 transgenic paddy rice is the 17 of wild rice, 6,28 respectively
With 2.9 times；The relative expression levels of four kinds of genes of N2 transgenic paddy rice is the 35 of wild rice, 3,52 and 1.6 respectively
Again (Fig. 5).
3 conclusions
Under drought stress, the germination percentage of MoHrip1 and N2 transgenic paddy rice seed is apparently higher than wild type, and seedling root length is bright
Aobvious longer than wild type；Arid is after 14 days, and transgenic paddy rice Seedling is more healthy than wild rice Seedling in phenotype, transgenic paddy rice blade
GA content is less than wild rice, MoHrip1 and N2 transgenic paddy rice blade ABA content and four kinds of arid positive correlation genes
It is significantly higher than wild rice.Illustrate that albumen exciton MoHrip1 and N2 gene proceed in Oryza sativa L. by regulation ABA content
Improve and improve the drought-resistant ability of Oryza sativa L. with ABA signal path.
Under drought stress, the drought resistance of plant is played important regulation effect, water stress profound influence plant by endogenous hormones
The content of the hormone such as inner gibberellin, abscisic acid.Aharoni N etc. thinks GA activity decrease in Caulis et Folium Lactucae sativae blade, affects its physiology and lives
Property, adapt to drought environment；Liang etc. think under drought condition, and ABA content increase causes pore to be closed, inhibited photosynthesis,
Resist drought stress.This research shows, during drought stress, in MoHrip1 and N2 transgenic paddy rice blade, GA content ratio is wild
Type is aobvious to be reduced, and ABA content is more notable than wild type to be increased.Illustrate MoHrip1 and N2 proceed to make Oryza sativa L. to drought stress produce
Obvious adaptability.
In plant leaf blade, the increase of ABA content promotes pore to close, and reduces water loss, maintains endocellular metabolism activity as far as possible
Carry out, strengthen the plant resistance to drought stress.ABA not only regulates and controls the expression of related gene affects stomatal movement, and also induction is anti-
The expression of drought related gene.Research shows, ABA synthesis related gene and ABA signal transduction pathway related gene can respond dry
Drought is coerced and is made expression relatively increase or less, to help plant to spend drought environment.This experiment to OsNCED2,
The relative expression levels of tetra-kinds of positive regulating genes of OsNCED3, OsZEP and OsbZIP23 analyzes, OsNCED2,
OsNCED3 and OsZEP is ABA synthesis related gene, and topmost two kinds of enzymes in coding ABA synthesis path, epoxy is beautiful
Cream colour matter cyclase and 9-epoxy significantly can be induced by drought stress along carotenoid dioxygenase, both enzymes, strengthen plant
Drought resistance.OsbZIP23 belongs to a member in bZIP family, and this family member is in plant drought reaction and hormone signal are conducted
Play an important role.The expression of these four gene substantially raises in MoHrip1 and N2 transgenic paddy rice, to drought environment
It is made that response.Proceeding to by regulation ABA content and the induction of ABA signal path related gene of MoHrip1 and N2 is described
Oryza sativa L. produces resistance to drought stress.
MoHrip1 and N2 gene can be expressed in Oryza sativa L..Transgenic seed germination percentage in 20%PEG solution is higher than
Wild type seeds, after germination, average root length is also long than wild type；When strain is by drought stress, transgenic paddy rice Seedling is withered state
Lower than wild rice, GA content relative reduction, ABA content raises relatively, and gene related to drought tolerance expression substantially raises.
These results all show, MoHrip1 and N2 albumen exciton can regulate plant in Oryza sativa L. and arid is made active responding,
Improve the drought-resistance ability of Oryza sativa L..Strong for cultivating drought resisting, the rice varieties that yield is high provides basis.
Embodiment described above is only to be described the preferred embodiment of the present invention, not carries out the scope of the present invention
Limiting, on the premise of designing spirit without departing from the present invention, technical scheme is made by those of ordinary skill in the art
Various deformation and improvement, all should fall in the protection domain that claims of the present invention determines.
Claims (2)
1. protein elicitor MoHrip1 and N2 application in the drought-resistant ability improving and improving plant, described albumen
The aminoacid sequence of matter exciton MoHrip1 as shown in SEQ ID NO:1 in sequence table, described protein elicitor N2
Aminoacid sequence as shown in SEQ ID NO:2 in sequence table.
Application the most according to claim 1, it is characterised in that described plant is Oryza sativa L..
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| CN112126655A (en) * | 2020-10-14 | 2020-12-25 | 河北省农林科学院棉花研究所（河北省农林科学院特种经济作物研究所） | Application of Asian cotton GaNCED3 gene in improving drought resistance of plants |
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