CN102260709B - Use of key gene OsLSD2 associated with formation of rice aerenchyma - Google Patents
Use of key gene OsLSD2 associated with formation of rice aerenchyma Download PDFInfo
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- CN102260709B CN102260709B CN201110200437A CN201110200437A CN102260709B CN 102260709 B CN102260709 B CN 102260709B CN 201110200437 A CN201110200437 A CN 201110200437A CN 201110200437 A CN201110200437 A CN 201110200437A CN 102260709 B CN102260709 B CN 102260709B
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- oslsd2
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- formation
- paddy rice
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
- C12N9/0026—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on CH-NH groups of donors (1.5)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
- Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants
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Abstract
The invention belongs to the technical field of genetic engineering and discloses the use of a key gene OsLSD2 associated with formation of rice aerenchyma. The rice key gene OsLSD2 controlling the formation of the rice aerenchyma can be used for increasing the height of plants and the nitrogen fertilizer utilization efficiency and/or yield of plants. The sequence registry number of the gene is AK111759. Through systemic study, the invention is the first to provide the biological functions of the key gene OsLSD2 controlling the formation of the rice aerenchyma. In the invention, an OsLSD2 overexpression material obtained by a transgenic means increases the expression of the OsLSD2 in laminae in roots greatly, so that the aerenchyma in the roots of rice plants change; and compared with a reference wide plant, the plants obtained by the transgenic material are taller and can better absorb and use nutrients, particularly nitrogen, and the nitrogen utilization efficiency and yield per plant are increased by over 30 percent.
Description
Technical field
The invention belongs to gene engineering technology field, relate to the application that paddy rice ventilating tissue forms key gene OsLSD2.
Background technology
Nitrogen is one of important macronutrient of crop, participates in the various metabolic processes of organism.It is the moity of a lot of living matters in the plant materials, such as: amino acid, protein, nucleic acid, enzyme, chlorophyll etc.Nitrogen accounts for 16% (Frink CR., Waggoner PE.and Ausubel JH.Nitrogen fertilizer:retrospect and prospect.Proc.Nati.Acad.Sci.USA.1999.96:1175～1180.) of the 1.5-2% and the plant total protein of plant materials dry weight respectively.At present, Chinese nitrogen fertilizer amount accounts for 30% (Peng Shaobing, Huang Jianliang, Zhong Xuhua of global nitrogen fertilizer amount; Yang Jianchang, Wang Guanghuo, Zou Yingbin; Zhang Fusuo, Zhu Qingsen, Roland Buresh; Christian Witt. improves the research strategy of Chinese rice field utilization rate of nitrogen fertilizer. Scientia Agricultura Sinica .2002,35 (9): 1095～1103.), become the big country of consumption of the first in the world.Wherein the amount of application of nitrogenous fertilizer surpasses other any farm crop in the rice terrace, and the loss amount of nitrogenous fertilizer accounts for 70% of total fertilization amount.China's ubiquity because the low a series of environmental problems that cause with a large amount of nitrogens loss of utilization rate of nitrogen fertilizer.Though paddy rice happiness ammonium crop there are some researches show that a certain amount of nitric nitrogen can promote the absorption of paddy rice to ammonium, and be main mainly with nitric nitrogen the later stage field and the drought rice of rice growth.The root system development of paddy rice is regulated and control by nitrate salt, and the mechanism of the short root growth of wherein main nitrate salt is the transportation distribution that nitrate salt has been regulated and control growth hormone.In recent years, the development of Protocols in Molecular Biology is very fast, for crop genetic and breeding work provide new means.People also attach great importance to biological method and improve utilization rate of nitrogen fertilizer to solve the nitrogenous fertilizer problem.The same with other aquatic crops; The oxygen process of secreting of rice root is its essential process that can in anaerobic environment, grow; This oxygen process of secreting must cause the paddy rice rhizosphere that nitrification in various degree takes place; If the nitrate salt in Gen Biao and rhizosphere generation can be absorbed rapidly by root system, will produce hormesis with other physiological processs to the nitrogen nutrition of crop, thereby significantly promote crop growth.Our test-results shows (Li et al.; 2008) the efficient kind of nitrogen is raised rice No. 6 and is secreted more oxygen through its flourishing ventilating tissue; Compare with contrast nitrogen poor efficiency kind land-reclaimable 57; Its root table enrichment more polynitration bacterium has produced more nitrate salt, accumulates more nitrate salt and total nitrogen thereby the efficient paddy rice poplar of nitrogen rice shows the ground No. 6 in equal paddy field, and nitrogen utilizes enzyme work to be significantly higher than contrast land-reclaimable 57.Ultimate capacity and nitrogen utilising efficiency poplar rice all are significantly higher than land-reclaimable 57 for No. 7.But if if the nitrate salt of root table can not be absorbed rapidly by root system, just do not have these hormesiss, and these nitrate salt will denitrifications and are lost.Our research shows that the efficient kind poplar of nitrogen rice has not only had its absorption (Fan et al 2005) simultaneously of flourishing ventilating tissue for No. 7 and utilized the ability of nitrate salt (Fan et al 2007) all to be significantly higher than land-reclaimable 57 again.This explanation nitrogen efficient rice varieties is in those kinds with adaptive faculty the best of self evolving from Lu Sheng to aquatic process; They have not only shown when tissue forms and have evolved or adaptive advantage, also optimize or have kept more Lu Sheng Nitrogen Absorption simultaneously and utilize characteristic.
Ventilating tissue (aerenchyma) is the set of interior air chambers of plant parenchyma or cavity.Many aquatic and hygrophytes all form ventilating tissue in rhizome, some other plant in anaerobic environment also differentiation produce or quicken the growth (Watkin etc., 1998) of ventilating tissue.Under the waterflooding condition, paddy rice increases gas degree of leading (Miyamoto et al2001) through forming ventilating tissue, and foundation portion induces to produce and prevents the excessive interlayer of oxygen (Colmer, 2003), transmits to tip of a root direction thereby prolong oxygen.But the different water rice varieties shows very big-difference.Colmer (2003) is comparing 7 upland rice; The ventilating tissue under the waterflooding condition of 3 paddy rice and 2 rice varieties of anti-deep water and the formation difference that prevents the interlayer that oxygen is excessive; Find; Part upland rice foundation portion can not form the interlayer that prevents that oxygen is excessive, and the oxygen of the tip of a root is secreted outward and existed significant breed difference.
The summary that Seago et al. (2005), Evans et al. (2003) carry out the formation and the classification of ventilating tissue.Ventilating tissue compares with schizogenesis property, and the research of dissolving natural disposition ventilating tissue is more relatively.Dissolve the formation of natural disposition ventilating tissue and receive cell programmatic dead (PCD) control (Gunawardena A, 2008 summaries).Marrow in the rice root, large-scale aqtocytolysis almost make all centrocytes disappear, and form hollowly, and the resistance that gas transport receives is reduced to minimum (Della etc., 1999).It is generally acknowledged that necrocytosis occurs in the middle part of cortex.The result of transmission electron microscope observing maize peel confluent monolayer cells shows that the disintegration of vacuole is the physical process of seeing the earliest (Campbell & Drew, 1983).As if the mesogloea of rice cell changes more early, can see in the anoxic 1d that its structure changes, cell walls disintegrates before whole cytoclasis (Webb & Jackson, 1986).The cytology of Kawai etc. (1998) research rice root finds that cell acidify is cytoclastic afterwards sign early.This means the destruction that acid inclusion that vacuole skin damage discharges can cause membrane integrity.Be different from the formation of xylem, ventilating tissue's cell walls is (the Gunawardena et al 2001) that degrades fully.This maybe with xyloglucan transglucosidase xet gene, pyruvic carboxylase pdc gene, relevant (Subbaiah & Sachs, 2003) such as phosphoglycerol dehydrogenase gpc genes.Muhlenbock et al 2007 utilizes Arabidopis thaliana lsd1; Eds1; Pad4-5 single mutant and eds1/lsd1; Pad4-5/sd1 double-mutant research ventilating tissue under hypoxia condition forms synthetic with ethene, thinks LSD1 negative gene control EDS1 and PAD4-5 expression of gene, and EDS1 and PAD4-5 gene are just being regulated and control the formation of root system ventilating tissue under synthesizing of ethene and the hypoxia condition.In the paddy rice from PBZ1 expression of gene position this gene participate in the PCD of paddy rice; The ventilating tissue that comprises leaf senile and root system forms; Participated in DNA Ladder through this gene of this gene discovery that suddenlys change and formed, all expressed (Kim et al 2008) with all cells that PCD takes place.
Summary of the invention
The purpose of this invention is to provide the application that paddy rice ventilating tissue forms key gene OsLSD2.
The object of the invention is realized through following technical scheme:
The application of the key gene OsLSD2 that paddy rice control ventilating tissue forms aspect increase plant plant height, raising plant nitrogen fertilizer utilising efficiency and/or output, the sequence accession number of this gene is AK111759.
Wherein, described plant optimization monocotyledons, further preferred paddy rice, corn or wheat, preferred especially paddy rice.
The coded product paddy rice ventilating tissue of the key gene OsLSD2 that paddy rice control ventilating tissue forms forms the application of key protein OsLSD2 aspect increase plant plant height, raising plant nitrogen fertilizer utilising efficiency and/or output.
Beneficial effect of the present invention:
1, the present invention provides the biological function of rice auxin transport protein gene OsLSD2 first through systematic study.
2, utilize the expression of special primer research OsLSD2 in paddy rice, find OsLSD2 on the ground, underground part all expresses (Fig. 1), with having improved nitrogen utilising efficiency (table 1) behind the OsLSD2 overexpression.
3, increased plant height and the tiller number of paddy rice behind the OsLSD2 overexpression, helped improving plant type of rice (Fig. 4) output is improved more than 30% (table 2).
The present invention makes OsLSD2 roll up (Fig. 2) at leaf expression through the OsLSD2 overexpression material that the transgenic means obtain; The ventilating tissue of rice plant root system is changed; Compare with the contrast wild-type; Transgenic line has obtained taller and bigger plant, helps paddy rice to nutrient especially absorption of nitrogen and utilization, improves the nitrogen utilising efficiency.
Description of drawings
Fig. 1 OsLSD2 is at paddy rice different sites (blade, root) expression characteristic, wherein, and 1: root 2: blade.
The Molecular Identification of Fig. 2 OsLSD2 overexpression material (O-1, O-2, O-3, O-4), four overexpression strains tie up in the blade expression amount than the obvious enhancing of wild-type.
The evaluation of Fig. 3 OsLSD2 overexpression material southern copy number,
Wherein, WT: wild-type, CK: blank; O-1, O-2, O-3, O-4: represent OsLSD2 overexpression material respectively.
Fig. 4 OsLSD2 overexpression material (O4) is compared the plant height raising with wild-type (force is educated round-grained rice 7), tiller number increases.
Fig. 5 OsLSD2 overexpression material (O4) and wild-type (force is educated round-grained rice 7) ventilating tissue's displaing micro picture.
Wherein, WT: wild-type, O-4: expression OsLSD2 overexpression material, Image to right is the ventilating tissue's displaing micro picture apart from tip of a root 1cm place, the left side is the ventilating tissue's displaing micro picture apart from tip of a root 1.5cm place.
Embodiment
Embodiment 1OsLSD2 gene (accession number is AK111759) expression characteristic in paddy rice
1) the extraction paddy rice force of total RNA is educated the round-grained rice seventh-seeded through mass ratio 30%NaCLO
2Sterilization, vernalization is cultivated two leaves wholeheartedly the time; Pick out rice plant of the same size, transplant to the 1/2 international paddy rice IRRI of the institute nutritive medium of pH5.5 after removing endosperm, four leaves are changed to the international paddy rice IRRI of institute pancebrin (Mao D R.The methods of plant nutrition research.Beijing:Beijing Agricultural University Press wholeheartedly the time; 1994.), cultivate a week after, get root and blade and place the freezing preservation of liquid nitrogen rapidly; Take by weighing 0.1g left and right sides sample, grind, grind active addition 1.5ml centrifuge tube with liquid nitrogen; Add 1ml Trizol reagent rapidly, add the 0.2mL chloroform, supernatant is drawn in centrifugal back; Add the 0.5mL Virahol, abandon supernatant after centrifugal, add 70% washing with alcohol deposition; RNA is dissolved in (volume ratio is 1 ‰) in the DEPC water, and the use mass ratio is 1.7% agarose gel electrophoresis detection RNA quality, and detects concentration and the purity of total RNA with spectrophotometer;
2) synthetic each the RNA sample 2 μ g of total cDNA add 50 μ molL
-1Oligo dT18 adds volume ratio 1 ‰ DEPC water and supplies 10 μ L, 70 ℃ of following water-bath 5min; After placing 5min on ice, add RNase inhibitor 0.5 μ L and 5xRT buffer5 μ L successively, 10mM dNTP 2.5 μ L; M-MLV ThermoScript II 1 μ L; Volume ratio 1 ‰ DEPC water are supplied 25 μ L, and behind 42 ℃ of water-bath 60min, (Oligo dT18 is synthetic by Nanjing Jin Sirui company for 70 ℃ of water-bath 10min termination reactions; The reverse transcription test kit is available from Fermentas company, Canada).
3) behind synthetic total cDNA first chain of sxemiquantitative PCR reverse transcription, be that template is carried out pcr amplification with it.The PCR reaction system is 20 μ L, comprises 10pmolL
-1Each 1 μ L of forward and reverse primer, 10x PCR buffer 2 μ L, 2.5mM dNTP 1.6 μ L, Taq enzyme 0.4 μ L, (primer is synthetic by Nanjing Jin Sirui company to supply 20 μ L with aqua sterilisa then; PCR reagent is available from Takara company, Dalian).It is different because of its concentration to add the template amount, is proofreaied and correct by the amount of internal control gene rice cell skelemin gene (OsActin), and the PCR program of gene OsActin and OsLSD2 is following: 94 ℃ of preparatory sex change 4min; 94 ℃ of sex change 30s; 55 ℃ of renaturation 30s, 72 ℃ are extended 30s, after 30 circulations; 72 ℃ of 7min, amplification PCR products is that 1.5% agarose gel electrophoresis detects through mass ratio.After the dyeing of Australia second shallow lake (EB), form images at gel imaging system.The sequence number of gene and design of primers such as following table:
*: reference; Xiaorong Fan, Lijun Jia, Yilin Li; Susan J.Smith; Anthony J Millerand Qirong Shen, 2007 Comparing nitrate storage and remobilization in two ricecultivars that differ in their nitrogen use efficiency, Journal of Experimental Botany58 (7): 1729-40
Find that through this gene is carried out expression analysis at paddy rice different sites (blade, root) OsLSD2 all expresses in root, blade, and expression amount is higher than root (Fig. 1) in blade.
The overexpression plant of embodiment 2OsLSD2 gene
1) extraction of total RNA is with embodiment 1;
2) total cDNA is synthetic with embodiment 1;
3) acquisition of the cDNA total length of OsLSD2 gene
Use the above paddy rice force that obtains to educate No. 7 total cDNA of round-grained rice and be template, design PCR primer, its PCR product comprise complete OsLSD2 and read frame (from initiator codon ATG to 3 ' end non-coding region), and primer sequence is:
OverLSD2-F:5 '-TTGAGGATCCGTGCCATTTACACCTC-3 ' (SEQ ID NO.5 contains BamH I restriction enzyme site)
OverLSD2-R:5 '-ATATGGTACCACAGACCTTGCGCCAT-3 ' (SEQ ID NO.6 contains Kpn I restriction enzyme site)
PCR 20 μ L systems: 2 μ L 2.5mM dNTP, 2uL 10x PCR buffer, 1 μ L OverLSD2-F, 1 μ LOverLSD2-R, 1 μ L cDNA, 13 μ L ddH
2O.
The PCR program is following: 94 ℃ of preparatory sex change 4min, and 98 ℃ of sex change 10s, 68 ℃ of renaturation are extended 2min; After 30 circulations, 72 ℃ of 10min, amplification PCR products detects through mass ratio 1% agarose gel electrophoresis; Its size is the 822bp fragment, purpose PCR product is cut glue reclaim after agarose electrophoresis is separated, and the fragment that reclaims is connected with the pMD-19 carrier; The enzyme disjunctor is TV 10 μ L, comprises 5 μ L and connects liquid, the pMD-19 carrier of 1 μ L; The PCR purified product of 3-4 μ L, water are supplied 10 μ L, and 16 ℃ of connections are spent the night then; Change over to again and be coated in the bacillus coli DH 5 alpha competent cell on the LB solid medium that contains peace benzyl 100 μ gmL-1 behind the growth 12h-14h; The picking positive bacteria is dropped into capable dna sequencing; OsLSD2 gene accession number is AK111759, and the cDNA full length sequence of OsLSD2 comprises ORFs (ORF) 552bp and 3 ' end non-coding region UTR270bp; It is subsequent use in-70 ℃ of preservations that the bacterium liquid that order-checking is correct adds equal-volume volume ratio 30% glycerine, obtains to contain the recombinant plasmid of goal gene OsLSD2cDNA full length sequence, called after OLSD2-T;
4) structure of overexpression carrier pUbi-LSD2
Use the above OLSD2-T plasmid that obtains to be template, OverLSD2-F (SEQ ID NO.5) and OverLSD2-R (SEQ IDNO.6) are primer, carry out pcr amplification OsLSD2 gene fragment; The PCR program is following: 94 ℃ of preparatory sex change 4min, and 98 ℃ of sex change 10s, 68 ℃ of renaturation are extended 2min; After 30 circulations; 72 ℃ of 10min, amplification PCR products detects through mass ratio 1% agarose gel electrophoresis, and PCR product size is 822bp.Purpose PCR product is cut glue after agarose electrophoresis is separated reclaim; Reclaim product and carry out double digestion with restriction enzyme BamHI, KpnI; While double digestion plant overexpression carrier pCAMBIA1300 (purchasing co.) plasmid in Biovector Science Lab; Reclaim PCR fragment and carrier that enzyme was cut then respectively, carrier is carried out reclaiming once more behind the dephosphorylation; The PCR fragment of through the T4 ligase enzyme linearizing carrier and enzyme being cut after reclaiming is connected under 4 ℃ spends the night; Be transformed in the escherichia coli DH5a competent cell; After being coated in the 12h that grows on the LB solid medium that contains kantlex 50 μ gmL-1; The positive bacterium colony of picking, extract plasmid through BamH I, Kpn I enzyme cut the checking clip size errorless after, this bacterium liquid is carried out dna sequencing; The bacterium liquid that will contain the correct clone of order-checking adds equal-volume volume ratio 30% glycerine in-70 ℃ of preservations, extracts positive colony plasmid called after pUbi-LSD2;
Through electric shocking method the pUbi-LSD2 plasmid is converted in the competent cell of agrobacterium tumefaciens EHA105 at last, is coated in and contains kantlex and Streptomycin sulphate is 50 μ gmL
-1The YEP solid medium on behind the growth 48h, the positive bacterium colony of picking extracts plasmid, through BamH I, Kpn I enzyme cut checking errorless after, bacterium liquid adds equal-volume volume ratio 30% glycerine in-70 ℃ of preservations, transgenic is subsequent use.
5) acquisition of transfer-gen plant
The commentaries on classics of above acquisition there is the Agrobacterium of pUbi-LSD2 plasmid, infects force and educate No. 7 rice callus tissues of round-grained rice, cultivated altogether 60 days, obtain T1 for transfer-gen plant through selecting to cultivate, break up, take root, refine seedling.
5.1) reagent and solution abbreviation
The abbreviation expression is write as follows by the used English institute of substratum among the present invention: 6-BA (6-benzyladenine); Car (Pyocianil); NAA (naphthylacetic acid); IAA (indolylacetic acid); 2,4-D (2,4 dichlorophenoxyacetic acid); AS (Syringylethanone); CH (caseinhydrolysate); L-pro (L-proline(Pro)); L-Glu (L-glutaminate); MES (2-morpholino b acid); N6 (N6 macroelement composition solution); B5 (B5 trace element components solution); AA (AA macroelement composition); Agar (agar).
5.2) solution and culture medium prescription
1) every liter of content of N6 substratum mother liquor (20 times):
2) B5 trace every liter of content of mother liquor (100 times):
3) organic mother liquor:
4) molysite (100 times):
5) AA macroelement mother liquor (every liter of content):
6) evoked callus and subculture medium (every liter of content):
7) rice callus tissue and Agrobacterium are total to culture medium (every liter of content):
8) resistant calli is selected substratum (every liter of content):
9) differentiation culture based formulas (every liter of content):
10) root culture based formulas (every liter of content):
11) culture medium prescription (AAM) (every liter of content) of suspension agroinfection callus group:
12) the YEP liquid culture based formulas (every liter of content) of Agrobacterium growth:
5.3) agriculture bacillus mediated rice conversion
Evoked callus: the rice paddy seed of peeling (14 on a dish) is gone into triangular flask; With volume ratio 70% alcohol immersion 1min (flooding seed); Outwell volume ratio 70% ethanol, soak 30min, clean 5-6 time until limpid with aqua sterilisa then with volume ratio 30% Youxiaolin.Push seed on the filter paper of sterilization with tweezers, suck dry moisture is educated the round-grained rice seventh-seeded to force at last and is placed on the inducing culture, cultivates 20-30d at 30 ℃ of illumination boxs.
Succeeding transfer culture: select the callus that splits away off of the yellow flexible of small rice grain size, transfer to succeeding transfer culture basal growth 7-14d with the tweezers of sterilization.
The preparation of Agrobacterium: picking changes the Agrobacterium EHA105 stoste 20 μ L that the pUbi-LSD2 plasmid is arranged, and is inoculated in 5ml and contains 50mgL
-1Streptomycin sulphate and 50mgL
-1In the YEP of kantlex (Sambrook, the etal. molecular cloning experiment guide .2001) liquid nutrient medium, 28 ℃ of shaken overnight.Get activation bacterium liquid 500 μ L, be inoculated in 5ml and contain in the fresh YEP substratum of identical microbiotic, continue to be cultured to light absorption value (OD600) 0.8-1.0 of bacterium liquid in the 600nm wavelength.
Infect callus and common cultivation: force is educated No. 7 rice callus tissues of round-grained rice from subculture medium, choose and put into centrifuge tube, the quantity amount of callus there was not 50ml centrifuge tube taper position to get final product (callus of selecting yellowish mellow and full flexible).Get cultured Agrobacterium bacterium liquid 1ml in the 1.5ml centrifuge tube, 4 ℃, 5000rpm, centrifugal 1min removes supernatant.With containing 200 μ molL
-1The substratum (AAM) of the 30ml suspension agroinfection callus group of Syringylethanone (As) is processed suspension-s with the thalline of collecting, and this suspension-s is poured in the callus of choosing, and infects 5min.Outwell liquid, callus is taken out, place on the aseptic petridish that contains thieving paper and drain 30-40min.Callus is placed (top pad last layer 9cm aseptic filter paper) on the common culture medium, 25 ℃ of dark cultivations 60 hours.
Washing bacterium and antibiotic-screening cultivates: with callus from taking out the culture medium altogether, with sterilized water clear 5 times, the vibration 5min that does not at every turn stop.Again with containing 500mgL
-1The sterilized water of Pyocianil (car) soaks 40-60min.Place at last and drain 2h on the aseptic filter paper.First round screening: the air dried callus changed over to contain 250mgL
-1Pyocianil (car) and 50mgL
-1Carry out the first time on the selection substratum of Totomycin (Hyg) and select, 30 ℃, illumination cultivation 14d; Second takes turns screening: have the initial callus of resistant calli to forward to length and contain 250mgL
-1Pyocianil (car) and 80mgL
-1On the selection substratum of Totomycin (Hyg), 30 ℃, illumination cultivation 10d transfers to then in the group training chamber and cultivates 4d.
The inducing differentiation and take root of resistant calli: the resistant calli of picking color cadmium yellow moves into and is equipped with in the differentiation jar of division culture medium; Put into the constant temperature culture chamber; Wait for seedling differentiation (about 30d, group training chamber culture condition is 24-30 ℃, and 14h light/8h is dark); Treat that seedling grows to about 5cm, put into root media strong sprout.
The exercise of transgenic seedling and transplanting: shoot root portion and cauline leaf broken up more intactly test tube and choose (seedling grows to the test tube top; Will in time uncap); Open and seal film, add an amount of sterilized water (preventing the long bacterium of substratum), about refining seedling 3d to 7d; Flush away agar is transplanted to the greenhouse and is carried out water planting or earth culture growth, detects then.
5.4) Totomycin rapid detection transgenic seedling obtains T0 for plant
Clip is also collected fresh green blade (otch is all left at two ends) long about seedling 1cm to be detected, lies against to contain Totomycin (80mgL
-1) on the substratum, 30 ℃, 16h/8h (light/dark) cultivates the 48h blade and still keeps the bud green positive plant that is, and the blade of negative seedling occur block downright bad (Zheng Ye. the foundation of rice high efficient transformation system and use .2008).Obtain 26 strain systems of positive T0 plant through hygromycin selection.In May, 2010 to November, Agricultural University Of Nanjing's decorated archway Wembledon tennis open competition chamber carried out the plantation of overexpression material is obtained T1 for seed, utilized a year May in December, 2010-2011 T2 to carry out the cell production experiment for material.
5.5) Molecular Identification of OsLSD2 overexpression strain system
Extraction transgenic line not homophyletic is total RNA of blade; The total cDNA of reverse transcription, carry out sxemiquantitative identify (extraction of total RNA, total cDNA's is synthetic; The sxemiquantitative PCR method is with embodiment 1), obtain overexpression outstanding effect O-1, O-2, O-3, O-4 transgenic line (Fig. 2).The obvious strain of several phenotypes of T0 generation system has been carried out the southern copy number identified that the result sees Fig. 3.
5.6) the numerous and physiological measurement of expansion of overexpression strain system
Can find out that by Fig. 4 OsLSD2 overexpression material (O4) is compared the plant height raising with wild-type (force is educated round-grained rice 7), tiller number increases.
The completion of embodiment 3 root paraffin sections
Choose wild-type (force is educated round-grained rice 7) and OsLSD2 overexpression material (O-4) the seedling seminal root (being article one root that sends behind the seed germination) sprouted 5-10 days; Get respectively apart from tip of a root 1-1.5cm; 1.5-2cm two sections roots; The preparation paraffin section, the ventilating tissue that examines under a microscope root system, the result sees Fig. 5.Compare root system ventilating tissue with wild-type (force is educated round-grained rice 7) by the visible OsLSD2 overexpression material (O-4) of Fig. 5 and become flourishing.
The acquisition of embodiment 4 yield datas
The experiment place is Hainan Le Dongxian, and the time is year May in December, 2010-2011, and experiment material is that force is educated No. 7 wild-types of round-grained rice and OsLSD2T2 for transgenic line, and it is following specifically to test implementation process:
1) vernalization: daytime bubble, after the flushing with clean water, evening, wind dried; Second day bubble again after the flushing with clean water, takes in one's arms warming evening.
2) sowing: vernalization is planted after 1 day and is sowed after subdivision shows money or valuables one carries unintentionally.Can not waterflooding, can not waterflooding after the germination, up to 1 leaf 1 heart stage (sowing 10 days).
3) seedling fertilizer is used: fertilising 5 jin of urea/mus on seedling bed during 1 leaf, 1 heart; Tillered 8 jin+7 jin urea/mus of composite fertilizer in 5-6 days afterwards; 15 jin/mu in preceding 3 days urea lifts seedlings;
4) rice transplanting fertilising
Rice transplanting on January 25,60 jin of composite fertilizer/mus of rice transplanting 2-3 days base manure were used day and are appended every mu in weedicide and 15 jin of urea of dressing of turnning green stage nitrogenous fertilizer on February 1, appended every mu in 15 jin of urea of tillering fertilizer nitrogenous fertilizer on February 7.
Note: plantation sub-district and density
6 row are multiply by in 10 strains, spacing in the rows 15cm, and line-spacing 20cm, April 30 gathered in the crops, the individual plant results, the calculating single plant yield of weighing after drying, the result sees table 1 and table 2.Can find out that from table 2 OsLSD2 overexpression material improves 30-60% than wild-type output, the Nitrogen Absorption amount increases than wild-type is obvious.
Table 1.OsLSD2 overexpression material (O-4) and wild-type phenotypic difference
| Phenotype | The strain of OsLSD2 overexpression is O-4 | WT |
| The individual plant tiller number | 10 | 7 |
| Single plant yield (g) | 31.05 | 14.2 |
| Plant total nitrogen (mg) | 948.7 | 189.3 |
The difference of table 2.OsLSD2 overexpression material yield and wild-type
| Strain system | Output (kg) | Output improves per-cent (%) |
| WT | 0.016 | ? |
| O-1 | 0.026 | 60.9％ |
| O-2 | 0.022 | 32.1％ |
| O-3 | 0.023 | 41.5％ |
[0100]?
| O-4 | 0.023 | 41.5％ |
Claims (2)
1. the application of the key gene OsLSD2 of paddy rice control ventilating tissue formation aspect increase paddy rice plant height, raising nitrogen fertilizer for paddy rice utilising efficiency and/or output, the sequence accession number of this gene is AK111759.
2. paddy rice is controlled the application of the formation key protein OsLSD2 of coded product paddy rice ventilating tissue aspect increase paddy rice plant height, raising nitrogen fertilizer for paddy rice utilising efficiency and/or output of the key gene OsLSD2 of ventilating tissue's formation, and the key gene OsLSD2 accession number that wherein said paddy rice is controlled ventilating tissue's formation is AK111759.
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| CN101392257B (en) * | 2008-11-10 | 2013-03-13 | 南京农业大学 | Genetic engineering application of nitrate transport protein genom OsNRT2.3 in Rice |
| EP2258865A1 (en) * | 2009-06-05 | 2010-12-08 | Universitätsklinikum Freiburg | Lysine-specific demethylase 1 (LSD1) is a biomarker for breast cancer |
| CN102260709B (en) * | 2011-07-18 | 2012-10-03 | 南京农业大学 | Use of key gene OsLSD2 associated with formation of rice aerenchyma |
-
2011
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- 2011-12-16 WO PCT/CN2011/084098 patent/WO2013010368A1/en active Application Filing
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| CN102260709A (en) | 2011-11-30 |
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