CN104120137A - Gene OsNAP for regulating and controlling senescence and drought resistant ability of rice leaves and application of gene OsNAP - Google Patents
Gene OsNAP for regulating and controlling senescence and drought resistant ability of rice leaves and application of gene OsNAP Download PDFInfo
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- CN104120137A CN104120137A CN201410362588.5A CN201410362588A CN104120137A CN 104120137 A CN104120137 A CN 104120137A CN 201410362588 A CN201410362588 A CN 201410362588A CN 104120137 A CN104120137 A CN 104120137A
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Abstract
The invention belongs to the field of plant genetic engineering and particularly relates to a gene OsNAP for regulating and controlling the senescence and drought resistant ability of rice leaves and an application of the gene OsNAP. The gene has a nucleotide sequence as shown in SEQ ID NO.1 and a cDNA (Complementary Deoxyribose Nucleic Acid) sequence as shown in SEQ ID NO.2. A protein of the gene has the sequence as shown in SEQ ID NO.3. The gene has a certain expression in tender rice leaves, the expression quantity of the gene is gradually increased along with the increment of aging degrees of the leaves, and the expression activity of the gene is up to the maximum when the aging degrees of the leaves are up to the later period. Through researching gene overexpressed and suppression expressed transgenic rice plants, people find that the leaf aging speed of the overexpressed transgenic rice plant is accelerated, meanwhile, the drought resistance of the overexpressed transgenic rice plant in a seedling stage is remarkably improved, and the leaf aging speed of the suppression expressed transgenic rice plant is remarkably reduced, so that the gene is proved to have the functions of regulating and controlling the senescence of rice leaves and improving the stress resistance.
Description
Technical field
The invention belongs to plant genetic engineering and Rice molecular breeding technical field, be specifically related to gene OsNAP and the application of an adjusting and controlling rice leaf senile and drought-resistant ability.
Background technology
Leaf senile is an only stage which must be passed by leaf development process, and it is accompanied by the variation of leaf color, is reflected in chlorophyllous forfeiture, finally causes the death of blade and comes off.Leaf senile can cause photosynthetic decline, causes the minimizing of carbon assimilation, thereby affects the output of farm crop.Therefore nearly decades, people studys reason and the mechanism of leaf senile generation always, the generation that hope can planned control leaf senile.
NAC transcription factor (
naM,
atAF and
cuC) be distinctive transcription factor in plant, a lot of research shows that they play an important role in the process of regulation and control leaf senile.A NAC gene NAM-B1 in wheat is subject to Senescence manipulation, in regulating grain albumen, in the content of zinc and iron, play a role (Uauy etc., A NAC gene regulating senescence improves grain protein, zinc, and iron content in wheat.Science, 2006,314:1298-1301).ANAC092/AtNAC2/ORE1 is a NAC gene in Arabidopis thaliana, and its expression amount is regulated and controled by leaf senile also, and it can just regulate and control the necrocytosis of age-dependent in Arabidopsis leaf; Oresara1 (ore1) mutant, owing to lacking the function of ANAC092/AtNAC2/ORE1 gene, shows the phenotype of Delaying Leaf-Senescence; ANAC092/AtNAC2/ORE1 also can be expressed by Salt Stress-induced, and the excised leaf of its mutant anac092-1 chlorophyll degradation speed under salt stress is processed declines, and shows the phenotype of Delaying Leaf-Senescence; In the overexpression plant of ANAC092/AtNAC2/ORE1, there are 170 gene activations to express, wherein a lot of genes can be by Salt Stress-induced, illustrate that ANAC092/AtNAC2/ORE1 is at the during Leaf Senescence of the Salt Stress-induced (Oh etc. that play an important role, Identification of three genetic loci controlling leaf senescence in Arabidopsis thaliana.Plant J, 1997,12:527-535; Kim etc., Trifurcate feed-forward regulation of age-dependent cell death involving miR164 in Arabidopsis.Science, 2009,323:1053-1057; Balazadeh etc., A gene regulatory network controlled by the NAC transcription factor ANAC092/AtNAC2/ORE1 during salt-promoted senescence.Plant J, 2010a, 62:250-264; Balazadeh etc., Salt-triggered expression of the ANAC092-dependent senescence regulon in Arabidopsis thaliana.Plant Signal Behav, 2010b, 5:733-735).ORS1 is the homologous gene of ORE1/ANAC092/AtNAC2, and in old and feeble tissue, expression amount is very high and can be by salt and H
2o
2induction, the early ageing of ORS1 overexpression plant leaf, mutant ors1-1 and the RNAi rotaring gene plant blade of ORS1 delay senility simultaneously, illustrate that ORS1 may be at salt and H
2o
2the during Leaf Senescence of induction plays a role (Balazadeh etc., ORS1, an H
2o
2-responsive NAC transcription factor, controls senescence in Arabidopsis thaliana.Mol Plant, 2011,4:346-360).VND-INTERACTING2 in Arabidopis thaliana (VNI2) the NAC transcription factor of also encoding, its expression amount is induced by ABA and leaf senile, by regulation and control RESPONSIVE TO DEHYDRATION (RD) and COLD-REGULATED (COR), mediate salt stress and leaf senile approach (Yang etc., The Arabidopsis NAC transcription factor VNI2integrates abscisic acid signals into leaf senescence via the COR/RD genes.Plant Cell, 2011,23:2155-2168).AtNAP only expresses in old and feeble blade, and its expression amount is along with the aging of blade is risen gradually; Mutant atnap all shows the phenotype of Delaying Leaf-Senescence under self-sow state and dark induction state, there is obvious early ageing phenomenon in the transfer-gen plant of overexpression AtNAP, and the NAP homologous gene of paddy rice, string bean can the complementary Arabidopsis Mutants atnap of allos holds green phenotype; Further research find AtNAP can with the promotor combination of SENESCENCE-ASSOCIATED GENE113 (SAG113), stomatal movement and rate-of-loss of coolant (Guo etc. when forming an ABA-AtNAP-SAG113 protein regulation chain and controlling leaf senile, AtNAP, a NAC family transcription factor, has an important role in leaf senescence.Plant J, 2006,46:601-612; Zhang etc., An abscisic acid-AtNAP transcription factor-SAG113 protein phosphatase 2C regulatory chain for controlling dehydration in senescing Arabidopsis leaves.Plant Physiol, 2012,158:961-969).Although the expression amount of a lot of NAC transcription factors can be raised by aging, their effects in leaf senile are also not very clear, need people and go research.
Paddy rice is one of main food crop of the mankind, in paddy rice, 60% to 100% carbon is all fixing during grain milk, thereby paddy rice can directly affect its output (Murchie etc. at the speed of intra vane aging breeding time, Interactions between senescence and leaf orientation determine in situ patterns of photosynthesis and photoinhibition in field-grown rice.Plant Physiol, 1999,119:553-564).Along with the development of biotechnology, in cultivation, to hold in the strategy of green property paddy rice, it is the measure of an economical and effective and protection of the environment that the means that adopt molecular genetic to regulate and control are carried out Delaying Leaf-Senescence.Therefore, good senescence-associated gene is improved, cultivated the rice varieties of Delaying Leaf-Senescence, for the output that increases paddy rice, had great significance.
Summary of the invention
The object of the invention is to overcome the scarce limit that prior art exists, gene and an application that can regulate and control leaf senile and improve drought resistance is provided, described gene is relevant with NAC transcription factor gene.
The NAC transcription factor that the invention provides a kind of energy adjusting and controlling rice leaf senile and raising drought resistance, this transcription factor gene is OsNAP gene, this gene source is in paddy rice.Its cDNA nucleotide sequence is as shown in 1-1783 position in SEQ ID NO:2; The sequence of its protein is as shown in SEQ ID NO:3.
The structure that the invention still further relates to described energy adjusting and controlling rice leaf senile and the expression vector of the genes involved OsNAP of raising drought resistance, described expression vector includes the nucleotide sequence as shown in 1-1895 position in SEQ ID NO:1.
The invention still further relates to and utilize the described rice leaf aging gene OsNAP expression vector rice transformation host that is correlated with.
OsNAP gene of the present invention can be building up in different expression vectors and bring into play its function, for example:
1), for the ease of genetically modified cell or plant are screened, can transform used carrier, as add the alternative mark (Bar gene, gus gene, GFP gene and Lux gene etc.) of plant;
2), in order to change the expression amount of goal gene OsNAP, can before its transcription initiation Nucleotide, use different promotors, as enhancement type promotor, inducible promoter, constitutive promoter, tissue-specific promoter, developmental stage specificity promoter etc.
3) expression vector with OsNAP gene of the present invention transforms different vegetable cells or tissue, to obtain the transgenic plant of the proterties such as Leaf senescence development change, drought-resistant ability enhancing.Wherein, the host who is converted can be monocotyledons, as paddy rice, corn, wheat and turfgrass etc.; Also can be dicotyledons, as Arabidopis thaliana, willow, soybean and cotton etc., but be not limited to the above species.
4) with the expression vector of OsNAP gene of the present invention except can transforming by Agrobacterium Ti-plasmids mediated method, can also transform different vegetable cells or tissue by methods such as Ri plasmid, plant viral vector, microinjections, and be bred as plant, to obtain the transgenic plant containing purposive shape.
Realizing concrete steps of the present invention is:
(1) structure of overexpression carrier p1301-OsNAP and phenotype analytical
1) take in rice varieties and spend 11 genomic dnas as template, obtain the genomic dna of OsNAP through pcr amplification, its nucleotide sequence is as shown in SEQ ID NO:1.
2) by step 1) in the DNA fragmentation that obtains be connected on plasmid pCAMBIA1301 (being so kind as to give by Australian CAMBIA), obtain p1301-OsNAP carrier, then imported the agrobacterium tumefaciens EHA105 (being so kind as to give by Australian CAMBIA) of agropine type, recycle agriculture bacillus mediated genetic transforming method and will in described carrier Introduced into Rice kind, spend 11, obtain transfer-gen plant.
3) by the method for qRT-PCR, detect the expression amount of OsNAP gene in transfer-gen plant and wild-type.
4) measure chlorophyll content and the Net Photosynthetic Rate of transfer-gen plant and wild-type blade in each in period, observe its phenotype and take pictures, analyze the difference of the old and feeble situation of transgenosis and wild-type plant leaf.
5) wild-type of normal growth to 4 leaf phase and transfer-gen plant are carried out to drought stress processing, observe its phenotype, relatively transgenosis and the resistance difference of wild-type plant to drought stress.
(2) structure of RNAi carrier and phenotype analytical
1) take that to spend the cDNA of 11 old and feeble blade RNA reverse transcriptions in rice varieties be template, through the nonconservative 3 '-UTR of pcr amplification OsNAP region, then be connected respectively to the upper (Yuan etc. of pMCG161 and pDS1301, Mitogen-activated protein kinase OsMPK6 negatively regulates rice disease resistance to bacterial pathogens.Planta, 2007,226:953-960), form pMCG161-OsNAP and pDS1301-OsNAP.Enzyme is cut pMCG161-OsNAP and pDS1301-OsNAP respectively again, and the carrier of the external source of pMCG161-OsNAP and pDS1301-OsNAP is interconnected to form to RNAi carrier.
2) by step 1) in the RNAi carrier that obtains import the agrobacterium tumefaciens EHA105 of agropine type, recycle agriculture bacillus mediated genetic transforming method and will in described carrier Introduced into Rice kind, spend 11, obtain transfer-gen plant.
3) measure the chlorophyll content of transfer-gen plant blade in each in period, observe its phenotype and take pictures, analyze the difference of the old and feeble situation of transgenosis and wild-type plant leaf.
4) with the expression amount of OsNAP and old and feeble Marker gene in the method detection transfer-gen plant of qRT-PCR, analyze their expression amount difference in transgenosis and wild-type plant leaf.
The invention has the advantages that:
(1) the present invention has identified a leaf senile correlation gene OsNAP, can be used as the Marker gene of studying leaf senile in paddy rice, for follow-up investigator provides reference.
(2) in the present invention, identify the method for leaf senile correlation gene, can be follow-up investigator reference is provided.
(3) transformed plant of the change Leaf senescence development that the present invention obtains provides new resource for genetically engineered and molecular breeding.
(4) the overexpression transformed plant that the present invention obtains can improve drought-resistant ability to a certain extent, for Rice Drought Resistence improvement provides new resource.
(5) expression vector with OsNAP gene of the present invention can transform different vegetable cells or tissue, to obtain the transgenic plant of the proterties such as senescence process change, drought-resistant ability enhancing.
Accompanying drawing explanation
Sequence table SEQ ID NO:1 is the nucleotide sequence of the present invention's OsNAP gene of cloning, and sequence length is 1895 bp.
Sequence table SEQ ID NO:2 is the full length cDNA sequence of the present invention's OsNAP gene of cloning; Length is 1783 bp; At the 172-1350 of this sequence bp section, be this gene CDS district, length is 1179 bp.
Sequence table SEQ ID NO:3 is the protein sequence of OsNAP gene, 392 amino acid of encoding.
Fig. 1: the collection of illustrative plates that is the initial carrier pCAMBIA1301 that is purchased of the present invention.
Fig. 2: be recombinant vectors prepared by the present invention, i.e. the design of graphics of expression vector p1301-OsNAP.Description of symbols in figure: LB, T-DNA left margin; Hyg, hygromycin phosphotransferase gene; 35S, CaMV 35S promoter; RB, T-DNA left and right sides circle.
Fig. 3: the design of graphics that is the OsNAP RNAi carrier prepared of the present invention.Description of symbols in figure: LB, T-DNA left margin; Hyg, hygromycin phosphotransferase gene; 35S, CaMV 35S promoter; Intron, paddy rice Waxy gene intron; GUS, beta-glucosiduronatase gene; RB, T-DNA left and right sides circle.
Fig. 4: be method with the qRT-PCR detected result to the expression amount of OsNAP gene in wild-type plant different times blade.Description of symbols in figure: SL1: the sword-like leave in boot stage; SL2: the sword-like leave in flowering period; SL3: the sword-like leave of milk stage; SL4: the sword-like leave of the stage of yellow ripeness.
Fig. 5: be method with the qRT-PCR detected result to the expression amount of OsNAP in wild-type (non-transgenic) and OsNAP gene overexpression transfer-gen plant.Description of symbols in figure: a figure in Fig. 5 and the RNA sample in b figure come from respectively transgenosis family line15, line17 and line19 and wild-type seedling leaf and the sword-like leave in ripe later stage.
Fig. 6: demonstration be the analysis of OsNAP overexpression transgenic rice plant leaf senile phenotype.Mark in figure: a in Fig. 6 figure and b scheme to represent respectively OsNAP overexpression transgenic paddy rice family and the wild-type rice plant photo of sword-like leave when 30 d and 40 d after heading respectively.
Fig. 7: be the sword-like leave of OsNAP overexpression transgenic paddy rice family and wild-type rice plant after heading each in period chlorophyll content mensuration.Error line represents the standard deviation repeating 4 times; " * " represents that the P value of t test is less than 0.05, significant difference; " * * " represents that the P value of t test is less than 0.01, and difference is extremely remarkable.
Fig. 8: be the sword-like leave of OsNAP overexpression transgenic paddy rice family and wild-type rice plant after heading each in period Net Photosynthetic Rate mensuration.Error line represents the standard deviation repeating 4 times; " * " represents that the P value of t test is less than 0.05, significant difference; " * * " represents that the P value of t test is less than 0.01, and difference is extremely remarkable.
Fig. 9: the phenotype analytical situation that is OsNAP RNAi transgenic rice plant of the present invention.Mark in figure: a figure in Fig. 9 represents the OsNAP RNAi transgenosis family (RNAi-1 and RNAi-2) of rear 50 d of heading and the whole strain photo of wild-type plant.B figure in Fig. 9 and c figure be the rear wild-type plant of 50 d of heading and the sword-like leave of OsNAP RNAi transgenosis family (RNAi-1 and RNAi-2) and the two leaf photos that fall respectively.
Figure 10: be the sword-like leave of transgenic paddy rice family RNAi-1 of the present invention, RNAi-2 and wild-type paddy rice and the mensuration of chlorophyll content of two leaves of falling.Error line represents the standard deviation repeating 3 times; " * * " represents that the P value of t test is less than 0.01, and difference is extremely remarkable.
Figure 11: be method with the qRT-PCR detected result to the expression amount of OsNAP and OsDOS in wild-type (non-transgenic) and OsNAP RNAi transgenic paddy rice family (RNAi-1 and RNAi-2) sword-like leave.
Figure 12: demonstration be wild-type (non-transgenic) and OsNAP overexpression transfer-gen plant Drought at seedling stage disposition.Description of symbols in figure: after drought stress rehydration, the survival rate of high expression level amount family line17 and line19 is significantly higher than low expression amount family line15 and wild-type contrast.
Embodiment
Embodiment 1: the acquisition of candidate segment
Utilize information biology website NCBI (http://www.ncbi.nlm.nih.gov/) software BlastP (Gish etc., Identification of protein coding regions by database similarity search.Nature Genet.1993,3:266-272) search Arabidopsis leaf senescence-specific albumin A tNAP is at the homologous sequence of rice genome, applicant be take the protein sequence of AtNAP and is basis, the NAC albumen of search and its homology in the full genome of paddy rice, obtain its homologous protein OsNAP, TIGR number is LOC_Os03g21060.Utilize its No. LOC search in TIGR (http://rice.plantbiology.msu.edu), obtain CDS, genome (its nucleotide sequence is as shown in sequence table SEQ ID NO:1) and the protein sequence (its sequence is as shown in sequence table SEQ ID NO:3) of OsNAP.Utilize the CDS sequence of OsNAP to compare in KOME (http://cdna01.dna.affrc.go.jp/cDNA/), obtain the cDNA sequence (its cDNA sequence is as shown in SEQ ID NO:2) of OsNAP.
Take that to spend 11 (from Institute of Crop Science, Chinese Academy of Agricultural Science) genomic dna in paddy rice be template, through the genomic dna of pcr amplification OsNAP.The DNA sequence dna of the primer of this fragment that increases is: forward primer F:5 '-ATCGggtcaccTTCGCCATGTGCAATTATGT-3 '; Reverse primer R:5 '-ATCGccatggCAGGGAGGTGTGTGTTGTGT-3 '.PCR response procedures: 94 ℃ of 5 min; 94 ℃ of 1 min, 58 ℃ of 3 min, 72 ℃ of 1 min, 30 circulations; 72 ℃ of 7 min.Reclaim after PCR product, be connected into pGEM-T carrier (purchased from Promega company); To connect product and transform bacillus coli DH 5 alpha competent cell, by resistance screening, enzyme, cut and the detection of checking order (for ordinary method), obtain the positive TA clone of OsNAP genomic dna.Positive TA clone is checked order (for ordinary method), and result shows, this positive TA clone is containing just like the nucleotide sequence shown in SEQ ID NO:1.
At middle 11 different development stages of spending, get blade extracted total RNA (SL1: the sword-like leave in boot stage; SL2: the sword-like leave in flowering period; SL3: the sword-like leave of milk stage; SL4: the sword-like leave of the stage of yellow ripeness), extract total RNA method according to the reagent specification sheets of this Trizol (Invitrogen company).The total RNA of 3 μ g processes 15 min by 1 units RNase-free DNase I (purchased from Invitrogen company) and pollutes to remove genomic dna, then adds 0.5 mg Oligo (dT)
15, 1 mM dNTPs, 10 mM dithiothreitol, 200 units SuperScript
tMiII ThermoScript II (purchased from Invitrogen company) is carried out reverse transcription, final volume 20 μ l.After 50 ℃ of water-bath 1 h, add 20 μ l ddH
2o dilutes reverse transcription product.QRT-PCR adopts the test kit of SYBR Green I (purchased from TaKaRa company) to carry out on Applied Biosystems 7500 Real-Time PCR System (purchased from Applied Biosystems company), and each sample of every secondary response arranges 3 repetitions.Response procedures: 95 ℃ of 10 s; 95 ℃ of 5 s, 60 ℃ of 36 s, 40 circulations.The Actin1 of usining judges that as interior mark OsNAP is at the middle expression of spending 11 different development stages.The qRT-PCR primer sequence of OsNAP gene is: forward primer F:5 '-aaccatttcatcgcgaacaac-3 '; Reverse primer R:5 '-cagtgacgatccctgcaagg-3 '.The qRT-PCR primer sequence of Actin1 gene is: forward primer F:5 '-tggcatctctcagcacattcc-3 '; Reverse primer R:5 '-tgcacaatggatgggtcaga-3 '.Result shows: along with the aging of blade, the expression amount of OsNAP gene rises (Fig. 4) gradually, and explanation is leaf senile correlation gene of OsNAP gene.
The structure of embodiment 2:OsNAP gene inhibition and overexpression carrier
(1) structure of overexpression carrier p1301-OsNAP:
1) by containing positive TA clone Nco I and the BstE II double digestion of OsNAP genomic dna in embodiment 1, be connected with the carrier pCAMBIA1301 skeleton fragment through Nco I and BstE II double digestion.PCAMBIA1301 carrier figure as shown in Figure 1.
2) by step 1) in connection product transform bacillus coli DH 5 alpha competent cell.
3) by resistance screening and enzyme, cut and detect confirmation, recombinant vectors p1301-OsNAP has inserted the nucleotide sequence as shown in SEQ ID NO:1 between the Nco I of carrier pCAMBIA1301 and BstE II site, and the T-DNA plot structure schematic diagram of recombinant vectors p1301-OsNAP as shown in Figure 2.
(2) structure of RNAi carrier:
1) get the middle blade of spending 11 agings, utilize Trizol reagent (purchased from Invitrogen company) extracted total RNA (extracting method is with reference to the operational manual of Trizol reagent).The total RNA of 3 μ g processes 15 min by 1 units RNase-free DNase I (purchased from Invitrogen company) and pollutes to remove genomic dna, then adds 0.5 mg Oligo (dT)
15, 1 mM dNTPs, 10mM dithiothreitol, 200 units SuperScript
tMiII ThermoScript II (purchased from Invitrogen company) is carried out reverse transcription, final volume 20 μ l.After 50 ℃ of water-bath 1 h, add 20 μ l ddH
2o dilutes reverse transcription product.
2) take that middle to spend the cDNA of 11 old and feeble blade RNA reverse transcriptions be template, through the nonconservative 3 '-UTR of pcr amplification OsNAP region.PCR response procedures: 94 ℃ of 5 min; 94 ℃ of 30 s, 58 ℃ of 30 s, 72 ℃ of 30 s, 30 circulations; 72 ℃ of 7 min.Primer sequence is: forward primer F:5 '-ATCggatccCCACCACCAACAACAACAAC-3 '; Reverse primer R:5 '-ATCggtaccCTCAGTCCCAGTGACGATCC-3 '.
3) reclaim after PCR product, be connected into pGEM-T carrier (purchased from Promega company).
4) will connect product and transform bacillus coli DH 5 alpha competent cell, by resistance screening, enzyme, cut and the detection of checking order (for ordinary method), obtain the positive TA clone who contains the nonconservative 3 '-UTR of OsNAP region.
5) positive TA clone being carried out to enzyme by Kpn I and BamH I cuts, then be connected respectively to the upper (Yuan etc. of pMCG161 and pDS1301, Mitogen-activated protein kinase OsMPK6 negatively regulates rice disease resistance to bacterial pathogens.Planta, 2007,226:953-960), form pMCG161-OsNAP and pDS1301-OsNAP.Again with Sac I and Spe I respectively enzyme cut pMCG161-OsNAP and pDS1301-OsNAP, the carrier of the external source of pMCG161-OsNAP and pDS1301-OsNAP is interconnected to form to RNAi carrier, the T-DNA plot structure schematic diagram of recombinant vectors as shown in Figure 3.
Embodiment 3: the conversion of expression vector
The above-mentioned expression vector building is imported to the agrobacterium tumefaciens EHA105 of agropine type, form and transform bacterial strain.Method shown in " agriculture bacillus mediated genetic transformation operational manual " that agriculture bacillus mediated genetic transforming method is mainly delivered with reference to the applicant's State Key Laboratory of Crop Genetic Improvent (support the army etc. by woods, the foundation of the agriculture bacillus mediated No. 8 high-efficient transgenic systems in Mudanjiang, Acta Agronomica Sinica, 2002,28:294-300).Concrete steps are as follows:
(1) reagent and solution abbreviation
In the present invention, the abbreviation of substratum plant hormone used is expressed as follows: 6-BA (6-BenzylaminoPurine, 6-benzyladenine); CN (Carbenicillin, Pyocianil); KT (Kinetin, kinetin); NAA (Napthalene acetic acid, naphthylacetic acid); IAA (Indole-3-acetic acid, indolylacetic acid); 2,4-D (2,4-Dichlorophenoxyacetic acid, 2,4 dichlorophenoxyacetic acid); AS (Acetosringone, Syringylethanone); CH (Casein Enzymatic Hydrolysate, caseinhydrolysate); HN (Hygromycin B, Totomycin); DMSO (Dimethyl Sulfoxide, dimethyl sulfoxide (DMSO)); N6max (N6 macroelement composition solution); N6mix (N6 Trace Elements solution); MSmax (MS macroelement composition solution); MSmix (MS Trace Elements solution).
(2) main solution formula
1) N6 substratum macroelement mother liquor (according to the preparation of 10 * concentrated solution):
Mentioned reagent is dissolved one by one, then under room temperature, with distilled water, be settled to 1000 ml.
2) N6 substratum trace element mother liquor (according to the preparation of 100 * concentrated solution)
Mentioned reagent is at room temperature dissolved and be settled to 1000 ml with distilled water.
3) molysite (Fe
2+eDTA) stock solution (according to the preparation of 100 * concentrated solution)
By 3.73 g b diammonium disodium edta (Na
2eDTA2H
2o) and 2.78 g FeSO
47H
2o dissolves respectively, mixes and is settled to 1000 ml with distilled water, to 70 ℃ of temperature, bathes 2 h, and 4 ℃ save backup.
4) VITAMIN stock solution (according to the preparation of 100 * concentrated solution)
Adding distil water is settled to 1000 ml, and 4 ℃ save backup.
5) MS substratum macroelement mother liquor (according to the preparation of 10 * concentrated solution)
Mentioned reagent is at room temperature dissolved, and be settled to 1000 ml with distilled water.
6) MS substratum trace element mother liquor (according to the preparation of 100 * concentrated solution)
Mentioned reagent is at room temperature dissolved, and be settled to 1000 ml with distilled water.
7) 2, the preparation of 4-D stock solution (1 mg/ml):
Take 2,4-D, 100 mg, with 1 ml 1 N potassium hydroxide, dissolve 5 min, then add after 10 ml distilled water dissolve completely and be settled to 100 ml, under room temperature, preserve.
8) preparation of 6-BA stock solution (1 mg/ml):
Take 6-BA 100 mg, with 1 ml 1 N potassium hydroxide, dissolve 5 min, then add after 10 ml distilled water dissolve completely and be settled to 100 ml, room temperature preservation.
9) preparation of naphthylacetic acid (NAA) stock solution (1 mg/ml):
Take NAA 100 mg, with 1 ml 1 N potassium hydroxide, dissolve 5 min, then add after 10 ml distilled water dissolve completely and be settled to 100 ml, 4 ℃ save backup.
10) preparation of indolylacetic acid (IAA) stock solution (1 mg/ml):
Take IAA 100 mg, with 1 ml 1 N potassium hydroxide, dissolve 5 min, then add after 10 ml distilled water dissolve completely and be settled to 100 ml, 4 ℃ save backup.
11) preparation of glucose stock solution (0.5 g/ml):
Take glucose 125 g, then with distilled water, dissolve and be settled to 250 ml, after sterilizing, 4 ℃ save backup.
12) preparation of AS stock solution:
Take AS 0.392 g, add DMSO 10 ml to dissolve, divide and be filled in 1.5 ml centrifuge tubes, 4 ℃ save backup.
13) 1N potassium hydroxide stock solution
Take potassium hydroxide 5.6 g, with distilled water, dissolve and be settled to 100 ml, room temperature preservation is standby.
(3) for the culture medium prescription of rice transformation
1) inducing culture
Adding distil water to 900 ml, 1N potassium hydroxide regulates pH value to 5.9, boil and be settled to 1000 ml, divide and install to 50 ml triangular flasks (25 ml/ bottle), sterilizing according to a conventional method after sealing (for example sterilizing 25 min at 121 ℃, following medium sterilization method is identical with the sterilising method of basal culture medium).
2) subculture medium
Adding distil water to 900 ml, 1N potassium hydroxide regulates pH value to 5.9, boils and is settled to 1000 ml, divides and installs to 50 ml triangular flasks (25 ml/ bottle), sealing, sterilizing as stated above.
3) pre-culture medium
Adding distil water to 250 ml, 1N potassium hydroxide regulates pH value to 5.6, sealing, sterilizing as stated above.
Use front heating for dissolving substratum and add 5 ml glucose stock solutions and 250 μ l AS stock solutions, (25 ml/ ware) in culture dish poured in packing into.
4) be total to substratum
Adding distil water to 250 ml, 1N potassium hydroxide regulates pH value to 5.6, sealing, sterilizing as stated above.
Use front heating for dissolving substratum and add 5 ml glucose stock solutions and 250 μ l AS stock solutions, (the every ware of 25 ml/) in culture dish poured in packing into.
5) suspension medium
Adding distil water to 100 ml, adjust pH to 5.4, divides and installs in the triangular flask of two 100 ml, sealing, sterilizing as stated above.
Before use, add the aseptic glucose stock solution of 1 ml and 100 μ l AS stock solutions.
6) select substratum
Adding distil water to 250 ml, regulates pH value to 6.0, sealing, sterilizing as stated above.
Before using, dissolve substratum, add 250 μ l HN (50 mg/ml) and 400 μ l CN (250 mg/ml) packing to pour (25 ml/ ware) in culture dish into.(note: selecting for the first time substratum Pyocianil concentration is 400 mg/l, selecting for the second time and later substratum Pyocianil concentration is 250 mg/l).
7) division culture medium
Adding distil water to 900 ml, 1 N potassium hydroxide regulates pH value to 6.0.Boil and be settled to 1000 ml with distilled water, dividing and install to 50 ml triangular flasks (50 ml/ bottle), sealing, sterilizing as stated above.
8) root media
Adding distil water to 900 ml, regulates pH value to 5.8 with 1N potassium hydroxide.Boil and be settled to 1000 ml with distilled water, dividing to install to and take root (25 ml/ pipe left and right) in pipe, sealing, sterilizing as stated above.
(4) agriculture bacillus mediated genetic transformation step
1) callus of induce
To in maturation, spend 11 rice paddy seeds to shell, 70% Ethanol Treatment 1 min successively then, 0.15% mercury chloride (HgCl
2) solution-treated 15 min, aqua sterilisa is transferred to seed on inducing culture after cleaning 4-5 time.By the dark cultivation of postvaccinal substratum 4 weeks, 26 ℃ of left and right of temperature.
2) callus subculture
Select the embryo callus subculture of glassy yellow, consolidation and relatively dry, be put in dark cultivation 2 weeks on subculture medium, 26 ℃ of left and right of temperature.
3) preculture
Select the embryo callus subculture of consolidation and relatively dry, be put in dark cultivation 2 weeks on pre-culture medium, 26 ℃ of left and right of temperature.
4) Agrobacterium is cultivated
First at the LA substratum of selecting with corresponding resistance, (preparation of LA substratum is with reference to J. Pehanorm Brooker etc., molecular cloning experiment guide, the third edition, Jin Dongyan etc. (translating), Science Press, 2002, Beijing) go up preculture Agrobacterium EHA105 (agrobacterium strains that this bacterial strain is openly used from CAMBIA company) 2 days, 28 ℃ of temperature; Subsequently, Agrobacterium is transferred in suspension medium, on 28 ℃ of shaking tables, cultivates 2-3 h.
5) Agrobacterium is infected
First regulate agrobacterium suspension to OD
600value for 0.8-1.0, is transferred to pre-incubated callus subsequently in agrobacterium suspension and soaks 30 min, callus is transferred on the filter paper that sterilizing is good and blots, and is then placed on and on common substratum, cultivates 3d, temperature 19-20 ℃.
6) callus washing and selection are cultivated
Aqua sterilisa washing callus is to cannot see Agrobacterium; Be immersed in containing 30 min in the aqua sterilisa of 400 mg/l CN; Shift callus blots to the good filter paper of sterilizing; Shift callus to selecting selection on substratum to cultivate 2-3 time, each 2 weeks.
7) differentiation
Kanamycin-resistant callus tissue is transferred on pre-division culture medium in dark place cultivation 5-7 d; Shift the callus of pre-differentiation culture to division culture medium, 30-40 d, 26 ℃ of culture temperature are cultivated in illumination (1500-2000 lx).
8) take root
Cut the root that differentiation phase produces, then transfer them to illumination in root media (1500-2000 lx) and cultivate 2-3 week, 26 ℃ of culture temperature.
9) transplant
Wash the residual substratum on root off, the seedling with good root system is proceeded to greenhouse.
Embodiment 4: the biometric authentication analysis of overexpression transgenic progeny adjusting and controlling rice Leaf senescence development
(1) applicant is first at paddy rice wild-type (non-transgenic) and T
2seedling stage of the OsNAP overexpression transfer-gen plant (line15, line17 and line19) in generation with the method validation of qRT-PCR the expression amount of OsNAP gene.Result shows: compare with wild-type, the expression amount of OsNAP is very high in line17 and line19, and little (a figure in Fig. 5) rises in line15.In the ripening stage, detected the expression amount of OsNAP gene in transfer-gen plant subsequently.Result shows: compare with wild-type, in line15, line17 and line19, the expression amount rising multiple of OsNAP gene is little, but trend identical with seedling stage (the b figure in Fig. 5).The expression amount that further illustrates OsNAP gene rises with the aging of blade.
(2) Phenotypic Observation
In heading in the time of latter 30 days, the blade tip flavescence of high expression level transgenosis family line17 and line19 sword-like leave, and low expression amount family line15 and wild-type are still green (a figure in Fig. 6).After heading, during 40 d, high expression level transgenosis family line17 and line19 sword-like leave blade are obviously than the blade of line15 and wild-type yellow (the b figure in Fig. 6).
(3) mensuration of chlorophyll content
The mensuration of chlorophyll content adopts acetone spectrophotometer method.Get approximately 0.04 g paddy rice wild-type (non-transgenic) and T
2the OsNAP overexpression transfer-gen plant (line15 in generation, line17 and line19) in chlorophyll extract, (extract is acetone to leaf sample: ethanol: water volume ratio=4.5:4.5:1), 4 ℃ of extractings are spent the night, after all bleaching, blade use spectrophotometer at the light absorption value of 663 nm and 645 nm place working samples, chlorophyll content calculates (Mao etc. by literature method, Two complementary recessive genes in duplicated segments control etiolation in rice.Theor Appl Genet, 2011, 122:373-383), with mg/g leaf fresh weight, represent.Result shows: after heading, during 20 d left and right, the chlorophyll content of high expression level transgenosis family line17 and line19 sword-like leave is compared and be there is no notable difference with wild-type with low express transgenic family line15; But during 30 d left and right, the chlorophyll content of line17 and line19 sword-like leave is starkly lower than line15 and wild-type after heading, after heading during 40 d left and right more obvious (Fig. 7).
(4) mensuration of Net Photosynthetic Rate
Applicant measures paddy rice wild-type (non-transgenic) and T with photosynthetic determinator CIRAS-2 (purchased from PP system company)
2the Net Photosynthetic Rate of OsNAP overexpression transfer-gen plant (line15, line17 and line19) the different development stage blade in generation, minute be the morning 9:00 to 11:00 or afternoon 3:00 to 5:00.Result shows: after heading, during 20 d left and right, the Net Photosynthetic Rate of high expression level transgenosis family line17 and line19 sword-like leave is compared and be there is no notable difference with wild-type with low express transgenic family line15; But during 30 d left and right, the Net Photosynthetic Rate of line17 and line19 sword-like leave is starkly lower than line15 and wild-type after heading, after heading during 40 d left and right more obvious (Fig. 8).Illustrate that the overexpression plant of OsNAP has accelerated the aging of blade.
The biometric authentication analysis of embodiment 5:RNAi transgenic progeny adjusting and controlling rice Leaf senescence development
(1) Phenotypic Observation
When wild-type plant leaf show obvious when yellow, the blade major part of transgenosis family RNAi-1 and RNAi-2 be all green (in Fig. 9 a).After heading during 50 d, the RNAi transgenosis family RNAi-1 of OsNAP and the Senescence of Flag Leaf of RNAi-2 obviously delay (b in Fig. 9), the two flavescence of leaf major part of wild-type simultaneously, and the blade of RNAi transgenosis family only has most advanced and sophisticated flavescence (c in Fig. 9).
(2) mensuration of chlorophyll content
The mensuration of chlorophyll content is shown in embodiment 4.Sample comes from the blade of the RNAi transgenosis family (RNAi-1 and RNAi-2) of paddy rice wild-type (non-transgenic) and OsNAP.Result shows: the chlorophyll content of 2 RNAi transgenosis familys (RNAi-1 and RNAi-2) blade is apparently higher than wild-type (Figure 10).Illustrate that the RNAi transfer-gen plant of OsNAP has delayed the aging of blade.
(3) applicant has detected the variation of OsNAP and OsDOS expression amount by the method for qRT-PCR in RNAi transgenosis family (RNAi-1 and RNAi-2).Result shows: compare with wild-type, in RNAi-1 and RNAi-2, the expression amount of OsNAP obviously declines; And in the blade of RNAi-1 and RNAi-2 OsDOS expression amount apparently higher than wild-type (Figure 11), the expression amount of OsNAP is lower, the chlorophyll content of sword-like leave and two leaves is higher, and OsDOS expression amount is also higher simultaneously, illustrates that the RNAi transfer-gen plant of OsNAP has delayed the aging of blade.The qRT-PCR primer sequence of OsDOS gene is: forward primer F:5 '-ATGATGATGATGGGGGAAGG-3 '; Reverse primer R:5 '-CTCACGGGGAGGTGAGACC-3 '.
Embodiment 6: the test of transfer-gen plant drought resistance of seedling
Overexpression family line15, line17 and line19 are germinateed at 1/2 MS substratum (root media in embodiment 3), after one week, seedling kind is entered in little red bucket.Plant grew to for 4 leaf phases under normal growth condition, drought stress 7 d that cut off the water supply, and then rehydration 3 d, observe phenotype, and take pictures.Result shows: drought stress 7 d cut off the water supply, after rehydration 3 d, in wild-type plant, spend after 11 yellow leafs witheredly, low expression amount family line15 blade also has a little green, and the blade major part of high expression level amount family line17 and line19 is all green (Figure 12).The overexpression transfer-gen plant that OsNAP is described can strengthen drought-resistant ability.
Claims (7)
1. OsNAP gene that derives from paddy rice application in regulation and control Leaf senescence development and drought-resistant ability, is characterized in that, described gene is one of following sequence:
1) genome nucleotide sequence shown in SEQ ID NO:1;
2) the full-length cDNA nucleotide sequence shown in SEQ ID NO:2.
2. the application of the proteins encoded of an OsNAP gene that derives from paddy rice in regulation and control Leaf senescence development and drought-resistant ability, is characterized in that, the sequence of described DNA encoding the protein is as shown in sequence table SEQ ID NO:3.
3. the application of gene OsNAP as claimed in claim 1, it is characterized in that, by containing the expression vector of gene OsNAP described in claim 1, proceed to paddy rice, thereby with this gene speeding-up blade senescence process of overexpression and/or raising Seedling drought-resistance ability, thereby also comprise by suppressing the expression Delaying Leaf-Senescence process of this gene.
4. a method that changes target plant Leaf senescence development, it is characterized in that, by improving the expression amount of gene OsNAP described in claim 1, carry out speeding-up blade senescence process or carry out Delaying Leaf-Senescence process by lowering the expression amount of gene OsNAP described in claim 1.
5. method as claimed in claim 4, is characterized in that, described target plant is monocotyledons or dicotyledons.
6. the method as described in claim 4 or 5, is characterized in that, described monocotyledons is paddy rice, corn, wheat and turfgrass.
7. the method as described in claim 4 or 5, is characterized in that, described dicotyledons is Arabidopis thaliana, willow, soybean and cotton.
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| CN108103075A (en) * | 2018-02-01 | 2018-06-01 | 南京农业大学 | A kind of switchgrass gene PvC3H29 for delaying plant senescence and its application |
| CN108605732A (en) * | 2017-01-06 | 2018-10-02 | 邵建平 | The living environment of Hybrid Rice late growth stage and sword-like leave early ageing and its comprehensive treatment |
| CN109134634A (en) * | 2018-11-07 | 2019-01-04 | 河北师范大学 | Plant senescence GAP-associated protein GAP and its encoding gene and application |
| CN111454987A (en) * | 2020-04-29 | 2020-07-28 | 河南大学 | Application of GhNAC091 gene in improving plant photosynthesis efficiency and strong light tolerance |
| CN116589545A (en) * | 2023-03-27 | 2023-08-15 | 华中农业大学 | Application of ONAC096 gene in controlling drought resistance of rice |
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| CN108605732A (en) * | 2017-01-06 | 2018-10-02 | 邵建平 | The living environment of Hybrid Rice late growth stage and sword-like leave early ageing and its comprehensive treatment |
| CN108103075A (en) * | 2018-02-01 | 2018-06-01 | 南京农业大学 | A kind of switchgrass gene PvC3H29 for delaying plant senescence and its application |
| CN108103075B (en) * | 2018-02-01 | 2021-07-13 | 南京农业大学 | Switchgrass gene PvC3H29 for delaying plant senescence and application thereof |
| CN108103076A (en) * | 2018-02-02 | 2018-06-01 | 南京农业大学 | A kind of rye grass transcription factor gene LpNACL for inhibiting leaf senile and its application |
| CN108103076B (en) * | 2018-02-02 | 2021-02-09 | 南京农业大学 | Ryegrass transcription factor gene LpNACL for inhibiting leaf senescence and application thereof |
| CN109134634A (en) * | 2018-11-07 | 2019-01-04 | 河北师范大学 | Plant senescence GAP-associated protein GAP and its encoding gene and application |
| CN111454987A (en) * | 2020-04-29 | 2020-07-28 | 河南大学 | Application of GhNAC091 gene in improving plant photosynthesis efficiency and strong light tolerance |
| CN111454987B (en) * | 2020-04-29 | 2021-12-07 | 河南大学 | Application of GhNAC091 gene in improving plant photosynthesis efficiency and strong light tolerance |
| CN116589545A (en) * | 2023-03-27 | 2023-08-15 | 华中农业大学 | Application of ONAC096 gene in controlling drought resistance of rice |
| CN116589545B (en) * | 2023-03-27 | 2024-04-02 | 华中农业大学 | Application of ONAC096 gene in controlling drought resistance of rice |
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