CN103320468A - UCH320 protein and application of coding gene thereof in adjusting and controlling plant growth and development - Google Patents
UCH320 protein and application of coding gene thereof in adjusting and controlling plant growth and development Download PDFInfo
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- CN103320468A CN103320468A CN201310261912XA CN201310261912A CN103320468A CN 103320468 A CN103320468 A CN 103320468A CN 201310261912X A CN201310261912X A CN 201310261912XA CN 201310261912 A CN201310261912 A CN 201310261912A CN 103320468 A CN103320468 A CN 103320468A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
- A01N37/44—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a nitrogen atom attached to the same carbon skeleton by a single or double bond, this nitrogen atom not being a member of a derivative or of a thio analogue of a carboxylic group, e.g. amino-carboxylic acids
- A01N37/46—N-acyl derivatives
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
- A01N65/40—Liliopsida [monocotyledons]
- A01N65/44—Poaceae or Gramineae [Grass family], e.g. bamboo, lemon grass or citronella grass
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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
Abstract
The invention discloses UCH320 protein and an application of encoding code thereof in adjusting and controlling plant growth and development. The application provided by the invention is an application of protein consisting of amino acid sequences shown by a sequence 1 in a sequence table or coding genes thereof in adjusting and controlling the plant growth and development. The plant growth and development is specifically embodied in at least one of (1) and (2): (1) the fructification rate of the seed; (2) the weight of the seed. Proven by experiment, in the development process of rice, the expression level of UCH320 protein in the rice is reduced by an RNA (Ribonucleic Acid) interference technology, the rice seed can be caused to show the following phenotype: compared with the wild type rice seed, the fructification rate is reduced, the fertile seed is also long and thin, and the weight of the seed is also obviously reduced. The invention lays foundation for finding out simpler ideas and methods for creating crops with high-yield characters.
Description
Technical field
The invention belongs to the molecular biology of plants technical field, relate to a kind of UCH320 albumen and the application of encoding gene in regulating growth of plants thereof.
Background technology
Since the seventies in last century, heterosis utilization is that China's Rice Production has been made great contribution.Face China's Economic development proposes " high yield, high-quality, efficient, safety, ecology " to agriculture production new great demand, can further excavate heterotic application potential with reply, just become the severe challenge of pendulum in face of the contemporary science man.
Heterotic formation is based on two different parents' combination.To on existing basis of using, further excavate its potentiality, strengthening hybrid vigour except needs forms the machine-processed research, also be badly in need of setting up effective means and create male-sterile character, in order to effectively enlarge screening and the outstanding application that is combined in the production of cross combination.
At present, the male-sterile character of widespread use comes from spontaneous mutation and proterties transformation strain thereof more in breeding work and production.The source of male-sterile character is very limited, is to enlarge the serious restrictive factor that the cross combination screening is particularly used.According to the rule of the present current international practice, all innovations with application potential all are subject to intellectual property protection.Therefore, seek thinking and the method for initiative Artificial Control crop seed size new, that have independent intellectual property right and output, become one of the key issue that can't avoid, need to be resolved hurrily that the countries and regions of wish to hold excavating hybrid vigour application potential initiative face.
For a long time, people utilize the method seed selection hybrid crop of conventional breeding always, and these are long in method cycles, it is slow to take effect, can not satisfy production development in the urgent need to.Gene engineering method is compared traditional method and had Some features: the seed selection cycle shortens, and fertility is relatively stable, and is affected by environment little, genotype is relied on few, low in the pollution of the environment.
Summary of the invention
The purpose of this invention is to provide a kind of UCH320 albumen and the application of encoding gene in regulating growth of plants thereof.
Application provided by the present invention is specially the protein (called after UCH320 albumen) that is comprised of the aminoacid sequence shown in the sequence in the sequence table 1 or its encoding gene (called after UCH320 gene) at regulating plant following 1)-2) at least a middle application:
1) seed-setting rate;
2) seed weight.
Above-mentioned application is embodied in: the described expression amount of UCH320 albumen in described plant that is comprised of the aminoacid sequence shown in the sequence in the sequence table 1 is lower, and the seed-setting rate of described plant is lower and/or seed weight is lighter; The described expression amount of UCH320 albumen in described plant that is comprised of the aminoacid sequence shown in the sequence in the sequence table 1 is higher, and the drought resistance seed-setting rate of described plant is higher and/or seed weight is heavier.
(UCH320 albumen) or its encoding gene (UCH320 gene) of being comprised of the aminoacid sequence shown in the sequence in the sequence table 1 have following I in seed selection)-II) application at least a plant variety also belongs to protection scope of the present invention in the purpose proterties:
I) the seed-setting rate improves or reduces;
II) seed weight increases or reduces.
In actual applications, when the plant variety of institute's seed selection is the plant variety that the seed-setting rate improves and/or seed weight increases, need the higher plant of described UCH320 expressing quantity is hybridized as the parent.When the plant variety of institute's seed selection is the plant variety that the seed-setting rate reduces and/or seed weight reduces, need the lower plant of described UCH320 expressing quantity is hybridized as the parent.
A further object of the present invention provides a kind of method of cultivating transgenic plant.
The method of cultivation provided by the present invention transgenic plant specifically can be following (A) or (B):
(A) cultivate have following b1)-b2) method of at least a transgenic plant in the purpose proterties, comprise the steps:
A) import the encoding gene of the protein that is formed by the aminoacid sequence shown in the sequence in the sequence table 1 in the purpose plant, obtain expressing the transgenic plant of described encoding gene;
B) from step a) gained transgenic plant, obtain comparing with described purpose plant, have following b1)-b2) at least a transgenic plant in the purpose proterties:
B1) the seed-setting rate improves;
B2) seed weight increases;
(B) cultivate have following d1)-d2) method of at least a transgenic plant in the purpose proterties, comprise the steps:
The encoding gene of the protein that c) in the purpose plant aminoacid sequence shown in the sequence 1 in by sequence table is formed suppresses to express, and obtains transgenic plant;
D) from step c) gained transgenic plant, obtain comparing with described purpose plant, have following d1)-d2) at least a transgenic plant in the purpose proterties:
D1) the seed-setting rate reduces;
D2) seed weight reduces.
In above-mentioned application or method, the encoding gene (UCH320 gene) of the described protein that is comprised of the aminoacid sequence shown in the sequence in the sequence table 1 (UCH320 albumen) is arbitrary described dna molecular in following (1) to (4):
(1) encoding sequence be in the sequence table sequence 2 from the dna molecular shown in 5 ' terminal the 102nd to 791 Nucleotide;
(2) dna molecular shown in the sequence 2 in the sequence table;
(3) the protein DNA molecule that the aminoacid sequence shown in the sequence 1 forms in the dna molecule hybridize that limits with (1) or (2) under stringent condition and the coding sequence table;
(4) dna molecular with the arbitrary restriction in (1)-(3) has the protein DNA molecule that the aminoacid sequence shown in the sequence 1 forms in 90% above homology and the coding sequence table.
Above-mentioned stringent condition can be with 6 * SSC, and the solution of 0.5%SDS 65 ℃ of lower hybridization, is then used 2 * SSC, and 0.1%SDS and 1 * SSC, 0.1%SDS respectively wash film once.
Wherein, sequence 2 is comprised of 974 Nucleotide, is the cDNA sequence of described UCH320 gene, and wherein the 102-791 position is encoding sequence (ORF); Protein shown in the sequence 1 in the tabulation of sequence 2 code sequences, sequence 1 is comprised of 229 amino-acid residues.
In aforesaid method (B), the encoding gene of the described protein that in the purpose plant aminoacid sequence shown in the sequence 1 in by sequence table is formed suppresses to express, and can be any method that reduces the expression of UCH320 gene described in the described purpose plant.
In the present invention, the encoding gene of the described protein that in the purpose plant aminoacid sequence shown in the sequence 1 in by sequence table is formed suppresses to express, and specifically realizes in the described purpose plant by changing over to as shown in the formula the dna fragmentation shown in (I):
SEQ
Forward-X-SEQ
Oppositely(I)
Described SEQ
ForwardThe 14-268 position Nucleotide of sequence 3 in the sequence table;
Described SEQ
OppositelySequence and described SEQ
ForwardThe sequence reverse complemental;
Described X is described SEQ
ForwardWith described SEQ
OppositelyBetween intervening sequence, on sequence, described X and described SEQ
ForwardAnd described SEQ
OppositelyAll not complementary.
In the present invention, the nucleotides sequence of the dna fragmentation shown in the described formula (I) is classified the 14-736 position of the sequence 3 in the sequence table as.
Further, the dna fragmentation shown in the described formula (I) is that form by the rnai expression carrier changes in the described purpose plant; The promotor that the dna fragmentation shown in the described formula of startup (I) is transcribed on the described rnai expression carrier is the Actin promotor.Concrete, described rnai expression carrier is for inserting the recombinant plasmid that obtains behind the RNA interference sequence (sequence 3) of described UCH320 gene at the multiple clone site place of pCAM23A carrier; More specifically, described rnai expression carrier is to prepare according to the method that comprises the steps: with the dna fragmentation shown in the sequence 3 in Spe I and the sequence table of Sal I double digestion, it is isocaudarner with process Xba I(Xba I with SpeI that glue reclaims rear) link to each other with the pCAM23A carrier framework large fragment of Sal I double digestion, obtain described rnai expression carrier.
In aforesaid method (A), the recombinant expression vector of the encoding gene that the encoding gene of the described protein that is comprised of the aminoacid sequence shown in the sequence in the sequence table 1 can be by containing described protein imports in the described purpose plant.
Described recombinant expression vector can be used existing plant expression vector construction.Described plant expression vector comprises the double base agrobacterium vector and can be used for the carrier etc. of plant micropellet bombardment, such as pGreen0029, pCAMBIA3301, pCAMBIA1300, pBI121, pBin19, pCAMBIA2301, pCAMBIA1301-UbiN or other derivative plant expression vector.Described plant expression vector also can comprise 3 ' end untranslated zone of foreign gene, namely comprises the dna fragmentation of polyadenylic acid signal and any other participation mRNA processing or genetic expression.The bootable polyadenylic acid of described polyadenylic acid signal joins 3 ' end of mRNA precursor.When using described gene constructed recombinant expression vector, before its transcription initiation Nucleotide, can add any enhancement type, composing type, organizing specific type or inducible promoter, such as cauliflower mosaic virus (CAMV) 35S promoter, ubiquitin gene Ubiquitin promotor (pUbi), stress induced promoter rd29A etc., they can use separately or be combined with other plant promoter; In addition, when using gene constructed recombinant expression vector of the present invention, also can use enhanser, comprise translational enhancer or transcriptional enhancer, these enhanser zones can be ATG initiator codon or neighboring region initiator codon etc., but must be identical with the reading frame of encoding sequence, to guarantee the correct translation of whole sequence.The source of described translation control signal and initiator codon is widely, can be natural, also can synthesize.Translation initiation region can be from transcription initiation zone or structure gene.For the ease of transgenic plant cells or plant being identified and screening, can process used recombinant expression vector, can produce enzyme or the gene of luminophor, the antibiotic marker thing with resistance or the anti-chemical reagent marker gene etc. of colour-change as adding the coding that in plant, to express.Also can not add any selected marker, directly with adverse circumstance screening transformed plant.
In the method (A) and method (B) of above-mentioned cultivation transgenic plant, import described purpose plant with carrying the described recombinant expression vector of described UCH320 gene or the described rnai expression carrier of described UCH320 gene, specifically can be: Ti-plasmids, Ri plasmid, plant viral vector, directly delivered DNA, microinjection, electricity are led by using, conventional biological method transformed plant cells or the tissue such as agriculture bacillus mediated, and the plant tissue that transforms is cultivated into plant.
In above-mentioned each application or each method, described plant namely can be monocotyledons, also can be dicotyledons.In the present invention, described plant is specially monocotyledon rice, as spending 11 in the rice varieties.
In the present invention, above-mentioned each use or each method described in the seed-setting rate be the percentage that real grain number accounts for total number (real grain number+unfilled gtains number) (reference " and Zhang Yi; Shen Fucheng. the relation of Weighing setting percentage and counting setting percentage. hybrid rice; 2006,21 (2): 64-68 ").
Also belong to protection scope of the present invention as shown in the formula the dna fragmentation shown in (I):
SEQ
Forward-X-SEQ
Oppositely(I)
Described SEQ
ForwardThe 14-268 position Nucleotide of sequence 3 in the sequence table;
Described SEQ
OppositelySequence and described SEQ
ForwardThe sequence reverse complemental;
Described X is described SEQ
ForwardWith described SEQ
OppositelyBetween intervening sequence, on sequence, described X and described SEQ
ForwardAnd described SEQ
OppositelyAll not complementary;
The nucleotide sequence of described dna fragmentation is specially the 14-736 position of the sequence 3 in the sequence table.
The recombinant vectors, recombinant bacterium, expression cassette or the transgenic cell line that contain described dna fragmentation also belong to protection scope of the present invention.
Described recombinant vectors both can be recombinant expression vector, also can be recombinant cloning vector.In one embodiment of the invention, the promotor that the described RNA interference sequence of startup is transcribed in the described recombinant expression vector is the Actin promotor, concrete, described recombinant expression vector is for inserting the recombinant plasmid that obtains behind the RNA interference sequence (sequence 3) of described UCH320 gene at the multiple clone site place of pCAM23A carrier; More specifically, described rnai expression carrier is to prepare according to the method that comprises the steps: with the dna fragmentation shown in the sequence 3 in Spe I and the sequence table of Sal I double digestion, it is isocaudarner with process Xba I(Xba I with Spe I that glue reclaims rear) link to each other with the pCAM23A carrier framework large fragment of Sal I double digestion, obtain described rnai expression carrier.
Experimental results show that, in the growth course of paddy rice, by the RNA perturbation technique UCH320 protein expression level in the paddy rice is reduced, can cause rice paddy seed to show following phenotype: to compare than wild-type rice paddy seed, setting percentage reduces, even the seed that can educate is also more tall and thin, the weight of seed also significantly reduces.The present invention finds out thinking and the method for more simply formulating the high crop yield proterties to lay a good foundation.
Description of drawings
Fig. 1 is the structure collection of illustrative plates of pUCCRNAi interference carrier.
Fig. 2 is part T
1In generation, change the PCR qualification result of the transgenic paddy rice of RNAi expression vector pCAM23A-UCH320 over to.Wherein, swimming lane M is dna molecular amount standard, and each band is followed successively by 5000,3000,2000,1000,750,500,300,200bp from big to small; Swimming lane 1-12 is for identifying positive plant.
Fig. 3 changes the PCR qualification result of the adjoining tree of pCAM23A empty carrier over to for part.Wherein, swimming lane M is dna molecular amount standard, and each band is followed successively by 5000,3000,2000,1000,750,500,300,200bp from big to small; Swimming lane 1-10 is for identifying positive plant.
Fig. 4 is each genetic stocks paddy rice spike of rice phenotype of UCH320 gene.Wherein, wt represents to spend No. 11 in the not genetically modified wild-type rice varieties; 320R13-1-1 and 320R28-13 are that two embodiment 1 identify positive T
1In generation, change the transgenic rice plant of RNAi expression vector pCAM23A-UCH320 over to.
Fig. 5 is each genetic stocks rice grain phenotype of UCH320 gene.Wherein, wt represents to spend No. 11 in the not genetically modified wild-type rice varieties; 320R13-6-1 is that an embodiment 1 identifies positive T
1In generation, change the transgenic rice plant of RNAi expression vector pCAM23A-UCH320 over to.
Fig. 6 is that each genetic stocks rice paddy seed weight of UCH320 gene changes ratio.Wherein, wt represents to spend No. 11 in the not genetically modified wild-type rice varieties; 24 " 320R-" is 24 strain embodiment 1 and identifies positive T
1In generation, change the transgenic rice plant of RNAi expression vector pCAM23A-UCH320 over to.
Fig. 7 is each genetic stocks rice paddy seed setting percentage of UCH320 gene.Wherein, wt represents to spend No. 11 in the not genetically modified wild-type rice varieties; 24 " 320R-" is 24 strain embodiment 1 and identifies positive T
1In generation, change the transgenic rice plant of RNAi expression vector pCAM23A-UCH320 over to.
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
The pUCCRNAi interference carrier: obtain from Chinese Academy of Sciences heredity professor Chu Chengcai of institute, be documented in " ten thousand is elegant clear etc. for Yan Peiqiang, Bai Xianquan. and use the RNAi technology and cultivate anti-TMV virus transgene tobacco. heredity, 2007,29 (8): 1018-1022 " in the literary composition.In the pUCCRNAi interference carrier, the recognition site of restriction enzyme Spe I and Bgl II is positioned at the intron upstream, and the recognition site of restriction enzyme BamH I and Xba I is positioned at the intron downstream.The structure collection of illustrative plates of pUCCRNAi interference carrier as shown in Figure 1.
PCAM23A carrier: Beijing DingGuo ChangSheng Biology Technology Co., Ltd.Be recorded in " Chi Zhengchang. the OsSG01 functional study of paddy rice meiotic gene and analysis. Yangzhou University,, Master's thesis in 2010 " in the literary composition.The promotor that is positioned at Xba I upstream that carries on the pCAM23A carrier is the Actin promotor.
Spend in the rice varieties No. 11: available from crop investigations institute of the Chinese Academy of Agricultural Sciences; Spend training by the Chinese Academy of Agricultural Sciences crop institute 1979 with No. five/Te Tepu of capital wind/good fortune brocade.Be recorded in " Ni Pichong. the Anther Culture new variety-in spend No. 11. Crop Germplasm Resources, 04 phase in 1989 ".
Agrobacterium EHA105: Beijing full formula gold biotechnology company limited.
Obtain substratum related in the process of transgenic plant among the following embodiment as follows:
1, rice callus is induced and subculture medium (japonica rice) NB minimum medium
2, AAM substratum
3, be total to culture medium
4, resistance screening substratum 1L is poured on the disposable flat board approximately 40-50
5, paddy rice division culture medium (japonica rice) 1L is poured on Boiling tube, approximately about 20
6, paddy rice root media (japonica rice)
| The 1/4MS inorganic salt | ? |
| The MS VITAMIN | ? |
| NAA | 0.5m?g/L |
| Paclobutrazol | 1m?g/L |
| Plant gel | 2.6g/L |
| pH | 5.8 |
The hormone mother liquor compound method that above each substratum is related:
(1) 0.5mg/ml2, the 4-D mother liquor join method: take by weighing 100mg2,4-D places in the small beaker; Adding a small amount of dehydrated alcohol makes it to dissolve fully; 2,4-D spirituous solution is slowly added in the water on the magnetic stirring apparatus, if there is precipitation, need to reconfigure; Water is settled to 200ml, 4 ℃ of preservations.
(2) 0.5mg/ml α-NAA mother liquor join method: take by weighing 100mgNAA and place in the small beaker; KOH solution dissolving NAA with 1M; Water is settled to 200ml, 4 ℃ of preservations.
(3) the 0.5mg/ml6-BA mother liquor join method: take by weighing 100mg6-BA and place small beaker; Add a small amount of concentrated hydrochloric acid, grind to form pasty state with glass rod, add again a small amount of concentrated hydrochloric acid, make it to dissolve fully; Dilute with water also is settled to 200ml, 4 ℃ of preservations.
(4) preparation of 100mM Syringylethanone (As): take by weighing 196.2mg As, directly dissolve with 5ml DMSO, and be settled to 10ml, be distributed into aseptic tubule ,-20 ℃ of stored frozen.
(5) 5mg/ml KT join method: take by weighing 100mg kinetin Kinetin, with a small amount of 1M KOH dissolving, dilute with water is settled to 20ml.Behind the filtration sterilization, be distributed in the aseptic tubule-20 ℃ of stored frozen.
The acquisition of embodiment 1, UCH320 transgenic plant and evaluation
UCH320 gene source related in the present embodiment is in paddy rice (Oryza.sativa L.), and its cDNA sequence is shown in sequence in the sequence table 2, and sequence 2 is comprised of 974 Nucleotide, and wherein the 102-791 position is encoding sequence (ORF); Protein (UCH320 albumen) shown in the sequence 1 in the tabulation of sequence 2 code sequences, sequence 1 is comprised of 229 amino-acid residues.
One, the structure of RNAi expression vector pCAM23A-UCH320
Design following RNAi primer sequence according to sequence in the sequence table 2:
RNAi-23A320-F:5 '-cc
ACT AGTATG GAG GAT GCT CAT TCC-3 ' (the underscore place is the recognition sequence of restriction enzyme site Spe I, is thereafter the 534-551 position of sequence 2)
RNAi-23A320-R:5 '-Tc
GGA TCCCAC AAC TTT CGA AAG AGC-3 ' (the underscore place is the recognition sequence of restriction enzyme site BamH I, is thereafter the reverse complemental of the 771-788 position of sequence 2)
Sequence 2 in the sequence table is carried out pcr amplification as template with primed RNA i-23A320-F and RNAi-23A320-R.Cut with restriction enzyme Spe I and BamH I enzyme and to reclaim the purpose fragment behind the PCR product, with its with link to each other (Bgl II and BamH I are isocaudarners) with pUCCRNAi carrier framework large fragment after Bgl II enzyme is cut with restriction enzyme Spe I, obtain middle interstitial granules 1.With restriction enzyme BamH I and the Xba I enzyme interstitial granules 1 that hits, reclaim the skeleton large fragment again, be connected into the above PCR product of cutting with restriction enzyme Spe I and BamH I enzyme (Xba I and Spe I are isocaudarners), obtain middle interstitial granules 2.Again with restriction enzyme Spe I and the Sal I enzyme interstitial granules 2 that hits, reclaim purpose fragment (748bp), link to each other (Xba I and Spe I are isocaudarners) with the pCAM23A carrier framework large fragment that Sal I enzyme is cut with process restriction enzyme Xba I, obtain recombinant plasmid.To show between the restriction enzyme site Xba I of pCAM23A carrier and Sal I the recombinant plasmid called after pCAM23A-UCH320 of the dna fragmentation shown in the sequence 3 in the insertion sequence table through order-checking.In RNAi expression vector pCAM23A-UCH320, the promotor that the dna fragmentation shown in the sequence 3 is transcribed in the initiating sequence table is the Actin promotor.Wherein, sequence 3 is comprised of 748 Nucleotide.Wherein, the 14-268 position is the forward sequence (consistent with the 534-788 position of sequence in the sequence table 2) of a fragment of described UCH320 gene, the 276-474 position is GA20 intron (intron) nucleotide sequence that comes from the pUCCRNAi carrier, and the 482-736 position is the reverse sequence (being the reverse complementary sequence of the 534-788 position of sequence 2 in the sequence table) of a fragment of described UCH320 gene.Separated to keep the stability of carrier between forward sequence and the reverse sequence by one section intron (intron) sequence; This system produces the dsRNA of band hairpin structure (hairpin) at the vegetable cell transcription, cause RNAi, thereby suppresses the expression of goal gene.
Two, the acquisition of transgenic paddy rice and evaluation
1, the acquisition of transgenic paddy rice
(1) preparation of rice conversion acceptor
A. the inducing culture of Rice Young Embryo callus
Take away the young fringe threshing of spending No. 11 in the rice varieties of spending about rear 12-15 days, float blighted grain with clear water, with 70% alcohol immersion 1-2 minute, then with being added with 1%(v/v) 1.25% the aqueous sodium hypochlorite solution (active chlorine content is 1.25%(w/v) of Tween20) soaked 90 minutes, carry out surface sterilization.(will often stir during sterilization) uses aseptic water washing 3-4 time, drains water for subsequent use.Extrude Rice Young Embryo with tweezers and dental scaler at aseptic filter paper and place on the solid inducing culture (NB minimum medium), 26 ℃ of dark evoked callus of cultivating.Approximately peel callus after 5-7 days, change on the freshly prepared subculture medium (NB minimum medium), succeeding transfer culture was used for cultivating altogether about 5 days under the same conditions.
B. the inducing culture of Mature Embryos of Rice callus
The Mature seed of rice that shells is used first 70% alcohol immersion 1-2 minute, then use the aqueous sodium hypochlorite solution (active chlorine content is 30%-40%(w/v) of 30%-40%) soaked 30 minutes, carry out surface sterilization (be preferably on the shaking table and carry out), aseptic water washing 3-4 time, again seed is placed on the aseptic filter paper behind the suck dry moisture, be placed on the mature embryo calli induction media (NB minimum medium) 26 ℃ of dark cultivations (can cultivate by light, light is cultivated and looked fast).Approximately after 20 days, peel the callus that the mature embryo scultellum grows, change on the mature embryo subculture medium (NB minimum medium), under the same conditions succeeding transfer culture.Per two all succeeding transfer culture once later on.Select succeeding transfer culture 4-5 days, the yellowish granular callus of color and luster cultivates altogether.
(2) conversion of Agrobacterium and cultivation
A. extracting and purifying plasmid
The bacillus coli DH 5 alpha bacterial classification that will contain RNAi expression vector pCAM23A-UCH320 that step 1 makes up and pCAM23A empty carrier is inoculated in respectively 5ml LB(and contains kantlex 50mg/L) in the liquid nutrient medium, 37 ℃, 200rpm shakes overnight incubation.The plasmid extraction kit of pressing V-GENE company extracts recombinant plasmid.
B. power taking is hit cup and is used soaked in absolute ethyl alcohol, dries.
C. Agrobacterium EHA105 electric shock preliminary treatment
I. Agrobacterium EHA105 is inoculated in 5ml YEP(and contains Streptomycin sulphate Sm50mg/L) in the liquid nutrient medium, 28 ℃, 200rpm concussion overnight incubation to OD600 value is 0.4.
Collect 1ml bacterium liquid in the II.1.5ml centrifuge tube, 4 ℃, 8000rpm, centrifugal 30s.
III. remove raffinate, precipitate with 200 μ l ddH
2O fully suspends, and 4 ℃, 8000rpm, centrifugal 30s.
IV. repeating step III is three times.
V. remove raffinate, precipitation ddH
2O fully suspends, and is electric shock Agrobacterium EHA105 competence.Add 200 μ l sterile glycerol mixings be placed on-80 ℃ for subsequent use.
D. shock by electricity
I. get plasmid (RNAi expression vector pCAM23A-UCH320 or pCAM23A empty carrier that step 1 makes up) to 200 μ l EHA105 competence, then the tip-tap mixing is transferred in the electric shock cup, puts on ice.
II. be ready to electric shock device (BioRad), voltage is 2.5V, pins shock button with hand, until a acoustic-electric hits complete.
III. room temperature adds the YEP liquid medium after leaving standstill 2min, and 28 ℃ leave standstill 1h, and then 28 ℃, 200rpm cultivates 2h.
The centrifugal 30s of IV.8000rpm collects bacterium liquid, precipitation ddH
2O suspends, and contains kantlex 50mg/L with the glass stick coating dull and stereotyped with the YEB solid medium that contains Streptomycin sulphate Sm50mg/L, cultivates 48h for 28 ℃.Scraping lawn Eddy diffusion is in the YEB liquid nutrient medium, is cultured to logarithmic growth late period at 28 ℃; Therefrom get again 0.5ml be forwarded in the same YEB liquid nutrient medium of 100ml behind the 2-3h to OD600 be about 0.5, with centrifugal 10 minutes of cultured restructuring Agrobacterium 4000g, precipitation suspended into the restructuring agrobacterium suspension with the AAM liquid nutrient medium of 100ml.
(3) the common cultivation of Rice Callus and Agrobacterium
Select the state that obtains in the step 1 preferably subculture put into the aseptic triangular flask of 100ml to the Rice Callus of certain hour (succeeding transfer culture 4-5 days, color and luster are yellowish, particulate state), then add the restructuring agrobacterium suspension (ensuring at least enough bacterium liquid contacts with material) that an amount of step 2 obtains, place 20min with the 80-100r/min room temperature.Take out callus, suck unnecessary bacterium liquid at aseptic filter paper, transferring to immediately the solid that is covered with one deck aseptic filter paper is total on the culture medium, all the time being close to the side of substratum during with callus induction and succeeding transfer culture still places down, callus should be put neatly, had better not stack each other 25 ℃ of dark culturing 3 days.
(4) screening of resistant calli
Callus after cultivating is altogether fully washed 4-6 time with sterilized water, becomes limpid until wash the aqueous solution of callus, is aseptic washing 4-5 time of the cephamycin cef of 300mg/L with adding concentration again, and 15-20min blots callus with aseptic filter paper at every turn.
Callus being placed on the screening culture medium that contains 25mg/L Totomycin Hygromycin screening changes over to after 14 days and continues screening on the screening culture medium that contains 50mg/L Totomycin Hygromycin.2 all generation.Most of callus is in rear 10 days brownization of the left and right sides of screening, and milky resistant calli then regrows out at the edge of brownization tissue.Select the general 6-8 of continuing week.
(5) differentiation of resistant calli
From the resistant calli that after the screening of two-three-wheel, grows, the resistant calli of selecting the milk yellow densification goes to the division culture medium that contains 50mg/L Totomycin Hygromycin and cultivates first dark the cultivation 3 days, after forward again 16-20h/d to, intensity of illumination 100-120 μ molm
-2s
-1Illumination condition under cultivate, further differentiate seedling after 30-40 days.
(6) take root, strong sprout and transplanting
When the bud of resistant calli differentiation grows to approximately 2-4cm, seedling is moved on on the root media, cultivate about two weeks.Select the seedling of high approximately 10cm, well developed root system, with warm water flush away substratum, in the greenhouse, transplant and bury.The water surface is not flood seedling as degree, if become a fine day, need to shade survives (being as the criterion with guttation) to seedling.
By aforesaid operations, final two kinds of transgenic seedlings that obtain to have hygromycin resistance namely change the RNAi expression vector pCAM23A-UCH320 of step 1 structure and the rice plant (T of pCAM23A empty carrier over to
1Generation).
2, the evaluation of transgenic paddy rice
T from step 1 acquisition
1In generation, change the transgenic paddy rice of RNAi expression vector pCAM23A-UCH320 over to, and change in the adjoining tree of pCAM23A empty carrier and extract respectively genomic dna.For the transgenic paddy rice that changes RNAi expression vector pCAM23A-UCH320 over to, carry out pcr amplification with primer 1 and primer 2, obtain the plant that size is about 460bp purpose band (with UCH320 forward sequence and GA20 intron sequences) through evaluation and be the positive plant that changes RNAi expression vector pCAM23A-UCH320 over to.For the adjoining tree that changes the pCAM23A empty carrier over to, use for primer 1 and primer 2 and carry out pcr amplification, show that through evaluation (PCR product size the is about 200bp) plant that contains (with the GA20 intron sequences) gene is the positive plant that changes the pCAM23A empty carrier over to.
Primer 1:5 '-ACTAGTAGATCTGATGGA-3 ';
Primer 2: 5 '-GGATCCCCTATATAATTTAAG-3 ' (reverse complementary sequence of the 461-481 position of sequence 3).
The T that part steps 1 obtains
1The generation change over to RNAi expression vector pCAM23A-UCH320 transgenic paddy rice qualification result as shown in Figure 2, the part change over to the pCAM23A empty carrier adjoining tree qualification result as shown in Figure 3.Identify through above-mentioned PCR, finally obtain 24 strain PCR and identify positive T
1In generation, change the transgenic rice plant of RNAi expression vector pCAM23A-UCH320 over to.
Identify positive T with 24 strain embodiment 1
1Generation changes in the transgenic paddy rice transfer-gen plant of RNAi expression vector pCAM23A-UCH320, the not genetically modified wild-type rice varieties spends No. 11, and the adjoining tree that changes the pCAM23A empty carrier over to that embodiment 1 obtains is experiment material.The planting seed of each experiment material is carried out vernalization (every kind of experiment material sowing 80-100 grain) in culture dish, the seedling replanting after the vernalization is emerged in flowerpot, then forwards the large field of Beijing suburb to and grows.Behind the spike of rice of gathering in the crops each experiment material plant, it is carried out the Analysis and Identification of following several respects:
1, spike of rice phenotype analytical
The result shows, compares for No. 11 with spending in the not genetically modified wild-type rice varieties, and 24 strain embodiment 1 identify positive T
1Dysplastic phenotype (Fig. 4) has all appearred in the spike of rice that generation changes the transgenic rice plant of RNAi expression vector pCAM23A-UCH320 over to.And the adjoining tree that changes the pCAM23A empty carrier over to that obtains for embodiment 1, spend in its spike of rice phenotype and the not genetically modified wild-type rice varieties No. 11 basically identical, no difference of science of statistics.
2, seed-setting rate and seed weight statistical study
The result shows, compares for No. 11 with spending in the not genetically modified wild-type rice varieties, and 24 strain embodiment 1 identify positive T
1On the spike of rice for the transgenic rice plant that changes RNAi expression vector pCAM23A-UCH320 over to a lot of shrivelled seeds (Fig. 5) are arranged.Seed on each experiment material plant spike of rice is carried out setting percentage and hundred grain weight statistical study, and its detailed results sees Table 1 and Fig. 6, Fig. 7.Wherein, Figure 6 shows that each experiment material rice paddy seed weight changes ratio, calculates with reference to table 1 result by following formula: rice paddy seed weight changes per hundred real grain weight of ratio=(per hundred real grain weight of per hundred real grain weight-wild-type samples of sample to be tested)/wild-type sample.Can find out from above result, compare for No. 11 with spending in the not genetically modified wild-type rice varieties, 24 strain embodiment 1 identify positive T
1Seed-setting rate and seed weight that generation changes the transgenic rice plant of RNAi expression vector pCAM23A-UCH320 over to all reduce, even the seed that can educate simultaneously is also more tall and thin.And for the adjoining tree that changes the pCAM23A empty carrier over to that embodiment 1 obtains, spend in its seed-setting rate and seed weight and the not genetically modified wild-type rice varieties No. 11 basically identical, no difference of science of statistics.
Seed setting rate and hundred grain weight statistical study on each experiment material plant spike of rice of table 1
| Plant | Real grain number | Real grain gross weight g | Per hundred real grain weight g | The unfilled gtains number | Total grain number | Setting percentage |
| 320R13-1-1 | 516 | 11.518 | 2.232171 | 1126 | 1642 | 31.43% |
| 320R13-1-2 | 534 | 11.354 | 2.126217 | 1212 | 1746 | 30.58% |
| 320R13-1-3 | 1015 | 22.041 | 2.171527 | 2165 | 3180 | 31.92% |
| 320R13-3-1 | 607 | 13.828 | 2.278089 | 1295 | 1902 | 31.91% |
| 320R13-3-2 | 714 | 16.352 | 2.290196 | 1400 | 2114 | 33.77% |
| 320R13-3-3 | 686 | 16.132 | 2.351603 | 1104 | 1790 | 38.32% |
| 320R13-3-4 | 614 | 13.689 | 2.229479 | 1123 | 1737 | 35.35% |
| 320R13-3-5 | 760 | 17.748 | 2.335263 | 1186 | 1946 | 39.05% |
| 320R13-4-1 | 473 | 10.38 | 2.194503 | 988 | 1461 | 32.38% |
| 320R13-4-2 | 172 | 3.47 | 2.017442 | 646 | 818 | 21.03% |
| 320R13-6-1 | 144 | 3.31 | 2.298611 | 642 | 786 | 18.32% |
| 320R13-6-2 | 190 | 4.19 | 2.205263 | 588 | 778 | 24.42% |
| 320R27-A | 31 | 0.66 | 2.129032 | 678 | 709 | 4.37% |
| 320R28-13 | 395 | 9.822 | 2.486582 | 2530 | 2925 | 13.50% |
| 320R28-B | 64 | 1.69 | 2.640625 | 1122 | 1186 | 5.40% |
| 320R29-1-1 | 70 | 1.653 | 2.361429 | 1458 | 1528 | 4.58% |
| 320R29-1-2 | 93 | 2.16 | 2.322581 | 630 | 723 | 12.86% |
| 320R29-1-3 | 106 | 2.643 | 2.493396 | 2190 | 2296 | 4.62% |
| 320R29-4-1 | 21 | 0.457 | 2.17619 | 2365 | 2386 | 0.88% |
| 320R29-4-2 | 107 | 2.83 | 2.64486 | 1235 | 1342 | 7.97% |
| 320R29-4-3 | 40 | 0.896 | 2.24 | 1450 | 1490 | 2.68% |
| 320R29-D | 1435 | 36.879 | 2.569965 | 1428 | 2863 | 50.12% |
| 320R29-F-1 | 90 | 2.12 | 2.355556 | 1610 | 1700 | 5.29% |
| 320R29-F-2 | 826 | 20.924 | 2.533172 | 961 | 1787 | 46.22% |
| wt | 2543 | 70.4411 | 2.77 | 23 | 2566 | 99.10% |
Annotate: 1,24 " 320R-" in the table are 24 strain embodiment 1 and identify positive T
1In generation, change the transgenic rice plant of RNAi expression vector pCAM23A-UCH320 over to; Wt represents to spend No. 11 in the not genetically modified wild-type rice varieties.2, " total grain number " in the table equals " real grain number (a meter seed is arranged) " and adds " unfilled gtains number (without a rice ghost) "; " setting percentage " equals " real grain number " divided by " total grain number ".
Claims (10)
1. the protein or the application of its encoding gene in regulating growth of plants that are formed by the aminoacid sequence shown in the sequence in the sequence table 1;
Described growth and development of plants is embodied in following 1)-2) at least a:
1) seed-setting rate;
2) seed weight.
2. the protein or its encoding gene that are comprised of the aminoacid sequence shown in the sequence in the sequence table 1 have following I in seed selection)-II) application at least a plant variety in the purpose proterties:
I) the seed-setting rate improves or reduces;
II) seed weight increases or reduces.
3. cultivate the method for transgenic plant, for following (A) or (B):
(A) cultivate have following b1)-b2) method of at least a transgenic plant in the purpose proterties, comprise the steps:
A) import the encoding gene of the protein that is formed by the aminoacid sequence shown in the sequence in the sequence table 1 in the purpose plant, obtain expressing the transgenic plant of described encoding gene;
B) from step a) gained transgenic plant, obtain comparing with described purpose plant, have following b1)-b2) at least a transgenic plant in the purpose proterties:
B1) the seed-setting rate improves;
B2) seed weight increases;
(B) cultivate have following d1)-d2) method of at least a transgenic plant in the purpose proterties, comprise the steps:
The encoding gene of the protein that c) in the purpose plant aminoacid sequence shown in the sequence 1 in by sequence table is formed suppresses to express, and obtains transgenic plant;
D) from step c) gained transgenic plant, obtain comparing with described purpose plant, have following d1)-d2) at least a transgenic plant in the purpose proterties:
D1) the seed-setting rate reduces;
D2) seed weight reduces.
4. arbitrary described application or method according to claim 1-3 is characterized in that: the encoding gene of the described protein that is comprised of the aminoacid sequence shown in the sequence in the sequence table 1 is arbitrary described dna molecular in following (1) to (4):
(1) encoding sequence be in the sequence table sequence 2 from the dna molecular shown in 5 ' terminal the 102nd to 791 Nucleotide;
(2) dna molecular shown in the sequence 2 in the sequence table;
(3) the protein DNA molecule that the aminoacid sequence shown in the sequence 1 forms in the dna molecule hybridize that limits with (1) or (2) under stringent condition and the coding sequence table;
(4) dna molecular with the arbitrary restriction in (1)-(3) has the protein DNA molecule that the aminoacid sequence shown in the sequence 1 forms in 90% above homology and the coding sequence table.
5. according to claim 3 or 4 described methods, it is characterized in that: the encoding gene of the described protein that in the purpose plant aminoacid sequence shown in the sequence 1 in by sequence table is formed suppresses to express, and is to realize by changing in the described purpose plant as shown in the formula the dna fragmentation shown in (I):
SEQ
Forward-X-SEQ
Oppositely(I)
Described SEQ
ForwardThe 14-268 position Nucleotide of sequence 3 in the sequence table;
Described SEQ
OppositelySequence and described SEQ
ForwardThe sequence reverse complemental;
Described X is described SEQ
ForwardWith described SEQ
OppositelyBetween intervening sequence, on sequence, described X and described SEQ
ForwardAnd described SEQ
OppositelyAll not complementary.
6. method according to claim 5, it is characterized in that: the nucleotides sequence of the dna fragmentation shown in the described formula (I) is classified the 14-736 position of the sequence 3 in the sequence table as.
7. arbitrary described method according to claim 3-6 is characterized in that: the dna fragmentation shown in the described formula (I) is that the form by the rnai expression carrier changes in the described purpose plant; The promotor that the dna fragmentation shown in the described formula of startup (I) is transcribed on the described rnai expression carrier is the Actin promotor.
8. arbitrary described application or method according to claim 1-7 is characterized in that: described plant is monocotyledons or dicotyledons.
9. as shown in the formula the dna fragmentation shown in (I):
SEQ
Forward-X-SEQ
Oppositely(I)
Described SEQ
ForwardThe 14-268 position Nucleotide of sequence 3 in the sequence table;
Described SEQ
OppositelySequence and described SEQ
ForwardThe sequence reverse complemental;
Described X is described SEQ
ForwardWith described SEQ
OppositelyBetween intervening sequence, on sequence, described X and described SEQ
ForwardAnd described SEQ
OppositelyAll not complementary;
The nucleotide sequence of described dna fragmentation is specially the 14-736 position of the sequence 3 in the sequence table.
10. the recombinant vectors, recombinant bacterium, expression cassette or the transgenic cell line that contain the described dna fragmentation of claim 9.
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