CN101993484B - Stress tolerance associated protein and coded gene and application thereof - Google Patents
Stress tolerance associated protein and coded gene and application thereof Download PDFInfo
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Abstract
The invention discloses a stress tolerance associated protein and a coded gene and application thereof. The protein provided by the invention is the protein selected from (a) the protein consisting of an amino acid sequence shown as sequence 1 in a sequence table or (b) the protein which is obtained by performing substitution and/or deletion and/or addition on the amino acid sequence shown as the sequence 1 through one or more amino acid residues, associated with plant stress tolerance and derived from the sequence 1. The protein is associated with stress tolerance, plant height and the sizes of millet sprays and kernels, can be used for culturing stress tolerant transgenic plants as well as transgenic plants with high growth speeds and large millet sprays and kernels and has important application value in the aspect of crop genetic improvement.
Description
Technical field
The present invention relates to a kind of stress tolerance correlative protein and encoding gene thereof and application.
Background technology
The adverse circumstance factors such as low temperature, arid, high temperature are the important factors that influences crop yield and quality, and according to estimates, only low temperature causes all over the world paddy rice underproduction 5-10% every year on average, and the serious cryogenic time (3-4 once) is caused underproduction 20-40%.
Under environment stress, can produce a series of responsing reactions in the plant materials, the variation that is accompanied by many Physiology and biochemistries and grows.Clear and definite plant is to the reaction mechanism of adverse circumstance, will provide the science argument for adversity gene engineering research and application.At present, the plant stress-resistance Journal of Sex Research is deep into cell, molecular level gradually, and combines with genetics and genetic engineering research, explores and improves plant growth characteristics with biotechnology, its objective is and improves the adaptive faculty of plant to adverse circumstance.
Under the adverse environmental factor of environment-stress such as arid, high salt and low temperature, plant can be made corresponding adjustment on molecule, cell and integral level, with the injury that reduces environment to the full extent and caused and survived.Many genes are expressed by stress-inducing, the product of these genes not only can be participated in the stress response of plant directly, and can regulate other Expression of Related Genes or participate in signal transduction path, thereby plant is avoided or reduce injury, strengthen coercing the resistance of environment.
Summary of the invention
The purpose of this invention is to provide a kind of stress tolerance correlative protein and encoding gene thereof and application.
Stress tolerance correlative protein provided by the invention from long-grained nonglutinous rice (Oryza sativa), is following (a) or (b):
(a) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 1;
(b) with the aminoacid sequence of sequence 1 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and relevant with plant stress tolerance by sequence 1 deutero-protein.
In order to make the protein in (a) be convenient to purifying, proteinic N-terminal or C-terminal that can the aminoacid sequence shown in the sequence 1 is formed in by sequence table connect label as shown in table 1.
The sequence of table 1 label
| Label | Residue | Sequence |
| Poly-Arg | 5-6 (being generally 5) | RRRRR |
| Poly-His | 2-10 (being generally 6) | HHHHHH |
| |
8 | DYKDDDDK |
| Strep- |
8 | WSHPQFEK |
| c- |
10 | EQKLISEEDL |
Above-mentioned (b) but in the protein synthetic, also can synthesize its encoding gene earlier, carry out biology again and express and to obtain.Proteinic encoding gene in above-mentioned (b) can be by the codon with one or several amino-acid residue of disappearance in the dna sequence dna shown in the sequence in the sequence table 2, and/or carry out the missense mutation of one or several base pair, and/or obtain at the encoding sequence that its 5 ' end and/or 3 ' end connects the label shown in the table 1.
The present invention also protects described proteic encoding gene or genomic gene.
Described encoding gene can be following 1) or 2) or 3) or 4) or 5) dna molecular:
1) in the sequence table sequence 2 from the dna molecular shown in 5 ' terminal the 73rd to 789 Nucleotide;
2) in the sequence table sequence 2 from the dna molecular shown in 5 ' terminal the 5th to 856 Nucleotide;
3) dna molecular shown in the sequence 2 in the sequence table;
4) under stringent condition with 1) or 2) or 3) the dna sequence dna hybridization that limits and the dna molecular of coded plant stress tolerance correlative protein;
5) with 1) or 2) or 3) or 4) dna sequence dna that limits has the dna molecular of 90% above homology and coded plant stress tolerance correlative protein
Described genomic gene can be following 1) or 2) or 3) or 4) dna molecular:
1) dna molecular shown in the sequence 3 in the sequence table;
2) dna molecular shown in the sequence 4 in the sequence table;
3) under stringent condition with 1) or 2) the dna sequence dna hybridization that limits and the dna molecular of coded plant stress tolerance correlative protein;
4) with 1) or 2) or 3) dna sequence dna that limits has the dna molecular of 90% above homology and coded plant stress tolerance correlative protein.
In the dna fragmentation shown in the sequence 2, being upstream primer from 5 ' terminal the 5th to 30 Nucleotide, is downstream primer from 5 ' terminal the 836th to 856 Nucleotide, is the open reading frame of gene from 5 ' terminal the 73rd to 789 Nucleotide.This gene does not have intron, so the sequence 2 of sequence table is on all four from the sequence 4 of 5 ' terminal the 5th to 856 Nucleotide and sequence table from 5 ' terminal the 1004th to 1855 Nucleotide.
The recombinant expression vector, expression cassette, transgenic cell line and the reorganization bacterium that contain described gene all belong to protection scope of the present invention.
Available existing plant expression vector construction contains the recombinant expression vector of described gene.Described plant expression vector comprises the double base agrobacterium vector and can be used for the carrier etc. of plant micropellet bombardment.Described plant expression vector also can comprise 3 ' end untranslated zone of foreign gene, promptly 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 plant expression vector, can add any enhancement type promotor or constitutive promoter before its transcription initiation Nucleotide, they can use separately or be used in combination with other plant promoter; In addition, when using gene constructed plant 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 be synthetic.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 plant expression vector, can produce enzyme or the gene of luminophor, antibiotic marker thing or the anti-chemical reagent marker gene etc. of colour-change with resistance as adding the coding that in plant, to express.From the security consideration of transgenic plant, can not add any selected marker, directly with adverse circumstance screening transformed plant.
Insert the recombinant plasmid that specific DNA fragment obtains between Kpn I that described recombinant expression vector specifically can be at pCAMBIA1300 and the Sal I restriction enzyme site; Described specific DNA fragment is the small segment that obtains with restriction enzyme Kpn I and Xhol double digestion pJIT163-AK068115; Described pJIT163-AK068115 inserts the recombinant plasmid that obtains between the BamH I of pJIT163 and the HindIII restriction enzyme site with sequence in the sequence table 2 from the dna molecular shown in 5 ' terminal the 5th to 856 Nucleotide.
The primer of described gene of increasing also belongs to protection scope of the present invention.
The present invention also protects a kind of method of cultivating transgenic plant, is described gene is imported in the purpose plant, obtains following (A) and/or (B) and/or transgenic plant (C):
(A) resistance of reverse is higher than the transgenic plant of described purpose plant;
(B) plant height is higher than the transgenic plant of described purpose plant;
(C) grain ear and/or grain are greater than the transgenic plant of described purpose plant.
Described gene specifically can import in the described purpose plant by described recombinant expression vector.Carry that described expression carrier can Ti-plasmids, Ri plasmid, plant viral vector, directly DNA conversion, microinjection, electricity be led, conventional biological method transformed plant cells or tissue such as agriculture bacillus mediated by using, and the plant transformed tissue cultivating is become plant.Described purpose plant both can be that monocotyledons also can be a dicotyledons, and described monocotyledons can be paddy rice, and was fine or 9311 as Japan.Described resistance of reverse specifically can be winter hardiness.
The invention provides a kind of new albumen that derives from paddy rice, this albumen is relevant with the size of resistance of reverse, the speed of growth and grain ear and grain, can be used for cultivating the resistance of reverse transfer-gen plant, also can be used for cultivating fast growth, transfer-gen plant that grain ear grain is big, aspect crop genetic improvement, have important use and be worth.
Description of drawings
Fig. 1 analyzes the expression level that the super hybridized rice parent trains short 64S different development stage, different tissues organ OsXGD under arid, low temperature, hot conditions for gene chip; 1,2,3 represent seedling stage, boot stage, full heading time; L represents blade, and P represents fringe; K represents contrast, and c represents low temperature, and h represents high temperature, and d represents arid.
Fig. 2 is Harpin-induced 1 domain of gene.
Fig. 3 is the electrophoretogram of pcr amplification product; M:200bp marker; The 1-4:PCR amplified production.
Fig. 4 is the collection of illustrative plates of pMD-18T carrier.
Fig. 5 is the electrophoretogram of each endonuclease bamhi in the construction of recombinant plasmid; M:500bp marker; 1:Kpn I and Xhol enzyme are cut pJIT163-AK068115; 2:Kpn I and Sal I enzyme are cut pCAMBIA1300.
Fig. 6 is that the PCR of transfer-gen plant among the embodiment 3 identifies collection of illustrative plates; M:marker; 1-14: the seedling of taking root and surviving, wherein swimming lane 5 is T13-1 strain system, swimming lane 13 is T17-1 strain system; 15: Japan's fine (negative control).
Fig. 7 is T among the embodiment 3
1For the upgrowth situation after the transfer-gen plant deepfreeze; DZ: empty carrier contrast; WT: Japanese fine; T13-1, T17-1: two strain systems of transfer-gen plant.
Fig. 8 is T among the embodiment 3
1Upgrowth situation, spike of rice and grain of rice photo for transfer-gen plant.
Fig. 9 is T among the embodiment 3
1For transfer-gen plant, T
2Spike of rice photo for transfer-gen plant.
Figure 10 is T among the embodiment 4
1Spike of rice photo for transfer-gen plant.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment if no special instructions, is ordinary method.Used test materials among the following embodiment if no special instructions, is to buy from routine biochemistry reagent shop and obtains.% among the following embodiment if no special instructions, is the quality percentage composition.Quantitative test in following examples all is provided with repeated experiments three times, results averaged.
Rice variety is trained short 64S (paddy rice PA64): available from rice in China cross-breeding center (China, Changsha); The Institute of Subtropical Agriculture, The Chinese Academy of Sciences guarantees to provide to the public; Reference: Liang Yongshu etc., Xian round-grained rice hand over short 64S/ Japan fine F2, F3 of combination training and F6 for the Other Main Agronomic Characters analysis, BULLETIN OF BOTANY Vol. Chinese Bulletin of Botany 2008,25 (1): 59-66.
Paddy rice Japan is fine: seed is available from rice in China cross-breeding center (China, Changsha); The Institute of Subtropical Agriculture, The Chinese Academy of Sciences guarantees to provide to the public; Reference: Liang Yongshu etc., Xian round-grained rice hand over short 64S/ Japan fine F2, F3 of combination training and F6 for the Other Main Agronomic Characters analysis, BULLETIN OF BOTANY Vol. Chinese Bulletin of Botany 2008,25 (1): 59-66.
Paddy rice 9311: The Institute of Subtropical Agriculture, The Chinese Academy of Sciences guarantees to provide to the public; Reference: Yang Dewei etc. are based on rice ear sprouting period QTL location and the genetic analysis of CSSL, Botany Gazette Chinese Bulletin of Botany 2010,45 (2): 189-197.
Columbia Arabidopis thaliana (the environmental Arabidopis thaliana of Colombia; Col-0): The Institute of Subtropical Agriculture, The Chinese Academy of Sciences guarantees to provide to the public; Reference: Wang Jinlan etc., the OsRAA1 ectopic expression promotes to bloom and hypocotyl elongation in Arabidopis thaliana, Science Bulletin, 2009 the 54th volume the 18th phase: 2819-2825.
The pJIT163 carrier: The Institute of Subtropical Agriculture, The Chinese Academy of Sciences guarantees to provide to the public; Reference: Feng-Yun Zhao, Xue-Jie Zhang
1, Ping-Hua Li
1, Yan-Xiu Zhao
1And Hui Zhang, Co-expression of the Suaeda salsa SsNHX1 and Arabidopsis AVP1 confer greater salt tolerance to transgenic rice than the single SsNHX1, Molecular Breeding, 1380-3743 (Print) 1572-9788 (Online), Volume 17, Number in July, 4/2006,341-353.
The discovery of embodiment 1, OsXGD gene
With the super hybridized rice is material, use the Affymetrix biochip technology and contain 51, the rice genome expression chip of 279 transcriptons (The GeneChip Rice Genome Array, Part Number900601), systematically analyzed the super hybridization rice parent and trained short 64S different development stage, different tissues organ complete genomic express spectra under arid, low temperature, hot conditions, found the gene relevant with the resistance of reverse height.Probe is (GeneChip Rice Genome Array Probe Set ID:Os.12119.1.S1_at) shown in the sequence 5 of sequence table.The results are shown in Figure 1 and table 1.
The relative expression quantity of each histoorgan of table 1 different treatment
| 1Lck 50.4 | 1Lc 1575.9 | 2Lck 76.4 |
| 2Lc 1078.8 | 2Lck 76.4 | 2Lh 742.0 |
| 2Lck 76.4 | 2Ld 1021.2 | 3Lck 197.6 |
| 3Lh 823.0 | 2Pck 18.7 | 2Pc 129.6 |
| 2Pck 18.7 | 2Pd 78.5 | 3Pck 163.2 |
| 3Pc 457.9 | 3Pck 163.2 | 3Pd 480.8 |
The OsXGD gene is positioned on No. 1 karyomit(e) of paddy rice.OsXGD albumen contains 238 amino-acid residues.CG content is 60.7%, intronless.The proteic iso-electric point of OsXGD is 10.08.The OsXGD gene contains Harpin-induced 1 domain, and important effect is arranged in the sensitivity response of plant, sees Fig. 2.This albumen of DANMAN analysis revealed is a hydrophilic protein.The possible promoter region of OsXGD gene is at ORF upstream bp more than 1200 place, some cis-acting elements are arranged, as cis-acting element involved in heat stress responsiveness, gibberellin-responsive element etc., 3 region are at terminator codon downstream bp more than 60 place.The gene chip data show this gene may be cold-resistant with paddy rice, heat-resisting, drought tolerance is relevant, at each histoorgan of paddy rice, each development growth all has the expression of certain level period, but also is subjected to the multiple adverse circumstance factor to induce high expression level.Initial analysis is-transcription factor gene (commander's gene).
The encoding sequence of OsXGD gene is held shown in the 73rd to 789 Nucleotide from 5 ' as the sequence 2 of sequence table, genomic gene is shown in the sequence 4 of the sequence 3 of sequence table and sequence table (sequence 3 and sequence 4 are reverse complementary sequence), and encoded protein matter (OsXGD albumen) is shown in the sequence 1 of sequence table.The OsXGD gene be subjected to high temperature, low temperature and drought-induced, period and histoorgan all significantly raise respectively growing.
The clone of embodiment 2, OsXGD gene and the structure of recombinant expression vector
One, the clone of OsXGD gene
Extracting the genomic dna of paddy rice PA64, is template (intronless) with the genomic dna, to carrying out pcr amplification, obtains pcr amplification product (OsXGD gene) with the primer of P1 and P2 composition;
P1 (forward primer): 5 '-
TCTAGACATCCATCACTCGTTCGATCTCTCCC-3 ';
P2 (reverse primer): 5 '-
CACGTGTGTGTCGTGTCGTTCCCAGCC-3 '.
PCR reaction conditions: 94 ℃/5min; 94 ℃/30s, 57 ℃/40s, 72 ℃/1min, 30 circulations; 72 ℃/10min.
The electrophoretogram of pcr amplification product (OsXGD gene) is seen Fig. 3.
Two, the structure of recombinant expression vector
1, the pcr amplification product that step 1 is obtained is connected on pMD-18T cloning vector (TA Cloning, safe day of Jinan and the bio tech ltd) (see figure 4), obtains pMD-18T-AK068115 (OsXGD), sequence verification.
2, pMD-18T-AK068115 is cut with BamH I (carrying on the T carrier) and HindIII enzyme, reclaim small segment; PJIT163 (pGreen, http://www.pgreen.ac.uk/) is cut with BamH I and HindIII enzyme, reclaim big fragment; Big fragment is connected with small segment, obtains pJIT163-AK068115 (OsXGD).PJIT163 has been with 2 * 35S promoter, among the pJIT163-AK068115 (OsXGD) goal gene and strong promoter is coupled together.
3, pJIT163-AK068115 is cut with Kpn I and Xhol enzyme, reclaim small segment; PCAMBIA1300 (CambiaLabs, http://www.cambia.org/daisy/bioforge_legacy/3725.html) is cut with KpnI and Sal I enzyme, reclaim big fragment; Big fragment is connected with small segment, obtains recombinant plasmid pCAMBIA1300-AK068115 (OsXGD).The electrophoretogram of each endonuclease bamhi is seen Fig. 5.
Recombinant plasmid pCAMBIA1300-AK068115 (OsXGD) is checked order, the result shows: the skeleton of this recombinant plasmid is pCAMBIA1300, between Kpn I and Sal I restriction enzyme site, inserted sequence 2 in 2 * 35S promoter and the sequence table from the OsXGD gene shown in 5 ' terminal the 5th to 856 Nucleotide, started the OsXGD gene by 2 * 35S promoter.
Three, the structure of control vector
PJIT163 is cut with Kpn I and Xhol enzyme, reclaim small segment; PCAMBIA1300 is cut with Kpn I and Sal I enzyme, reclaim big fragment; Big fragment is connected with small segment, obtains control plasmid.
The acquisition of embodiment 3, transgenic paddy rice and evaluation
Inducing culture: NB+2,4-D (2.0mg/mL)+6-BA (0.2mg/mL)+Sucrose (30g/L)+Agar (10g/L).
Subculture medium: NB+2,4-D (2.0mg/mL)+CHL (0.3g/L)+Maltose (30g/L)+Agar (8g/L).
Screening culture medium: L3+2,4-D (2.0mg/mL)+Pro (0.5g/L)+Gln (0.5g/L)+cephamycin (500mg/L)+Pyocianil (400mg/L)+Totomycin (25mg/L)+Sucrose (30g/L)+Agar (8g/L); PH=5.8.
Pre-differentiation substratum: NB+Pro (0.5g/L)+CHL (0.3g/L)+6-BA (1.0mg/mL)+NAA (2.0mg/mL)+ABA (5.0mg/mL)+Pyocianil (200mg/L)+Sucrose (30g/L)+Agar (10g/L); PH=5.8.
Division culture medium: NB+Pro (0.5g/L)+CHL (0.3g/L)+6-BA (2.0mg/mL)+KT (1.0mg/mL)+Agar (10g/L)+NAA (0.5mg/mL)+IAA (0.5mg/mL)+Pyocianil (200mg/L)+Maltose (30g/L); PH=5.8.
Root media: MS+NAA (0.5mg/L)+Maltose (30g/L)+Agar (10g/L); PH=5.8.
One, the acquisition of transgenic paddy rice
1, the preparation of reorganization Agrobacterium and callus
With freeze-thaw method (Ca++buffer) recombinant plasmid pCAMBIA1300-AK068115 (OsXGD) is imported agrobacterium tumefaciens EHA105 (purchasing in TIANGEN Biotech (Beijing) Co., Ltd.), the reorganization Agrobacterium of acquisition.The reorganization Agrobacterium is drawn LB plate (LB+50mg/L kantlex+34mg/L paraxin), choose single bacterium colony two days later and draw the full ware of LB plate (LB+50mg/L kantlex+34mg/L paraxin), after spending the night bacterium is resuspended in 50ml and is total in the substratum (NBM+0.1mM Syringylethanone), transfer OD
600=0.5, be reorganization Agrobacterium bacterium liquid.
Select healthy Japanese fine seed and peel off clever shell, place 37 ℃ of incubators to spend the night.After taking out, puts in seed the triangular flask of sterilization, be 75% ethanol surface sterilization 2min with volume fraction earlier, with aseptic water washing 1 time, the 0.1%HgCl 12min that sterilizes with aseptic water washing 5 times, puts into the former liquid disinfectant 40min of clorox again, aseptic water washing 5 times, in the sterilization filter paper on dry, be inoculated into then on the inducing culture, allow embryo half the contact substratum.Every ware 18-20 grain places 25-26 ℃ of dark down the cultivation, with evoked callus.After 20 days, select callus surperficial dry and comfortable, compact structure, the bud head of removing in grain and the callus forwards on the subculture medium, and at this time nutrition is absorbed and deliquescing in the grain, succeeding transfer culture 1 time (14 days).Select the callus of surperficial dry and comfortable compact structure, on the filter paper of sterilization, air-dryly turn white to the surface.
2, reorganization agrobacterium mediation converted
Callus transferred in the reorganization Agrobacterium bacterium liquid soak 30min, rock once every 5min.Use aseptic water washing callus 5 times not muddy then,, air-dryly on the filter paper of sterilization turn white to the callus surface with sterilization filter paper suck dry moisture to liquid.Callus is transferred to common substratum (NBM+0.1mM Syringylethanone; Useful liquid is total to substratum wetted filter paper on it), 25-26 ℃ of dark down the cultivation 3 days.
3, screening and culturing
Callus is changed in the triangular flask of the bacterium of going out, not muddy with bacterium water flushing 5 times to liquid, soak 30min with the aqua sterilisa that is added with 500mg/L saitomycin and 400mg/L Pyocianil again, rock once every 5min.With sterilization filter paper suck dry moisture, air-dryly on the filter paper of sterilization turn white to the callus surface.25-26 ℃ of dark down cultivation on screening culture medium, the callus integral body that will grow kanamycin-resistant callus tissue after 20 days changes in the new screening culture medium.
4, differentiation culture
After twice screening with the callus global transfer that grows kanamycin-resistant callus tissue in the screening culture medium in pre-differentiation substratum, 25-26 ℃, 14h illumination cultivation (light intensity 1000-1500lx) had callus to turn green in 3-7 days successively.Forward in the division culture medium breaking up the callus that turns green in the substratum in advance, place 25-26 ℃, 14h illumination cultivation (light intensity 1000-1500lx), per 20 days replacing one subcultures.
5, take root and transplant
During green height of seedling 5-8cm, transfer to root media, 25-26 ℃, 14h illumination cultivation (light intensity 1000-1500lx) 3-4 week.The little transplantation of seedlings that to take root and survive is cultured to maturation to earth.
The seedling that survives is got blade, extracts genomic dna, carries out PCR with Totomycin primer (P3 and P4) and identifies, the evaluation collection of illustrative plates is seen Fig. 6.PCR identifies that the male plant is for changeing OsXGD trans-genetic hybrid rice T
0For plant.
P3：5’-ACCTGCCTGAAACCGAACTG-3’；
P4 5’-CTGCTCCATACAAGCCAACC-3’。
T
1T is shown in representative
0The seed that produces for selfing and by plant that it grew up to.T
2T is shown in representative
1The seed that produces for selfing and by plant that it grew up to.
Two, change the acquisition of empty carrier contrast paddy rice
Replace recombinant plasmid pCAMBIA1300-AK068115 with control plasmid, other same step 1 obtains to change empty carrier contrast paddy rice T
0For plant.
Three, the evaluation of transgenic paddy rice
1, winter hardiness is identified
Detect the T that changes two of OsXGD trans-genetic hybrid rice strain systems (T13-1 and T17-1) respectively
1The T of generation, commentaries on classics empty carrier contrast paddy rice (DZ)
1The winter hardiness of generation and Japanese fine paddy rice (WT), each strain are 30 strains.
Rice paddy seed is after planting placed growth cabinet, grew into earlier for 3 leaf phases under 22 ℃, 6 ℃ of deepfreezes are 7 days then, recover growth 10 days down at 22 ℃ again, take pictures.Photo is seen Fig. 7.Transfer-gen plant is survived mostly, and changes empty carrier plant and the almost whole withered and yellow death of wild-type plant.
2, growth and production performance are identified
With T
1Be seeded in field (each strain is 30 strains) for seed (T13-1 or Japan fine or change empty carrier contrast paddy rice), observe when receiving fringe and take pictures, photo is seen Fig. 8 A, and behind the results spike of rice, the photo of spike of rice is seen Fig. 8 C and Fig. 8 D.With T
1Be seeded in flowerpot (each strain is 30 strains) for seed (T17-1 or Japan fine or change empty carrier contrast paddy rice), observe when receiving fringe and take pictures, photo is seen Fig. 8 B.The phenotype of changeing empty carrier contrast paddy rice is with Japanese fine consistent.
The result shows: the transfer-gen plant growth is quickened, and increases, increases nearly one times, and grain ear and grain all enlarge markedly.
3, production performance is identified
T with the warm and fine T13-1 of Japan
1Generation and T
2, grain ear is observed and taken pictures behind the results grain ear in the field (each strain is 30 strains) for planting seed, photo is seen Fig. 9.
The result shows: grain ear, the grain of transfer-gen plant all enlarge markedly.
The acquisition of embodiment 4, transgenic paddy rice and evaluation
Replace Japan fine with rice varieties 9311, other method obtains transgenic paddy rice and changes empty carrier contrast paddy rice with embodiment 3.
With 9311, the T of transgenic paddy rice
1T for seed and commentaries on classics empty carrier contrast paddy rice
1, grain ear is observed and taken pictures behind the results grain ear in the field (each strain is 30 strains) for planting seed, photo is seen Figure 10.The phenotype of changeing empty carrier contrast paddy rice is with Japanese fine consistent.
The result shows: grain ear, the grain of transfer-gen plant all enlarge markedly.
Claims (3)
1. method of cultivating transgenic plant is that the proteinic gene that the aminoacid sequence shown in the sequence in the sequence table 1 is formed is imported in the purpose plant, obtain following (A) and (B) with (C) transgenic plant:
(A) winter hardiness is higher than the transgenic plant of described purpose plant;
(B) plant height is higher than the transgenic plant of described purpose plant;
(C) grain ear and/or grain are greater than the transgenic plant of described purpose plant;
Described purpose plant is a paddy rice.
2. method according to claim 1 is characterized in that: described gene is following 1) or 2) or 3) dna molecular:
1) in the sequence table sequence 2 from the dna molecular shown in 5 ' terminal the 73rd to 789 Nucleotide;
2) in the sequence table sequence 2 from the dna molecular shown in 5 ' terminal the 5th to 856 Nucleotide;
3) dna molecular shown in the sequence 2 in the sequence table.
3. method according to claim 1 and 2 is characterized in that: described gene is to import in the described purpose plant by recombinant expression vector;
Described recombinant expression vector is for inserting the recombinant plasmid that specific DNA fragment obtains between the Kpn of pCAMBIA1300 I and Sal I restriction enzyme site; Described specific DNA fragment is the small segment that obtains with restriction enzyme Kpn I and Xhol double digestion pJIT163-AK068115; Described pJIT163-AK068115 inserts the recombinant plasmid that obtains between the BamH I of pJIT163 and the Hind III restriction enzyme site with sequence in the sequence table 2 from the dna molecular shown in 5 ' terminal the 5th to 856 Nucleotide.
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| US20040123343A1 (en) * | 2000-04-19 | 2004-06-24 | La Rosa Thomas J. | Rice nucleic acid molecules and other molecules associated with plants and uses thereof for plant improvement |
| CN1680552A (en) * | 2004-04-06 | 2005-10-12 | 北京未名凯拓农业生物技术有限公司 | Reverse-tolerant concerned gene of rice and its coding protein and use |
| CN1772899A (en) * | 2005-05-09 | 2006-05-17 | 中国科学院植物研究所 | Wild rice drought-resisting gene and its coded protein and application |
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| CN1680552A (en) * | 2004-04-06 | 2005-10-12 | 北京未名凯拓农业生物技术有限公司 | Reverse-tolerant concerned gene of rice and its coding protein and use |
| CN1772899A (en) * | 2005-05-09 | 2006-05-17 | 中国科学院植物研究所 | Wild rice drought-resisting gene and its coded protein and application |
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