CN101280008A - Protein related to cold resistance of plant, coding genes and application thereof - Google Patents
Protein related to cold resistance of plant, coding genes and application thereof Download PDFInfo
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Abstract
The invention discloses a plant cold resistance protein, the coding gene and application thereof. The protein has formula (a) or (b): (a)a protein composed of the amino acid sequence in list 1 of the sequence table; 2)a protein derivated from (a), which is from the substitution and /or deletion and/ or addition of one or more amino acid residues on the amino acid sequence in list 1 in the sequence table and is capable of improving the cold resistance of plants. The method of cultivating cold-resistant plants provided by the invention is of great theoretical and practical significance to the research on the functioning of the cold resistance molecules of plants, the seed selection and breeding of breeds, as well as the breeding of the cold resistance molecules of plants, thus providing an economic, quick and effective approach for improving the cold resistance of plants. The invention is of broad application and market prospect in agriculture.
Description
Technical field
The present invention relates to a kind of albumen relevant and encoding gene and application with the plant resistance to cold.
Background technology
Paddy rice is an important crops, the bud phase damage to plants caused by sudden drop in temperature be influence China's middle and lower reach of Yangtze River early rice growing area and northeast, northwest rice district and one season of the Yunnan-Guizhou Plateau rice district Rice Production one of important factor.Paddy rice bud phase such as experience damage to plants caused by sudden drop in temperature, to cause slow, the minimizing of tillering of young rice seedlings growth, severe patient even large-area stiff seedling, seedling death phenomenon also can occur finally causes the reduction significantly of rice yield, therefore presses for to cultivate cold-resistant rice varieties of budding time.Common wild-rice is ancestors' kind of Asia cultivated rice, and wild-rice is in being evolved into the process of cultivated rice, and through natural selection and artificial selection, gene diversity reduces, the allelotrope number reduces.According to statistics, the allelotrope number of cultivated rice is about 60% (Sun C Q of wild-rice, Wang X K, Li Z C, Yoshimura A.Comparison of the geneticdiversity of common wild rice (Oryza rufipogon Griff.) and cultivated rice (O.sativa L.) using RFLP markers.Theor Appl Genet, 2001,102:157-162), thus the hereditary bottleneck problem that causes current rice variety selective to face.Therefore from (common wild-rice Oryzarufipogon Griff.) genome of rice near edge wild species, excavate and utilize the excellent gene of in cultivated rice, having lost or having weakened, and they are applied to have important theoretical meaning and more practical value in the rice breeding production, also be an effective way that solves a current rice breeding difficult problem.
Dongxiang, Jiangxi common wild-rice is one of the most northern wild-rice in habitat that distributes in the world at present, has extremely strong resistance to cold, but the low temperature of its subterraneous stem ability-12.8 ℃ and safe overwintering (Chen D Z, Xiao Y Q, Zhao SX, Xiong H J, Pi Y H, Luo L J.Studies on cold tolerance of seedling and heading stagein Dongxiang wild rice.Acta Agric Jiangxi, 1996,8:1-6 (in Chinese); Chen D Z, XiaoY Q, Zhao S X, Pi Y H, Xiong H J, Luo L J.Genetic study on the cold tolerance ofDongxiang wild rice at the seedling stage.Acta Agric Jiangxi, 1997,9:56-59 (inChinese)), and this resistance of none tool of current cultivated rice, so Dongxiang, Jiangxi common wild-rice is the ideal material of rice cold tolerance Journal of Sex Research.Liu et al. (Liu F X, Sun C Q, Tan L B, Li D J, Fu Y C, Wang XK.Identification and mapping of quantitative trait loci controlling cold-tolerance ofChinese common wild rice (O.rufipogon Griff.) at booting to flowering stages.ChineseScience Bulletin, 2003,48:2068-2071) reported that 3 quantitative trait locus from Dongxiang, Jiangxi common wild-rice (QTL) can improve cultivated rice receptor parent (osmanthus is towards No. 2) the booting resistance to cold in flowering period, further confirmed from the common wild-rice of Dongxiang, Jiangxi, to excavate the feasibility of cold-resistant gene.
China's wild-rice aboundresources; from wild-rice, excavate, locate and clone cold-resistant genes involved and not only provide new gene and new technology for cultivating super cold-resistant new variety, and to the protection of strengthening China's wild-rice genetic resources, resources advantage become economic advantages have great importance.
Summary of the invention
The purpose of this invention is to provide a kind of albumen and the encoding gene thereof relevant with the plant resistance to cold.
The protein name relevant with the plant resistance to cold provided by the present invention is LTT1, derives from Oryza common wild-rice (O.rufipogon Griff.), is following (a) or protein (b):
(a) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 1;
(b) with the amino acid residue sequence of sequence in the sequence table 1 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and relevant with the plant resistance to cold by (a) deutero-protein.
In order to make the LTT1 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-tag II | 8 | WSHPQFEK |
| c- |
10 | EQKLISEEDL |
Above-mentioned (b) but in the LTT1 synthetic, also can synthesize its encoding gene earlier, carry out biology again and express and to obtain.The encoding gene of LTT1 in above-mentioned (b) can be by lacking sequence in the sequence table 2 codon of one or several amino-acid residue in the dna sequence dna shown in the 5 ' terminal 64-570 bit base, 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.
Above-mentioned and encoding gene plant resistance to cold associated protein also belongs to protection scope of the present invention.
Specifically can be following 1 with the encoding gene of plant resistance to cold associated protein) or 2) or 3) gene:
1) its encoding sequence be in the sequence table sequence 2 from 5 ' terminal 64-570 position deoxyribonucleotide;
2) its nucleotide sequence is the sequence 2 in the sequence table;
3) the proteic dna molecular that under stringent condition, can have the aminoacid sequence of sequence 1 in the sequence table with the dna sequence dna hybridization and the coding of 2 qualifications of sequence in the sequence table.
Above-mentioned stringent condition can be that (or 0.1 * SSC), the solution of 0.1%SDS is hybridized under 65 ℃ and washed film with 0.1 * SSPE in DNA or RNA gel blot experiment.
Increase above-mentioned LTT1 full length gene or arbitrary segmental primer to also belonging to protection scope of the present invention.
Contain above-mentioned and recombinant vectors, transgenic cell line and reorganization bacterium plant resistance to cold associated protein encoding gene and also belong to protection scope of the present invention.
Available existing plant expression vector construction contains the recombinant expression vector of LTT1 gene.Described plant expression vector comprises the double base agrobacterium vector and can be used for the carrier etc. of plant micropellet bombardment, as pCAMBIA1300, pBI121, pBin19, pCAMBIA2301, pCAMBIA1301-UbiN or other plant expression vector of deriving.Conventional biological methods such as the plant expression vector that carries the present invention and plant resistance to cold associated protein encoding gene LTT1 can lead by Ti-plasmids, Ri plasmid, plant viral vector, directly DNA conversion, microinjection, electricity, agriculture bacillus mediated are transformed in vegetable cell or the tissue.By the plant transformed host both can be monocotyledonss such as paddy rice, also can be dicotyledonss such as Arabidopis thaliana.
When using the gene constructed recombinant plant expression vector of LTT1, before its transcription initiation Nucleotide, can add any enhancement type, composing type, organizing specific type or inducible promoter, as cauliflower mosaic virus (CAMV) 35S promoter, general living plain gene Ubiquitin promotor (pUbi) etc., 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 in plant, express enzyme or the gene (gus gene, luciferase genes etc.) of luminophor, antibiotic marker thing (gentamicin marker, kantlex marker etc.) or the anti-chemical reagent marker gene (as anti-weedkiller gene) etc. that can produce colour-change with resistance as adding.From the security consideration of transgenic plant, can not add any selected marker, directly with adverse circumstance screening transformed plant.
Described recombinant expression vector specifically can be and insert the recombinant plasmid pCAMBIA1300-35S-LTT1 that the 64-570 position deoxynucleotide from 5 ' end of sequence 2 obtains in the sequence table between the multiple clone site of pCAMBIA1300-35S; Described pCAMBIA1300-35S is with HindIII and XbaI double digestion carrier pBI121 and carrier pCAMBIA1300 respectively, and the big fragment after connecting the pBI121 enzyme and cutting the 35S promoter that obtains and pCAMBIA1300 enzyme and cut promptly obtains pCAMBIA1300-35S.
Another object of the present invention provides a kind of method of cultivating cold-resistant plant.
The method of the cold-resistant plant of cultivation provided by the present invention is that above-mentioned encoding gene LTT1 with plant resistance to cold associated protein is imported in the plant, obtains the plant that resistance to cold improves.
Described plant can be monocotyledons or dicotyledons.
Described plant specifically can be paddy rice or Arabidopis thaliana.
The present invention has cloned the gene LTT1 relevant with the plant resistance to cold from the common wild-rice of Dongxiang, Jiangxi, and changes in the paddy rice it over to its T
1In generation, changes the comparison of LTT1 gene plant and shows stronger resistance to cold according to strain Japan is fine.4-5 ℃ of subzero treatment 5d, behind the 7d that recovers under the normal condition to grow, T
1In generation, changeed LTT1 gene plant well-grown, and the leaf look dark green, and the seedling rate of living is 100%, plant height 8.37 ± 0.51cm; And the adjoining tree of same treatment (changeing the Japanese fine of pCAMBIA1300-35S) growing way is very poor, and plant height 2.9 ± 0.53cm is obviously short in T
1In generation, changeed the LTT1 gene plant, and the seedling rate of living is 50%.
The characteristics that the method for the cold-resistant plant of cultivation of the present invention has is simple to operate, the cycle is short are suitable for applying.This method has important theory and practical significance for the seed selection and the plant resistance to cold molecular breeding of plant resistance to cold Molecular Study, cold-resistant kind, for the resistance to cold that improves plant provides an economy, approach fast and effectively.The present invention has wide application and market outlook at agriculture field.
Description of drawings
Fig. 1 is for being the band of template pcr amplification product with Dongxiang, Jiangxi common wild-rice, IL112 and osmanthus towards No. 2 genomic dna
Fig. 2 is the band of template pcr amplification product for the genomic dna with 20 rice varieties
Fig. 3 A is the T that changes pCAMBIA1300-35S-LTT1 over to
0PCR detected result for transfer-gen plant and contrast strain
Fig. 3 B is the T that changes the LTT1 gene over to
1Bud phase resistance to cold qualification result for transfer-gen plant and contrast strain
Embodiment
Method therefor is ordinary method if no special instructions among the following embodiment, and the primer synthesizes and examining order is finished by Beijing AudioCodes biotechnology limited liability company.
Following rice varieties is as if no specified otherwise, all available from national seed resource storehouse.
At first backcrossed and selfing towards No. 2 in Dongxiang, Jiangxi common wild-rice and super high-yielding rice varieties osmanthus, made up with osmanthus towards being for No. 2 that the height of genetic background is for backcross population (BC
4F
2Colony), this colony is carried out bud phase resistance to cold identify, simultaneously in contrast with Lijiang xintuanheigu.Concrete authentication method is: with osmanthus towards No. 2, high for backcross population (BC
4F
2Colony) and the seed of Lijiang xintuanheigu use the chlorine bleach liquor of 5% (volumn concentration) to soak behind the 20min respectively with flushing with clean water 3-4 time, 37 ℃ of presoaking and germinating 1d, subsequently seed is placed on the filter paper that glass test tube soaks, test tube is put into the illumination cultivation chamber, and (daytime, temperature was 28 ℃, night, temperature was 25 ℃, illumination every day, respectively be 12h interlunation, relative humidity 83%), when treating that bud length is grown to the 5mm left and right sides behind the seed germination, each rice varieties selects 10 healthy and strong consistent buds to place diameter 4cm respectively, in the glass test tube of high 9.5cm, test tube is placed 4-5 ℃ of refrigerator subzero treatment 5d, then the young shoot after the subzero treatment is moved to and recover growth 7d in the illumination cultivation chamber, measure high for backcross population (BC
4F
2Colony), osmanthus is towards the seedling rate alive of No. 2 and Lijiang xintuanheigu, carries out the resistance to cold evaluation with the seedling rate of living [the seedling rate of living=(the seedling number of living/confession examination seedling number) * 100%] as the index of bud phase resistance to cold, the screening budding time is cold-resistant by force to be IL112.The result shows that the seedling rate alive of IL112 is 100%, and its bud phase is cold-resistant stronger.
Being donor subsequently with IL112, is that acceptor makes up F with osmanthus towards No. 2
2:3Colony carries out bud phase resistance to cold according to the method described above to its colony and identifies, adds up the seedling rate alive of each strain system.Further resistance to cold analysis revealed, the 4-5 ℃ of low temperature of IL112 and offspring's ability 9d thereof has extremely strong bud phase resistance to cold.
Extract above-mentioned F
2:3The genomic dna of each individual plant of colony carries out ssr analysis, obtains the genotype of each strain system, and utilizes QTXMAP17 software to carry out the resistance to cold qtl analysis, and the result is the 1st, 2,5,6,7 with 10 karyomit(e)s on all detect and the relevant QTL of bud phase resistance to cold.For these QTL of Fine Mapping, find and the relevant gene of paddy rice bud phase resistance to cold, be that material carries out the chip (full genome chip (GeneChip of the paddy rice of Affimetrix company with IL112 and osmanthus towards No. 2
Rice Genome Array, article No.: 900599) hybridization, on the basis of chip data analysis, in conjunction with the comparative genomics analysis, full genomic level top sieve be selected in have genome difference between Japanese warm and fine 93-11 differential expression genes as candidate's goal gene.According to the fine genome sequence of Japan, there is the zone design primer p18 (sequence 3 and sequence 4) of genome difference at the two, be that template is carried out pcr amplification with Dongxiang, Jiangxi common wild-rice, IL112 and osmanthus towards No. 2 genomic dna respectively.Concrete pcr amplification reaction system is: oryza sativa genomic dna template 20ng, Taq Plus archaeal dna polymerase 0.5U, 2.0 μ l, 10 * PCR damping fluid (100mM TrisCl pH9.0,500mM KCl, 15mM Mg
2+, 1%Triton X-100), 100 μ MdNTPs, each 0.2 μ M of forward and reverse primer, DEPC water postreaction system to 20 μ l.The PCR reaction conditions is: 94 ℃ of 3min of elder generation; 94 ℃ of 1min then, 58 ℃ of 1min30sec, 72 ℃ of 2min, totally 35 circulations; 72 ℃ of 10min again.Pcr amplification product is carried out 1% agarose gel electrophoresis detect, concrete detected result as shown in Figure 1.Wherein 1,2 and 3 to be respectively with Jiangxi Dongxiang Wild Rice, IL112 and osmanthus be the pcr amplification product band of template towards No. 2 genomic dna, DL2000 is dna molecular amount standard (day root biochemical technology company limited, the article No.: Cat#HT402-01) of 100-2000bp.The result shows, the identical band that in Dongxiang, Jiangxi common wild-rice and IL112, increases, and its size is about 1000bp; And osmanthus does not have amplified band in No. 2.Reclaim band that amplification obtains and it is checked order, sequencing result shows that its nucleotide sequence is shown in sequence in the sequence table 2, and with its called after LTT1, its amino acid sequence coded is shown in sequence in the sequence table 1.
In order to verify whether gene LTT1 is relevant with paddy rice bud phase resistance to cold, be primer with p18, to F
2:3Colony has carried out genotype detection, and in conjunction with the phenotypic evaluation result who carries out previously, with the relation of T-Test methods analyst genotype and bud phase resistance to cold, the result shows, has significant correlation (P＜0.01) with bud phase resistance to cold when the p18 amplified production infiltrates separately.
In order further to verify the dependency of gene LTT1 and paddy rice bud phase resistance to cold, according to the method described above 125 rice varieties and local race have been carried out the resistance to cold evaluation of bud phase, and 20 rice varieties of picked at random (10 cold-resistant kinds therefrom, 10 resistance to cold difference kinds, concrete kind sees Table 3) extract its genomic dna respectively, with p18 primer, carry out pcr amplification, detecting its genotype, simultaneously towards No. 2 in contrast with IL112 and osmanthus.The concrete detection as shown in Figure 2.Wherein, swimming lane 1 is the pcr amplification product band of template for the genomic dna with IL112; Swimming lane 2-11 is the pcr amplification product band of template for the genomic dna with 10 cold resistant paddy rice kinds; Swimming lane 12 is for being the pcr amplification product band of template with osmanthus towards No. 2 genomic dna; Swimming lane 13-22 is the pcr amplification product band of template for the genomic dna with the rice varieties of 10 resistance to cold differences; DL2000 is dna molecular amount standard (day root biochemical technology company limited, the article No.: Cat#HT402-01) of 100-2000bp.The result shows that in 10 cold resistant paddy rice kinds, 9 kinds all amplify the band identical with IL112, and stripe size is about 1000bp; In the rice varieties of 10 resistance to cold differences, 9 kinds all do not amplify any band, only have a kind can amplify the band identical with IL112.
Simultaneously phenotypic evaluation is carried out towards No. 2 bud phase resistance to cold in above 20 rice varieties, IL112 and osmanthus, the result is as shown in table 4.
The bud phase resistance to cold qualification result of table 4 paddy rice
Rice varieties seedling rate alive
IL112 100％
746 100％
Cloud peak No. 7 100%
Fragrant round-grained rice glutinous 100%
Late 88-1 100%
Still the continent 10 100%
Close positive 111 100%
Syzygy 35 100%
REIMEL 100％
IR66746-76-3-2 100％
9505-138 100％
Osmanthus is towards No. 2 0%
03A-11 0％
03A-9 0％
In excellent 13 0%
37760 0％
Water source 349 0%
Water source 332 0%
Water source 287 0%
VISTA 0％
TP34 0％
SR64446-1 0％
Three repetitions are established in experiment, and the data in the table 4 are three multiple mean values.With the relation of T-Test methods analyst genotype and bud phase resistance to cold, the correlation analysis result is as shown in table 5.The result shows that there are utmost point significant correlation (P＜0.01) in gene LTT1 and paddy rice bud phase resistance to cold.
The correlation analysis of table 5.p18 and rice cold tolerance
| Molecule marker | R Square | Significance F | Ratio1 | Ratio2 |
| The PCR product of p18 | 0.5664 | 2.2074E-05 ** | 90％ | 10％ |
Ratio1 is cold resistant paddy rice kind number/10 that have molecule marker to infiltrate; Ratio2 is not cold resistant paddy rice kind number/10 that have molecule marker to infiltrate; *: p＜0.01.
Above result shows that gene LTT1 is and the relevant gene of paddy rice bud phase resistance to cold, and this gene is the gene of a Unknown Function.
The acquisition of embodiment 2, LTT1 transgenic paddy rice and resistance to cold thereof are identified
One, the structure of LTT1 plant expression vector
Full length cDNA sequence according to the fine LOC_Os01g51670 of Japan designs primer, and introduces restriction enzyme KpnI and SacI recognition site and protection base respectively at the primer two ends, and primer sequence is as follows: CDS-PF:5 '-
CGGGGTACCCCGTCTGAACTCCAAACATCAGTAAGC-3 ' (sequence 5) (band underscore base is restriction enzyme KpnI recognition site and protection base); CDS-PR:5 '-
CGAGCTCGTGACTTCAAATAATTTTTCAGGATATT-3 ' (sequence 6) (band underscore base is restriction enzyme SacI recognition site and protection base).
Utilize TRIZOL reagent to extract Dongxiang, Jiangxi common wild-rice through total RNA of the bud phase of 4 ℃ of subzero treatment 12h, with this RNA is template, use SuperScriptII ThermoScript II (Invitrogen, Cat no.18064-014) carries out reverse transcription and obtain cDNA, with this cDNA is template, under the guiding of primer CDS-PF and CDS-PR, the encoding sequence of RT-PCR amplifying rice LTT1 gene.After reaction finishes, amplified production is carried out 1% agarose gel electrophoresis to be detected, reclaim and the dna fragmentation of purifying 995bp checks order, sequencing result shows, the deoxynucleoside acid sequence of the dna fragmentation that amplification obtains be in the sequence table sequence 2 from 5 ' end 1-995 position nucleotide sequence.
With HindIII and XbaI double digestion carrier pBI121 (available from Promega company) and carrier pCAMBIA1300 (available from Australian CAMBIA company) respectively, big fragment after connecting the pBI121 enzyme and cutting the 35S promoter that obtains and pCAMBIA1300 enzyme and cut promptly obtains plant expression vector pCAMBIA1300-35S.
The dna fragmentation of above-mentioned 995bp is cloned between the KpnI and SacI restriction enzyme site of plant expression vector pCAMBIA1300-35S multiple clone site, obtains containing the recombinant expression vector of oryza sativa l. TT1 gene, called after pCAMBIA1300-35S-LTT1.
Two, rice transformation
Recombinant expression vector pCAMBIA1300-35S-LTT1 and pCAMBIA1300-35S that step 1 is made up transform the fine mature embryo callus of Japan with particle bombardment respectively, carry out 2 with the NB substratum that contains the 50mg/L Totomycin and take turns screening, the every wheel screened 20-30 days, the callus that screening obtains obtains genetically modified rice plant through pre-differentiation, differentiation, uses T
0Representative is shown, T
0The seed that produces for selfing and use T by the plant that it grew up to
1Representative is shown.
Three, the PCR of transgenic paddy rice identifies
Cultivate the T that pCAMBIA1300-35S-LTT1 is changeed in a strain
0For the paddy rice transfer-gen plant, extract above-mentioned T
0For the genomic dna of transgenic paddy rice, and, under the guiding of primer 1 (sequence 7) and primer 2 (sequence 8), carry out pcr amplification as template.Simultaneously in contrast fine with not genetically modified Japan.The PCR reaction conditions is: 94 ℃ of 5min of elder generation; 94 ℃ of 30sec then, 58 ℃ of 45sec, 72 ℃ of 1min, totally 35 circulations; 72 ℃ of 10min again.After reaction finishes, pcr amplification product is carried out 1% agarose gel electrophoresis detect.Concrete detected result as shown in Figure 3A.Wherein, swimming lane 1 is for changing the T of recombinant expression vector pCAMBIA1300-35S-LTT1 over to
0Agarose gel electrophoresis result for the transfer-gen plant pcr amplification product, swimming lane 2 is the fine pcr amplification result's of not genetically modified Japan agarose gel electrophoresis result, DL2000 is dna molecular amount standard (day root biochemical technology company limited, the article No.: Cat#HT402-01) of 100-2000bp.The result shows, changes the T of recombinant expression vector pCAMBIA1300-35S-LTT1 over to
0Amplify the band that size is about 1Kb for transfer-gen plant, and do not have band in the fine plant of the not genetically modified Japan of contrast.
According to embodiment 1 described bud phase resistance to cold authentication method to the positive T of above-mentioned PCR qualification result
0The offspring in generation, i.e. T
1Carry out resistance to cold for transfer-gen plant and identify, simultaneously to change the T of pCAMBIA1300-35S
0The T that obtains for paddy rice
1For transfer-gen plant in contrast.Each strain is 10 strains.Concrete detected result is shown in Fig. 3 B.Wherein, CK is the T that grows under the normal condition
1In generation, changeed pCAMBIA1300-35S plant (Control) and T
1In generation, changeed pCAMBIA1300-35S-LTT1 plant (OE1); Cold is through 4-5 ℃ of subzero treatment 5d, the T under the normal condition behind the recovery growth 7d
1In generation, changeed pCAMBIA1300-35S plant (Control) and T
1In generation, changeed pCAMBIA1300-35S-LTT1 plant (OE1).
The result shows, T
1Show stronger resistance to cold for the transfer-gen plant comparison according to strain.4-5 ℃ of subzero treatment 5d, behind the 7d that recovers under the normal condition to grow, T
1For the transfer-gen plant well-grown, the leaf look dark green, and the seedling rate of living is 100%, plant height 8.37 ± 0.51cm; And the adjoining tree of same treatment (changeing the Japanese fine of pCAMBIA1300-35S) growing way is very poor, and plant height 2.9 ± 0.53cm is obviously short in T
1In generation, changeed the LTT1 gene plant, and the seedling rate of living is 50%.
Sequence table
<160>8
<210>1
<211>168
<212>PRO
＜213〉common wild-rice (O.rufipogon Griff.)
<400>1
Met Ser Ser Leu Arg Arg Glu Ala Pro Pro Ile Ser Asp Tyr Glu Ala
1 5 10 15
Leu Asp Gly Ser Gly Lys Cys Thr Asp Glu Pro Ser Cys Ser Ser Asp
20 25 30
Pro Ser Lys Asp Ser Ser Ser Cys Thr Ser Ala Phe Ala Phe Thr Ile
35 40 45
Leu Ala Ile Asn Cys Gly Ala Ala Ile Tyr His Ser Arg Arg Asp Pro
50 55 60
Trp Ser Val Ala Phe Val Leu Ala Ala Phe Leu Met Leu Ile Ser Leu
65 70 75 80
Phe Cys Ala Leu Arg Leu Phe Glu Ser Leu Pro Arg Ser Ser Pro Arg
85 90 95
Arg Ser His Val Lys Ala Gly Val Trp Val Leu Ser Thr Val Leu Thr
100 105 110
Ile Leu Phe Thr Tyr Arg Val Ala Ala Leu Met Pro Phe Pro Val Ala
115 120 125
Val Val Val Trp Ala Met Ser Val Phe Thr Ile Leu Ala Gly Phe Tyr
130 135 140
Met Phe Phe Val Cys Ser Asp Glu Val Lys Ala Ala Pro Glu Glu Arg
145 150 155 160
Pro Ala Lys Val Ser Asp Met Ala
165
<210>2
<211>995
<212>DNA
＜213〉common wild-rice (O.rufipogon Griff.)
<400>2
tctgaactcc aaacatcagt aagctagata tcccttccta cgccgaaggc agaagcgtcg 60
acgatgtcga gtctccgcag ggaagctcca ccgatctctg actacgaggc actagatggt 120
tccggcaaat gcaccgacga accatcatgc tcctcggatc ctagcaaaga cagctcgtcg 180
tgtacatcgg ccttcgcctt caccatcctt gccatcaact gcggcgcagc tatctaccat 240
tcgcggcgtg acccctggtc agtcgcgttt gtgttggccg ccttcctcat gctcatctcg 300
ctcttctgcg cgctccgctt gttcgagagc ttgcctcgca gctccccccg cagatcccat 360
gtcaaagccg gtgtttgggt cctttccacg gtgctcacaa tcttgttcac ctacagagta 420
gctgcgctta tgcctttccc agtcgccgtt gtcgtgtggg ctatgtccgt cttcacaatt 480
cttgcaggat tctacatgtt cttcgtttgc agcgacgagg tcaaggcggc gccagaggag 540
aggccggcaa aagtgtctga catggcttga gccctggggc cctggctgtg caacccctgt 600
ggaaacgtca gtgctccact gctgggaccc gagacggaga cgccggcaac atcataattc 660
cccagcagtg tgtgtcccga ttgcacaacc ggaacaaaag ggattgtgaa aagatcgatt 720
cctcagctgt gattgcaagt gctcctccac cctgcgcagc tgtatggttt tgaccgagtt 780
tcaggaccaa aaacggttcc tccgccctgt gctgattcac cggtagttcc tgttgataca 840
gtatcttcca aagcctctgg ttttcagaat gatcaaagga accacagaag atacactagg 900
gctcggatta cagtacaacc tgtaactttc cagtttcggc cacaaattgt tccgagtgat 960
gtcatatgta atatcctgaa aaattatttg aagca 995
<210>3
<211>18
<212>DNA
＜213〉artificial sequence
<400>3
<210>4
<211>20
<212>DNA
＜213〉artificial sequence
<400>4
<210>5
<211>36
<212>DNA
＜213〉artificial sequence
<400>5
CGGGGTACCCCG TCTGAACTCCAAACATCAGTAAGC 36
<210>6
<211>35
<212>DNA
＜213〉artificial sequence
<400>6
cgagctcgtg acttcaaata atttttcagg atatt 35
<210>7
<211>18
<212>DNA
＜213〉artificial sequence
<400>7
tacttctaca cagccatc 18
<210>8
<211>18
<212>DNA
＜213〉artificial sequence
<400>8
Claims (10)
1, a kind of albumen is following (a) or protein (b):
(a) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 1;
(b) with the amino acid residue sequence of sequence in the sequence table 1 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and relevant with the plant resistance to cold by (a) deutero-protein.
2, the described proteic encoding gene of claim 1.
3, encoding gene according to claim 2 is characterized in that: described proteic encoding gene is following 1) or 2) or 3) gene:
1) its encoding sequence be in the sequence table sequence 2 from 5 ' end 64-570 position deoxyribonucleotide;
2) its nucleotide sequence is a sequence 2 in the sequence table;
3) dna molecular that under stringent condition, can have the amino acid residue sequence of sequence 1 in the sequence table with the dna sequence dna hybridization and the coding of 2 qualifications of sequence in the sequence table.
4, the recombinant expression vector that contains claim 2 or 3 described genes.
5, recombinant expression vector according to claim 4 is characterized in that: the carrier that sets out of described recombinant expression vector is pCAMBIA1300-35S;
Described pCAMBIA1300-35S is with HindIII and XbaI double digestion carrier pBI121 and carrier pCAMBIA1300 respectively, and the big fragment after connecting the pBI121 enzyme and cutting the 35S promoter that obtains and pCAMBIA1300 enzyme and cut promptly obtains pCAMBIA1300-35S.
6, the transgenic cell line that contains claim 2 or 3 described genes.
7, the reorganization bacterium that contains claim 2 or 3 described genes.
8, total length or arbitrary segmental primer of amplification claim 2 or 3 described genes are right.
9, a kind of method of cultivating cold-resistant plant is that claim 2 or 3 described encoding genes are changed in the host plant, obtains the plant that resistance to cold improves.
10, method according to claim 9 is characterized in that, described host plant is a monocotyledons, is preferably paddy rice.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103834653A (en) * | 2012-11-22 | 2014-06-04 | 中国农业大学 | Cold induced promoter p-LTT1 for rice and application thereof |
| CN107418957A (en) * | 2017-09-11 | 2017-12-01 | 浙江师范大学 | Control rice cold tolerance gene C OLD2 and its application |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100494403C (en) * | 2006-07-18 | 2009-06-03 | 中国农业大学 | Method of assisting screening for cold resistant paddy rice and its special primer |
| CN100526465C (en) * | 2007-03-12 | 2009-08-12 | 华中农业大学 | Raising plant cold endurance and salt tolerance by means of transcription factor gene SNAC2 of rice |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103834653A (en) * | 2012-11-22 | 2014-06-04 | 中国农业大学 | Cold induced promoter p-LTT1 for rice and application thereof |
| CN103834653B (en) * | 2012-11-22 | 2016-02-24 | 中国农业大学 | Rice Cold evoked promoter p-LTT1 and application thereof |
| CN107418957A (en) * | 2017-09-11 | 2017-12-01 | 浙江师范大学 | Control rice cold tolerance gene C OLD2 and its application |
| CN107418957B (en) * | 2017-09-11 | 2020-08-18 | 浙江师范大学 | Cold-resistant gene COLD2 for controlling rice and application thereof |
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