CN111763672B

CN111763672B - Rice low-temperature inducible expression promoter Poscold10 and application thereof

Info

Publication number: CN111763672B
Application number: CN202010618772.7A
Authority: CN
Inventors: 许蓉芳; 李娟�; 秦瑞英; 刘小双; 廖圣祥
Original assignee: Rice Research Institute of Guangdong Academy of Agricultural Sciences
Current assignee: Rice Research Institute of Guangdong Academy of Agricultural Sciences
Priority date: 2020-06-30
Filing date: 2020-06-30
Publication date: 2022-03-25
Anticipated expiration: 2040-06-30
Also published as: CN111763672A

Abstract

The invention provides a plant low temperature inducible expression promoter Poscold10 and its application. The present invention also provides expression cassettes, plant expression vectors, host bacteria and transformants containing the promoter. Specifically, the present invention applies the above promoter in plant genetic engineering. The promoter provided by the present invention can specifically drive the high-strength expression of exogenous genes in plants under low temperature conditions. By linking the promoter of the present invention to the gene to be expressed and using the vector to transfer it into the plant, it is possible to drive the expression of the foreign gene in various parts of the plant under low temperature conditions.

Description

Rice low-temperature inducible expression promoter Poscold10 and application thereof

Technical Field

The present invention relates to biotechnology and plant genetic engineering technology. Specifically, the invention relates to a plant low-temperature inducible expression promoter and application thereof, wherein the promoter can drive a target gene to express in a plant in a rice transgenic regulation system.

Background

Rice is one of important food crops in the world, and in recent years, due to the early-nino phenomenon of climate, the rice is subjected to extreme climate of drought or flooding in the growth stage, so that the yield of the rice is seriously influenced. The method for improving the resistance of crops to extreme climate by the plant genetic engineering method has important application prospect. The regulation and control of the promoter on the gene expression suggest that the expression of the stress-resistant gene is activated by changing or adding the promoter through genetic engineering, so that the stress resistance of crops is improved.

Low temperature is a common environmental stress factor encountered by plants, has wide and profound effects on the plants, and is affected in various stages from seed germination, vegetative growth, reproductive growth to flowering and fruiting. In addition, physiological and biochemical metabolic processes such as photosynthesis, respiration, absorption and transportation of water and nutrient elements, activity of various enzymes and conversion of organic matters are affected by low temperature, and too low temperature or continuous lower than optimal growth temperature can have important influence on plant/crop production and directly affect yield. Crop losses caused by low-temperature damage are as high as billions of dollars worldwide every year, so that how to overcome the low-temperature damage and cultivate low-temperature-resistant genetically engineered crops is a hot problem for molecular design and breeding of crops.

A complex low-temperature stress signal response network system exists in plants. After the plants are stressed by low temperature, a series of genetic modifications can be carried out through the system, and the metabolism and the growth of the plants are adjusted to adapt to the influence of the adverse factors. By using the key gene in the response network, the low-temperature tolerance of the plant can be effectively improved. However, in the current genetic engineering operation, a constitutive promoter is mostly used to drive the key node genes to be over-expressed, and although the obtained transgenic plant can show strong low-temperature tolerance, unnecessary expression brought by the constitutive promoter often causes the dwarfing, development delay and material energy waste of the plant, so that the potential practical application and popularization are not facilitated. Therefore, the use of low temperature inducible promoters to activate key regulatory genes only under low temperature stress will be one of the main development directions for improvement of low temperature resistant gene engineering.

Disclosure of Invention

The invention aims to provide a promoter for driving exogenous genes to express under a low-temperature induction condition, a transformant obtained by the promoter and application of the promoter. Herein, reference to "plant" is to a monocotyledonous plant, such as rice, wheat, maize, barley, sorghum or oats, preferably rice. Furthermore, the promoter of the present invention is a promoter having high strength (or referred to as high inducible expression efficiency).

In order to achieve the aim, the invention provides a plant low-temperature inducible expression promoter which comprises a DNA sequence shown as SEQ ID No. 1 in a sequence table. The DNA sequence shown in SEQ ID No. 1 in the sequence list is a rice low-temperature inducible expression promoter derived from Nipponbare (Oryza sativa L cv. Nipponbare), and is called Poscold10 or Poscold10 herein. Specifically, the inventors of the present application found that a 2100bp DNA sequence including a transcription initiation site upstream of the LOC _ Os12g05210 gene of Oryza sativa L cv. Nipponbare has a function of driving specific expression of a target gene under low temperature conditions, and isolated, cloned, and identified the function of the DNA sequence.

Preferably, the DNA sequence of the plant low-temperature inducible expression promoter provided by the invention is the sequence shown in SEQ ID No. 1, namely Poscold10 or Poscold 10.

In another aspect, the invention provides a plant low temperature inducible expression promoter, the DNA sequence of which has at least 80% homology with the DNA sequence shown in SEQ ID No. 1; or the plant low-temperature inducible expression promoter is a mutant or allele or derivative generated by adding, substituting, inserting or deleting one or more nucleotides in the DNA sequence shown in SEQ ID No. 1; or the plant low-temperature inducible expression promoter has a product hybridized with the DNA sequence shown in SEQ ID No. 1. The plant low-temperature inducible expression promoter sequences have the same functions as the DNA sequences shown in SEQ ID No. 1, namely driving the target genes to express in the plants.

In another aspect, the invention also provides an expression cassette comprising the plant low-temperature inducible expression promoter.

In still another aspect, the present invention also provides a recombinant expression vector comprising the plant low temperature inducible expression promoter described above, wherein the plant low temperature inducible expression promoter is linked upstream of a gene sequence to be expressed; preferably, the gene to be expressed is a Gus gene, the recombinant expression vector is pCAMBIA1391-Poscold10, and the recombinant expression vector is a recombinant expression vector obtained by constructing a sequence shown in SEQ ID No:1, namely Poscold10 or a promoter Poscold10 in pCAMBIA1391 and is named pCAMBIA1391-Poscold10 herein. Or the gene to be expressed may be any gene having an ability to improve the cold resistance of crops. The promoter drives the cold-resistant gene to be intensively expressed under the cold condition, so that the function of improving the cold-resistant property of crops is realized.

In another aspect, the present invention also provides a host bacterium, wherein the host bacterium comprises the plant low-temperature inducible expression promoter provided by the present invention, the expression cassette provided by the present invention, or the recombinant expression vector provided by the present invention; preferably, the host bacterium is agrobacterium tumefaciens.

In another aspect, the present invention provides a transformant comprising the plant low-temperature inducible expression promoter, the expression cassette, the recombinant expression vector, or the host bacterium provided by the present invention. Wherein, the transformant is preferably a transgenic cell line, callus or plant.

In another aspect, the invention provides the application of the plant low-temperature inducible expression promoter in culturing transgenic plants. The application comprises the steps of connecting the plant low-temperature inducible expression promoter provided by the invention to the upstream of a gene sequence to be expressed of a vector (for example, placing the promoter sequence in front of a target gene), so as to construct a recombinant expression vector, and transforming the recombinant expression vector into plant cells, tissues or organs for cultivation.

And preferably the application may be for improving the growth characteristics of plants which are monocotyledonous plants such as rice, wheat, maize, barley, sorghum or oats, preferably rice.

The DNA sequence of the promoter provided by the invention is (same as SEQ ID No:1 in the sequence table):

CTTGGGCCCGTGTCAGTGAGGTGGCGGCGGCAATGGCGTCGGCAGCGGGTGGTTCGTGGTGGGCATCCTTCTTCAACCTCTGGTTATTCTGTAGCATTGCCCACAGCTCAAGCACTCAACTGTTATGTTGAGAAAACGAAGGCAGAAGAGATAAAATGAAGCTAGAGCTGGAAATGGTTGACCATGACCGGCTTTCAGACATAGAAAATTCATTCAAGAAGTTGTCTAAGCAGAAAAAAGAGACTGATGACATTAGAAATTGGTTCATAGGAACAGTAGTTGTAGCTGTGTTCCTTCTCATGAAGCTATTTGATAATATGTACTATGCTTGCCATTGTTGATTTTGTACATGTCAATTACCAGAATTAATGGATGGGGATCACTCAGCATGTTGATGTCTCAGCAACCTATCCATGTGAATTCTGTTTATCTATCTCCCACTATTTGAAACTCTAAATTACAGCTTCTTCAGATGGGTTGTCTAACTTATCCTTCAATTTTAGATTCTTGCTAAGTTCTAGTTCGTTCTGTTTGGCTGAAAATTTGCAGAATTCTGTTTATCTATCTCCCACTGTCTGAAACTCTGAATTACAACACACTAGTACGAGTATGTTTGTTTCAGTCTTTCAGAATCATGTAAATTATACTTTTTTTCAGTAATTATTGCCCATGTGTCTAGCACTCTACTCCAGTGATTCCTCAACTTTTACATCTATAGCTGGATTGCTGTTCATTGAAAACACTTGCTTGTAACCATTGTTGTGCTGCTGATCACTCCACAGAATGATATATATTTTACAGGAGATCGAGCAAAAACATCAGACAAAATATTAATGGCTCTCAATACGACATATATATCATGCTCTGTTCACAGCAGACAAAAATTAAGTCAAAAGCTCCATTCAACGATAGATCATGCCCTGTAAACAGCATACCAAAAAACGGACGACAGATGAGCTTAAAATTTTCCATTCAAAGATTAAAGTCTCCAGCAAAAGCTTGTGCTTTTCTCACAAATTCATTACAAGAAACATGGCAGATTGAATTGTTAATTACAAAATAACTAGACCATATAGTGGAGTAGTGTTTGAGCCGTAATGGGGGCCTAGGCTGCGCCATGGCAGCGGGGGAGGTTAGGTACTTGCTTGAGCTGTCGAGGGAGGGGGATAGGGGACACAGAATACCATGACATCTTCGCTCGTGAATGCGGCGGCGTTGTTGGTGACACTGTCGCGGACAGCAGATGGATGGGAAAAAATCGGCACCGGCAGAGGCATGGATGCGGGGCGAGCGCGCCGTTTCTCCAGTACCGCGACCACGGACGGCAGATGGATGGGAAAAAACCGTTCGGCGGGGCGTGGATGCGGCGCAACTGCTCCGTTTCTGCGCTCAGATGGAGGAATAGATGAGAAAGAACCGATGCGGGGAGGGAGGAAAGAAAGAGCGCAGCGAGGGAGGAAAGCGGCGGCGTGTGACTCCGGAATTGTTCCGCTTCTATTTGTTGTTTTCCAATCGTATCCTTTCAAGTTGGACGGCCAGATTTAATCTGGTACCTCCTAGTACCAGTACTGGCTGGTACAGACTGCCGGACGGGAGTAAAACTCTTCATAGATAGATGAATCTAGATAGATAATCAATATGAATATAGAAAATGCTATAATGACTTACATTGTGAAATGGAGAGAGTAGTTCGAAAGCATGCGCATAGAAAAGAAAGAAATAGGTCGGAAAAGTGAAGGAAAAAAAAACTCAGCCTATAAGTAGGGTGCACGTTTATAGTTTCACTAAGTGTTTATACTGTGTTTCTTGATAGAAAATAATACGGGCTTTAAATTTCCCGTGACGCTATCGATGCAATTCATGCAACCAACCAGGCGTGCTGCGTGCAACACCTGGAACAAATCCTTTCGGCGTTCGTTGCACCTGGACACACACCTGCACATGACGTGGCCCCCTATCCATCCATCCCTTACCGCCTTCCTCTCCACGCGTCGCCTTCCGCCTCCGCCACCTCCTCCCCCTCTCCACACACTCTTCTTCTCCACTCCTCCGCCGCTCGCCTCGCCGCGCGCCGCTTATATACCCGCGTCTCTGCGCTCA

in summary, the present inventors found, extracted and identified a 2100bp DNA sequence including 2000bp and 100bp upstream of the transcription initiation site in Oryza sativa L cv. Nipponbare, and named Poscold10 (SEQ ID No:1 in the sequence Listing). The sequence is connected to a plant binary expression vector pCAMBIA1391 after enzyme digestion to obtain a corresponding recombinant plasmid (namely, a recombinant expression vector), the recombinant plasmid is used for transforming an agrobacterium tumefaciens strain EHA105, and then the rice is transformed by an agrobacterium-mediated method to obtain a transgenic rice plant. The GUS expression quantitative detection of the obtained transgenic rice shows that the overall expression level of the GUS gene is improved after the transgenic plant is subjected to low-temperature induction treatment, so that the 2100bp sequence is proved to have the activity of driving the gene expression, and the GUS gene driven by the promoter is expressed after the rice is subjected to low-temperature induction treatment.

The promoter sequence of the invention can be connected with a plant binary expression vector and used for replacing a constitutive promoter. Moreover, the promoter sequence can be connected with a required target gene to construct a recombinant plant expression vector, and after transformation and low-temperature induction treatment, the promoter sequence can drive the target gene to specifically express in a plant, so that the expression quantity of an exogenous target gene in the plant is improved, and the transgenic effect is increased.

Technical effects

The cloned rice promoter Poscold10 can regulate and control the centralized expression of genes in plants, and has obvious value in practical application. The promoter is used for carrying out gene modification on crop varieties, and if the promoter is used for driving a target gene to express in plants, the growth characteristics, especially the cold resistance, of rice can be improved, so that practical and effective cold-resistant plant varieties can be cultivated.

Drawings

Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:

FIG. 1 is a schematic representation of the construction of the Poscold10 promoter into the pCAMBIA1391 vector plasmid, wherein A is a schematic representation of pCAMBIA1391 and B is a schematic representation of pCAMBIA1391-Poscold10, wherein the use of the Poscold10 promoter to drive GUS gene expression downstream thereof is shown;

FIG. 2 is a schematic diagram showing the results of enzyme digestion verification of the promoter of the present invention.

FIG. 3 shows Poscold10 of 30-day-old plant tissue staining pattern of GUS transgenic plant. After the rice plant which normally grows at the temperature of 30 ℃ is dyed for 24 hours, no Gus activity exists in roots, stems and leaves, and after the rice plant is treated at the low temperature of 4 ℃ for 24 hours and is dyed for 24 hours, Gus strong expression exists in the roots, stems and leaves.

FIG. 4 shows the change in the relative expression levels of POscold10 and PUBI as a control after 0h, 12h and 24h of the low-temperature treatment.

Detailed Description

The invention is illustrated below with reference to specific examples. It will be understood by those skilled in the art that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention in any way.

The experimental procedures in the following examples are conventional unless otherwise specified. The biochemical reagents, consumable materials, and the like used in the following examples are all commercially available products unless otherwise specified.

Obtaining Poscold10 promoter containing enzyme cutting site

Step 1, design of primers

The applicant unexpectedly found a gene sequence with low temperature induction property in the research, named Poscold10, and the specific sequence is shown in the sequence table. The applicant designs an amplification primer according to the sequence of the rice Poscold10, and designs the enzyme cutting site of the primer according to the characteristics of the selected vector and the target gene.

In this example, a rice binary expression vector pCAMBIA1391 (part A in FIG. 1, from CAMBIA, publicly used vector, component supervision and inspection test center for transgenic biological products of department of agriculture of the academy of agricultural sciences of Anhui province) was taken as an example, and the target gene was a Gus gene. The specifically designed primers are: the forward primer (SEQ ID No:2) has PstI and cleavage site (CTGCAG) at 5 'end, the reverse primer (SEQ ID No:3) has BamHI cleavage site (GGATCC) at 5' end, and the primer sequences are as follows:

a forward primer: 5'-CTGCAGCTTGGGCCCGTGTCAGTGAGGT-3' PstI

Reverse primer: 5'-GGATCCTGAGCGCAGAGACGCGGGTATA-3' BamHI

Synthesized by Shenzhen Hua DageneCo.

Step 2, obtaining a promoter Poscold10

The DNA of a rice variety Nipponbare is taken as a template, a forward primer and a reverse primer are utilized to amplify a promoter Poscold10, and the following amplification program is adopted according to a conventional PCR system:

pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 2min for 30s, and circulation for 35 times; finally, extension is carried out for 10min at 72 ℃.

Recovering a target fragment amplified by PCR, wherein the length of the target fragment is 2100bp, connecting the target fragment to a PGEM-T-Easy vector (purchased from Promega corporation and mixed according to the proportion in the vector specification), transforming escherichia coli XL-Blue competent cells according to a cold shock method, activating the competent cells, transferring the target fragment into the activated competent cells, then obtaining positive clones through colony PCR screening, selecting monoclonal shake bacteria liquid to extract plasmids, and performing double enzyme digestion verification by PstI and BamHI, as shown in figure 2. The identified positive clones were sent to Invitrogen for sequencing. The correct clone is verified to be the promoter Poscold10 to be obtained, and the nucleic acid sequence of the clone is shown as SEQ ID No. 1.

Construction of plant expression vectors and transformation of Agrobacterium

A plasmid was extracted from the positive clone obtained in the procedure of "obtaining promoter Poscold 10" above, and digested with PstI and BamHI to recover the promoter Poscold10 fragment. Meanwhile, pCAMBIA1391 is linearized by PstI and BamHI, pCAMBIA1391 is recovered, the Poscold10 fragment and pCAMBIA1391 fragment are connected by T4 ligase (purchased from TaKaRa), a plant expression vector pCAMBIA1391-Poscold10 (FIG. 1B) with a promoter Poscold10 fused with a Gus gene is obtained, and the plant expression vector is transferred into Agrobacterium tumefaciens (Agrobacterium tumefaciens) EHA105 (rice component supervision and inspection test center of agricultural division transgenic biological products of the academy of agricultural sciences, Anhui) by a freeze-thaw method.

Gus reporter gene driven by promoter Poscold10 to be expressed in rice

Step 1: agrobacterium-mediated genetic transformation of rice

After the glumes of the mature rice seeds are removed, the seeds are soaked in 70% alcohol for 1min, and the alcohol is poured off. Seeds were soaked for 40min (150r/min) with 1 drop of Tween 20 in 50% sodium hypochlorite (stock solution available chlorine concentration greater than 4%). And pouring off sodium hypochlorite, and washing for 5 times by using sterile water until the solution is clear and has no sodium hypochlorite taste. The seeds were soaked in sterile water overnight. The embryos were detached along the aleurone layer with a scalpel seed and inoculated onto callus induction medium. And after dark culture for 11 days at the temperature of 30 ℃, separating the callus from endosperm and embryo, and pre-culturing the primary callus with good bud removal state and vigorous division for 3-5 days for agrobacterium transformation.

The agrobacterium tumefaciens which are transferred with the recombinant expression vector in the process of 'construction of plant expression vector and transformation of agrobacterium tumefaciens' are adopted to carry out agrobacterium-mediated genetic transformation, and 20 strains of Poscold10 are obtained: : GUS transgenic rice plants, genetic transformation, transformant selection, and transgenic Plant regeneration are performed by methods such as those proposed by Yongbo Duan (Yongbo Duan, Chenguang ZHai, et al. an effective and high-throughput protocol for Agrobacterium mediated transformation based on phosphorinanoisomerase reactivity selection in Japonica rice Plant (Oryza sativa L.) [ J ]. Plant Cell Report, 2012.DOI 10.1007/s 00299-012-.

Step 2, Poscold10: : low temperature stress treatment of GUS plants and Low temperature response Activity of Poscold10

From transgenic plant poscoled 10: : GUS plants and PUBI as a control: : in GUS plants (GUS genes are driven by a constitutive promoter corn PUBI promoter commonly used in the current genetic engineering, and the components of transgenic biological products of department of agriculture of the academy of agricultural sciences of Anhui province are preserved in a rice group in a supervision, inspection and test center), 4 strains T1 are selected to substitute for promoter activity identification. Plants growing for 30 days are respectively placed at 4 ℃ for 24h for low-temperature treatment, and parallel materials are placed in a normal growth environment at 30 ℃ as a control.

Gus staining: the material which is processed at low temperature of 4 ℃ for 24h is taken, and the root, the stem and the leaf of the material are respectively dyed and photographed. The results show that under the condition of inducing at the low temperature of 4 ℃ for 24 hours, roots, stems and leaves can be stained into obvious visible blue (see fig. 3, it needs to be noted that fig. 3 is a color picture, but a gray image is modified to adapt to the requirements of patent law, but a stained part can still be seen), and the promoter can drive the GUS gene to be strongly expressed when being treated at the temperature of 4 ℃ for 24 hours.

Quantitative analysis of Gus gene: taking whole plant materials treated for 0h, 12h and 24h as samples, extracting total RNA of the samples by using a plant total RNA extraction kit of Tiangen Biochemical technology Limited, carrying out reverse transcription by using a Fastquant RT kit of Tiangen Biochemical technology Limited to obtain cDNA, taking the cDNA as a template, taking an ACTIN gene as an internal reference, taking SuperReal PreMix Plus real-time fluorescent quantitative PCR premixed solution of Tiangen Biochemical technology Limited as a reaction reagent, and detecting GUS gene expression intensity driven by POscold10 and a PUBI promoter through qRT-PCR reaction on a PRISM7500 fluorescent PCR instrument of ABI. Wherein, the quantitative qRT-PCR primer for calibrating the ACTIN gene is as follows:

ACTIN upstream primer: 5'-CCTTCAACACCCCTGCTATG-3' the flow of the air in the air conditioner,

ACTIN downstream primer: 5'-CAATGCCAGGGAACATAGTG-3'

The qRT-PCR primers used for detecting GUS gene expression are as follows:

gus upstream primer: 5'-TACGGCAAAGTGTGGGTCAATAATCA-3'

Gus downstream primer: 5'-CAGGTGTTCGGCGTGGTGTAGAG-3'

The results show that in the absence of low temperature induction, poscoled 10: : the expression quantity of the GUS gene in the GUS plant is only one percent of the activity of driving GUS by PUBI, while the POscold10 expression activity is obviously improved along with low-temperature treatment, after the low-temperature treatment is carried out for 24 hours, the expression activity of POscold10 reaches nearly 40 times of that of untreated plants, and is more than half of the expression quantity of the GUBI, and the result is shown in figure 4. This result indicates that POscold10 is a low-temperature-inducible promoter with high low-temperature-inducible strength.

The above description of the specific embodiments of the present invention is not intended to limit the present invention, and those skilled in the art may make various changes and modifications according to the present invention without departing from the spirit of the present invention, which is defined by the scope of the appended claims.

Sequence listing

<110> institute of Paddy Rice of agricultural science institute of Anhui province

<120> rice low-temperature inducible expression promoter Poscold10 and application thereof

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2100

<212> DNA

<213> promoter

<400> 1

cttgggcccg tgtcagtgag gtggcggcgg caatggcgtc ggcagcgggt ggttcgtggt 60

gggcatcctt cttcaacctc tggttattct gtagcattgc ccacagctca agcactcaac 120

tgttatgttg agaaaacgaa ggcagaagag ataaaatgaa gctagagctg gaaatggttg 180

accatgaccg gctttcagac atagaaaatt cattcaagaa gttgtctaag cagaaaaaag 240

agactgatga cattagaaat tggttcatag gaacagtagt tgtagctgtg ttccttctca 300

tgaagctatt tgataatatg tactatgctt gccattgttg attttgtaca tgtcaattac 360

cagaattaat ggatggggat cactcagcat gttgatgtct cagcaaccta tccatgtgaa 420

ttctgtttat ctatctccca ctatttgaaa ctctaaatta cagcttcttc agatgggttg 480

tctaacttat ccttcaattt tagattcttg ctaagttcta gttcgttctg tttggctgaa 540

aatttgcaga attctgttta tctatctccc actgtctgaa actctgaatt acaacacact 600

agtacgagta tgtttgtttc agtctttcag aatcatgtaa attatacttt ttttcagtaa 660

ttattgccca tgtgtctagc actctactcc agtgattcct caacttttac atctatagct 720

ggattgctgt tcattgaaaa cacttgcttg taaccattgt tgtgctgctg atcactccac 780

agaatgatat atattttaca ggagatcgag caaaaacatc agacaaaata ttaatggctc 840

tcaatacgac atatatatca tgctctgttc acagcagaca aaaattaagt caaaagctcc 900

attcaacgat agatcatgcc ctgtaaacag cataccaaaa aacggacgac agatgagctt 960

aaaattttcc attcaaagat taaagtctcc agcaaaagct tgtgcttttc tcacaaattc 1020

attacaagaa acatggcaga ttgaattgtt aattacaaaa taactagacc atatagtgga 1080

gtagtgtttg agccgtaatg ggggcctagg ctgcgccatg gcagcggggg aggttaggta 1140

cttgcttgag ctgtcgaggg agggggatag gggacacaga ataccatgac atcttcgctc 1200

gtgaatgcgg cggcgttgtt ggtgacactg tcgcggacag cagatggatg ggaaaaaatc 1260

ggcaccggca gaggcatgga tgcggggcga gcgcgccgtt tctccagtac cgcgaccacg 1320

gacggcagat ggatgggaaa aaaccgttcg gcggggcgtg gatgcggcgc aactgctccg 1380

tttctgcgct cagatggagg aatagatgag aaagaaccga tgcggggagg gaggaaagaa 1440

agagcgcagc gagggaggaa agcggcggcg tgtgactccg gaattgttcc gcttctattt 1500

gttgttttcc aatcgtatcc tttcaagttg gacggccaga tttaatctgg tacctcctag 1560

taccagtact ggctggtaca gactgccgga cgggagtaaa actcttcata gatagatgaa 1620

tctagataga taatcaatat gaatatagaa aatgctataa tgacttacat tgtgaaatgg 1680

agagagtagt tcgaaagcat gcgcatagaa aagaaagaaa taggtcggaa aagtgaagga 1740

aaaaaaaact cagcctataa gtagggtgca cgtttatagt ttcactaagt gtttatactg 1800

tgtttcttga tagaaaataa tacgggcttt aaatttcccg tgacgctatc gatgcaattc 1860

atgcaaccaa ccaggcgtgc tgcgtgcaac acctggaaca aatcctttcg gcgttcgttg 1920

cacctggaca cacacctgca catgacgtgg ccccctatcc atccatccct taccgccttc 1980

ctctccacgc gtcgccttcc gcctccgcca cctcctcccc ctctccacac actcttcttc 2040

tccactcctc cgccgctcgc ctcgccgcgc gccgcttata tacccgcgtc tctgcgctca 2100

<210> 2

<211> 28

<212> DNA

<213> Artificial sequence

<400> 2

ctgcagcttg ggcccgtgtc agtgaggt 28

<210> 3

<211> 28

<212> DNA

<213> Artificial sequence

<400> 3

ggatcctgag cgcagagacg cgggtata 28

Claims

1. A plant low-temperature inducible expression promoter Poscold10 is characterized in that the sequence of the plant low-temperature inducible expression promoter Poscold10 is shown as SEQ ID No. 1.

2. An expression cassette comprising the plant cold inducible expression promoter poscoled 10 of claim 1.

3. A recombinant expression vector comprising the plant cold inducible expression promoter poscoled 10 of claim 1, wherein said plant cold inducible expression promoter poscoled 10 is linked upstream of the gene sequence to be expressed in the vector.

4. The recombinant expression vector of claim 3, wherein the gene to be expressed is a Gus gene or a cold tolerance gene, the recombinant expression vector is pCAMBIA1391-Poscold10, and pCAMBIA1391 is a plant binary expression vector.

5. A host bacterium comprising the plant low temperature inducible expression promoter Poscold10 of claim 1, the expression cassette of claim 2, or the recombinant expression vector of claim 3 or 4, wherein the host bacterium is Agrobacterium tumefaciens.

6. A transformant comprising the plant low temperature inducible expression promoter poscoled 10 of claim 1, the expression cassette of claim 2, or the recombinant expression vector of claim 3 or 4, or the host bacterium of claim 5, wherein the transformant is a non-plant cell.

7. Use of the plant promoter for low temperature inducible expression of claim 1 in growing transgenic plants, comprising: connecting the plant low-temperature inducible expression promoter Poscold10 of claim 1 to the upstream of a gene sequence to be expressed in a vector, thereby constructing a recombinant expression vector; and transforming the recombinant expression vector into plant cells, tissues or organs for cultivation, wherein the plant is rice.