CN104480119B - Plant salt stress inducible gene OsSIR1 and its encoding proteins and application - Google Patents

Abstract

The invention discloses a kind of plant salt stress inducible gene OsSIR1 and its application, its encoding amino acid sequence of plant salt tolerance related gene such as SEQ ID NO:Shown in 1, its nucleotide sequence of the gene of the albumen such as SEQ ID NO are encoded:Shown in 2, or its degenerate sequence.Suppress the expression of rice Os SIR1 by RNAi methods, it is possible to increase survival rate, growth rate and biomass under the salt stress of transgenic rice plant.OsSIR1 genes of the present invention are that the crop for cultivating salt tolerance raising provides the foundation.

Description

Plant salt stress inducible gene OsSIR1 and its encoding proteins and application

Technical field

The invention belongs to biological technical field, it is related to a kind of plant salt stress inducible gene OsSIR1 and its application.

Background technology

The soil salinization is a worldwide problem, according to UNESCO（UNESCO）And food and agricultural organization （FAO）Incomplete statistics, about 1,000,000,000 hectares of the saline and alkaline land area in the whole world.At present, NORTHWEST CHINA, northeast and strand ground are distributed in The saline-alkali wasteland in area and saline and alkaline obstacle total cultivated area wherein nearly 200,000,000 mu with agricultural use potentiality, account for me more than 500,000,000 mu More than the 10% of state total cultivated area.It is saline and alkaline that there is material impact for growth and development of plants, it is cause crop failure important Factor.Soil salination is to cause China western and that coastal middle-and-low-yielding fields and large area soil resource are difficult to effectively to utilize is direct Reason.Although salt-soda soil can be irrigated by moisture, soil improvement be carried out using chemical improvement agent, usually because cost is huge Greatly, take effect less and be difficult to.By conventional breeding seed selection Salt And Alkali Tolerance high-yield variety, often the cycle is long, while being not easy to take Obtain preferable result.By biotechnology cultivate resistance to rice varieties against the current be make full use of China salt-soda soil, alleviate shortage of water resources, Ensure that rice high yield, stable yields be most economical, fast and effectively approach.

Plant is grown and right in transcriptional level by adjusting a series of activation and suppressing molecular web influence The response of the stress such as arid, low temperature, salt stress and pest and disease damage.If the defense reaction of plant and environment stress reaction mechanism Be metabolized for increase and more multi-energy is consumed by sustained activation, plant.Therefore, plant evolution goes out a set of adaptation mechanism and is existed with ensureing plant These reactions are closed under normal growth developmental state, most important of which means are exactly to suppress environment stress phase using repressor The expression of correlation gene.Transcription inhibition suppresses plant defense and environment stress related gene expression under non-environment stress, passes through Suppress some activity of the sub- GAP-associated protein GAP of activation in defence and environment stress, to prevent because reaction is excessively acutely and to itself Cell produces injury.

There are EAR (ethylene-responsive element binding in numerous plant transcription factor families Factor-associated amphiphilic repression) motif (L/FDLNL/F (x) P) repressor, such as ERF, The transcription factor family such as MYB, ABI3/VP1, NIM1-INTERACTING1 and MADS member includes EAR motifs and has The function of repressor.

Functional analysis is carried out by the EAR genes to Under Salt Stress in Rice induced expression, it has been found that paddy rice is received into salt stress Induced expression EAR genes OsSIR1 (Oryza sativa Salt Induced EAR 1) transgenosis can be improved after silence The salt tolerance of paddy rice.

The content of the invention

It is an object of the invention to provide an EAR genes OsSIR1 by salt stress induced expression from paddy rice (Oryza sativa Salt Induced EAR 1), the salt tolerance of transgenic paddy rice will can be improved after paddy rice SIR1 silences.

The plant salt stress inducible gene that the present invention is providedOryza sativa Salt Induced EAR 1, referred to as OsSIR1, from paddy rice (Oryza sativa), its encoding proteins amino acid sequence such as SEQ ID NO:Shown in 1.

SEQ ID NO:1 sequence is made up of 284 amino acid residues.

The present invention is also provided SEQ ID NO:1 amino acid sequence by one or several amino acid residues substitution And/or missing and/or addition and related to plant stress tolerance by SEQ ID NO:Protein derived from 1 sequence.

In order that described OsSIR1 albumen is easy to purifying, can be by SEQ ID NO:Amino acid sequence composition shown in 1 Protein amino terminal or the upper label as shown in table 1 of carboxyl terminal connection.

The sequence of the label of table 1

It is above-mentioned by SEQ ID NO:Protein can be artificial synthesized derived from 1 sequence, also can first synthesize its encoding gene, then enter Row biological expression is obtained, and its encoding gene can be by by SEQ ID NO:One or several amino are lacked in DNA sequence dna shown in 2 The codon of sour residue, and/or the missense mutation of one or several base-pairs is carried out, and/or connected at its 5 ' end and/or 3 ' ends The coded sequence of the label shown in table 1 is obtained.

The present invention also provides a kind of coding the plant salt stress inducible gene OsSIR1, its nucleotide sequence such as SEQ ID NO:Shown in 2, or its degenerate sequence.

Meanwhile, the present invention also provides such as SEQ ID NO:DNA sequence dna shown in 2 has more than 90% homology, and encodes resistance to The DNA molecular of inverse property GAP-associated protein GAP.

The present invention be additionally provided under strict conditions with SEQ ID NO:DNA sequence dna shown in 2 hybridizes and encodes the egg White DNA molecular；Also provide simultaneously has more than 90% homology with the DNA molecular, and encodes the DNA of stress tolerance correlative protein Molecule；The stringent condition can be in 6 × SSC, in the solution of 0.5% SDS, 65^oHybridize under C, then with 2 × SSC, 0.1% SDS and 1 × SSC, 0.1% SDS respectively washes film once.

The present invention also provides the recombinant expression carrier of the gene.

The recombinant expression carrier of the gene can be contained with existing plant expression vector construction.

The plant expression vector is including double base agrobacterium vector with the carrier that can be used for plant micropellet bombardment etc..

During using the gene constructed recombinant plant expression vector, can be plus any one before its transcription initiation nucleotides Enhanced promoter or constitutive promoter, such as cauliflower mosaic virus（CAMV）The ubiquitin promoter of 35S promoter, corn （Ubiquitin）, they can be used alone or are used in combination with other plant promoters.

For the ease of transgenic plant cells or plant are identified and screened, plant expression vector used can be carried out Processing, such as adds the coding that can be expressed in plant to produce the enzyme of color change or the gene of luminophor（Gus gene, Luciferase genes etc.）, resistant antibiotic marker（Gentamicin label, kanamycins label etc.）Or it is anti- Chemical reagent marker gene（Such as anti-herbicide gene）Deng.From the security consideration of genetically modified plants, any selectivity can be not added with Marker gene, directly screens transformed plant with adverse circumstance.

The recombinant expression carrier is the recombinant plasmid for obtaining the MCS of gene insertion pC5300.

The present invention also provides gene described in any of the above（OsSIR1）Expression cassette, transgenic cell line and recombinant bacterium.

Expand the gene（OsSIR1）The primer pair of total length or any fragment falls within protection scope of the present invention.

The present invention also provides application of the described gene in the genetically modified plants that salt tolerance is improved are cultivated.

Above-mentioned application is will to encode the plant salt stress inducible gene fragment to import purpose plant（Such as plant cell or group Knit）In, obtain genetically modified plants of the salt tolerance higher than the purpose plant.Specifically, the recombinant expression carrier is imported into mesh Plant in, obtain genetically modified plants salt tolerance be higher than the recipient plant.

Experiment shows, by artificial tiny RNA method (artificial microRNA) by encoding regulator of the present invention Plant salt stress inducible geneOsSIR1 The expression part of gene suppresses, it is possible to increase transgenic paddy rice salt tolerance, and raising turns base Because of the survival rate under the salt stress of rice plant, growth rate and biomass.Coded plant salt stress of the present invention induces base CauseOsSIR1Gene is to cultivate other there is the crop that salt tolerance is improved to provide the foundation.

Brief description of the drawings

Fig. 1 is identified for OsSIR1 transfer-gen plants PCR.

Fig. 2 is identified for OsSIR1 transfer-gen plants real-time quantitative PCR.

Fig. 3 is that water planting control and salt stress process wild type and OsSIR1 transgenic lines Ri-1.

Fig. 4 is the survival rate that water planting control and salt stress process wild type and OsSIR1 transgenic lines Ri-1 and Ri-2.

Fig. 5 is the plant height that water planting control and salt stress process wild type and OsSIR1 transgenic lines Ri-1 and Ri-2.

Fig. 6 is earth culture experiment contrast and salt stress treatment wild type and OsSIR1 transgenic lines Ri-1.

Fig. 7 is the survival of earth culture experiment contrast and salt stress treatment wild type and OsSIR1 transgenic lines Ri-1 and Ri-2 Rate.

Fig. 8 is the strain of earth culture experiment contrast and salt stress treatment wild type and OsSIR1 transgenic lines Ri-1 and Ri-2 It is high.

Fig. 9 is the ground of earth culture experiment contrast and salt stress treatment wild type and OsSIR1 transgenic lines Ri-1 and Ri-2 Part single-strain fresh weight.

Specific embodiment

Come that the present invention is furture elucidated below by the detailed description of specific embodiment, but be not to limit of the invention System, only illustrates.

Experimental technique in following embodiments, unless otherwise specified, is conventional method, test material used, such as without Specified otherwise, is what is be commercially available from routine biochemistry reagent shop, and experiment is respectively provided with three repetitions, results averaged.

Embodiment 1：The clone of OsSIR1 cDNA

With PatMatch1.2 (www.arabidopsis.org) search Nipponbare transcription factor databases (Plant Transcription Factor Database v2.0, http://planttfdb.cbi.pku.edu.cn), at 2424 There are 374 transcription factors to contain EAR motifs (DLNxxP or LXLXL) in rice transcription factor.In order to study EAR transcription factors Function in salt stress response, we analyze the expression of EAR genes under condition of salt stress.We are from Rice Oligo Array Database (http://www.ricearray.org/) obtain paddy rice EAR gene tables under salt stress and collating condition The data for reaching, are control with the sample without salt treatment, and screening is raised more than 2 times of gene by salt stress induced expression, we Obtain the EAR genes that 71 salt stresses induce more than 2 times.These gene major parts do not have studies have reported that, in the EAR of unknown function In gene, we choose 40 maximum genes of induced expression amplitude and carry out gene function checking by transgenic method, to turning The Salt Tolerance Analysis experiment of gene plant confirms that one of EAR genes can improve the salt tolerance of transfer-gen plant, Wo Menming Entitled OsSIR1 (Oryza sativa salt induced EAR1).Although OsSIR1 completes the water of gene order-checking Gene order in rice varieties Nipponbare and 9311 is, it is known that still at present not to the open report of its biological function.For The function of research OsSIR1, we clones and then are verified its function in salt stress regulation and control by transgene method. According to Nipponbare Rice Genome Sequence information (http://rice.plantbiology.msu.edu/), design OsSIR1 bases Because special primer is as follows：

Sense primer：5’- ATGGAAGCAGACGCGAGCCATAC -3’；

Anti-sense primer：5’- CTACTCGGCCCACAAGAGTGG -3’.

A monthly age rice leaf 0.2g is taken, liquid nitrogen grinding, TRIzol methods extract total serum IgE.Take 2 μ g total serum IgEs use SuperScript II RT reverse transcriptase carries out reverse transcription, synthesizes the chains of cDNA first, as template, is specifically drawn with above-mentioned Thing enters performing PCR reaction.After PCR primer is reclaimed through electrophoretic separation, pEASY-T is cloned into（The full formula gold limited public affairs of biotechnology in Beijing Department）, pEASY-T-OsSIR1ORF is named as, it is sequenced.

Sequencing result shows, the nucleotide sequence such as SEQ ID NO of the fragment:Shown in 2, SEQ ID NO are encoded:Shown in 1 Protein.

Embodiment 2：The silence of rice Os SIR1

First, the structure of OsSIR1 silent carriers

This programme takes the artificial tiny RNA method silence OsSIR1 complementary with OsSIR1 cDNA sequences.

1st, with plasmid pNW55 as masterplate, KOD FX archaeal dna polymerases are used and with following primer amplified.

Reaction 1：Sense primer：5’- CTGCAAGGCGATTAAGTTGGGTAAC -3’；

Anti-sense primer：5’- TGGTGACAAAGTTTAGTCTATGACTGCTGCTGCTACAGCC -3’.

Reaction 2：Sense primer：5’- AGTCATAGACTAAACTTTGTCACCAGGAGATTCAGTTTGA -3’；

Anti-sense primer：5’- AATCATAGACTAATCTTAGTCACAGAGAGGCAAAAGTGAA -3’.

Reaction 3：Sense primer：5’- CTGTGACTAAGATTAGTCTATGATTCCTGCTGCTAGGCTG -3’；

Anti-sense primer：5’- GCGGATAACAATTTCACACAGGAAACAG -3’.

After PCR primer electrophoretic separation, the difference bp DNA fragmentations of gel extraction 256,87,259.

2. above-mentioned 3 different length DNA fragmentations are mixed as template, using KOD FX archaeal dna polymerases and primer 5 '- CTGCAAGGCGATTAAGTTGGGTAAC -3’；5 '-GCGGATAACAATTTCACACAGGAAACAG -3 ' PCR are expanded, After PCR primer electrophoretic separation, the bp DNA fragmentations of gel extraction 554.

3. the 2nd step is obtained into DNA fragmentation restriction enzyme BamHI and KpnI digestion, after electrophoretic separation, cut glue Reclaim 255 bp DNA fragmentations.

4th, with restriction enzyme BamHI and KpnI digestion pC5300 (Ubiquitin promoters) (PLoS ONE 2008, 3(3):E1829), skeleton is reclaimed.

5th, the fragment connection that the fragment and step 4 for obtaining step 3 are obtained, converts bacillus coli DH 5 alpha, and sequencing confirms just Really, pC-OsSIR1 is obtained.

2nd, the acquisition of genetically modified plants

1st, using electric shocking method by recombinant expression carrier pC-OsSIR1 import Agrobacterium AGL0 (ATCC BAA-100, www.atcc.org)。

5th, the Agrobacterium AGL0 containing pC-OsSIR1 is infected into the embryonic type callus that the induction of Nipponbare wild type is produced, Then the screening resistant calli in MS culture mediums (containing 30mg/L hygromycin), per 15 days generations, altogether 3 generation, then will Resistant calli induces into whole plant, and rice transplanting harvests the T1 of genetically modified plants for seed in crop field.

3rd, the Molecular Detection of genetically modified plants

CTAB methods extract leaf DNA, with-the GAGCATATACGCCCGGAGTC -3 ' of pC-OsSIR1 specific primers 5 ' and 5 '-CTCTCGGAGGGCGAAGAATC -3 ' PCR detections, as a result show there is specific amplification band in transfer-gen plant, and wild Without specific amplified band (Fig. 1), this shows that pC-OSIR1 carrier segments have been introduced into transfer-gen plant to raw type DNA.

TRIzol methods extract 2 week old wild types and OsSIR1 transfer-gen plants (Ri-1, Ri-2Ri-1) blade total serum IgE, 2 μ g total serum IgEs are taken, with 2 u RQ1 RNase-free DNase (Promega), 37 °C of 30 min for the treatment of, plus terminating reaction After liquid, with polyA as primer, using 1 hour synthesis chain of cDNA first of M-MLV reverse transcriptase 42 °C, then with cDNA as mould Plate, be with Ubiquitin (primer 5 '-CCATCCTCAAGCTGCTTACC-3 ' and 5 '-GACTGGCAAGACCATTACCC-3 ') Internal reference, PCR method detection OsSIR1 genes (- the TTCAAGCACCCGTCGTACCG -3 ' of primer 5 ' and 5 ' - GAAGTTGAGGTGCGCGTTCC -3 ') expression, as a result as shown in Figure 2.Relative to Wild type control plants, OsSIR1 genes Expression is reduced to 0.27 and 0.18 times of wild type in pC-OsSIR1 transfer-gen plants (Ri-1, Ri-2).This shows turning The expression of OsSIR1 is by part silence in gene plant.

Embodiment 3：OsSIR1 transgenic rice plants salt tolerance is detected

First, water planting rice plant salt tolerance detection

After wild type and the treatment 3 days of 42 °C of OsSIR1 transgenic paddy rice seeds, soaked seed 24 hours in water, 37 °C of vernalization, The consistent seed of rudiment is chosen in following nutrient solutions, 28 °C are cultivated 4 days, are then located in the nutrient solution containing 100 mM NaCl Reason 4 days.As in Figure 3-5, wild type and OsSIR1 transgenic rice plants are not significantly different from result in untreated control. After 100 mM NaCl treatment, wild type and the transfer-gen plant speed of growth in processing procedure slow down, and gradually wilting, death, right Physical signs statistics shows that WT lines survival rate is 34%, plant height 12.8cm；And OsSIR1 transgenic lines Ri-1 and Ri- 2 survival rate 52% and 66%；Ri-1 and Ri-2 plant heights are respectively 16.7 and 17.8 cm.This show water planting salt stress treatment in, OsSIR1 transgenic rice plant salt tolerances are significantly higher than wild type control.

Culture formula of liquid：The mg/L of four water-calcium nitrate 945, the mg/L of potassium nitrate 506, the mg/L of ammonium nitrate 80, phosphoric acid The mg/L of potassium dihydrogen 136, magnesium sulfate 493 mg/L, 2.5 ml of iron salt solutions/L, micro- mother liquor 5 ml/L, pH= 6.0。

Iron salt solutions：Ferrous sulfate heptahydrate 2.78g, disodium ethylene diamine tetraacetate（EDTA.Na）3.73g, distilled water 500ml, pH=5.5.

Micro- mother liquor：The mg/l of KI 0.83, boric acid 6.2 mg/L, manganese sulfate 22.3mg/L, sulfuric acid Zinc 8.6mg/L, sodium molybdate 0.25mg/L, copper sulphate 0.025mg/L, cobalt chloride 0.025mg/L.

2nd, earth culture rice plant salt tolerance detection

After wild type and the treatment 3 days of 42 °C of OsSIR1 transgenic paddy rice seeds, soaked seed 24 hours in water, 37 °C of vernalization, The consistent seed kind of rudiment is chosen in the small basin equipped with Nutrition Soil, one layer of preservative film moisturizing is covered, after coming up, removal Preservative film, after 2 weeks, the small basin of rice cultivation seedling is put into the water containing 100 mM NaCl and is soaked 1 week, then pours out salt solution, A running water is changed daily, is taken a picture after 1 week, count physiological phenotype.As Figure 6-9, in the treatment of no salt stress to according to the facts In testing, OsSIR1 transgenic paddy rice strains Ri-1 and Ri-2 grew no notable difference, by 100 mM NaCl treatment 1 week Rehydration afterwards, the survival rate of Wild type control plants only has 37%, and the survival rate of OsSIR1 transgenic paddy rice strains Ri-1 and Ri-2 Respectively 58% and 65%, it is significantly higher than wild type control.As shown in FIG. 8 and 9, OsSIR1 turns for measurement plant height and fresh weight result The plant height of trans-genetic hybrid rice strain Ri-1 and Ri-2 is 25.8 and 26.3 cm, is significantly higher than the 20.1 of Wild type control plants cm;The single-strain fresh weight of OsSIR1 transgenic paddy rice strains Ri-1 and Ri-2 is 151 and 162 mg, also significantly greater than wild type pair According to the mg of single-strain fresh weight 87.Result above shows that in earth culture experiment, compared with non-transgenic wild type control, OsSIR1 turns base Because the salt tolerance of rice strain is significantly improved.

<110>Biological Technology institute, Chinese Academy of Agricultural Sciences

<120>Plant salt stress-inducing OsSIR1 genes and its encoding proteins and application

<130> 1

<160> 2

<210> 1

<211> 284

<212> PRT

<213> Oryza sativa

<400> 1

Met Glu Ala Asp Ala Ser His Thr Pro Thr Thr Ser Ser Ser Val Ser

Val Ser Phe Ser Ser Ser Ser Leu Ser Thr Ser Ser Ser Thr Ser Ser

Leu Val Asp Asn Gly Ala Gln Asp Arg Pro Lys Ser Ser Lys Pro Lys

His Ala Ala Lys Lys Arg Lys Arg Ala Ala Ala Glu Glu Pro Ala Asn

Ala Ala His Gly Ala Gly Glu Asp Thr Ser Ser Cys Ser Thr Asp Asp

Asn Ala Ala Ala Ser Gly Lys Ala Gln Ala Gly Gly Gly Gly Gly Gly

Val Asp Ser Ser Ser Thr Cys Thr Ala Ala Ser Ala Pro Arg Ser Gly

Phe Lys His Pro Ser Tyr Arg Gly Val Arg Arg Arg Ser Trp Gly Lys

Trp Val Ser Glu Ile Arg Glu Pro Arg Lys Lys Ser Arg Ile Trp Leu

Gly Thr Phe Pro Thr Ala Glu Met Ala Ala Arg Ala His Asp Val Ala

Ala Leu Ala Ile Lys Gly Arg Asn Ala His Leu Asn Phe Pro Asp Ser

Ala His Glu Leu Pro Arg Pro Glu Ser Thr Ser Pro Ala Asp Ile Gln

Ala Ala Ala Ala Lys Ala Ala Ala Glu Val Arg Cys Glu Glu Glu Ser

Ser Pro Ser Ser Ser Pro Thr Ala Glu Gln Pro Glu Glu Glu Ala Ala

Cys Pro Asp Thr Val His Ala Asp Gly Gly Gln Asp Asn Ala Leu Phe

Asp Leu Pro Asp Leu Leu Leu Asp Leu Arg Asp Gly Leu Trp Trp Ser

Pro Val Trp Pro Ala Ala Leu Ala Ala Glu Glu Tyr Asp Gly Gly Asp

Ala Val Val Leu Asn Glu Pro Leu Leu Trp Ala Glu

<210> 2

<211> 855

<212> DNA

<213> Oryza sativa

<400> 2

atggaagcag acgcgagcca tacacccacc acctcctcct ccgtctccgt ctccttctcc 60

tcctcgtcgc tgtccacttc ctcctccacc tcctccctcg tcgacaatgg cgcgcaagac 120

cggcccaaga gctccaagcc caaacacgcc gccaagaagc gcaagagagc ggccgcggag 180

gaacctgcca atgccgccca cggcgcaggg gaggatacca gcagctgcag caccgacgac 240

aacgcggcgg cgagcggcaa ggcgcaggcg ggcggcggcg gcggcggcgt cgacagcagc 300

agcacctgca ccgccgcctc ggcgccgagg agcggcttca agcacccgtc gtaccgcggc 360

gtgcgccgcc ggagctgggg gaagtgggtg tcggagatcc gggagccccg caagaagtcg 420

cgcatctggc tgggtacctt ccccaccgcg gagatggcgg cgcgcgccca cgacgtggcc 480

gcgctcgcca tcaagggccg gaacgcgcac ctcaacttcc cggacagcgc ccacgagctg 540

ccccgcccgg agtccacctc cccggcagac atccaggccg ccgccgccaa ggctgccgcc 600

gaggtgcggt gcgaggagga gtcgtcgccg tcgtcgtcgc ccaccgccga gcaacccgag 660

gaggaagccg cctgccctga cacggtgcac gccgacggcg gccaggacaa tgctctcttc 720

gacctacccg accttcttct cgacctacgg gacgggctct ggtggtcgcc ggtgtggccg 780

gcggcactgg cggccgagga gtacgacggc ggcgacgccg tcgtgctcaa tgagccactc 840

ttgtgggccg agtag 855

Claims (3)

1. applications of the plant salt stress inducible gene OsSIR1 in the enhanced genetically modified plants of salt resistance ability, it is by artificial Tiny RNA method is by the plant salt stress inducible gene in plantOsSIR1 The expression part of gene suppresses, and base is turned to improve Because of Salt Resistance of Rice, wherein the gene coded protein amino acid sequence such as SEQ ID NO:Shown in 1.

2. application as claimed in claim 1, it is characterised in that the nucleotide sequence of the gene such as SEQ ID NO:Shown in 2, Or its degenerate sequence.

3. application as claimed in claim 1 or 2, it is characterised in that：The plant is paddy rice.