CN102964438A - Stress-resistance-related protein PpLEA3-23 of plant as well as coding gene and application of protein - Google Patents
Stress-resistance-related protein PpLEA3-23 of plant as well as coding gene and application of protein Download PDFInfo
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Abstract
The invention discloses a drought-resistance-related protein of a plant as well as a coding gene and application of the protein. The drought-resistance-related protein of the plant is shown as (a) or (b), wherein the (a) refers to a protein with an amino acid sequence shown by a sequence II in a sequence table; and the (b) refers to a protein which is related to drought resistance of the plant and is derived from the sequence II in a manner that the amino acid sequence in the sequence II is subjected to replacement and/or deficiency and/or addition of one or more amino acid residues. Drought stress experiment results show that after being subjected to drought treatment for 22 days, transgene PpLEA3-23 strains, including Line 6, Line 7 and Line 8, with stable genetic transformation grow normally, and the wilting rates of the strains are respectively 14.2%, 21.5% and 15.3% while the wilting rate of a control plant with an empty vector reaches up to 84.3%; and after being re-watered for 7 days, the transgene strain Line 8 can reach a survival rate of 73% while the control plant with the empty vector does not survive, thereby proving that the gene PpLEA3-23 and the protein coded by the gene can remarkably improve the drought resistance of rice. The drought-resistance-related protein as well as the coding gene and application of the protein has important theoretical and practically significances to cultivation of new varieties of drought-resistance plants, particularly drought-resistance rice.
Description
Technical field
The present invention relates to a plant resistance relevant protein and encoding gene thereof and application, particularly a kind of plant drought resistance associated protein PpLEA3-23 and encoding gene and application that derives from bryophyte.
Background technology
Bryophyte is the land pioneer plant that occur the ancient ordovician period before 500,000,000 years, in its life history take the gametophyte of " cormus " as the Major Nutrient growth phase." cormus " blade is comprised of monolayer cell, only locates to be comprised of several confluent monolayer cells at its " middle rib ", without the weave construction of the regulation and control water metabolisms such as transfusion tissue and pore, is keeping the significantly feature of waterplant.Owing to lack water conduction and regulator control system, therefore, when original " bryophyte " dried up logging in, must at first coerce greatly in the face of two: water deficit, temperature shock.Huge selective pressure forces bryophyte to be evolved out to be different from the adverse circumstance coping mechanism of " vascular plant " (fern, spermatophyte).
The Stress Factors such as the arid in the physical environment, saline and alkaline, low temperature can cause the extensive underproduction of farm crop to the material impact that grown of plant when serious, and cultivating the resistance of reverse crop is one of major objective of plant husbandry.Current, the crop varieties that has resistance of reverse by the genetic engineering breeding acquisition is a kind of effective method.And the technical bottleneck problem of most critical is screening and the function discovery of effective adversity gene in the method.
Summary of the invention
The purpose of this invention is to provide a kind of albumen relevant with plant drought resistance and encoding gene and application.
The albumen relevant with plant drought resistance provided by the present invention, name is called PpLEA3-23, derives from small liwan moss (Physcomitrella patens), is following (a) or protein (b):
(a) protein that is formed by the aminoacid sequence shown in the sequence in the sequence table 2;
(b) with the aminoacid sequence of sequence 2 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and relevant with plant drought resistance protein of being derived by sequence 2.
For the ease of the purifying of PpLEA3-23 albumen, label as shown in the table on N-terminal that can the protein that the amino acid residue sequence of sequence 2 forms in by sequence table or C-terminal connect.
Table: the sequence of label
| Label | Residue | Sequence |
| Poly-Arg | 5-6(is generally 5) | RRRRR |
| Poly-His | 2-10(is generally 6) | HHHHHH |
| |
8 | DYKDDDDK |
| Strep-tag?II | 8 | WSHPQFEK |
| c- |
10 | EQKLISEEDL |
Above-mentioned (a) but in PpLEA3-23 albumen synthetic, also can synthesize first its encoding gene, carry out again biological expression and obtain.The encoding gene of the PpLEA3-23 albumen 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 1, 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.Above-mentioned (b) but in the protein synthetic, also can synthesize first its encoding gene, carry out again biological expression and obtain.
The nucleic acid molecule of described PpLEA3-23 albumen of encoding also belongs to protection scope of the present invention.
Described nucleic acid molecule can be DNA, such as cDNA, genomic dna or recombinant DNA; Described nucleic acid molecule also can be RNA, such as mRNA, hnRNA or tRNA etc.
In one embodiment of the invention, described nucleic acid molecule is specially the gene (called after PpLEA3-23) of the described PpLEA3-23 albumen of coding; Described PpLEA3-23 gene is following 1) to 4) in arbitrary described dna molecular:
1) encoding sequence is the dna molecular shown in the sequence 1 in the sequence table;
2) dna molecular shown in the sequence 1 in the sequence table;
3) under stringent condition with 1) or 2) dna molecule hybridize that limits and the dna molecular of the described PpLEA3-23 albumen of encoding;
4) with 1) or 2) or 3) dna molecular that limits has the dna molecular of 90% above homology and the described PpLEA3-23 albumen of encoding.
Above-mentioned stringent condition can be with 6 * SSC, and the solution of 0.5%SDS 65 ℃ of lower hybridization, is then used 2 * SSC, and 0.1%SDS and 1 * SSC, 0.1%SDS respectively wash film once.
Wherein, the sequence 1 in the sequence table is comprised of 561 Nucleotide, and its open reading frame (ORF) is from 5 ' terminal 1-561 position Nucleotide, the protein (PpLEA3-23 albumen) shown in the sequence 2 in the code sequence tabulation.
The recombinant vectors, expression cassette, transgenic cell line or the recombinant bacterium that contain above-mentioned nucleic acid molecule also belong to protection scope of the present invention.
Described recombinant vectors can be recombinant expression vector, also can be recombinant cloning vector.
Described recombinant expression vector can be used existing expression vector establishment.Described expression vector also can comprise 3 ' end untranslated zone of foreign gene, namely comprises the dna fragmentation of polyadenylic acid signal and any other participation mRNA processing or genetic expression.The bootable polyadenylic acid of described polyadenylic acid signal joins 3 ' end of mRNA precursor.When using described gene constructed recombinant expression vector, can add any enhancement type, composing type, organizing specific type or inducible promoter before its transcription initiation Nucleotide, they can use separately or be combined with other promotor; In addition, when using gene constructed recombinant expression vector of the present invention, also enhanser be can use, translational enhancer or transcriptional enhancer comprised.For the ease of transgenic plant cells or plant being identified and screening, can process used plant expression vector, as be added in the plant to express and to produce the enzyme of colour-change or the gene of luminophor (gus gene, GFP gene, luciferase genes etc.), have the antibiotic marker thing (gentamicin marker, kantlex marker etc.) of resistance or anti-chemical reagent marker gene (such as anti-weedkiller gene) etc.From the security consideration of transgenic plant, can not add any selected marker, directly with adverse circumstance screening transformed plant.
In the present invention, the promotor of the described PpLEA3-23 genetic transcription of startup is specially corn Ubiquitin promotor in the described recombinant expression vector.
More specifically, described recombinant expression vector is started the recombinant plasmid of described PpLEA3-23 genetic transcription for the described PpLEA3-23 gene of insertion between the multiple clone site of the plasmid pCambia1390 that is inserted with described corn Ubiquitin promotor obtains by described corn Ubiquitin promotor.
Further, in one embodiment of the invention, described recombinant expression vector prepares according to the method that comprises the steps:
(1) described corn Ubiquitin promotor (its nucleotide sequence can shown in sequence in the sequence table 3) sequence is inserted between the multiple clone site Hind III and Kpn I of plasmid pCambia1390, obtains intermediate carrier;
(2) described PpLEA3-23 gene is inserted between the multiple clone site BamH I and Spe I of described intermediate carrier, obtains described recombinant expression vector.
Described expression cassette is by the promotor that can start described PpLEA3-23 genetic expression, described PpLEA3-23 gene, and transcription termination sequence forms.
Described PpLEA3-23 albumen, or described nucleic acid molecule, or described recombinant expression vector, expression cassette or recombinant bacterium are at following a1) or a2) in application also belong to protection scope of the present invention:
A1) regulating plant drought resistance;
A2) plant variety of seed selection drought resistance raising.
In one embodiment of the invention, described regulating plant drought resistance is specially the raising plant drought resistance.The method of the plant variety that described seed selection drought resistance improves specifically can comprise the step that the plant that described PpLEA3-23 expressing quantity is higher breeds as the parent.
Another object of the present invention provides a kind of method of cultivating the transgenic plant of drought resistance raising.
The method of the transgenic plant that cultivation drought resistance provided by the present invention improves can comprise that specifically the gene with the described PpLEA3-23 albumen of coding imports the step that obtains transgenic plant in the purpose plant; Described transgenic plant are compared with described purpose plant, and drought resistance improves.
In aforesaid method, the expression amount of described PpLEA3-23 albumen in described transgenic plant is higher than the expression amount in the described purpose plant.The gene (PpLEA3-23 gene) of described PpLEA3-23 albumen of encoding concrete following 1) to 4) in arbitrary described dna molecular:
1) encoding sequence is the dna molecular shown in the sequence 1 in the sequence table;
2) dna molecular shown in the sequence 1 in the sequence table;
3) under stringent condition with 1) or 2) dna molecule hybridize that limits and the dna molecular of the described PpLEA3-23 albumen of encoding;
4) with 1) or 2) or 3) dna molecular that limits has the dna molecular of 90% above homology and the described PpLEA3-23 albumen of encoding.
Above-mentioned stringent condition can be with 6 * SSC, and the solution of 0.5%SDS 65 ℃ of lower hybridization, is then used 2 * SSC, and 0.1%SDS and 1 * SSC, 0.1%SDS respectively wash film once.
Described PpLEA3-23 gene specifically can import in the described purpose plant by above-mentioned arbitrary described recombinant expression vector, obtains described genetically modified organism.
In aforesaid method, described plant can be monocotyledons, also can be divided into dicotyledons.
In one embodiment of the invention, described plant is monocotyledons, is paddy rice, and it is fine to be specially paddy rice (Oryzasativa L.) kind Japan.
The primer of described PpLEA3-23 full length gene or its arbitrary fragment of increasing is to also belonging to protection scope of the present invention.
Experimental results show that, during PpLEA3-23 gene importing wild-type paddy rice provided by the present invention is Japanese fine, obtain transgenic paddy rice, in the drought stress experiment, turning PpLEA3-23 gene plant growth conditions all will be significantly better than empty carrier adjoining tree and wild-type plant, arid was processed 22 days, turn PpLEA3-23 gene strain Line6, Line7, the Line8 growth is normal, wilted percent is respectively 14.2%, 21.5% and 15.3%, and the wilted percent of empty carrier adjoining tree is up to 84.3%, after the rehydration 7 days, turn PpLEA3-23 gene strain Line1, Line6, Line7, the survival rate of Line8 and Line10 can reach 57% respectively, 71%, 64%, 73% and 49%, and empty carrier adjoining tree and wild-type plant illustrate that all without survival the albumen of PpLEA3-23 gene provided by the invention and its coding can significantly improve the drought tolerance of paddy rice.The present invention particularly cultivates the drought resisting paddy rice and has important theory and practical significance cultivating the drought-resistant plant new variety.
Description of drawings
Fig. 1 is the two dimensional electrophoresis of small liwan moss (Physcomitrella patens) cormus protein.Wherein, A is the contrast of processing without arid; B is the plant that arid was processed 3 days.Arrow indication place is PpLEA3-23 albumen, and this albumen abundance in the plant that arid is processed significantly increases.
Fig. 2 is the structure iron of recombinant expression vector PpLEA3-23-pCUBI1390.Wherein, Gene represents the PpLEA3-23 gene.
Fig. 3 is for turning PpLEA3-23 trans-genetic hybrid rice T
1Electrophorogram for the PCR Molecular Detection of part plant DNA.Wherein, swimming lane M is dna molecular amount standard; The positive contrast of swimming lane P (PpLEA3-23-pCUBI1390 carrier); Swimming lane CK is the strain (empty carrier contrast) that changes empty carrier pCUBI1390 over to; Swimming lane 1-9 is for turning the positive strain of PpLEA3-23 trans-genetic hybrid rice.
Fig. 4 is for turning PpLEA3-23 gene T
2RT-PCR expression analysis for positive strain.Wherein the Actin gene is confidential reference items, and CK is the strain (empty carrier contrast) that changes empty carrier pCUBI1390 over to, and 1-5 is 5 and turns PpLEA3-23 gene strain.
Fig. 5 turns the upgrowth situation of PpLEA3-23 gene plant in the drought tolerance experiment.Wherein, A is plant phenotype before arid is processed; B is that arid is processed plant phenotype after 22 days; C is arid rehydration plant phenotype after 7 days; D is that arid is processed plant wither rate statistics (ordinate zou is wilted percent %) after 22 days.Among the A-D, strain Line1,6,7,8,10 is 5 and turns PpLEA3-23 trans-genetic hybrid rice strain through the screening inheritance stability; CK is the strain (empty carrier contrast) that changes empty carrier pCUBI1390 over to.
Fig. 6 is the surviving rate statistics of rehydration each rice strain after 7 days.Wherein, strain Line1,6,7,8,10 is 5 and turns PpLEA3-23 trans-genetic hybrid rice strain through the screening inheritance stability; CK is the strain (empty carrier contrast) that changes empty carrier pCUBI1390 over to.
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
Small liwan moss (Physcomitrella patens): be recorded in " Geng Xuke etc.; 2008 plant genes are practiced shooting---the application of model plant small liwan moss. biology circular; 43(4): 13-15 " in the literary composition, the public can obtain from Biological Technology institute, Chinese Academy of Agricultural Sciences.
Paddy rice (Oryza sativa L.) kind Japan is fine: be recorded in " A draft sequence of the rice genome (Oryza sativa L.ssp.Japonica.Science.2002 Apr 5; 296 (5565): 92-100 " in the literary composition, the public can obtain from Biological Technology institute, Chinese Academy of Agricultural Sciences.
PCambia 1390 carriers: can be available from Cambia, Queensland.Australia.
Agrobacterium AGL1: available from Beijing ancient cooking vessel state biotechnology limited liability company.
Used minimum medium prescription such as following table:
One, with the acquisition of plant drought resistance associated protein PpLEA3-23
Normal cultivation group: small liwan moss (Physcomitrella patens) cormus that will be grown on the BCD substratum is transferred on the filter paper that soaks, and puts into the 500ml beaker, seals with thieving paper.24 ± 1 ℃ of temperature, cultivate in the culturing room of relative humidity 30 ± 5%.The small liwan moss material of cultivating after 3 days is used for experiment afterwards.
The arid treatment group:: at 10 filter paper of crystallizing dish middle berth through sterilising treatment.Choose the reasonable small liwan moss protonema of growing way material, glassine paper is transferred to together with the small liwan moss material in the crystallizing dish that is covered with filter paper, with the sealed membrane sealing, be put into culturing rack (24 ± 1 ℃ of temperature, relative humidity 30 ± 5%) and cultivate.When processing about 3 days, small liwan moss material dehydration degree can reach more than 90%.With the material preservation of processing, the experiment after being used for.
Take the small liwan moss cormus of normal cultivation group and arid treatment group as experiment material, adopt phenol extraction method to extract respectively protein.Two groups of protein that extract are separated with fluorescence difference gel electrophoresis (DIGE) by classical IEF/SDS-PAGE two-dimensional electrophoresis (2DE) respectively.Concrete steps are carried out according to the experimental implementation guide that Amersham Bioscience provides.
The protein pattern that obtains passes through respectively ImageMaster 2D Platinum(Amersham Bioscience) and DeCyder 2D Software, Version 6.5(Amersham Bioscience) analyze.Compare with normal cultivation group, arid treatment group abundance changes the separated acquisition of protein spots above 2 times.All differential protein spots are used respectively trypsin treatment, and institute's polypeptide that obtains is identified with mass spectrograph (MALDI TOF/TOF MS) and database (NCBI, small liwan moss genome).
The result shows that compare with normal cultivation group, arid treatment group has an albumen to raise 10 times (Fig. 1) at the two-dimensional electrophoresis (2DE) of classics.By mass spectroscopy, obtain its aminoacid sequence (shown in sequence in the sequence table 2), with this protein called after PpLEA3-23.
Two, with the acquisition of plant drought resistance associated protein PpLEA3-23 encoding gene
At first the total RNA of small liwan moss cormus of arid treatment group in the extraction step one obtains cDNA with the RNA reverse transcription.Take this cDNA as template, adopt primer 1 and primer 2 to carry out pcr amplification, the PCR product is carried out 0.8% agarose gel electrophoresis detect, obtain the band that molecular weight is about 0.56Kbp.Reclaim test kit (TIANGEN) with sepharose and reclaim this fragment.Should reclaim fragment and pMD19-T Simple(Takara) be connected, to connect product and transform the bacillus coli DH 5 alpha competent cell, carboxylic Bian penicillin resistance label screening positive colony according on the pMD19-T Simple carrier obtains containing the recombinant plasmid that reclaims fragment.With the universal primer M13 on this recombinant plasmid it is carried out nucleotide sequencing, sequencing result shows that the gene that increases is comprised of 561 deoxyribonucleotides, shown in sequence 1, its open reading frame (ORF) for sequence 1 in the sequence table from terminal the 1st to 561 deoxyribonucleotide of 5 ＇, with this unnamed gene PpLEA3-23.The protein of PpLEA3-23 gene coding amino acid sequence shown in sequence in the sequence table 2.The recombinant cloning vector called after PpLEA3-23-pMD19 that has connected sequence 1.
Primer 1:5 '-GTT
GGATCCATGGCTTCGTACCAGAGC-3 ' (underscore partly is the recognition sequence of restriction enzyme site BamH I, and sequence thereafter is the 1-18 position of sequence 1)
Primer 2: 5 '-GG
ACTAGTCTACTGCTTCGGAGTC-3 ' (underscore partly is the recognition sequence of restriction enzyme site Spe I, and sequence thereafter is the reverse complementary sequence of the 546-561 position of sequence 1)
One, turns acquisition and the evaluation of PpLEA3-23 trans-genetic hybrid rice
1, the structure of recombinant expression vector PpLEA3-23-pCUBI1390
(1) structure of intermediate carrier pCUBI1390
Take corn gene group DNA as template, carry out pcr amplification with primer 3 and primer 4, obtain two ends respectively with the corn Ubiquitin promotor (the 879-2864 position of GENBANK:AY572837.1) of restriction enzyme site Kpn I and Hind III.
Primer 3:5 '-
GGTACCCCTCTAGTGCAGAAGTAACACCA-3 ' (underscore partly is the recognition site of Kpn I, and sequence thereafter is the 868-890 position of GENBANK:AY572837.1)
Primer 4:5 '-
AAGCTTTCTAGTGCAGTGCAGCGTGAC-3 ' (underscore partly is the recognition site of Hind III, and sequence thereafter is the reverse complementary sequence of the 2849-2869 position of GENBANK:AY572837.1)
The two ends that above-mentioned pcr amplification obtains are carried out double digestion with the corn Ubiquitin promoter sequence of restriction enzyme site Kpn I and Hind III with restriction enzyme Kpn I and Hind III respectively, enzyme is cut product link to each other with the skeleton large fragment of the plant binary expression vector pCambia 1390 of the same double digestion of process, obtain recombinant plasmid.The recombinant plasmid that shows the 868-2869 position of having inserted GENBANK:AY572837.1 between the restriction enzyme site Kpn I of pCambia 1390 and Hind III through order-checking is positive, with its called after intermediate carrier pCUBI1390.
(2) structure of recombinant expression vector PpLEA3-23-pCUBI1390
Recombinant cloning vector PpLEA3-23-pMD19 with restriction enzyme BamH I and Spe I double digestion embodiment 1 acquisition, reclaim purpose fragment PpLEA3-23 gene, and it is linked to each other the acquisition recombinant plasmid with skeleton large fragment through the described intermediate carrier pCUBI1390 of step (1) of same double digestion.Order-checking is shown that to have inserted in the sequence table recombinant plasmid of the PpLEA3-23 gene shown in the sequence 1 between the restriction enzyme site BamH I of described intermediate carrier and Spe I positive, with its called after PpLEA3-23-pCUBI1390(plasmid structure iron as shown in Figure 2).In recombinant expression vector PpLEA3-23-pCUBI1390, the PpLEA3-23 gene is positioned at corn Ubiquitin promotor downstream, and described corn Ubiquitin promotor starts transcribing of described PpLEA3-23 gene.This recombinant expression vector has card and receives mycin and two selection markers of Totomycin (pCambia 1390 carriers carry).
2, turn acquisition and the evaluation of PpLEA3-23 trans-genetic hybrid rice
(1) turns the acquisition of PpLEA3-23 trans-genetic hybrid rice
The recombinant expression vector PpLEA3-23-pCUBI1390 that step 1 is obtained changes among the Agrobacterium AGL1 with the mode that shocks by electricity.Restructuring Agrobacterium after transforming with primer 1 and primer 2 amplification PpLEA3-23 gene, is used primer 3 and primer 4 amplification corn Ubiquitin promoter sequences.To show through evaluation and contain simultaneously the PpLEA3-23 gene (sequence 1 in the sequence table, the purpose stripe size is about 560bp) and the restructuring Agrobacterium called after AGL1/PpLEA3-23-pCUBI1390 of corn Ubiquitin promotor (the 879-2864 position of GENBANK:AY572837.1, the purpose stripe size is about 2000bp).The contrast (empty carrier contrast) that changes intermediate carrier pCUBI1390 over to is set simultaneously, gained restructuring Agrobacterium called after AGL1/pCUBI1390.
To recombinate Agrobacterium AGL1/PpLEA3-23-pCUBI1390 or AGL1/pCUBI1390 places 28 ℃ of cultivations, treat that it is between 0.120～0.140 the time that its concentration reaches the OD600 value, infect the fine callus of rice varieties Japan with Agrobacterium bacterium liquid, infected 30 minutes, during frequently shake with have gentle hands.Callus is transferred on the common culture medium, and lucifuge was cultivated 3～4 days altogether.Cultivate altogether finish after, callus transferred to selects to cultivate 10～15 days follow-up generations once on the substratum.Picking is transferred on the pre-division culture medium from former callus surface growth new resistant calli out, and pre-differentiation culture is 7 days under 28 ℃ of dark.Select creamy white, ganoid callus is transferred on the division culture medium, 28 ℃ of differentiation culture: cultivated 3 days under dark first, then cultivated 15～20 days under lasting cold light shines.To transfer on the root media from the seedling that resistant calli bears again, continuous light was cultivated 15 days, and the seedling that upgrowth situation is good is directly transferred to field planting.Gather in the crops then transgenic paddy rice seed autumn, be T
1Generation.
Results T
1Behind seed, be sowed on the MS minimum medium that contains Totomycin (30mg/L) and screen.Treat T
1Move on in the land for growing field crops when growing to the 4-6 leaf for plant and grow.With T
1After the individual plant results, each single-strain seed is sowed respectively, continues screening to observe T with Totomycin
2The separation case in generation, what obtain genetic stability turns PpLEA3-23 gene strain (T
2Generation) and the strain (T that changes empty carrier pCUBI1390 over to
2Generation).
(2) turn the evaluation of PpLEA3-23 trans-genetic hybrid rice
That selects that step (1) obtains turns PpLEA3-23 gene strain and changes each 20 strain of strain of empty carrier pCUBI1390 over to, carries out respectively PCR, RT-PCR Molecular Identification.Japanese in contrast fine with not genetically modified wild paddy rice simultaneously.
PCR checking: extract T with the CTAB method
1For oryza sativa genomic dna, use 6 pairs of PpLEA3-23 genes of primer 5 primers to carry out pcr amplification, use primer 3 and 4 couples of corn Ubiquitin of primer promotor to increase.
Primer 5:5 '-ATGGCTTCGTACCAGAGC-3 ' (the 1-18 position of sequence 1)
Primer 6:5 '-CTACTGCTTCGGAGTC-3 ' (reverse complementary sequence of the 546-561 of sequence 1)
The result shows, T
1The strain that generation turns the PpLEA3-23 gene has all amplified the big or small PpLEA3-23 gene purpose band that is about 560bp, and changes the fine PpLEA3-23 gene purpose band that all do not amplify of strain and wild paddy rice Japan of empty carrier pCUBI1390 over to.The strain that turns PpLEA3-23 gene strain and change empty carrier pCUBI1390 over to has all amplified the corn Ubiquitin promotor purpose band that size is about 2000bp, and the wild paddy rice Japan fine corn Ubiquitin promotor purpose band that all do not amplify.Part T
1For the PpLEA3-23 gene amplification result of strain as shown in Figure 3.
RT-PCR checking: extract T with the Trizol method
2For paddy rice RNA.Obtain cDNA after the reverse transcription, analyze template as RT-PCR, the primer of target gene PpLEA3-23 is primer 7 and primer 8.According to the paddy rice actin sequences Design confidential reference items primer of having reported, actin-F and actin-R.The PCR response procedures is: 94 ℃ of denaturation 3min; 94 ℃ of sex change 30s; 58 ℃ of annealing 30s; 72 ℃ are extended 40s; 30 circulations; 72 ℃ are extended 5min.
Primer 7:5 '-AGAAATGGGAGCAGACGAAAC-3 ' (the 191-211 position of sequence 1)
Primer 8:5 '-TTCACGCTCTGGTAGGCAT-3 ' (reverse complementary sequence of the 518-536 position of sequence 1)
actin-F：5′-GATACTCCCTCACAACAACCGC-3′
actin-R：5′-TGACCATCAGGCATCTCATAGC-3′
The result shows, T
2The expression amount that generation respectively turns PpLEA3-23 gene in the PpLEA3-23 gene strain is higher and close each other, and changes the fine expression that does not all detect the PpLEA3-23 gene of strain and wild paddy rice Japan of empty carrier pCUBI1390 over to.Part T
2For the RT-PCR detected result of the PpLEA3-23 gene of strain as shown in Figure 4.
Two, turn the drought resisting Function Identification of PpLEA3-23 trans-genetic hybrid rice
With growth conditions consistent (one month plant of rear growth sprouts), the T of the genetic stability that obtains in the step 1
2It is experiment material that 5 of generation turn PpLEA3-23 gene strain (Line1, Line6, Line7, Line8 and Line10), water after saturated, stop to water (A among Fig. 5) treats that the soil table has just become dry in the basin to be considered as the arid beginning, be designated as the 1st day, routine observation plant phenotype.(blade wilting degree calculates statistics wilted percent when arid is processed the 22nd day: Stress treatment was observed during at arid 14:00 at noon seedling stage, wilted percent=curling strain number/strain sum * 100%) carrying out rehydration after processes, rehydration is observed each plant phenotype after 7 days, and statistics plant survival rate.The strain that changes empty carrier pCUBI1390 over to is set simultaneously contrasts (CK) as empty carrier, it is Japanese fine as wild-type contrast (WT) that not genetically modified rice varieties is set.Experiment repeats 3 times, and the survival rate statistics is averaged.
The result shows, the strain that changes empty carrier pCUBI1390 when arid is processed the 10th day over to is wilted, yellow leaf, it is not obvious to turn PpLEA3-23 trans-genetic hybrid rice strain water shortage status, the fine blade of strain and wild-type paddy rice Japan that changes empty carrier pCUBI1390 in the time of the 22nd day over to dries up, and turn PpLEA3-23 trans-genetic hybrid rice strain blade slightly wilt (B among Fig. 5), empty carrier contrast (CK), turn PpLEA3-23 trans-genetic hybrid rice strain Line1 and the curling wilting of Line10 plant, its wilted percent is respectively 84.3%, 70.5% and 82.4%, and turn PpLEA3-23 trans-genetic hybrid rice strain Line6, Line7, the Line8 growth is normal, and wilted percent is respectively 14.2%, 21.5% and 15.3%(Fig. 5 in D).After the rehydration 7 days, empty carrier control group and wild-type control group rice plant all can not bring back to life, and the most of plant that turns the PpLEA3-23 trans-genetic hybrid rice all can be brought back to life (C among Fig. 5), concrete survival rate of plant statistics is shown in Fig. 6 and table 1,5 turn in the PpLEA3-23 gene strain, and the surviving rate of Line8 is the highest, can reach 73%, the surviving rate that turns PpLEA3-23 gene strain Line10 is minimum, still can reach 49%.Above-mentioned presentation of results is compared with empty carrier control group and wild-type control group, and the drought resistance that turns the PpLEA3-23 trans-genetic hybrid rice is significantly improved.
Three repeated experiments statistics (units: %) of each rice plant survival rate of table 1
Claims (10)
1. protein is following (a) or (b):
(a) protein that is formed by the aminoacid sequence shown in the sequence in the sequence table 2;
(b) with the aminoacid sequence of sequence 2 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and relevant with plant drought resistance protein of being derived by sequence 2.
2. the nucleic acid molecule of coding claim 1 described protein.
3. nucleic acid molecule according to claim 2 is characterized in that: described nucleic acid molecule is the gene of the described protein of coding claim 1; Described gene is following 1) to 4) in arbitrary described dna molecular:
1) encoding sequence is the dna molecular shown in the sequence 1 in the sequence table;
2) dna molecular shown in the sequence 1 in the sequence table;
3) under stringent condition with 1) or 2) dna molecule hybridize and the described protein DNA molecule of coding claim 1 that limit;
4) with 1) or 2) or 3) dna molecular that limits has 90% above homology and the described protein DNA molecule of claim 1 of encoding.
4. the recombinant vectors, expression cassette, transgenic cell line or the recombinant bacterium that contain claim 2 or 3 described nucleic acid molecule.
5. recombinant vectors according to claim 4, it is characterized in that: described recombinant vectors is recombinant expression vector or recombinant cloning vector.
6. recombinant vectors according to claim 5 is characterized in that: the promotor that starts described genetic transcription in the described recombinant expression vector is the Ubiquitin promotor.
7. protein claimed in claim 1, or claim 2 or 3 described nucleic acid molecule, or claim 4 or 5 or 6 described recombinant expression vectors, expression cassette or recombinant bacterium are at following a1) or a2) in application:
A1) regulating plant drought resistance;
A2) plant variety of seed selection drought resistance raising.
8. a method of cultivating the transgenic plant of drought resistance raising comprises that the gene with the described protein of coding claim 1 imports the step that obtains transgenic plant in the purpose plant; Described transgenic plant are compared with described purpose plant, and drought resistance improves.
9. application according to claim 7, or method claimed in claim 8 is characterized in that: described plant is monocotyledons or dicotyledons.
10. application according to claim 9 or method, it is characterized in that: described monocotyledons is paddy rice.
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| CN201210499263.2A CN102964438B (en) | 2012-11-29 | 2012-11-29 | Stress-resistance-related protein PpLEA3-23 of plant as well as coding gene and application of protein |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105801679A (en) * | 2016-04-15 | 2016-07-27 | 首都师范大学 | Application of protein PpLEA3-2 in regulating and controlling plant stress resistance |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102532287A (en) * | 2010-12-13 | 2012-07-04 | 首都师范大学 | Stress-resistant protein PpLEA3-17 of bryophyte as well as encoding gene and application thereof |
| CN102584965A (en) * | 2011-01-10 | 2012-07-18 | 首都师范大学 | Bryophyte reversal-resistant protein PpLEA3-20 and encoding gene and application thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102532287A (en) * | 2010-12-13 | 2012-07-04 | 首都师范大学 | Stress-resistant protein PpLEA3-17 of bryophyte as well as encoding gene and application thereof |
| CN102584965A (en) * | 2011-01-10 | 2012-07-18 | 首都师范大学 | Bryophyte reversal-resistant protein PpLEA3-20 and encoding gene and application thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105801679A (en) * | 2016-04-15 | 2016-07-27 | 首都师范大学 | Application of protein PpLEA3-2 in regulating and controlling plant stress resistance |
| CN105801679B (en) * | 2016-04-15 | 2019-04-09 | 首都师范大学 | Application of the protein PpLEA3-2 in regulation stress resistance of plant |
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| CN102964438B (en) | 2014-05-07 |
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