CN102260339B - Rice OsWRKY7 protein, and encoding gene and application thereof - Google Patents

Abstract

The invention discloses a rice OsWRKY7 protein, and an encoding gene and application thereof. The protein is a protein 1) or a protein 2), wherein the protein 1) has amino acid sequences shown as a sequence 2 in a sequence table; and the protein 2) has an amino acid residue sequence which is derived from the sequence 2 by substituting and/or losing and/or adding one or more amino acid residues and has the same function. The ultraviolet B and cold resistance of T2-generation plants of a plant WRKY transcription factor overexpressed transgenic strain is enhanced, while the ultraviolet B and cold resistance of antisense suppression transgenic T2-generation plants is reduced, which indicates that the gene mediates ultraviolet and cold defense responses of plants.

Description

Rice Os WRKY7 albumen and encoding gene thereof and application

Technical field

The present invention relates to a kind of rice Os WRKY7 albumen and encoding gene and application.

Background technology

Can activate corresponding signal transduction path after the various external worlds of plant perception coerce arrives transcription factor, is participated in increasing or the expression of retarding effect factor gene, reached the purpose that opposing is coerced by transcription factor.From structural analysis of protein, transcription factor generally by DNA in conjunction with territory (DNA-binding domain, BD), (nuclear localization signal, NLS) these 4 functional areas are formed for transcriptional regulatory domain (transcription regulation domain) (comprise and activating or the inhibition territory) and nuclear localization signal.Transcription factor combine with cis element in the gene promoter by these functional areas or with the expression of other transcription factor or interactions between protein with regulatory gene.Transcription factor might be in the joint of unlike signal pathway, participates in replying that difference is coerced.External, transcription factor generally can combine with special cis element, and some expression of gene are regulated and control in the environment around this combination may be subjected in plant materials and the influence of other cofactor.Therefore, understand the mechanism of the physiology flexibility of growth and development of plants and adaptation external environment, be necessary to study in great detail all kinds of transcription factors function in vivo from molecular level.

Summary of the invention

An object of the present invention is to provide a kind of rice Os WRKY7 albumen and encoding gene thereof.

A kind of protein provided by the present invention, name is called OsWRKY7, derives from paddy rice (Oryza sativa subsp.japanica var.Zhonghua 17), is following 1) or 2) described protein:

1) protein of forming by the amino acid residue sequence of the sequence in the sequence table 2;

2) with 2 amino acid residue sequences of the sequence in the sequence table through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and have identical function by 1) deutero-protein.

Wherein, the sequence in the sequence table 2 is made up of 246 amino-acid residues; The replacement of described one or several amino-acid residue and/or disappearance and/or interpolation are meant replacement and/or the disappearance and/or the interpolation of no more than ten amino-acid residues.

Described proteinic encoding gene OsWRKY7 also is a scope of protection of the invention.

Described encoding gene is following 1)-3) in arbitrary described dna molecular:

1) dna molecular shown in the sequence 1 in the sequence table;

2) under stringent condition with 1) dna sequence dna hybridization that limits and dna molecular with identical function;

3) with 1) dna sequence dna that limits has 70% at least, have 75% at least, have 80% at least, have 85% at least, have 90% at least, have 95% at least, have 96% at least, have 97% at least, have 98% or have 99% homology at least and have the dna molecular of identical function at least.

Above-mentioned stringent condition is at 0.1 * SSC, in the solution of 0.1%SDS, hybridizes under 65 ℃ and washes film.

Wherein, the sequence 1 in the sequence table is made up of 741 deoxynucleotides, and deoxynucleotide is the coding region from 5 ' terminal 1-741 position.

The recombinant vectors, reorganization bacterium, transgenic cell line or the expression cassette that contain described encoding gene also are the scope of protection of the invention.

The described encoding gene total length that increases or arbitrary segmental primer are to also being the scope of protection of the invention; Described primer is to as follows: a primer sequence is shown in sequence in the sequence table 4, and another primer sequence is shown in sequence in the sequence table 5.

The application of above-mentioned encoding gene in cultivating resistance enhanced plant also is the scope of protection of the invention; The application of above-mentioned protein in cultivating resistance enhanced plant also is the scope of protection of the invention.

Another object of the present invention provides a kind of method of cultivating resistance enhanced plant.

The method of cultivation resistance enhanced plant provided by the invention is above-mentioned encoding gene to be imported the purpose plant obtain transgenic plant, and the resistance of described transgenic plant is higher than described purpose plant.

Described resistance is ultraviolet radiation resisting and/or anti-low temperature.

The mode of described uviolizing is: described ultraviolet ray is a UV-B; Described ultraviolet light intensity is 1.28 μ mol m ^-2s ^-1, described irradiation time is 3 hours, the distance of described ultraviolet light source and described transgenic plant is 0.5 meter; Described low temperature is 4 ℃.

Described purpose plant is dicotyledons or monocotyledons, and described monocotyledons is specially paddy rice.

The present invention experimental results show that, the transfer-gen plant that OsWRKY7 is imported the paddy rice acquisition has tolerance ultraviolet B and cold characteristic, and under these conditions, this gene can be fast, express consumingly, and Antisense Suppression transgenosis T2 weakens ultraviolet B and cold resistance for plant, show this gene mediated plant to ultraviolet B and cold defense response.

Description of drawings

Fig. 1 is the Subcellular Localization of OsWRKY7

Fig. 2 is the transcriptional activation activity analysis of OsWRKY7 and determining of transcription activating domain

Fig. 3 is OsWRKY7 and W box specific combination

Fig. 4 is the expression of OsWRKY7 under coercing

Fig. 5 is the carrier structure synoptic diagram of pCoU-OsWRKY7

Fig. 6 is the carrier structure synoptic diagram of pCam35S-OsWRKY7A

Fig. 7 is the acquisition and the detection of OsWRKY7 transgenic paddy rice

Fig. 8 is the analysis of OsWRKY7 transfer-gen plant to ultraviolet B resistance

Fig. 9 is the analysis of OsWRKY7 transfer-gen plant to cold (4 ℃) resistance

Figure 10 is the analysis of OsWRKY7 genetic expression in the analysis of OsWRKY89 genetic expression in the OsWRKY7 transfer-gen plant and the OsWRKY89 transfer-gen plant

Figure 11 is OsWRKY7 and the combining of OsWRKY89 promotor

Embodiment

In order to understand the present invention better, give further instruction by the following examples, but be not limitation of the present invention.

Experimental technique among the following embodiment if no special instructions, is ordinary method.

Percentage composition among the following embodiment if no special instructions, is the quality percentage composition.

Restriction enzyme that relates in the experimentation and T ₄Dna ligase is produced by precious biotechnology (Dalian) company limited, consults and uses specification sheets and uses.

The acquisition of embodiment 1, commentaries on classics OsWRKY7 paddy rice

One, the clone of OsWRKY7 cDNA

1, the separation of OsWRKY7 cDNA

According to the consequence devised primer WRKY7F and the WRKY7R of database analysis, WRKY7F:5 '-CGCGCGATGTCGTCGCTGTAC-3 ' (sequence 4); WRKY7R5 '-TCAAGGAAGCAGCAGCGAGGTG-3 ' (sequence 5) amplifies the cDNA of OsWRKY7 from paddy rice by RT-PCR with the LaTaq archaeal dna polymerase.

Specific as follows: as will to spend 17 (Oryza sativa subsp.japanica var.Zhonghua 17 in 21 days the paddy rice of normal light cycle growth, Haihua Wang, Junjie Hao, Xujun Chen, Zhongna Hao, Xia Wang, Yonggen Lou, Youliang Peng, and Zejian Guo, Overexpression of rice WRKY89 enhances ultraviolet B toleranceand disease resistance in rice plants.Plant Mol Biol, 65:799-815,2007; The public can obtain from China Agricultural University.) blade, extract RNA with Triozol (purchasing company) in Clontech, carry out the reverse transcription PCR amplification.(MJ Research carries out in USA) at DNA thermal cycler PTC-220 in amplification.Amplification condition is: 94 ℃ of pre-sex change 4 minutes; Again 94 ℃ 40 seconds, 64 ℃ 40 seconds, 72 ℃ 50 seconds, 30 circulations were extended 10 fens down at 72 ℃ at last.The PCR product that recovery obtains, send to order-checking, the nucleotide sequence that sequencing result has sequence 1 in the sequence table for this PCR product, sequence 1 be the coding region from 5 ' terminal 1-741 position Nucleotide, with the unnamed gene of this PCR product is OsWRKY7, the albumen called after OsWRKY7 of this genes encoding, this proteic aminoacid sequence are sequence 2 in the sequence table.

This PCR product is connected on the pMD-18T carrier, constitutes carrier pMD-OsWRKY7,, contain the nucleotide sequence of ordered list sequence 1 in this carrier through order-checking.

The search of database is with the BLAST instrument on the NCBI.Primary Structure Analysis shows that the proteic homology of the WRKY of OsWRKY7 and paddy rice or other species is not high, illustrates that it is a new WRKY member.OsWRKY7 has an amino-acid residue to change in quite conservative WRKYGQK territory, becomes K by Q and becomes WRKYGKK; The genome sequence of OsWRKY7 is classified sequence 3 in the sequence table as, form by 945 deoxynucleotides, contain two introns (from 5 of sequence 3 ' the 306th to 409 deoxynucleotides of end, from 5 of sequence 3 ' the 575th to 674 deoxynucleotide of end) and three exons (from 5 of sequence 3 ' the 1st to 305 deoxynucleotides of end, from 5 of sequence 3 ' the 410th to 574 deoxynucleotide of end, from 5 of sequence 3 ' the 675th to 945 deoxynucleotide of end).

The primer table

WRKY7F 5 '-CGCGCGATGTCGTCGCTGTAC-3 ' (sequence 4)

WRKY7R 5 '-TCAAGGAAGCAGCAGCGAGGTG-3 ' (sequence 5)

WRKY7BH1F 5’-TAATGGATCCCGCGCGATGTCGTCGCTGTAC-3’(BamH I)

WRKY7ER1R 5’-TAATGAATTCCAAGGAAGCAGCAGCGAGGTG-3’(EcoR I)

WRKY7Xh1R 5’-TAATCTCGAGAGGAAGCAGCAGCGAGGTG-3’(Xho I)

W7B1F 5’-TATAGGATCCATGTCGTCGCTGTAC-3’(BamH I)

W7B1R 5’-TAATGGATCCTCAAGGAAGCAGCAGCG-3’(BamH I)

W7C1B1R 5’-TAATGGATCCGAGGCGGCCGTGCGGCG-3’(BamH I)

W7C1E1F 5’-TAATGAATTCTGGAGGAAGTACGGCAAG-3’(EcoR I)

W7E1F 5’-TAATGAATTCGCGATGTCGTCGCTG-3’(EcoR I)

W7N1B1R 5’-TAATGGATCCGTCGTCGTCGTCGTCGAC-3’(BamH I)

W7N2B1R 5’-TAATGGATCCCCGGTAGCCGTCGTCCAG-3’(BamH I)

W7N2E1F 5’-TAATGAATTCATGGCTGCCGTCGCCGAC-3’(EcoR I)

W7PstF 5’-TATACTGCAGATGTCGTCGCTGTAC-3’(Pst I)

W7Xh1R 5’-TATACTCGAGAGGAAGCAGCAGCGAG-3’(Xho I)

W89PE1F 5’-AATTGAATTCTGGAGTACATCCATCCGAGC-3’(EcoR I)

W89PXB1R 5’-TATATCTAGAGTTGATCACTACTTGACAGTC-3’(XbaI)

W7E1F 5’-TAATGAATTCGCGATGTCGTCGCTG-3’(EcoR I)

W7B1R 5’-TAATGGATCCTCAAGGAAGCAGCAGCG-3’(BamH I)

2, the Subcellular Localization of OsWRKY7

PBluescript-SK-GFP makes up as follows: the GFP gene (GenBank number is AY596277) with synthetic is a template, primer is: F:5 '-TAATAAGCTTATGAGTAAAGGAGAAGAACT-3 ', R:5 '-TAATGAATTCAAGGATCATGTG ATCTCTC-3 ', the PCR product (AY596277 67-781 position Nucleotide) that amplification obtains are inserted between the Hind III of pBluescript SK (purchasing the company in Clontech) and the EcoR I recognition site and obtain pBluescript-SK-GFP.

PCam35S-GFP makes up as follows: the small segment that obtains after the pBluescript-SK-GFP of above-mentioned acquisition is cut with Pst I and Sal I enzyme inserts between the recognition site of the Pst I of pCam35S and Sal I and obtains pCam35S-GFP;

Wherein, pCam35S makes up as follows: be template with pCAMBIA1301 (purchasing the company in Clontech) carrier 1), carry out pcr amplification with primer 5 '-ACGAATTCGGTC CCCAGATTAGCC-3 ' and 5 '-CCGAGCTCGTCCCCCGTGTTC-3 ', cut the PCR product with EcoR I and Sac I enzyme and obtain endonuclease bamhi, endonuclease bamhi is inserted between pCAMBIA1301 carrier EcoR I and the Sac I restriction enzyme site obtains recombinant vectors; 2) genome with cowpea (available from the food market) is a template, with primer 5 '-AATCTGCAGAGCTTTCGTTCGTATCATCG-3 ' and primer 5 '-AATAAGCTTCGATTGATGCATGTTGTCAA-3 ' amplification, obtains the PCR product; 3) with 2) the PCR product and 1 that obtains) after the recombinant vectors that obtains passes through Pst I and HindIII double digestion respectively, reclaim corresponding fragment and connect, obtain carrier pCam35S.

PCam35S-OsWRKY7-GFP makes up as follows: the pMD-OsWRKY7 carrier with above-mentioned acquisition is a template, with primer WRKY7BH1F and WRKY7Xh1R, amplification obtains the PCR product, reclaim this PCR product, the fragment of cutting acquisition through BamH I and Xho I enzyme is inserted between the BamH I of pCam35S-GFP carrier and Sal I recognition site and obtains fusion vector pCam35S-OsWRKY7-GFP again.Sequence verification, sequencing result show that pCam35S-OsWRKY7-GFP obtains inserting between the BamH I of pCam35S-GFP and Sal I recognition site from 5 ' terminal 1-738 position Nucleotide of sequence in the sequence table 1.

The particle gun that pCam35S-OsWRKY7-GFP is used for onion (Allium cepa) epidermic cell transforms, and compares with pCam35S-GFP.Concrete steps are as described below:

1) preparation of plasmid: extract purifying pCam35S-OsWRKY7-GFP with test kit, it is standby to get 2.5 μ g after quantitatively.

2) preparation of onion: the white onion entocuticle of tearing (1cm * 1cm), on the interior side, place to contain penbritin (Amp, 100 μ g m ^-1) improved MS substratum (1 * MS salt, 1 * Gamborg ' s B5 VITAMIN, 30g L ^-1Sucrose, 2% agar, pH 5.7) standby.

3) bronze suspension preparation: take by weighing 60mg bronze (diameter 1.0 μ m) in the 1.5mL centrifuge tube; Add 1000 μ L dehydrated alcohols, vibration suspension 1-2 minute; 10,000rpm is centrifugal, 10 seconds; Abandon supernatant, repeat once; With the aseptic two distillation H of 1000 μ L ₂O suspends, and vibrates 1-2 minute; 10,000rpm is centrifugal, 10 seconds; Abandon supernatant, repeat once; With the aseptic two distillation H of 1000 μ L ₂O suspends, and vibrates 1-2 minute, obtains uniform bronze suspension, and it is standby to show usefulness or-20 ℃ of preservations.

4) embedding of bronze and DNA: get the uniform bronze suspension 50 μ L that step 3) obtains, add 1) the 2.5 μ g plasmids that obtain, mixing; Add 20 μ L 0.1mol L ^-1Spermidine, mixing; Drop by drop add 50 μ L 2.5mol L lightly ^-1CaCl ₂, and jiggle frequently; Gentle concussion is after 3-5 minute on the oscillator, and room temperature was placed 10 minutes; 10,000rpm is centrifugal, 10 seconds, abandons supernatant, dehydrated alcohol rinsing 2 times; Add 60 μ L dehydrated alcohols and suspend, mixing obtains bronze-dna complex.

5) bombardment receptor material: pressure membrane and bombardment film are soaked taking-up after 1-2 hour in 70% alcohol, in Bechtop, dry; Metal baffle with 70% alcohol-pickled after, on spirit lamp, sterilize; Get bronze-dna complex that 10 μ L step 4) obtain, evenly coat the mid-way of bombardment film, airing is not coated on the whole film, and size should be consistent with the pore diameter range on the carrier fixed ring; Be installed on the launching device; Can split the lower end that film is installed to the gas acceleration tube; Target material focuses on the middle part of culture dish, puts into vacuum chamber, takes off the culture dish lid; Vacuumize pointer to 26; Put helium in the gas acceleration tube, pressure reaches in the time of can splitting the pressure that disk can bear in pipe, can split film and break; Gas is flushed on the bombardment film, and carrier moves downward, and blocked by metal baffle, and following metallic particles sees through the mesh of metal baffle, directive target cell; Can split film pressure is 1,000p.s.i..Plate is sealed with Parafilm, secretly cultivates after 18 hours for 22 ℃, observes, also gathers image down with 40 times of mirrors of laser confocal scanning microscope (Bio-Rad MRC 1024).LASER Light Source is argon-krypton laser (Argonm-Krypton), and excitation wavelength is set at 488nm, also has a passage to be used for transmission microscopic examination under the visible light in addition.Image overlay and other processing are carried out with Confocal Assistant and Adobe Photoshop 6.0 softwares.

With pCam35S-GFP is contrast, the result as shown in Figure 1, A among Fig. 1, B and C are pCam35S-GFP transient expression photo, D, E, F are pCam35S-OsWRKY7-GFP transient expression photo.As can be seen from the figure, A and D are green fluorescence under blue light excites among Fig. 1; C and F are the onion cell under the visible light among Fig. 1; B is the stack of A and C among Fig. 1, and E is the stack of D and F among Fig. 1, shows that OsWRKY7 is positioned (photo amplifies 40 * 10 times and takes pictures under laser confocal microscope) in the nucleus.

3, the analysis of the transcriptional activation activity of OsWRKY7 and transcription activating domain determines

PMD-OsWRKY7 carrier with above-mentioned acquisition is a template, with primer W7E1F and W7B1R amplification, the PCR product is cut the fragment that obtains through EcoR I and BamH I enzyme, be inserted into yeast GAL4DNA in conjunction with (binding domain, BD) carrier pGBKT7 (is the pBD carrier; Purchase company in Clontech) EcoR I and BamH I recognition site between, obtain carrier pBD-OsWRKY7.There is dislocation-free OsWRKY7 coding region among the sequence verification pBD-OsWRKY7.

Increase each deletion fragment the primer to as follows:

Adopting uses the same method makes up deleted carrier pBD-Δ C1 (from the 1st of the aminoterminal of sequence 2 to 223 amino acids residues), pBD-Δ C2 (from the 1st of the aminoterminal of sequence 2 to 141 amino acids residues), pBD-Δ N1 Δ C2 (from the 38th of the aminoterminal of sequence 2 to 141 amino acids residues), pBD-Δ N1 Δ C1 (from the 38th of the aminoterminal of sequence 2 to 223 amino acids residues), pBD-Δ N1 (from the 38th of the aminoterminal of sequence 2 to 246 amino acids residues), pBD-Δ N2 (from the 142nd of the aminoterminal of sequence 2 to 246 amino acids residues), pBD-Δ N2 Δ C1 (from the 142nd of the aminoterminal of sequence 2 to 223 amino acids residues), and the carrier of pBD-N1 (from the 1st of the aminoterminal of sequence 2 to 37 amino acids residues) encoding gene: the amplimer of each deletant coding region is to as follows:

PBD-Δ C1:W7E1F/W7C1B1R; PBD-Δ C2:W7E1F/W7N2B1R; PBD-Δ N1 Δ C2:W7N2E1F/W7N2B1R; PBD-Δ N1 Δ C1:W7N2E1F/W7N2B1R; PBD-Δ N1:W7N2E1F/W7B1R; PBD-Δ N2:W7C1E1F/W7B1R; PBD-Δ N2 Δ C1:W7C1E1F/W7C1B1R; PBD-N1:W7E1F/W7N1B1R; PMD-OsWRKY7 carrier with above-mentioned acquisition is a template.

The cDNA fragment that above-mentioned amplification obtains is cut with the enzyme enzyme that corresponding primer carries, and is inserted into respectively between the C end same enzyme recognition site of pGBDKT7 (available from Clontech company), makes up each deletion mutant carrier, sequence verification.

With the carrier difference transformed yeast strains A H109 (available from Clontech company) of above-mentioned structure, then respectively at SD substratum (6.7g L ^-1Yeast does not have the amino acid nitrogenous source, and the 2g agar powder, pH 5.8) and the SD-Trp-Ade-His substratum (the SD substratum adds 0.06g L ^-1Leu-Trp-Ade-His (available from Clontech company), add 0.06g L ^-1Leu (available from Clontech company)) cultivated 3 days for 30 ℃.Carry out alpha-galactosidase activity then and measure, alpha-galactosidase activity is measured with test kit (available from Clontech company), is undertaken by supplier's specification sheets, and substrate is PNP-α-Gal (p-nitrophenyl α-D-galactopyranoside).The positive contrast of yeast transformant (pBD-53) that negative contrast of transformant (pBD) that obtains with pGBDKT7 empty carrier transformed yeast strains A H109 and pGBDKT7-53 (available from Clontech company) transformed yeast strains A H109 obtain.

The alpha-galactosidase activity measuring principle: PNP-α-Gal hydrolysis under the effect of alpha-galactosidase produces semi-lactosi and p-NP, and the latter has absorbance value at 410nm.

The result shows the yeast well-grown on SD-Trp-Ade-His (SD-Trp-A) substratum that carries pBD-OsWRKY7 as shown in Figure 2, illustrates that OsWRKY7 has the ability of transcriptional activation.The transformant (pBD-Δ C1) of the transformant (pBD-Δ N1) of disappearance N end 37aa (1-37aa) and disappearance C end 23aa (223-246aa) can be grown, but their betagalactosidase activities with carry the remarkable decline of comparing of pBD-OsWRKY7 transformant; And N end and C are held transformant (pBD-Δ N1 Δ C2, the 38-141aa of disappearance simultaneously; PBD-Δ N2 Δ C1,142-223aa; PBD-Δ N1 Δ C1 38-223aa) can not grow at the SD-Trp-Ade-His substratum; And the 37aa of N end and the transformant (pBD-N1) of Gal4-BD fusion can be grown on the selection substratum, and have betagalactosidase activity, show that the 37aa of N end and C end 23aa are the transcriptional activation zones of OsWRKY7.

A is OsWRKY7 total length and different disappearance Δ C1 (aa 1-223) among Fig. 2, Δ C2 (aa 1-138), Δ N1 Δ C2 (aa 38-141), Δ N1 (aa 38-246), Δ N2 (aa 142-246), Δ N2 Δ C1 (aa 142-223), Δ N1 Δ C1 (aa 38-223), N1 (aa 1-37) is inserted into yeast GAL4DNA in conjunction with domain vector pGBKT7.

B is with above-mentioned carrier transformed yeast strains A H109 among Fig. 2, selects to cultivate last 30 ℃ of cultivation results after 3 days at SD-Trp-A (SD-Trp-Ade-His) or SD-Leu (SD-Leu-Trp-Ade-His).Wherein CK is pBD, and position 1 is pBD-OsWRKY7, and position 2 is pBD-Δ C1, and position 3 is pBD-Δ C2, position 4 is pBD-Δ N1 Δ C2, and position 5 is pBD-Δ N1, and position 6 is pBD-Δ N2, position 7 is pBD-Δ N2 Δ C1, and position 8 is pBD-Δ N1 Δ C1, and position 9 is pBD-N1.

C is that alpha-galactosidase activity is measured among Fig. 2.Measuring method is undertaken by supplier's specification sheets, and substrate is PNP-α-Gal (p-nitrophenyl α-D-galactopyranoside).With the positive contrast of yeast transformant (substratum SD-Leu) of negative contrast of pGBKT7 empty carrier transformant and pBD-53, get the mean value of the value that draws in independently testing for three times respectively.Numerical value is respectively pBD-53:100, pBD-OsWRKY7:93.483, pBD-Δ C1:70.235, pBD-Δ C2:61.703, pBD-Δ N1 Δ C2:12.374, pBD-Δ N1:48.657, pBD-Δ N2:43.874, pBD-Δ N2 Δ C1:13.067, pBD-Δ N1 Δ C1:12.522, pBD-N1:38.144, pBD:12.072.Negative control does not have enzyme value alive.

The above results shows that the 37aa of OsWRKY7 albumen n end and C end 23aa are the transcriptional activation zones of OsWRKY7.

4, OsWRKY7 combines with the W box

In order in bacterium, to express OsWRKY7 albumen, with primer WRKY7BH1F and WRKY7ER1R, pMD-OsWRKY7 with above-mentioned acquisition is a template, the fragment that the PCR product that obtains is obtained after BamH I and EcoR I enzyme are cut is inserted between the BamH I of pGEX-4T carrier (purchasing the company in Clontech) and EcoRI recognition site and is obtained pGST-OsWRKY7, sequence verification, the result is ' obtaining between the BamH I of terminal 1-738 position Nucleotide insertion pGEX-4T carrier and EcoRI recognition site from 5 sequence table sequence 1 for pGST-OsWRKY7.

With pGST-OsWRKY7 and contrast empty carrier pGEX-4T difference Transformed E .coli BL21 (DE3) bacterial strain (purchasing company), be inoculated into 5ml then and contain 100mg L in Takara ^-1In the LB substratum of Amp, 37 ℃ of overnight shakings are cultivated 180rpm.Getting 1ml bacterium liquid is inoculated into the fresh LB substratum of 100ml and (contains 100mg L ^-1Amp) further cultivate in, treat the OD of culture ₆₀₀Reach 0.5 to 0.7, add 0.4mmol L ^-1The IPTG inducible protein is expressed.Through 37 ℃ cultivated in 4 o'clock or 25 ℃ of incubated overnight after, 4 ℃ of centrifugal collection thalline are with STE (100mmol L ^-1NaCl, 10mmol L ^-1TrisHCl, 1mmol L ^-1EDTA, pH 8.0) wash once, be suspended in 100mmolL then ^-1Phosphoric acid buffer (PBS) (contains 1mmol L ^-1DTT, 1mmol L ^-1PMSF, 1%Triton X-100) in.Lysate is 12, and 000g 4 ℃ times centrifugal 15 minutes, collects the purifying that supernatant liquor is used for inducible protein.

With Sepharose 4B-GST post albumen is carried out purifying.Purification process is by supplier's specification sheets.Protein sample (supernatant liquor of collection) is added in the pillar of handling well, add 0.1%Triton-100, (concentration respectively is 1mmol L to add PMSF and DTT ^-1), room temperature incubation 20 minutes, and stir gently frequently; 4 ℃, centrifugal 5 minutes of 500g removes supernatant; Wash pillar 3 times with the PBS damping fluid of 10 times of pillar volumes; Add 200 μ l elutriant (10mmol L ^-1Reduced glutathion, 50mmol L ^-1Tris-Cl pH 8.0 contains each 1mmol L of PMSF and DTT ^-1), 20-25 ℃ of incubation 15 minutes, 4 ℃, centrifugal 5 minutes of 500g draws supernatant liquor (adding glycerine to 10%) and preserves and obtain recombinant protein.

Determining the protein quantity with bovine serum albumin as standard protein.Recombinant protein purity is passed through SDS-PAGE, (Alphainnotech USA) detects with the Alphaimage analytical system.

It is the characteristic feature of transcription factor that transcription factor combines with specific cis element.WRKY class transcription factor can combine with the W box specificity that contains the TGAC core sequence.Utilize this characterized OsWRKY7 whether can with the combination of W box, thereby determine whether OsWRKY7 is WRKY family transcription factor.

The synthetic probe P22 that contains the sequence (TGAC and its complementary sequence GTCA) of 4 W boxes:

5 '-GATCTG TGACTGGTC TGACGCCA GTCAC GTCATG-3 ' sports CT as probe with the GA among the W box TGAC among the P22, and the TC among its complementary sequence GTCA sports AG, i.e. mutant probe mP22:

5′-GATCTGTCTCTGGTCTCTCGCCAGAGACGAGATG-3′。After the P22 probe of mark and the reorganization OsWRKY7 protein binding, the speed that moves in electrophoresis slows down, and can present the band of a hysteresis; The sudden change probe mP22 can not with the OsWRKY7 protein binding.Concrete experimental technique is as follows:

Earlier the recombinant protein that pGST-OsWRKY7 is expressed carries out the protein competition experiment, and the reaction system of protein competition experiment is 20 μ l altogether, comprises recombinant protein (perhaps not adding albumen), the 0.2pmol[α of the pGST-OsWRKY7 expression of 800ng purifying ^-32P] end-labelled oligonucleotide probe (P22 or mP22), DNA binding buffer liquid (glycerine 5%, EDTA 1mmol L ^-1, DTT 1mmol L ^-1, NaCl 50mmol L ^-1, poly (dI-dC) 50ng mL ^-1, TrisHCl 10mmol L ^-1, pH 7.5), wherein, add the system of mark P22 and prepare five parts, a copy of it does not add albumen, and the system preparation of adding mark mP22 probe is a, and during preparation, the adding probe adds albumen then earlier.Do not add adding mark P22 and not add unlabelled probe in the proteic system, all the other four parts systems of adding mark P22 are carried out following processing respectively: (2) do not add unmarked probe; (3) adding is the unmarked P22 of 10 times of mol ratios of label probe; (4) adding is the unmarked P22 of 100 times of mol ratios of label probe; (5) adding is the unmarked mP22 of 100 times of mol ratios of label probe.In the system of adding mark mP22 probe, do not add any unlabelled probe.Behind the above-mentioned system mixing, be incubated 1 minute down at 25 ℃ earlier, then at 0.5 * tbe buffer liquid (45mmolL ^-1Tris, 45mmol L ^-1Boric acid, 1mmol L ^-1EDTA) carry out electrophoresis in, gum concentration 6%.Wrap glue with preservative film behind the electrophoresis, and mould phosphorus screen (Amersham Pharmacia, UK), scanning.

The result as shown in Figure 3, among the figure 10 *, 100 * respectively show that the unmarked probe that is added in the reaction system is 10 times of label probe mole number, 100 times; "+" and "-" is illustrated respectively in and adds and do not add reactant in the reaction system; Band after " S " expression albumen and the probe specific combination, " F " expression does not have protein-bonded probe band.Figure is OsWRKY7 and the situation that combines of probe, add 10 times of unlabelled P22 because the combining of unmarked P22 and OsWRKY7, combine active (the 3rd swimming lane is played on a left side) that shows the P22 probe reduced OsWRKY7 and mark adds 100 times of unlabelled P22 and almost completely stops interaction (left the 4th swimming lane) between the P22 probe of OsWRKY7 and mark.In contrast, the mutant probe mP22 of same concentrations can not compete combine (the 5th swimming lane is played on a left side) between the P22 probe of OsWRKY7 and mark effectively.The probe mP22 of sudden change can not interact with OsWRKY7 on (left side rise the 6th swimming lane), thus prove OsWRKY7 can with W box element specific combination.The result shows that reorganization OsWRKY7 can combine (second swimming lane is played on a left side) strongly with the P22 probe of mark.

5, the adverse circumstance abduction delivering of OsWRKY7

1) paddy rice is cultivated with adverse circumstance and handles

Spend 17 seeds to soak 1-2 days down at 37 ℃ in the rice varieties, the back vernalization 1 day that shows money or valuables one carries unintentionally is sowed then in nutrition soil, cultivates in the greenhouse.28 ℃ of (daytime)/22 ℃ (night), the natural light cycle.Seedling was used for various processing in 25 days, in the different time sampling, got blade and extracted RNA.

Cold (cold) handles: spend 17 plant to put incubator in the rice varieties of growing 21 days, keep 4 ℃ (cold), dark culturing is got blade respectively at 0,1,3,6,12, behind the 24h and is extracted total RNA.

Ultraviolet (UV) is handled: spend 17 plant being used for germ-resistant ultraviolet lamp (20w/m in the rice varieties of growing 21 days ²) shone 15 minutes down, handle as all wave band ultraviolet.Get blade respectively at 0,1,3,4,5,6,12, behind the 24h and extract total RNA.

2) RNA extracts with Northern and analyzes

Extract the total RNA that spends 17 plant leaf samples in the above-mentioned adverse circumstance inductive rice varieties.With total RNA (10 μ g) electrophoresis classification in 1.2% denaturing formaldehyde agarose gel, be transferred to Hybond-N then ⁺On the nylon membrane, with [α ^-32P]-the OsWRKY7 specific probe (sequence 1 is from the strand nucleotide sequence of 5 ' terminal 1-420 position Nucleotide in the sequence table) of mark is 65 ℃ of hybridization down.Under 65 ℃, add 0.1%SDS with 2 * SSC and washed 5 fens, under same temperature, add 0.1%SDS and washed again 5 fens with 0.1 * SSC.Mould phosphorus screen (Amersham Pharmacia, UK).

The result as shown in Figure 4, the result shows, OsWRKY7 is subjected to UV-B and coldly induces back 3 hours to 12 hours expression amounts to increase, to 24 hours expression amounts return to induce before.Do the applied sample amount contrast with rRNA.

Two, change acquisition and the detection of OsWRKY7 paddy rice

1, the acquisition of overexpression vector

A: the building process of overexpression vector pCoU-OsWRKY7:

Cut pUCmT-OsWRKY7 with BamH I and Kpn I enzyme, obtain pCoU-OsWRKY7 between the BamH I of the endonuclease bamhi insertion pCoU carrier that obtains and Kpn I recognition site, sequence verification, pCoU-OsWRKY7 for sequence table sequence 1 ' terminal 1-738 position Nucleotide is inserted into (and then the 738th the Nucleotide back of the OsWRKY7 among the pCoU-OsWRKY7 be exactly the strong terminator that has on the pCoU) that obtains between the recognition site of the BamH I of pCoU carrier and Kpn I, and the carrier structure synoptic diagram of pCoU-OsWRKY7 the results are shown in Figure 5 from 5.

The building process of above-mentioned pUCmT-OsWRKY7: the pMD-OsWRKY7 with above-mentioned acquisition is a template, increase with primer W7B1F and W7Xh1R, the PCR product that obtains is connected to pUCmT carrier (purchasing the company in TAKARA) and obtains pUCmT-OsWRKY7, sequence verification, pUCmT-OsWRKY7 contain ordered list sequence 1 from 5 ' terminal 1-738 position Nucleotide.

The building process of above-mentioned pCoU carrier:

1) be template with corn agricultural university 108 (available from the Beijing Jinse nonghua seed industry Technology Co., Ltd) genome, use the pfu enzymatic amplification with primer 5 '-TAAAGCTTGTTTCAAACTACCTGGTGG-3 ' and 5 '-ATGTGAGCTCTCTAGAGTCGACCTGCAGAAG-3 ', obtain PCR product (being the Ubiquitin promotor), this PCR product is cut the back with Sac I enzyme reclaim fragment;

2) pCam35S with above-mentioned acquisition cuts with the EcoRI enzyme earlier, will give prominence to the base benefit with the Klenow enzyme again and put down, and cuts with Sac I enzyme again, reclaims big fragment;

3) fragment that step 1) is reclaimed is connected to step 2) on the big fragment that reclaims, the recombinant plasmid that obtains is called pCoU, sequence verification, the result contains the Ubiquitin promotor among the pCoU.

The antisense vector pCam35S-OsWRKY7A of B:OsWRKY7 makes up:

PMD-OsWRKY7 with above-mentioned acquisition is a template, increase with primer W7PstF and W7B1R, the PCR product that obtains after cutting, Pst I and BamH I enzyme is obtained endonuclease bamhi, this endonuclease bamhi inserted between the Pst I of pCam35S carrier and BamH I recognition site obtain recombinant vectors, sequence verification, this recombinant vectors is for oppositely having inserted the Nucleotide shown in the sequence 1 in the sequence table between the Pst of pCam35S carrier I and BamH I site, with this recombinant vectors called after pCam35S-OsWRKY7A, be the antisense vector of OsWRKY7.The carrier structure synoptic diagram of pCam35S-OsWRKY7A is seen shown in Figure 6.

2, change acquisition and the detection of OsWRKY7 paddy rice

1) acquisition of commentaries on classics OsWRKY7 paddy rice

The pCoU-OsWRKY7 that the pCam35S-OsWRKY7A that the B of step 1 is obtained and the A of step 1 obtain imports respectively and obtains the reorganization bacterium among the agrobacterium tumefaciens EHA105 (purchasing the company in Clontech) respectively

EHA105/pCam35S-OsWRKY7A and reorganization bacterium EHA105/pCoU-OsWRKY7, through sequence verification,

Contain the Nucleotide shown in the sequence 1 in the ordered list among the EHA105/pCam35S-OsWRKY7A,

Contain among the EHA105/pCoU-OsWRKY7 ordered list sequence 1 ' terminal 1-738 position Nucleotide illustrates that the reorganization bacterium all successfully constructs from 5.

EHA105/pCam35S-OsWRKY7A and EHA105/pCoU-OsWRKY7 are transformed into respectively and spend 17 in the paddy rice, concrete grammar is: spend 17 seeds to carry out surface sterilization after 1 minute with 70% ethanol in the ripe paddy rice, clorox with 20% (v/v) was sterilized 30 minutes, cultivated (MS interpolation 0.5g L on the NBI substratum ^-1Glutamine, 0.5g L ^-1Prolineamide, 2.0mg L ^-12,4-D; PH 5.8), grow callus after general 14 days, callus is peeled to be put on the new NBI substratum cultivate, can infect after 4 days; The bacterium liquid of the bacterium liquid of EHA105/pCam35S-OsWRKY7A and EHA105/pCoU-OsWRKY7 is evenly coated in respectively on the solid YEP flat board cultivated 2-3 days, scrape bacterium in liquid culture medium (interpolation 2g L the MS substratum altogether from flat board with aseptic spoon ^-1Inositol, 0.3g L ^-1CEH (enzymic hydrolysis casein), 100 μ mol L ^-1Syringylethanone) in to cell concn be 3 * 10 ⁷Cfu mL ^-1, callus is put into bacterium infected 20 minutes, callus is poured on the bacterium liquid that blots the surface on the clean filter paper, move on on the Nbco substratum and to cultivate that (the NBI substratum adds μ mol L ^-1Syringylethanone); Cultivate after 2 days, callus is transferred to the last cultivation of screening culture medium NBs, and (the NBI substratum adds 500mg L ^-1Cephamycin, 30mg L ^-1Totomycin); By the time callus goes out green back (general 30 days) and moves to division culture medium and (add 2.0mg L in the MS substratum ^-16-BAP (six benzyladenines), 0.5mg L ^-1Naphthylacetic acid, 30g L ^-1Sorbyl alcohol and 30mg L ^-1Totomycin) go up cultivation, the little seed of acquisition is planted up to receiving in the greenhouse.Obtain 11 strain T0 altogether for changeing pCam35S-OsWRKY7A paddy rice and 16 strain T0 for changeing the pCoU-OsWRKY7 paddy rice.

T0 is T1 generation for the seed of transfer-gen plant selfing gained with by the plant that this seed grows up to.T1 is T2 generation for the seed of transfer-gen plant selfing gained with by the plant that this seed grows up to.

To gather in the crops seed for changeing pCam35S-OsWRKY7A paddy rice and T0 for changeing the pCoU-OsWRKY7 paddy rice from T0 respectively, after planting obtain T1 respectively for changeing pCam35S-OsWRKY7A paddy rice and T1 for changeing the pCoU-OsWRKY7 paddy rice, gather in the crops seed for changeing pCam35S-OsWRKY7A paddy rice and T1 for changeing the pCoU-OsWRKY7 paddy rice from T1 again, after planting obtain T2 respectively for changeing pCam35S-OsWRKY7A paddy rice and T2 for changeing the pCoU-OsWRKY7 paddy rice.

Adopting uses the same method spends in 17 paddy rice during empty carrier pCam35S and pCoU changed over to respectively, obtain T0 respectively for changeing pCam35S paddy rice and T0, gather in the crops the final T2 of acquisition of seed and sowing again for changeing pCam35S paddy rice and T2 for changeing the pCoU paddy rice for changeing the pCoU paddy rice.

2) evaluation of commentaries on classics OsWRKY7 paddy rice

T2 being carried out normal growth respectively and carries out ultraviolet B irradiation 3 hours for changeing the pCam35S-OsWRKY7A paddy rice, recover growth after 7 days, is contrast with T2 for spending 17 (wild-types) in commentaries on classics pCoU-OsWRKY7 paddy rice and the rice varieties.

Extract ultraviolet B irradiation 3 hours respectively, the 7 strain T2 that recover growth after 7 days are for changeing pCam35S-OsWRKY7A paddy rice (being numbered A1, A2, A3, A4, A6, A7) and 5 strain T2 for the RNA that changes pCoU-OsWRKY7 paddy rice (being numbered S1, S8, S14, S18, S19), carry out Northern and analyze, used probe is OsWRKY7 specific probe (sequence 1 from 5 ' the strand nucleotide sequence of terminal 1-741 position Nucleotide).The result as shown in Figure 7, A figure is the result of normal condition growth, B figure recovers the result who grows after 7 days for ultraviolet B shone 3 hours.ZH represents to spend in the rice varieties 17 (wild-types) among the figure, and S1, S8, S14, S18, S19 represent the plant of overexpression, and A1, A2, A3, A4, A6, A7 represent the Antisense Suppression plant, does the applied sample amount contrast with rRNA.

Find out from figure B, compare that the middle OsWRKY7 expression amount of three overexpression plant (T2 is for changeing the pCoU-OsWRKY7 paddy rice) that is numbered S8, S14, S18 obviously strengthens, and proves that the overexpression plant successfully constructs with wild-type; Antisense Suppression plant (T2 is for changeing the pCam35S-OsWRKY7A paddy rice) the OsWRKY7 expression amount that is numbered A1, A2, A3, A4, A6, A7 does not have noticeable change, shows that the OsWRKY7 expression amount is suppressed.

Embodiment 2, the Function detection of changeing the OsWRKY7 paddy rice

1, the OsWRKY7 transfer-gen plant is to the analysis of ultraviolet B resistance

Respectively with T2 for changeing pCam35S-OsWRKY7A paddy rice (being numbered S1, S8, S14, S18) and T2 under the natural lighting condition, the grow plant of 21 days seedling ages of commentaries on classics pCoU-OsWRKY7 paddy rice (being numbered A1, A2, A3, A6), the commentaries on classics plant of the 21 days seedling ages of growing under field conditions (factors) is exposed to ultraviolet B (light intensity 1.28 μ mol m continuously ^-2s ^-1) following 3 hours, ultraviolet B lamp is from 0.5 meter of plant.Dark repair 24 hours, continuous light recovers then, light intensity 30Pmolm ^-2s ^-1Take pictures with digital camera after 7 days.Is contrast for the T2 that changes the pCoU paddy rice and obtained by embodiment 1 for changeing the pCam35S paddy rice with the T2 that spends 17 in the wild-type, obtained by embodiment 1, and contrast all adopts 17 strains to average.

Ultraviolet B handles back dark adatpation 10 minutes, amplifies adjustable luminoscope (PAM-2000, Heinz Walz, Effeltrich, Germany) chlorophyll fluorescence of mensuration the 2nd leaf (beginning number from above) with portable pulse.The maximum quantum yield of PSII photochemistry (Fv/Fm) calculates by (Genty B, Briantais JM, Baker NR. (1989) .The relationship between the quantum yield of photosynthetic electron transportand quenching of chlorophyll fluorescence.Biochim Biophys Acta, method 990:87-92).Fv/Fm=(Fm-F0)/Fm, wherein F0 is the initial fluorescence of chlorophyll, and Fm is the chlorophyll maximum fluorescence, and Fv is the variable fluorescence of chlorophyll.

The experiment triplicate, the result takes the mean.The result as shown in Figure 8, among the figure A for 21 days T2 of growth for changeing pCoU-OsWRKY7 paddy rice and T2 for changeing the pCam35S-OsWRKY7A rice seedling with ultraviolet B pre-irradiation; B is for ultraviolet B irradiation 3 hours, secretly cultivate 1 day, the comparison of illumination cultivation after 6 days again among the figure; Among the figure C in the contrast of different time point determining, spend 17, T2 is for changeing pCoU-OsWRKY7 paddy rice and T2 for the 2nd leaf Fv/Fm value of changeing the pCam35S-OsWRKY7A rice plant.A1 among the figure, A2, A3, A6 are that T2 is for three plant that change the pCam35S-OsWRKY7A paddy rice; ZH is for spending 17 (wild-types) in the contrast; S1, S8, S14, S18 are that T2 is for the plant that changes the pCoU-OsWRKY7 paddy rice.

As seen from the figure, be numbered S1, S8, S14, the T2 of S18 is respectively 0.824 for changeing the 0th hour the Fv/Fm value of pCoU-OsWRKY7 rice plant behind uv irradiating, 0.828,0.828 and 0.824, the 24th hour Fv/Fm value behind uv irradiating is respectively 0.774,0.766,0.784 and 0.771, the 48th hour Fv/Fm value behind uv irradiating is respectively 0.731,0.732,0.731 and 0.731, the 72nd hour Fv/Fm value behind uv irradiating is respectively 0.706,0.720,0.723 with 0.715, the 96th hour Fv/Fm value behind uv irradiating is respectively 0.691,0.701,0.714 and 0.697.As seen from the figure, uv irradiating after 7 days plant still keep survival, Fv/Fm value is reduction slowly, but remains on a higher level.

Be numbered A1, A2, A3, the T2 of A6 for change pCam35S-OsWRKY7A paddy rice the 0th hour behind uv irradiating the Fv/Fm value be respectively 0.821,0.830,0.820 and 0.825, the 24th hour Fv/Fm value behind uv irradiating is respectively 0.671,0.681,0.712 and 0.680, the 48th hour Fv/Fm value behind uv irradiating is respectively 0.597,0.594,0.627 and 0.601, the 72nd hour Fv/Fm value behind uv irradiating is respectively 0.508,0.504,0.514 with 0.504, the 96th hour Fv/Fm value behind uv irradiating is respectively 0.451,0.426,0.475 and 0.439.As seen from the figure, all dead after 7 days at uv irradiating.

Wild-type contrast (ZH) behind uv irradiating the 0th hour, the 24th hour, the 48th hour, the 72nd hour and the 96th hour Fv/Fm value are respectively 0.824,0.714,0.674,0.577 and 0.523.As seen from the figure, wild-type behind uv irradiating 7 days dead substantially, wild-type Fv/Fm fall greater than T2 for changeing the pCam35S-OsWRKY7A paddy rice, and less than T2 for changeing the pCam35S-OsWRKY7A paddy rice.

The above results shows that OsWRKY7 is relevant with the ultraviolet resistance of paddy rice.

T2 is for changeing pCoU paddy rice and T2 for spending the result of 17 plant not have marked difference in result who changes the pCam35S paddy rice and the wild-type.

2, the OsWRKY7 transfer-gen plant is to the analysis of cold (4 ℃) resistance

Respectively with T2 for changeing pCam35S-OsWRKY7A and T2 for changeing under the natural lighting condition, the grow plant of 21 days seedling ages of pCoU-OsWRKY7 rice plant, transfer to that 4 ℃ of incubators are dark cultivated after 24 o'clock the recovery illumination cultivation 6 days.Observe the phenotype variation and take pictures, and survey the chlorophyll content variation, method is as follows: get the paddy rice seedling leaf of handling respectively at 24 o'clock, claim fresh weight, change the ethanol of adding 10mL 80% in the test tube over to, lucifuge vibration at room temperature; Take out 1mL spectrophotometric instrumentation A at 0,1,3,6,12,24,48 hour time point ₆₆₄And A ₆₄₇Value, the sample after at every turn having surveyed is still refunded in the former test tube; Chlorophyllous total mole number formula: (7.93A ₆₆₄+ 19.53A ₆₄₇)/fresh weight calculates.Mole number with 48 hours time points is a total mole number, calculates the per-cent that each time point accounts for total mole number.Is contrast for the T2 that changes the pCoU paddy rice and obtained by embodiment 1 for changeing the pCam35S paddy rice with the T2 that spends 17 plant in the wild-type, obtained by embodiment 1.

The experiment triplicate, the result takes the mean.

The result as shown in Figure 9, among the figure A for 21 days T2 of growth for change pCoU-OsWRKY7 paddy rice, T2 for change pCam35S-OsWRKY7A rice seedling and wild-type rice seedling with 4 ℃ of processing before; B is 4 ℃ of dark cultivations of plant 1 day, the comparison of illumination cultivation after 6 days again; C is the plant chlorophyll changing value at different time point determinings among the figure.A1 among the figure, A2, A3, A6 are that T2 is for changeing the pCam35S-OsWRKY7A paddy rice; ZH is for spending 17 in the contrast; S1, S8, S14, S18 are that T2 is for changeing the pCoU-OsWRKY7 paddy rice.

As seen from the figure, T2 for change the pCoU-OsWRKY7 paddy rice 4 ℃ handle 7 days after plant still keep survival, chlorophyll changes numerical value and obviously compares according to slow, reached 70% in 24 hours, be lower than 80% of ZH, and T2 is all dead after 7 days 4 ℃ of processing for changeing the pCam35S-OsWRKY7A rice plant, it is obviously fast than ZH that chlorophyll changes numerical value, substantially reached 90% in 24 hours, and showed that OsWRKY7 was relevant to cold resistance with paddy rice.T2 is for changeing pCoU paddy rice and T2 for spending the result of 17 plant not have marked difference in result who changes the pCam35S paddy rice and the wild-type.

3, the research of OsWRKY7 and OsWRKY89

1) analysis of OsWRKY7 genetic expression in the analysis of OsWRKY89 genetic expression and the OsWRKY89 transfer-gen plant in the OsWRKY7 transfer-gen plant

Respectively will by the T2 of above-mentioned acquisition for change the pCam35S-OsWRKY7A paddy rice, by the T2 of above-mentioned acquisition for changeing the pCoU-OsWRKY7 paddy rice, changeing pCam35S-ds89 T2 for paddy rice (Haihua Wang, Overexpression of rice WRKY89 enhances ultraviolet B tolerance and disease resistance in rice plants, Plant Mol Biol, 65 (6): 799-815,2007; The public can obtain from China Agricultural University.), change pCoU-OsWRKY89T2 for paddy rice (Haihua Wang, Overexpression of rice WRKY89enhances ultraviolet B tolerance and disease resistance in rice plants, Plant Mol Biol, 65 (6): 799-815,2007; The public can obtain from China Agricultural University.) and contrast in spend 17, elegant water 11 (Oryzasativa subsp.japanica var.Xiushui 11; Haihua Wang, Junjie Hao, Xujun Chen, Zhongna Hao, Xia Wang, Yonggen Lou, Youliang Peng, and Zejian Guo, Overexpression of rice WRKY89enhances ultraviolet B tolerance and disease resistance in rice plants.Plant Mol Biol, 65:799-815,2007; The public can obtain from China Agricultural University.) planting seed in the little basin that nutrition soil is housed, each strain system kind of two basins, in the seedling sampling of growth basin after 21 days under the natural lighting condition, seedling ultraviolet B irradiation (the light intensity 1.28 μ mol m of another basin ^-2s ^-1, the UV-B lamp is from 0.5 meter of plant) and 15 minutes, dark repair was taken a sample after 6 hours.Carry RNA, the variation of Northern hybridization analysis OsWRKY89 gene mRNA amount, used probe is OsWRKY89 full-length cDNA (the 26-817 position of genbank AY781112).

As shown in figure 10, A changes for OsWRKY89 expression of gene in contrast and the commentaries on classics OsWRKY7 paddy rice among the figure; B changes for contrasting and changeing OsWRKY7 trans-genetic hybrid rice ultraviolet B processing back OsWRKY89 expression of gene among the figure, used probe [α- ³²P]-the OsWRKY89 specific probe (the 26-817 position of genbank AY781112) of mark.C changes for OsWRKY7 expression of gene in contrast and the commentaries on classics OsWRKY89 paddy rice among the figure; D changes for B outside contrast and the commentaries on classics OsWRKY89 rice purple handles back OsWRKY7 expression of gene among the figure, used probe [α- ³²P]-the OsWRKY7 specific probe (from the strand nucleotide sequence of the 1-741 position of sequence 1 Nucleotide) of mark.Do the applied sample amount contrast with rRNA; S8, S14, S18 are OsWRKY7 overexpression plant; A1, A2, A3 are OsWRKY7 Antisense Suppression plant; ZH is for spending 17 in the contrast; XS is the elegant water 11 of contrast; OS7, OS8, OS9, OS11 are OsWRKY89 overexpression plant; D3, D4, D8 are that OsWRKY89 interferes plant.

OsWRKY89 expresses increase in OsWRKY7 overexpression plant (T2 that is numbered S8, S14, S18 is for changeing the pCoU-OsWRKY7 paddy rice) as seen from the figure, and whether does not all detect the expression of OsWRKY89 in Antisense Suppression plant (T2 that is numbered A1, A2, A3 is for changeing the pCam35S-OsWRKY7A paddy rice) under the situation that ultraviolet B handles; At OsWRKY89 overexpression (the commentaries on classics pCoU-OsWRKY89T2 that is numbered OS7, OS8, OS9, OS11 is for paddy rice) with interfere plant (the commentaries on classics pCam35S-ds89T2 that is numbered D3, D4, D8 is for paddy rice,) middle OsWRKY7 expression does not all have to change, and OsWRKY7 expression amount increase after being subjected to ultraviolet B processing.Show that the OsWRKY7 gene can work in the upstream of OsWRKY89 gene, OsWRKY7 albumen may be regulated and control the OsWRKY89 genetic transcription.

2) OsWRKY7 combines with the promotor of OsWRKY89

Vector construction: from rice varieties, spend in 17 genomic dnas amplification to obtain the promotor of OsWRKY89 gene A TG upstream 1470bp as the OsWRKY89 gene with W89PE1F and W89PXB1R primer, be inserted between the EcoR I and Xba I recognition site of pHISi carrier (available from Clontech company) after this PCR product cut with EcoRI and Xba I enzyme, enzyme is cut and is identified and sequence verification.The carrier called after pHIS-OsWRKY89P that sequencing result is correct.With transformed yeast behind the carrier pHIS-OsWRKY89P usefulness Xho I linearization for enzyme restriction, obtain to have the sub yeast (, making the OsWRKY89 promotor be incorporated into yeast HIS upstream) of yeast report of OsWRKY89P-HIS for homologous recombination.

The PCR product that the complete coding region of OsWRKY7 obtains after with W7E1F and W7B1R primer amplification is cut through EcoR I and BamH I enzyme, being inserted into yeast GAL4DNA activated carrier pGAKT7 (is the pAD carrier, available from Clontech company) EcoR I and the recognition site of BamH I between, make up the pAD-OsWRKY7 carrier, this carrier enzyme is cut identified that also there is dislocation-free in sequence verification vector encoded district.

OsWRKY7 combines checking with the promotor of OsWRKY89 gene: preparation of yeast competence and method for transformation are the same.Yeast with yeast report that has OsWRKY89P-HIS prepares competence, and the pAD-OsWRKY7 carrier is transformed, and (the SD substratum adds 0.06g L to be coated on SD+His ^-1Leu-Trp-Ade (available from Clontech company)) (add 5mmol L ^-1Grow on the flat board of 3-AT (three ammonia azoles), pAD transforms in contrast with empty carrier.

Get single bacterium colony and on substratum, grow, take pictures with general camera.As shown in figure 11,1 expression reports that with OsWRKY89P-HIS transformed yeast (the OsWRKY89 promotor is only arranged) can grow among the figure on the SD+His substratum, but is containing 5mmol L ^-1Can not grow on the SD+His screening culture medium (SD+His+3AT) of 3-AT; 2 expressions can be grown on the SD+His substratum after the pAD control vector being transformed in the yeast that contains above-mentioned report among the figure, but are containing 5mmol L ^-1Can not grow on the SD+His screening culture medium of 3-AT; 3 expressions transform the pAD-OsWKRY7 carrier in the yeast contain above-mentioned report after (existing OsWRKY89 promotor has OsWRKY7 again) among the figure, not only can grow on the SD+His substratum, are containing 5mmol L ^-1Also can normal growth on the SD+His screening culture medium of 3-AT.The above results show OsWRKY7 can with the combining of OsWRKY89 gene promoter, OsWRKY7 may directly activate the OsWRKY89 genetic transcription.

Claims (6)

1. the application of protein in cultivating resistance enhanced plant of forming by the amino acid residue sequence of the sequence in the sequence table 2; Described resistance is ultraviolet radiation resisting and/or anti-low temperature; Described plant is a paddy rice.

2. the application of proteinic encoding gene in cultivating resistance enhanced plant of forming by the amino acid residue sequence of the sequence in the sequence table 2; Described resistance is ultraviolet radiation resisting and/or anti-low temperature; Described plant is a paddy rice.

3. application according to claim 2 is characterized in that: described encoding gene is the dna molecular shown in the sequence 1 in the sequence table.

4. a method of cultivating resistance enhanced plant is the proteinic encoding gene that the amino acid residue sequence of the sequence in the sequence table 2 is formed to be imported the purpose plant obtain transgenic plant, and the resistance of described transgenic plant is higher than described purpose plant; Described resistance is ultraviolet radiation resisting and/or anti-low temperature; Described purpose plant is a paddy rice.

5. method according to claim 4 is characterized in that: the mode of described uviolizing is: described ultraviolet ray is a UV-B; Described ultraviolet light intensity is 1.28 μ mol m ^-2s ^-1, described irradiation time is 3 hours, the distance of described ultraviolet light source and described transgenic plant is 0.5 meter; Described low temperature is 4 ℃.

6. according to claim 4 or 5 described methods, it is characterized in that: described encoding gene is the dna molecular shown in the sequence 1 in the sequence table.

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