CN1831010A - Regulatory factor for anti-reverse transcription of corn, and its coding gene and application thereof - Google Patents

Abstract

The invention discloses the corn resisting transcription and adjusting factor and coding gene and appliance. The corn resisting transcription adjusting factor is among the proteid of the following amino acid residue: 1) the SEQ ID No:1 of the sequence list; 2)the amino acid residue sequence of the SEQ ID No:1 of the sequence list are replaced, appended by one to ten amino acid residue and the proteid acting with the LTRE arranging component to improve the capability of resisting contradictorily of the plane. The adjusting factor can be acted on the LTRE arranging component of the several resisting abiotic adversity dependency gene promoter zone and adjust several the expression of the resisting abiotic adversity dependency gene and improve the ability of the plane resisting the abiotic adversity such as the drought, the low temperature and the salting. So the LBF is important to breed the resisting athwart plant species particularly the corn and improve the yield of the crop particularly the corn.

Description

Anti-reverse transcription of corn regulatory factor and encoding gene and application

Technical field

The present invention relates in the plant and coerce relevant transcriptional regulator and encoding gene thereof and application, particularly relate to the transcriptional regulator of abiotic stress such as anti-low temperature, arid and salt marsh in the corn and encoding gene thereof and its and improve application in the plant cultivating resistance.

Background technology

Chilling injury is the normal natural disaster that takes place in the agriculture production, has greatly limited the plantation of rabi and the results of late-autumn crops.

Studies show that, under cold condition, plant can be transferred intravital defence system energetically and resist external environment stress, at this moment will great changes will take place in plant materials, and after one's own heart proteic synthetic, metabolic transformation, (Hans J, Adaptations to environmental stresses., the Plant Cell such as accumulation of degeneration-resistant border material, 1995,7:1009-1111).Plant is not only to rely on a certain, two functional genes to finish to the opposing of low temperature stress, there are a lot of albumen to participate in the reaction that plant is resisted low temperature stress, at present, more than 100 of the genes relevant have been found with low temperature stress, their synergies are regulated and control physiology, biochemistry and the metabotic change of plant, improve the resistance of plant to low temperature stress, existing result of study has also proved this viewpoint.As people such as Steponkus COR15a gene transformation (Steponkus P.L. in Arabidopis thaliana, Mode of action of the COR15a gene on the freezing toleranceof Arabidopsis.PNAS, 1998,1998:14570-14575), people such as Zhu B are HVA1 gene transformation (Zhu B in paddy rice, Overexpression of a 1-pyrroline-5-carboxylate synthetase geneand analysis of tolerance to water-and salt-stress in transgenic rice., Plant Sci, 1998,139:41-48), though the cold performance of the transformed plant that obtains through aforesaid method increases, actual effect is unsatisfactory.

For improving the resisting abiotic adverse circumstance performance of plant, people have carried out a large amount of researchs about plant resisting abiotic adverse circumstance gene, and begin to turn to research with the closely-related transcriptional modulatory gene of abiotic stress, because transcription factor can be regulated and control the expression with degeneration-resistant relevant series of genes, and not merely be a gene in action, therefore transcription factor is playing an important role aspect the ability of raising plant stress-resistance border, and becomes the research focus gradually.In recent years, obtained gratifying achievement in research, as DREB1A (the DRE binding factor) factor, can be in conjunction with DRE (Droughtresponse element) cis element, regulation and control RD29A, RD17, ERD10, KIN1, the isogenic expression of COR15a (Qiang Liu, Two transcription factors, DREB1 and DREB2, with an EREBP/AP2DNA binding domain separate two cellular signal transduction pathways indrought-and low-temperature-responsive gene expression, respectively, inArabidopsis.Plant Cell, 1998,10:1391-1406; Mie Kasuga, Improving plant drought, salt, and freezing tolerance by gene transfer of a single stess-inducibletransciption factor., Nature Biotechnology, 1999,17:287-291); ABF (ABREbinding factor) factor, can be in conjunction with ABRE (ABA response element) cis element, regulation and control RD28B, RAB18, the isogenic expression of ICK1 (Joung-youn Kang, Arabidopsis Basic Leucine ZipperProteins That Mediate Stress-Responsive Abscisic Acid Signaling., Plant Cell, 2002,14:343-357).Transgenic experiments result shows that above-mentioned transcription factor can significantly improve the resistivity that plant is coerced abiotic stress.

With the research of cold-resistant relevant transcriptional modulatory gene at first from the model plant Arabidopis thaliana, people such as Kisten R. are separated to Arabidopis thaliana CBF1 (the C-repeat binding factor) factor, CBF1 can interact with the C-repeat cis element, regulation and control COR6.6, COR15a, COR47, the isogenic expression of COR78 (Kirsten R, ArabidopsisCBF1 Overexpression Induces COR Genes and Enhances Freezing Tolerance., Science, 1998,280:104-106), transgenic experiments result shows that the cold performance of changeing the Arabidopis thaliana that the CBF1 gene is arranged is significantly improved.In addition, find after deliberation all to contain low temperature response element (LTRE) with the promoter region of cold-resistant relevant a plurality of functional genes, as the COR15a gene, the COR78/LTI78/RD29A gene, KIN1, KIN2 gene and RAB18 gene (Liu Qiang, Zhang Guiyou, Chen Shouyi. the structure of plant transcription factor and regulating and controlling effect. Science Bulletin, 2000,45 (14): 1465-1474).Recently, people such as Ming-Jun Gao are separated to the factor with the interactional BNCBFs of LTRE cis element (Brassica napus CBFs) from rape, BNCBFs is subjected to low temperature induction to express (Ming-Jun Gao, Regulation and characterization of four CBF transcriptionfactors from Brassica napus.Plant Molecular Biology, 2002,49:459-471).But at present with the interactional trans factor of LTRE cis element in monocotyledons, especially in important farm crop corn, yet there are no research report.

Summary of the invention

The purpose of this invention is to provide an anti-reverse transcription of corn regulatory factor and encoding gene thereof.

Anti-reverse transcription of corn regulatory factor provided by the present invention, name is called LBF (LTRE Binding Protein), derives from Zea corn (Zea mays L.), is the protein with one of following amino acid residue sequences:

1) the SEQ ID № in the sequence table: 1;

2) with SEQ ID № in the sequence table: 1 amino acid residue sequence is through replacement, disappearance or the interpolation of one to ten amino-acid residue and have to interact with the LTRE cis element and improve the protein of plant stress-resistance performance.

SEQ ID № in the sequence table: 1 is made up of 267 amino-acid residues, with LTRE bonded DNA core sequence be " CGAC ".

The encoding gene of above-mentioned anti-reverse transcription of corn regulatory factor (LBF) is one of following nucleotide sequence:

1) SEQ ID № in the sequence table: 2 dna sequence dna;

2) SEQ ID № in the code sequence tabulation: the polynucleotide of 1 protein sequence;

3) under the rigorous condition of height can with SEQ ID № in the sequence table: the nucleotide sequence of the 2 dna sequence dnas hybridization that limit.

The rigorous condition of described height can be that (or 0.1 * SSC), the solution of 0.1%SDS is hybridized and washed film with 0.1 * SSPE under 65 ℃.

SEQ ID № in the sequence table: 2 by 1042 based compositions, and its open reading frame is that coding has SEQ ID № in the sequence table: the protein of 1 amino acid residue sequence from 5 ' end 117-the 920th bit base.

Contain expression carrier of the present invention, transgenic cell line and host bacterium and all belong to protection scope of the present invention.

Arbitrary segmental primer is to also within protection scope of the present invention among the amplification LBF.

Utilize plant expression vector, the encoding gene importing vegetable cell with LBF of the present invention can obtain environment stress tolerance enhanced transgenic cell line and transfer-gen plant.

When using LBF to make up plant expression vector, before its transcription initiation Nucleotide, can add any enhancement type promotor or inducible promoter.For the ease of transgenic plant cells or plant being identified and screening, can process used plant expression vector, as adding selected marker's (gus gene, luciferase genes etc.) that can in plant, express or antibiotic marker thing (gentamicin marker, kantlex marker etc.) with resistance.From the security consideration of transgenic plant, can not add any selected marker, directly with adverse circumstance screening transformed plant.

Carry LBF of the present invention plant expression vector can Ti-plasmids, Ri plasmid, plant viral vector, directly DNA conversion, microinjection, electricity be led, conventional biological method transformed plant cells or tissue such as agriculture bacillus mediated by using, and the plant transformed tissue cultivating is become plant.By the plant transformed host both can be monocotyledonss such as corn, paddy rice, wheat, also can be dicotyledonss such as soybean, rape, cotton.

The present invention separates from important farm crop corn, the clone obtains the conjugated protein LBF of LTRE, belong to AP1 class transcription factor, it is the transcriptional regulator that has with the LTRE binding specificity, can regulate and control more adverse circumstance Expression of Related Genes, the bonded DNA of LBF institute core parts sequence is " CGAC ", this and the CBF1 that has reported, DREB1A, BNCBF, transcription factors such as osDREB are obviously different, their bonded DNA core sequences are " CCGAC ", experimental results show that this regulatory factor can act on the LTRE cis-acting elements in a plurality of resisting abiotic adverse circumstance related gene promoters district, regulate and control a plurality of resisting abiotic adverse circumstance Expression of Related Genes, improve plant arid, low temperature, the resistivity of abiotic stresses such as salt marsh.LBF is to cultivating the particularly anti-contrary corn variety of plant with adverse resistance kind, and particularly maize yield is significant to improve farm crop.

The present invention will be further described below in conjunction with drawings and Examples.

Description of drawings

Fig. 1 be LBF in yeast cell with the binding specificity analytical results of LTRE

Fig. 2 is the external bonded functional analysis of LBF and LTRE (EMSA experiment) result

Fig. 3 is LBF and LTRE bonded DNA core sequence analytical results

Fig. 4 is that LBF is in intravital LTRE binding specificity of yeast and transcriptional activation function checking

Fig. 5 is the expression of LBF gene under adverse environmental factors such as salt, arid, hydrogen peroxide, ABA, low temperature

Fig. 6 is the specifically expressing situation of LBF in the maize immature embryos growth course

Fig. 7 is the cold-resistant test-results of LBF transgenic arabidopsis

Fig. 8 is the anti-salt test result of LBF transgenic arabidopsis

Fig. 9 is the drought resisting test-results of LBF transgenic arabidopsis

Embodiment

Method therefor is ordinary method if no special instructions among the following embodiment, and the primer and dna fragmentation are synthetic by match Parkson, Beijing bio-engineering corporation, and described examining order is finished by Shanghai Bo Ya Bioisystech Co., Ltd.

The acquisition of embodiment 1, LBF gene

One, the structure in maize immature embryos cDNA library

1, the extraction of the total RNA of maize immature embryos and mRNA's separates

Get the rataria 1g of the neat 319 pollination backs the 17th day (17dpp) of corn variety, total RNA with the above-mentioned maize immature embryos of RNAgents Total RNAIsolation System test kit (Promega company) extraction uses PolyATtract mRNA Isolation System (Promega company) to isolate mRNA then from total RNA of above-mentioned maize immature embryos.

2, the structure in corn 17dpp rataria cDNA library

The mRNA of the corn 17dpp rataria that obtains with step 1 is a template, with GibcoBRL Corporation's Super ScriptTMPlasmid System for cDNA Synthesis and Plasmid Cloning test kit and strict cDNA library of operating structure corn 17dpp rataria by the test kit specification sheets, obtaining storage capacity at last is 5.2 * 10 ⁶The cDNA library of the corn 17dpp rataria of cfu.

Two, from corn 17dpp rataria cDNA library, screen the LBF encoding gene with the yeast one-hybrid method

1,4mer LTRE bait carrier and 4mer mutant LTRE (mLTRE) save the structure of carrier

1) the synthetic LTRE functional element (two sections complementary DNA fragments) of taking advantage of a situation, sequence is as follows:

LTRE(+)：5’-ATTTCATGGCCGACCTGCTTTTT-3’

LTRE(-)：5’-AAAAAGCAGGTCGGCCATGAAAT-3’

Respectively get LTRE (+) and LTRE (-) that 20 μ L concentration are 1 μ g/ μ L, mixing, add NaOAc and 100 μ L dehydrated alcohols that 4 μ L concentration are 3M, centrifugation DNA after placing 30 minutes under-20 ℃, to precipitate with 70% ethanol and wash once, after drying makes the ethanol volatilization then, add 6.5 μ L sterilized waters and 1 μ L, 10 * T ₄Polynucleotide kinase (Promega company) damping fluid is annealed, and annealing conditions is: 88 ℃ of 2min, 65 ℃ of 10min, 37 ℃ of 10min, 25 ℃ of 5min; The ATP and the 1 μ L T that add 1.5 μ L 20mM again ₄Polynucleotide kinase, 37 ℃ of reactions be after 2 hours, with phenol chloroform (blending ratio is 1: 1) with each extracting of chloroform is once, and usefulness dehydrated alcohol deposit D NA.Add 2 μ L, 10 * ligase enzyme damping fluid, 1 μ L ligase enzyme (5units/ μ L), 17 μ L sterilized waters, 16 ℃ connect 12-24 hour, carrying out 2% agarose gel electrophoresis detects, obtained the dna fragmentation of big or small about 80bp, (ancient cooking vessel state company) reclaims this fragment with dna fragmentation fast purifying/recovery test kit, and it is cloned into through the SpeI enzyme cuts and mend among the flat carrier pBSK+ (Clontech company), obtain containing the take advantage of a situation recombinant vectors of double chain DNA fragment of functional element bonded DNA core parts sequence of transcription factor and LTRE, called after pL4 through order-checking.

2) the synthetic LTRE that the contains sudden change functional element of taking advantage of a situation, composition sequence is as follows: mLTRE (+): 5 '-ATTTCATGattagttTGCTTTTT-3 ' and mLTRE (-): 5 '-AAAAAGCAaactaatCATGAAAT-3 ', use same quadrat method, obtain containing the take advantage of a situation recombinant vectors of double chain DNA fragment of functional element bonded DNA core parts sequence of the transcription factor of sudden change and LTRE, called after pmL4.

3) plasmid vector pL4 and pmL4 are all used purifying behind BamHI and the Xba I double digestion, more respectively with through the same carrier pRS315His (Leu that handles ⁺) (Wilson TE et al, Identification of the DNA bindingsite for NGFI-B by genetic selection in yeast.Science 252:1296-300 (1991); Use T ₄Dna ligase connects, and obtains containing the bait carrier pRSL4 (Leu of LTRE dna fragmentation ⁺) and contain the segmental redemption carrier of mLTREDNA pRSmL4 (Leu ⁺).

2, corn 17dpp rataria cDNA library screening

Preparation yeast yWAM2 (Leu ^-, His ^-, Trp ^-) (Cheng X, Boyer JL, Juliano RL.Selectionof peptides that functionally replace a zinc finger in the Sp1 transcriptionfactor by using a yeast combinatorial library.Proc.Natl.Acad.Sci.USA 1997,94:14120-14125) competent cell is bait carrier pRSL4 (Leu ⁺) be transformed into yeast strain yWAM2 (Leu ^-, His ^-, Trp ^-) in, method for transformation carries out with reference to Two Hybrid System TRAFO protocol, obtains to be integrated with the yeast strain yL4 (His of pRSL4 ^-, Trp ^-); Use yeast strain yL4 (His then ^-, Trp ^-) the corn 17dpp rataria cDNA library of step 1 structure is screened, method for transformation is the same, and transformant is coated onto His ^-Select on the substratum (Sigma company), cultivated 3-5 days down at 28 ℃, after treating that yeast grows, extract yeast plasmid, but extracting method reference literature (Christine Guthrie, Method I:Quick Plasmid DNAPreparations from Yeast, Methods in Enzymology, 1991,194:322) carry out; Then with yeast plasmid Transformed E .coli DH5 α, extract the plasmid of positive colony and carry out restriction analysis with Not I and Sal I, show the dna segment that can cut out about 1.2Kb, the positive colony that is obtained is carried out sequential analysis, the result show obtained with bait carrier in the LTRE proteic encoding gene in library that functional element combines of taking advantage of a situation, this gene has SEQ ID № in the sequence table: 2 polynucleotide sequence, by 1042 based compositions, its open reading frame is that coding has SEQ ID № in the sequence table: the protein of 1 amino acid residue sequence from 5 ' end 117-the 920th bit base.The complete sequence of this gene of search is not found identical sequence in GeneBank.Its coded albumen is carried out the amino acid residue sequence analysis, find that it has and the identical conserved domain of AP1 class transcription factor, holding 60-the 123rd amino acids residue from N is the conserved domain of AP1 class transcription factor, proof utilizes the method for yeast one-hybrid to obtain in the corn encoding gene with the interactional trans factor of LTRE cis element, with its called after LBF, with the albumen called after LBF of this coded by said gene.

The binding specificity analysis of embodiment 2, LBF and LTRE

One, LBF in yeast cell with the binding specificity analysis of LTRE

With recombinant vectors pL4 and pmL4 transformed yeast yWAM2 (Leu ^-, His ^-, Trp ^-), the yeast strain yWAM2/pmL4 that obtains being integrated with the yeast strain yWAM2/pL4 of pL4 and be integrated with pmL4, again the plasmid that contains the LBF gene that obtains through the sieve storehouse among the embodiment 1 is transformed and transform recombination yeast yWAM2/pL4 and yWAM2/pmL4 respectively, then under 28 ℃, at His ^-Select to cultivate 3-5 days on the substratum, (A is recombination yeast yWAM2/pL4 to the result among the figure as shown in Figure 1, B is recombination yeast yWAM2/pmL4 among the figure), have only recombination yeast yWAM2/pL4 to grow, and recombination yeast yWAM2/pmL4 can not grow, show that LBF can specific combination LTRE element in the yeast body, thereby activate reporter gene HIS3 expression of gene, so can be at His ^-Select to grow on the substratum, because LBF can not combine with mLTRE, can not activate reporter gene HIS3 expression of gene on the contrary, thereby at His ^-Select to grow on the substratum, prove that LBF has the binding specificity of LTRE in the yeast body.

Two, the external bonded functional selection of LBF and LTRE (EMSA experiment)

1, obtains the LBF albumen of purifying

The full-length gene of LBF is cloned among the prokaryotic expression carrier pGEX4T-1 (Amersham Pharmacia Biotech company), then with recombinant expression vector Transformed E .coli BL21, under 37 ℃, with 0.3mM IPTG abduction delivering 2-3 hour, expression product is carried out the SDS-PAGE electrophoresis detection, expression has molecular weight to be about the albumen of 56KD, conforms to the LBF (29.4KD) of expection and the proteic size of 26KD GST that merges.MicroSpin according to Amersham PharmaciaBiotech company ^TMGST Purification ModuLe protocol carries out purifying to the LBF albumen of expressing, and is used for the EMSA experiment.

2, isotopic labeling LTRE and mLTRE probe

The LTRE probe sequence is: 5 '-ATTTCATGGCCGACCTGCTTTTT-3 '

The mLTRE probe sequence is: 5 '-ATTTCATGattagttTGCTTTTT-3 '

Adopt DNA 5 ' the End-Labeling System of Promega company that LTRE and mLTRE probe are carried out isotopic labeling, reaction system is: LTRE (or mLTRE) probe 1 μ L, T ₄PNK 10 * buffer 5 μ L, γ- ³²P-ATP3 μ L, T ₄PNK (10U/ μ L) 2 μ L, H ₂O 39 μ L; Reaction conditions is: 37 ℃ 20 minutes; Add 2 μ L 0.5MEDTA, 68 ℃ of deactivations in 10 minutes; 37 ℃ 10 minutes; 4 ℃ of preservations are standby.

3, LBF albumen and DNA association reaction and non-sex change SDS-PAGE detect

LBF albumen and DNA bonded reaction system are: 5 * binding buffer liquid (contains 125mM HEPES-KOH pH7.6; 50% glycerine; 250mM KCl) 4 μ L, LBF (the purified LBF albumen of step 1 preparation) about 4 μ g (9 μ L), 1M DTT 1 μ L, the LTRE of step 2 mark (or mLTRE) probe 1 μ L, H ₂O 4 μ L.It is mixed the back ice bath reacted in 30 minutes, add 3 μ L sample-loading buffers (sterilized water that contains 0.025% tetrabromophenol sulfonphthalein) then, carry out native polyacrylamide gel electrophoresis and detect, the prescription of 5.4% polyacrylamide gel is: 30% acrylamide 9mL, and 10 * electrophoretic buffer 5mL (contains glycine 142.7g/L, EDTA 3.92g/L, Tris 30.28g/L), 50% glycerine 2.5mL, deionized water 33mL, 10%APS 400 μ L, TEMED 25 μ L.After treating gelling, with 1 * electrophoretic buffer electrophoresis, first prerunning 10 minutes (300V) is gone up sample then, continues electrophoresis 1 hour (300V); Electrophoresis finishes the back with the sticking glue down of filter paper, will press the X-ray sheet 1 hour than after sealing with preservative film; Develop a film, developed photographic fixing 5 minutes 2 minutes.(swimming lane 1 is a LBF albumen to the result as shown in Figure 2, swimming lane 2 is a LBF albumen), a tangible electrophoretic band has appearred in swimming lane 1, be the result that LTRE is blocked by LBF albumen, on the contrary, mLTRE is not blocked by LBF albumen then, shows that LBF expression of gene product is at the external binding specificity that has LTRE equally.

Embodiment 3, LBF and LTRE bonded DNA core sequence are analyzed

The core sequence possible to LTRE " GCCGACC " carries out rite-directed mutagenesis respectively, the mutant nucleotide sequence of m1-m7 following (base of band underscore is the mutational site):

LTRE：5’-ATTTCATG GCCGACCTGCTTTTT-3’(CK)

m1：5’-ATTTCATG aCCGACCTGCTTTTT-3’

m2：5’-ATTTCATGG tCGACCTGCTTTTT-3’

m3：5’-ATTTCATGGC tGACCTGCTTTTT-3’

m4：5’-ATTTCATGGCC aACCTGCTTTTT-3’

m5：5’-ATTTCATGGCCG gCCTGCTTTTT-3’

m6：5’-ATTTCATGGCCGA tCTGCTTTTT-3’

M7:5 '-ATTTCATGGCCGAC tTGCTTTTT-3 ' uses the method identical with embodiment 2 to carry out the EMSA experiment, the result as shown in Figure 3, swimming lane 1-7 is respectively the product that combines of LBF and m1, m2, m3, m45, m5, m6, m7 probe sequence, swimming lane CK is contrast, the product that combines of expression LBF and LTRE probe sequence shows that LBF and LTRE bonded DNA core sequence are " CGAC ".

In order further to prove conclusively LBF and LTRE cis element bonded specificity, the cis element that carries out the m1-m7 of single base mutation respectively is building up to pRS315His (Leu ⁺) on, transformed yeast (method is with embodiment 1,2).At His ^-Select to cultivate on the substratum, cultivated 3 days for 28 ℃, respectively counting, the result is under the on all four situation of transformation efficiency, m1 and m7 and wild-type LTRE (CK) growth colony number basically identical, the colony number of the last growth of m2 is about 30% of wild-type, and m3, m4, m5, m6 then do not have colony growth.Though show that the base mutation among the m2 is influential with combining of LTRE to LBF, LBF still can combine with LTRE, this experiment in vitro result with EMSA is consistent, proves that the bonded DNA of LBF institute core sequence is " CGAC ".

Embodiment 4, the LBF transcriptional activation test in yeast cell

The cDNA of LBF total length is cloned into Yeast expression carrier YepGAP (Trp ⁺) (Qiang Liu, Twotranscription factors, DREB1 and DREB2, with an EREBP/AP2DNA binding domainseparate two cellular signal transduction pathways in drought-and low-temperature-responsive gene expression, respectively, in Arabidopsis.PlantCell, 1998,10:1391-1406), obtain containing the recombinant yeast expression vector of LBF full-length gene, called after YepGAPLBF, then YepGAPLBF is transformed the recombination yeast yWAM2/pL4 that contains the LTRE element among the embodiment 2 respectively and contain among the recombination yeast yWAM2/pmL4 of mLTRE element, and at His ^-Select on the substratum, cultivated 3-5 days for 28 ℃, (A is that YepGAPLBF transforms the recombination yeast yWAM2/pmL4 that contains the mLTRE element to the result as shown in Figure 4, B is that YepGAPLBF transforms the recombination yeast yWAM2/pL4 that contains the LTRE element), show with the yeast that contains the LTRE element of YepGAPLBF conversion and can grow, and the yeast that contains the mLTRE element can not be grown, show that LBF can specific combination LTRE element in the yeast body, thereby activate the expression of reporter gene HIS3, so can select to grow on the substratum at His-, because LBF can not combine with mLTRE, can not activate reporter gene HIS3 expression of gene on the contrary, thereby at His ^-Select can not grow on the substratum, therefore, LBF not only has the binding specificity with LTRE in the yeast body, and has transcriptional activation function.

Embodiment 5, the LBF expression specificity analysis under the abiotic stress condition and in the rataria growth course

One, the expression specificity analysis of LBF under the abiotic stress condition

1, the processing of corn material

Get corn seed, imbibition 24hr plants in flowerpot, under 28 ℃, light application time 12hr/ days condition, cultivates about 20 days, treats that maize seedling grows three leaves, one core, carries out the processing of following different condition:

1) damage to plants caused by sudden drop in temperature processing: maize seedling is placed 2 ℃ of incubators, under 12hr/ days illumination conditions, cultivates 48hr, take out and the flush away root on soil, use liquid nitrogen flash freezer ,-80 ℃ of preservations are standby.

2) salt marsh is handled: respectively maize seedling is placed 0.6%, 0.8%, in 1% the NaCl solution, under 12hr/ days illumination conditions, cultivated 3 days, take out the soil on the flush away root also, use liquid nitrogen flash freezer ,-80 ℃ of preservations are standby.

3) arid is handled: respectively maize seedling being placed water content is 8% (mixed 920g dry ground and 80mL water), in 10%, 13% the soil, under 12hr/ days illumination conditions, cultivates 3 days, takes out the soil on the flush away root also, uses liquid nitrogen flash freezer, and-80 ℃ of preservations are standby.

4) ABA handles: respectively maize seedling is placed 10 ^-4M, 10 ^-5M, 10 ^-6(after getting 5mgABA usefulness 0.1N KOH dissolving, be settled in the 95mL water and be 10 in the ABA solution of M ^-4M), under 12hr/ days illumination conditions, cultivate 24hr, take out and the flush away root on soil, use liquid nitrogen flash freezer ,-80 ℃ of preservations are standby.

5) H ₂O ₂Handle: respectively maize seedling is placed 10mM H ₂O ₂(1.13mL 30%H ₂O ₂/ L), 60mM H ₂O ₂(6.78mL30%H ₂O ₂/ L), 150mM H ₂O ₂(14.95mL 30%H ₂O ₂In/L) the aqueous solution, under 12hr/ days illumination conditions, cultivate 24hr, take out and the flush away root on soil, use liquid nitrogen flash freezer ,-80 ℃ of preservations are standby.

6) water treatment (HCK): maize seedling is directly placed water, under 12hr/ days illumination conditions, cultivate 24hr ,-80 ℃ of preservations are standby.

7) Dui Zhao processing (CK): directly get without the maize seedling of any processing in contrast frozen in-80 ℃.

2, the removal of the extraction of RNA and DNA in the corn material

1) each the about 200mg of corn material that above-mentioned different methods handles that learns from else's experience, liquid nitrogen grinding is with the operating process extraction RNA of RNAgents TotalRNA Isolation System test kit (Promega company) and reference reagent box specification sheets.

2) RNA is dissolved in the 85 μ L water, adds 5 μ L RQ1 RNA Free DNase (1U/ μ L) (Promega company) and 10 * damping fluid, 10 μ L, 37 ℃ of insulation 5min, the pollution of removing DNA.

3) the phenol chloroform extracting of adding 100 μ L is once got supernatant, with isopyknic isopropanol precipitating RNA,, RNA is dissolved in the 50 μ L sterilized waters in that the RNA precipitation is washed once with 70% ethanol,

4) quantitatively the concentration of RNA is 1 μ g/ μ L.

3, RT-PCR legally constituted authority one template DNA concentration

1) RNA through the corn material of different treatment that extracts with step 2 is a template, and its cDNA is synthesized in reverse transcription, and reaction system and reaction conditions are: get Oligo dT ₁₈1 μ L (0.5 μ g/ μ L), RNA 5 μ L (1 μ g/ μ L), dNTP1 μ L (10mM) and water 27 μ L, 65 ℃ of 5min behind the mixing, 0 ℃ of 2min; Add 5 * First-Strand damping fluid, 10 μ L, DTT (100mM) 5 μ L and RNase Inhibitor 10U (40U/ μ L), 42 ℃ of 2-5min behind the mixing; Add SuperScipt II 1 μ L (200u/ μ L) (Gibcol BRL company), 42 ℃ of 50min behind the mixing, 70 ℃ of 15min deactivations, standby.

2) go up the maize actin gene of logining (Maize Actin1, Accession NO.J01238) according to GenBank and design following primer:

mAct1?F：5’-CAC?CTT?CTA?CAA?CGA?GCT?CCG-3’

mAct1?R：5’-TAA?TCA?AGG?GCA?ACG?TAG?GCA-3’

The cDNA through the corn material of different treatment that obtains with step 1) is a template, under primer mAct1 F and mAct1R guiding, carry out pcr amplification, the PCR reaction system is: template 1 μ L, 2 * PCR damping fluid, 10 μ L, 10mM dNTP1 μ L, 10 μ M mAct1 F, 1 μ L, 10 μ M mAct1 R, 1 μ L, Taq 1U, aqua sterilisa 6 μ L.The PCR reaction conditions is: 94 ℃ of 2min of elder generation; 94 ℃ of 30sec again, 55 ℃ of 30sec, 72 ℃ of 30sec, totally 30 circulations; At last, 72 ℃ of 5min.After reaction finishes, carry out agarose gel electrophoresis and detect, show that having amplified size is the band of 405bp,, then can amplify the band (containing the intron sequences that length is 107bp) of 512bp if be template with the genomic dna of vegetable material.According to the electrophoresis result of PCR product template DNA is diluted then, adjust the consumption of template DNA,, make the content basically identical of template cDNA in every microlitre solution until the amount basically identical of the DNA band that goes out with mAct1 F and mAct1 R primer amplification.

4, the pcr amplification of LBF gene

1) the LBF gene order design primer that obtains according to embodiment 1, primer sequence is as follows:

Primer 1:5 '-CTT GAG CAC GTG CCT GTG AA-3 '

Primer 2: 5 '-TTA ACT TTG CTA GTA GTA GCT CC-3 '

The cDNA through the corn material of different treatment with the unified concentration of step 3 is a template, under the guiding of primer 1 and primer 2, carry out pcr amplification, the PCR reaction system is: template 1 μ L, 2 * PCR damping fluid, 10 μ L, 10mMdNTP 1 μ L, 10uM mAct1 F 1 μ L, 10uM mAct1 R 1 μ L, Taq 1U, aqua sterilisa 6 μ L.The PCR reaction conditions is: 94 ℃ of 2min of elder generation; 94 ℃ of 30sec then, 55 ℃ of 30sec, 72 ℃ of 30sec, totally 30 circulations; Last 72 ℃ of 5min.Carry out 1% agarose gel electrophoresis after reaction finishes and detect, (swimming lane A-P represents respectively the result: A:CK1 (without any processing), B:2 ℃ of subzero treatment, C:8%H as shown in Figure 5 ₂The O arid is handled D:10%H ₂The O arid is handled E:13%H ₂The O arid is handled F:HCK (water treatment), G:10 ^-6M ABA, H:10 ^-5M ABA, I:10 ^-4M ABA, J:10mM H ₂O ₂, K:60mM H ₂O ₂, L:150mM H ₂O ₂, M:0.6%NaCl, N:0.8%NaCl, O:1%NaCl, P:CK is contrast), electrophoresis result shows that the LBF expression of gene is induced by adverse environmental factors such as low temperature, ABA and hydrogen peroxide.

Two, the expression specificity analysis of LBF in the maize immature embryos growth course

With the method identical with step 1, extract the RNA of the 15th, 17,21, the 23 and 25 day rataria in corn pollination back respectively, analyze the LBF gene at the expression of rataria between the growth period with the RT-PCR method, the result as shown in Figure 6, swimming lane 1-5 represents the 15th, 17,21,23 and 25 day LBF expression of gene situation respectively, and the expression amount that shows LBF is along with the growth of rataria obviously increases, and cell dewaters gradually in this and the seed development process, osmotic stress strengthens, and the adverse circumstance enhancing presents direct corresponding relation.

The Arabidopis thaliana of embodiment 6, LBF gene transforms and the Physiological Analysis of transfer-gen plant is tested

One, the detection of conversion of the Arabidopis thaliana of LBF gene and transfer-gen plant

1, the conversion of the structure of plant expression vector and Agrobacterium

The LBF gene clone in carrier pBI121, is obtained plant expression vector, called after pBI121-LBF; With pBI121-LBF CaCl ₂Method transformed into escherichia coli JM109, upgrading grain carry out enzyme and cut evaluation, choose the positive colony order-checking, show the recon that has obtained to be integrated with plant expression vector pBI121-LBF, and it is transformed among the Agrobacterium LBA4404.

2, Arabidopis thaliana transforms

1) cultivation of Arabidopis thaliana

The Arabidopis thaliana seed is carried out earlier 2-3 days vernalization treatment, every then basin sowing 7-10 grain seed (nutrition soil and vermiculite were by mixing in 2: 1) under 4 ℃; Place the greenhouse to cultivate (22 ℃, illumination 16h/ days), treat that Arabidopis thaliana is extracted just mossy out after, cut off mossy just, treat that it extracts more time mossy out, and minority can be used for following conversion when beginning to bear pods.

2) cultivation of Agrobacterium

Picking transforms the single colony inoculation of the reorganization Agrobacterium that pBI121-LBF is arranged in 3mL YEB (Kan 50mg/L, Rifampin 50mg/L) liquid nutrient medium, and 28 ℃ of 250rpm cultivated 30 hours; Go in fresh YEB (Kan 50mg/L, the Rifampin 50mg/L) liquid nutrient medium of 200mL by switching in 1: 400,28 ℃ of 250rpm cultivated about 14 hours, surveyed OD ₆₀₀≈ 1.5; Centrifugal 10min collects thalline under 4 ℃ of 7500rpm; Resuspended thalline is in penetrating fluid (1/2MS salt+5% sucrose, pH5.7,121 ℃ of sterilization 15min of diploid long-pending (400mL); With adding final concentration before is 0.044uM6-BA, VB6 1mg/L, and VB1 10mg/L, SILWET 0.02%).

3) arabidopsis thaliana transformation

The bud of Arabidopis thaliana is immersed step 2) in contain in the penetrating fluid of the Agrobacterium of recombinating, vacuumize (25 IN Hg kept 5 minutes); After conversion finishes, plant is put plastics bag, horizontal positioned can normally be cultivated its growth under low light intensity after 24-48 hour.

4) collecting seed screens

Collect the seed of transformed plant, claim the 25-30mg seed to put into the 1.5mL centrifuge tube, add 1mL 75% ethanol (containing 0.05%Tween 20), on shaking table, shake 10 minutes (300rpm), the centrifugal supernatant that goes; Add 1mL 95% ethanol and wash once the centrifugal supernatant that goes; Repeat once; In super clean bench, add the 0.3mL dehydrated alcohol, move on on the aseptic filter paper, dry up; The seed that dries up is spread on the 1/2MS flat board (Kan 50mg/L); 4 ℃, put 2 days after, be that 16h/ cultivates all over the world at 22 ℃, illumination condition; With positive plant (T ₀Generation) is transplanted in the basin and cultivates, and collect seed and carry out T ₁The generation screening.

3, PCR detects transgenic arabidopsis

The extraction of transgenic arabidopsis genomic dna may further comprise the steps:

1) gets 0.1-0.2g transgenic arabidopsis leaf, in liquid nitrogen, grind, be transferred in the 1.5mL centrifuge tube.

2) add 0.7mL CTAB (contain Tris 100mM, NaCl 1.4M, 20mM EDTA, CTAB 2%, mercaptoethanol 0.1%), 60 ℃ of water-baths 30 minutes every 10 minutes, are put upside down once.

3) add 0.7mL phenol chloroform (1: 1), put upside down several times, centrifugal 5 minutes of 10000rpm shifts the new centrifuge tube of supernatant to, adds isopyknic chloroform isoamyl alcohol (24: 1), mixing, centrifugal 5 minutes of 10000rpm.Shift the new centrifuge tube of supernatant to.

4) add isopyknic Virahol, put upside down mixing, centrifugal 10 minutes of 10000rpm removes supernatant, washes once with 70% ethanol, drains, and the DNA precipitation is dissolved in the 50 μ L sterilized waters, is used for PCR and detects.

According to the gene order design primer of 35S promoter sequence and LBF among the pBI121, primer sequence is as follows:

Primer 3:(forward primer) 5 '-TCTGCCGACAGTGGTCCCAA-5 '

Primer 4:(reverse primer) 5 '-TTAACTTTGCTAGTAGTAGCTCC-5 '

Genomic dna with transgenic arabidopsis is a template, under the guiding of primer 3 and primer 4, carries out the PCR reaction, and the PCR reaction system is (20 μ L): transfer-gen plant DNA 1 μ L (20ng-50ng), 10 * PCR damping fluid, 2 μ L, MgCl ₂(2.5mM) 2 μ L, Taq enzyme 0.2 μ L, dNTP (2.5mM) 2 μ L, primer 3 and primer 4 each 10 μ M add sterilized water to 20 μ L.The PCR reaction conditions is: earlier 94 ℃ 5 minutes; Again 94 ℃ 45 seconds, 60 ℃ 45 seconds, 72 ℃ 60 seconds, totally 35 circulations; Last 72 ℃ were extended 5 minutes.Reaction finishes to carry out 1% agarose gel electrophoresis and detects, and obtains transforming the positive plant (amplifying the purpose band of about 1000bp size) of LBF gene.

Two, the Physiological Analysis of LBF transgenic arabidopsis plant experiment

1, cold-resistant experiment

LBF transfer-gen plant and transfer-gen plant are not placed-6 ℃, kept 6 hours; Transfer to then and continue under the normal growth conditions to cultivate, the result shows: the survival rate of transfer-gen plant is 95%, and not genetically modified plant survival rate is 5%, and LBF can obviously improve the cold performance of plant, (A is a transfer-gen plant, and B is transfer-gen plant not) as shown in Figure 7.

2, anti-salt experiment

Place the NaCl of 600mM to soak 3 hours LBF transfer-gen plant and transfer-gen plant not; 22 ℃, illumination cultivation 24 hours; Transfer to and continue under the normal growth conditions of Arabidopis thaliana to cultivate, the result shows: the survival rate of transfer-gen plant is 80%, and not genetically modified plant survival rate is 15%, and LBF can obviously improve the anti-salt property of plant, (A is a transfer-gen plant, and B is transfer-gen plant not) as shown in Figure 8.

3, drought resisting experiment

LBF transfer-gen plant and transfer-gen plant not placed do not feed water under the normal growth conditions of Arabidopis thaliana cultured continuously 15-20 days, the result shows: the survival rate of transfer-gen plant is 80%, not genetically modified plant survival rate is 10%, LBF can obviously improve the drought resisting performance of plant, (A is a transfer-gen plant, and B is transfer-gen plant not) as shown in Figure 9.

Sequence table

<160>2

<210>1

<211>267

<212>PRT

＜213〉Zea corn (Zea mays L.)

<400>1

Met?Asp?Thr?Ala?Gly?Leu?Val?Gln?His?Ala?Thr?Ser?Ser?Ser?Ser?Thr

1 5 10 15

Ser?Thr?Ser?Ala?Ser?Ser?Ser?Ser?Ser?Glu?Gln?Gln?Ser?Arg?Lys?Ala

20 25 30

Ala?Trp?Pro?Pro?Ser?Thr?Ala?Ser?Ser?Pro?Gln?Gln?Pro?Pro?Lys?Lys

35 40 45

Arg?Pro?Ala?Gly?Arg?Thr?Lys?Phe?Arg?Glu?Thr?Arg?His?Pro?Val?Phe

50 55 60

Arg?Gly?Val?Arg?Arg?Arg?Gly?Ala?Ala?Gly?Arg?Trp?Val?Cys?Glu?Val

65 70 75 80

Arg?Val?Pro?Gly?Arg?Arg?Gly?Ala?Arg?Leu?Trp?Leu?Gly?Thr?Tyr?Leu

85 90 95

Ala?Ala?Glu?Ala?Ala?Ala?Arg?Ala?His?Asp?Ala?Ala?Ile?Leu?Ala?Leu

100 105 110

Gln?Gly?Arg?Gly?Ala?Gly?Arg?Leu?Asn?Phe?Pro?Asp?Ser?Ala?Arg?Leu

115 120 125

Leu?Ala?Val?Pro?Pro?Pro?Ser?Ala?Leu?Pro?Gly?Leu?Asp?Asp?Ala?Arg

130 135 140

Arg?Ala?Ala?Leu?Glu?Ala?Val?Ala?Glu?Phe?Gln?Arg?Arg?Ser?Gly?Ser

145 150 155 160

Gly?Ser?Gly?Ala?Ala?Asp?Glu?Ala?Thr?Ser?Gly?Ala?Ser?Pro?Pro?Ser

165 170 175

Ser?Ser?Pro?Ser?Leu?Pro?Asp?Val?Ser?Ala?Ala?Gly?Ser?Pro?Ala?Ala

180 185 190

Ala?Leu?Glu?His?Val?Pro?Val?Lys?Ala?Asp?Glu?Ala?Val?Ala?Leu?Asp

195 200 205

Leu?Asp?Gly?Asp?Val?Phe?Gly?Pro?Asp?Trp?Phe?Gly?Asp?Met?Gly?Leu

210 215 220

Glu?Leu?Asp?Ala?Tyr?Tyr?Ala?Ser?Leu?Ala?Glu?Gly?Leu?Leu?Val?Glu

225 230 235 240

Pro?Pro?Pro?Pro?Pro?Ala?Ala?Trp?Asp?His?Gly?Asp?Cys?Cys?Asp?Ser

245 250 255

Gly?Ala?Ala?Asp?Val?Ala?Leu?Trp?Ser?Tyr?Tyr

260 265

<210>2

<211>1042

<212>DNA

＜213〉Zea corn (Zea mays L.)

<400>2

caagctcgag?acaagaaacc?agaaccagct?cactcctcac?tccacttcca?ctcccaacag 60

caagctcaag?cagtcagtca?ccggcagggg?tcagggtcac?agtcacagca?gcagccatgg 120

acacggccgg?cctcgtccag?cacgcgacct?cctcgtcttc?cacctccacc?tcggcgtcgt 180

cgtcctcgtc?cgagcagcag?agccgcaagg?cggcgtggcc?gccgtcgacc?gcttcctcac 240

cacagcagcc?gcccaagaag?cgccccgcgg?ggcgcacaaa?gttccgggag?acgcggcacc 300

cggtgttccg?cggcgtgcgg?cggcggggcg?ccgcgggccg?gtgggtgtgc?gaggtgcgcg 360

tcccggggag?gcgcggcgcg?cggctgtggc?tcggcaccta?cctcgccgcc?gaggcggcgg 420

cgcgcgcgca?cgacgccgcg?atactcgccc?tgcagggccg?cggcgcgggg?cgcctcaact 480

tcccggactc?cgcgcggctg?ctcgccgtgc?cgcccccgtc?cgcgctcccg?ggcctggacg 540

acgcccgccg?cgcggcgctc?gaggccgtcg?cggagttcca?gcgccgctct?gggtccgggt 600

ccggggccgc?cgacgaagcg?acctcgggcg?cgtctcctcc?ctcctcgtcg?ccgtcgctgc 660

cggacgtttc?tgcggctggc?tcgccggcgg?cggcgcttga?gcacgtgcct?gtgaaggccg 720

acgaagcagt?ggcgttggac?ttggacggcg?acgtgttcgg?gcccgactgg?ttcggggaca 780

tgggcctgga?gttggatgcg?tactacgcca?gcctcgcgga?agggttgctc?gtggagccgc 840

cgccgccgcc?ggcggcctgg?gatcatggag?actgctgtga?ctccggagct?gcggacgtcg 900

cgctctggag?ctactactag?caaagttaac?aataataagc?ttgacagcca?accccaaaag 960

ccccccaact?gattgtattc?acctctgtaa?caaaattcaa?attgatttcc?cagcaaatga 1020

acttcaaaaa?aaaaaaaaaa?aa 1042

Claims (10)

1, anti-reverse transcription of corn regulatory factor is the protein with one of following amino acid residue sequences:

1) the SEQ ID № in the sequence table: 1;

2) with SEQ ID № in the sequence table: 1 amino acid residue sequence is through replacement, disappearance or the interpolation of one to ten amino-acid residue and have to interact with the LTRE cis element and improve the protein of plant stress-resistance performance.

2, anti-reverse transcription regulatory factor according to claim 1 is characterized in that: described anti-reverse transcription regulatory factor and LTRE bonded DNA core sequence are CGAC.

3, the gene of the described anti-reverse transcription of corn regulatory factor of coding claim 1.

4, gene according to claim 3, it is characterized in that: it has one of following nucleotide sequence:

1) SEQ ID № in the sequence table: 2 dna sequence dna;

2) SEQ ID № in the code sequence tabulation: the polynucleotide of 1 protein sequence;

3) under the rigorous condition of height can with SEQ ID № in the sequence table: the nucleotide sequence of the 2 dna sequence dnas hybridization that limit.

5, gene according to claim 4 is characterized in that: it has SEQ ID № in the sequence table: 2 nucleotide sequence.

6, the plant expression vector that contains claim 3 or 4 or 5 described anti-reverse transcription of corn regulatory factor genes.

7, the transgenic cell line and the host bacterium that contain claim 3 or 5 or 5 described anti-reverse transcription of corn regulatory factor genes.

8, arbitrary segmental primer in amplification claim 3 or the 4 or 5 described genes.

9, a kind of method of cultivating resistance raising plant is to make up the plant expression vector that contains claim 3 or 4 or 5 described genes, again the plant expression vector that makes up is transformed the purpose plant.

10, method according to claim 9 is characterized in that: described purpose plant is corn, paddy rice, wheat, soybean, rape or cotton.

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