CN104892737A

CN104892737A - Plant stress tolerance related protein GmNF-YA15, coding gene and applications thereof

Info

Publication number: CN104892737A
Application number: CN201410078461.0A
Authority: CN
Inventors: 徐兆师; 马有志; 冯志娟; 郑炜君; 陈明; 李连城
Original assignee: Institute of Crop Sciences of Chinese Academy of Agricultural Sciences
Current assignee: Institute of Crop Sciences of Chinese Academy of Agricultural Sciences
Priority date: 2014-03-05
Filing date: 2014-03-05
Publication date: 2015-09-09
Anticipated expiration: 2034-03-05
Also published as: CN104892737B

Abstract

The present invention discloses a plant stress tolerance related protein GmNF-YA15, a coding gene and applications thereof. The protein is the following (a) or (b): (a) a protein comprises an amino acid sequence represented by the sequence 1 in the sequence list; and (b) the protein is formed by carrying out substitution and/or deletion and/or addition of one or a plurality of amino acid residues on the amino acid sequence represented by the sequence 1, is related to the plant stress tolerance, and is derived from the sequence 1. According to the present invention, the GmNF-YA15 is cloned from soybean and is subjected to induced expression under mannitol, salt, drought, hydrogen peroxide and ABA, the encoded protein is localized on the nucleus, and the transcription expression of the gene containing the CCAAT-box cis element can be specifically regulated; and with the GmNF-YA15 of the present invention, the drought tolerance of plants can be improved, the foundation can be provided for the artificial control of the expression of the stress resistance and stress tolerance related gene, and the important effect is provided in the cultivation of the stress resistance and stress tolerance enhanced plant breeding.

Description

Plant stress tolerance correlative protein GmNF-YA15 and encoding gene thereof and application

Technical field

The present invention relates to biological technical field, particularly relate to a kind of plant stress tolerance correlative protein GmNF-YA15 and encoding gene thereof and application.

Background technology

The environment stresses such as arid, high salt and low temperature seriously govern growth, the growth of soybean.Therefore, understand soybean to the response of adverse environmental factor and signal transduction mechanism, improve the resistance of soybean varieties, become one of vital task of soybean heredity research and breed improvement.

A series of responsing reaction can be produced in plant materials, along with many Physiology and biochemistries and change developmentally under environment stress.Specify the reaction mechanism of plant to adverse circumstance, science argument will be provided for adversity gene engineering research and application.At present, plant stress-resistance Journal of Sex Research is deep into cell, molecular level gradually, and combines with genetics and genetic engineering research, and exploration biotechnology improves plant growth characteristics, its objective is and improves plant to the adaptive faculty of adverse circumstance.

Under the adverse environmental factor of the environment-stress such as arid, high salt and low temperature, plant can make corresponding adjustment in molecule, cell and integral level, to reduce the injury existence that environment causes to the full extent.Many genes are expressed by stress-inducing, the product of these genes can not only participate in the stress response of plant directly, and the expression of other genes involved can be regulated or participate in signal transduction path, thus plant is avoided or reduces injury, strengthen the resistance to stressful environmental.To coerce relevant gene product and can be divided into two large classes: the product of first kind genes encoding comprises ionophorous protein, aquaporin, osmotic factor (sucrose, proline(Pro) and trimethyl-glycine etc.) synthetic enzyme etc. participate in the gene product that plant stress is replied directly; The product of Equations of The Second Kind genes encoding comprises participation and coerces relevant signal transmission and the protein factor of Gene expression and regulation, as protein kinase, transcription factor etc.Wherein, play an important role in the gene expression regulation that transcription factor is replied at plant stress.

Transcription factor also referred to as trans-acting factor, be can with the DBP of cis-acting elements generation specific effect in eukaryotic gene promoter region, by between them and and other associated protein between interaction, activate or suppress transcribe.The DNA land of transcription factor determines the specificity that it is combined with cis-acting elements, and transcription regulatory region determines it and plays activation or restraining effect to genetic expression.In addition, himself activity is also subject to the impact of the effect such as nuclear location and oligomerization.

At present known in plant to coerce relevant transcription factor and mainly contain: AP2 (APETALA2)/EREBP (the element responsive to ethylene associated proteins with AP2 structural domain, ethylene responsive element binding protein) transcription factor family, bZIP(basic region/leucine zipper motif transcription factors containing basic region and leucine zipper) class transcription factor, WRKY transcription factor family containing conservative WRKY aminoacid sequence, CBF(CCAAT binding factor in conjunction with the main nuclear factor of CCAAT-box) class transcription factor, MYC family containing basic helix-loop-helix (bHLH) and leucine zipper and there is the MYB family of tryptophane bunch (Trp cluster).These transcription factor families, except the water Stress responses of WRKY family not involved in plant, other four families all participate in the environment stress reaction of regulating plant to arid, high salt and low temperature etc.NF-Y is the transcription factor of a class in conjunction with cis-acting elements CCAAT-box, special identification in conjunction with the cis-acting elements CCAAT-box in the promotor of many eukaryote composing types, inducibility and cell cycle dependant gene or enhanser, and then in the expression of these genes of transcriptional level control.The heterozygosis tripolymer that NF-Y is made up of NF-YA, NF-YB and NF-YC tri-different subunits.The histone fold motif (HFM) of NF-YB and NF-YC interacts and makes it to form dimer, then forms heterotrimeric NF-Y mixture in conjunction with NF-YA, thus in conjunction with CCAAT-box, regulates transcribing of its target gene.NF-YA has two structural domains at least for the combination of protein: structural domain (Q-rich domain) and the interactional structural domain of subunit (subunit interaction domain) of being rich in glutamine.NF-YB also has two protein bound structural domains: histone fold motif (histone-fold motif) and TATA associated proteins (TATA-binding protein) binding domains (TBP-binding domain).NF-YC has three binding domains of proteins: histone fold motif, TBP binding domains and be rich in the structural domain of glutamine.The structural amino acid sequence of NF-YA and NF-YC and histone fold motif homology, NF-YB and H2B histone fold motif is correlated with, and NF-YC is correlated with in H2A histone fold motif, this motif is made up of three α spirals and two rings, is responsible for the dimeric formation of H2A/H2B.

At present, the report about the function of NF-Y transcription factor in plant is less, and all play an important role (Nelson et al, 2007) in drought stress.Nelson etc. think, with the ZmNF-YB2 of Arabidopis thaliana AtNF-YB1 transcription factor homolog, under the condition of lack of water, the transgenic corns of process LAN ZmNF-YB2 obviously can strengthen drought resistance, because ZmNF-YB2 can be that multiple arid relevant parameters changes with plant, comprise chlorophyll content, gas port degree of leading, leaf temperature, minimizing wilt and maintain photosynthesis, thus improving drought resistance (Nelson, Peter P.Repetti, Tom R.Adams, Jingrui Wu, 2007).The research of Wen-Xue Li, Youko Oono etc. confirms, the expression of AtNF-YA5 is subject to the induction of arid and ABA process.Its promotor GUS is analyzed and shows that part induced reaction occurs in transcriptional level.NF-YA5 has a target site miR169, and under drought condition, miR169 expresses and is suppressed.NF-YA5 has very high expression at microtubule tissue and guard cell, therefore, utilizes a transcription factors critical to promote the expression of multiple functional gene, thus strengthens the resistance of plant, has become the engineered study hotspot of plant stress-resistance.

Summary of the invention

An object of the present invention is to provide a kind of plant stress tolerance correlative protein GmNF-YA15 and encoding gene thereof.

Protein provided by the invention, be the nuclear factor albumen in conjunction with CCAAT-box, name is called GmNF-YA15, derives from Glycine soybean (Glycine max L.), is following (a) or (b):

A protein that () is made up of the aminoacid sequence shown in sequence in sequence table 1;

(b) by the aminoacid sequence of sequence 1 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and the protein that by sequence 1 derived relevant to plant stress tolerance.

The protein that in sequence table, the amino acid residue sequence of sequence 1 is made up of 217 amino-acid residues, from aminoterminal 123-148 amino acids residue sequence for do region mutually with NFYB/C, being possible nuclear localization signal from aminoterminal 162-188 amino acids residue sequence, is conservative NF-YA structural domain from aminoterminal 123-183 amino acids residue sequence.

In order to make the GmNF-YA15 a) be convenient to purifying, the N-terminal of the protein that the aminoacid sequence shown in sequence 1 forms or C-terminal label as shown in table 1 can be connected in by sequence table.

The sequence of table 1. label

Label	Residue	Sequence
			Poly-Arg	5-6(is generally 5)	RRRRR
Poly-His	2-10(is generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

Above-mentioned 1) GmNF-YA15 in can synthetic, also can first synthesize its encoding gene, then carries out biological expression and obtain.The encoding gene of the GmNF-YA15 in above-mentioned 1 is by the codon lacking one or several amino-acid residue in the DNA sequence dna shown in 5 ' end 150-803 bit base by sequence in sequence table 2, and/or carry out the missense mutation of one or several base pair, and/or the encoding sequence connecting the label shown in table 1 is held to obtain at its 5 ' end and/or 3 '.

The DNA molecular of above-mentioned albumen of encoding also is the scope of protection of the invention.

Above-mentioned DNA molecular is following 1)-5) in any one DNA molecular:

1) coding region for shown in sequence in sequence table 2 DNA molecular;

2) coding region is for sequence 2 is from the DNA molecular shown in 5 ' end the 147 to 803 Nucleotide;

3) coding region is for sequence 2 is from the DNA molecular shown in 5 ' end the 150 to 803 Nucleotide;

4) under strict conditions with 1) or 2) or 3) DNA sequence dna that limits hybridizes and the DNA molecular of encode resistance of reverse and/or oxidation-resistance associated protein;

5) with 1) or 2) or 3) DNA sequence dna that limits has more than 90% homology, and the DNA molecular of coding resistance of reverse and/or oxidation-resistance associated protein.

Above-mentioned stringent condition can be in the solution of 6 × SSC, 0.5%SDS, hybridizes, then use 2 × SSC under 65oC, and 0.1%SDS and 1 × SSC, 0.1%SDS respectively wash film once.

Recombinant vectors containing above-mentioned DNA molecular, expression cassette, transgenic cell line or recombinant bacterium are also the scope of protection of the invention.

Above-mentioned recombinant vectors is that above-mentioned DNA molecular is inserted expression vector, obtains the carrier of expressing above-mentioned protein.

Available existing plant expression vector construction contains the recombinant expression vector of described gene.Described plant expression vector comprises double base agrobacterium vector and can be used for the carrier etc. of plant micropellet bombardment.Described plant expression vector also can comprise 3 ' end untranslated region of foreign gene, namely comprises the DNA fragmentation of polyadenylation signals and any other participation mRNA processing or genetic expression.The bootable polyadenylic acid of described polyadenylation signals joins 3 ' end of mRNA precursor, as Agrobacterium crown-gall nodule induction (Ti) plasmid gene (as kermes synthetic enzyme Nos gene), plant gene (as soybean storage protein genes) 3 ' hold the non-translational region of transcribing all to have similar functions.When using described gene constructed recombinant plant expression vector, any one enhancement type promotor or constitutive promoter can be added before its transcription initiation Nucleotide, as the ubiquitin promoter (Ubiquitin) of cauliflower mosaic virus (CAMV) 35S promoter, corn, they can be used alone or are combined with other plant promoter; In addition, when using gene constructed plant expression vector of the present invention, also enhanser can be used, comprise translational enhancer or transcriptional enhancer, these enhanser regions can be ATG initiator codon or neighboring region initiator codon etc., but must be identical with the reading frame of encoding sequence, to ensure the correct translation of whole sequence.The source of described translation control signal and initiator codon is widely, can be natural, also can be synthesis.Translation initiation region can from transcription initiation region or structure gene.For the ease of identifying transgenic plant cells or plant and screening, can process plant expression vector used, the coding can expressed in plant as added can produce enzyme or the gene (gus gene, luciferase genes etc.) of luminophor, the antibiotic marker thing (gentamicin marker, kantlex marker etc.) with resistance or the chemical resistance reagent marker gene (as anti-weedkiller gene) etc. of colour-change.From the security consideration of transgenic plant, any selected marker can not be added, directly with adverse circumstance screening transformed plant.

In an embodiment of the present invention, expression vector is YEP-GAP, corresponding recombinant vectors is YEP-GAP-GmNF-YA15, YEP-GAP-GmNF-YA15 is the recombinant plasmid multiple clone site that above-mentioned DNA molecular inserts YEP-GAP obtained, BamHI and the XhoI enzyme be preferably the sequence 2 of sequence table holds the DNA fragmentation shown in the 150 to 803 Nucleotide to insert YEP-GAP from 5' cuts the recombinant vectors obtained between recognition site;

Expression vector is pBI121, corresponding recombinant vectors is pBI121-GmNF-YA15, pBI121-GmNF-YA15 is the recombinant plasmid multiple clone site that above-mentioned DNA molecular inserts pBI121 obtained, and is preferably and holds the Sma I of the insertion of the DNA fragmentation shown in the 150 to 803 Nucleotide pBI121 and SacI enzyme to cut the recombinant vectors obtained between recognition site from 5' the sequence 2 of sequence table.

The primer pair of above-mentioned DNA molecular or its any fragment of increasing.

Described primer pair is GmNF-YA15 – BI and GmNF-YA15 – XI or GmNF-YA15-121F and GmNF-YA15-121R:

GmNF-YA15-BI：5'-CGCGGATCCATGGTTTGGACAGTGT-3'

GmNF-YA15-XI：5'-CCGCTCGAGTTAGGATGCCCTATCT-3'

GmNF-YA15-121F：5'-TCCCCCGGGATGGTTTGGACAGTGTTACGTG-3'；

GmNF-YA15-121R：5'-GCCGAGCTCTTAGGATGCCCTATCTGATGAAG-3'；

The application in regulating plant resistance of reverse or regulating plant oxidation-resistance of above-mentioned protein, above-mentioned DNA molecular or above-mentioned recombinant vectors, expression cassette, transgenic cell line or recombinant bacterium is also the scope of protection of the invention.

In above-mentioned application, described regulating plant resistance of reverse is for improving plant stress tolerance; Described regulating plant oxidation-resistance is for improving plant anti-oxidation;

In above-mentioned application, described resistance of reverse is salt tolerance and/or drought tolerance;

Described plant is dicotyledons or monocotyledons; Described dicotyledons is specially Arabidopis thaliana.

Another object of the present invention is to provide a kind of method of cultivating transgenic plant.

Method provided by the invention, is imported in object plant by above-mentioned DNA molecular, obtains transgenic plant; The resistance of reverse of described transgenic plant and/or oxidation-resistance are higher than described object plant.

In aforesaid method, above-mentioned DNA molecular imports described object plant by above-mentioned recombinant vectors;

Described resistance of reverse is drought tolerance and/or salt tolerance;

Described salt tolerance is embodied in NaCl and coerces down, and the main root length of described transgenic plant is greater than described object plant;

Described drought tolerance is embodied in PEG and coerces down, and the main root length of described transgenic plant is greater than described object plant;

Described oxidation-resistance is embodied in H ₂o ₂coerce down, the main root length of described transgenic plant is greater than described object plant.

Described object plant is dicotyledons or monocotyledons; Described dicotyledons is specially Arabidopis thaliana.

Above-mentioned albumen is also the scope of protection of the invention as the application of transcription factor.

Experiment of the present invention proves, the present invention clones and obtains gene GmNF-YA15 from soybean, it expresses under the induction of N.F,USP MANNITOL, salt, drought, hydrogen peroxide and ABA, the protein localization of coding is in tenuigenin, special regulation and control can contain the transcriptional expression of the gene of CCAAT-box cis element (core sequence: CCAAT), and GmNF-YA15 of the present invention can improve the drought tolerance of plant, for manual control is degeneration-resistant and the expression of the gene of resistance to retrocorrelation provides the foundation, play an important role in the plant breeding of cultivating resistance and resistance of reverse enhancing.

Accompanying drawing explanation

Fig. 1 is the sequence analysis result of GmNF-YA15 and Arabidopis thaliana amino acid AtNF-YA5 aminoacid sequence

Fig. 2 is fluorescence real-time quantitative (Real-time) the PCR collection of illustrative plates that GmNF-YA15 is expressed by stress-inducing

Fig. 3 is the positioning result of GmNF-YA15 in onion epidermis cell

Fig. 4 is the goal gene of Molecular Detection in transgenic arabidopsis

Fig. 5 be wildtype Arabidopsis thaliana and turn GmNFYA-15 Arabidopis thaliana salt resistance, drought resistance compares with oxidation-resistance.

Embodiment

The experimental technique used in following embodiment if no special instructions, is ordinary method.

Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.

% in following embodiment, if no special instructions, is mass percentage.Quantitative test in following examples, all arranges and repeats experiment for three times, results averaged.

The acquisition of embodiment 1, gene GmNF-YA15

One, the acquisition of gene GmNF-YA15

Soybean iron rich No. 8 (national germplasm resource bank, the numbering ZM242 of about 20 days will be grown in sandy soil; Be documented in as in Publication about Document: Wang Caijie, Sun Shi, Wu Baomei, Chang Ru town, the pedigree analysis of Chinese establishing in large scale soybean varieties since Han Tian rich .20 forties in century.China's oil crops journal.2013,35 (3): 246-252, the public can obtain from Institute of Crop Science, Chinese Academy of Agricultural Science) four leaf phase seedling Osmotic treatment 2 hours, with liquid nitrogen flash freezer ,-80 DEG C save backup.Adopt Quikprep Micro mRNA Purification Kit(Pharmacia) carry out the separation of mRNA.ThermoScript II XL(AMV is used in first chain cDNA synthesis).Adopt SMART method synthesis ds cDNA, PCR primer carries out 1.0% agarose gel electrophoresis detection.

The sequence 2 in the nuclear factor C race full length gene sequence nucleotide sequence table of soybean CCAAT-box is obtained by the method for 5 ' RACE and 3 ' RACE.

In this sequence table, the unnamed gene shown in sequence 2 is GmNF-YA15, its open reading frame is 5 ' end the 150th to 803 Nucleotide of the sequence 2 from sequence table, the protein designations of this genes encoding is GmNF-YA15, the aminoacid sequence of this albumen is the sequence 1 in sequence table, sequence 1 is made up of 218 amino-acid residues, has conservative histone fold motif.

Above-mentioned sequence 2 also can pass through synthetic.

The sequence of GmNF-YA15 is compared on Genabnk, has higher homology (Fig. 1), and in soybean, do not find homologous protein with the albumin A tNF-YC11 in Arabidopis thaliana, proves that GmNF-YA15 albumen is a new albumen.

Two, real-time fluorescence quantitative PCR analyzes the expression characterization of GmNF-YA15

1, Stress treatment

Seedling age is rich No. 8 seedling of the iron of 10 days, carries out following process:

(1) Osmotic treatment (Fig. 2 A): potted plant soybean seedling is taken out the moisture blotted on root, be placed on dry filter paper, arid is cultivated after 30 minutes, 1 hour, 2 hours, 5 hours, 12 hours, 24 hours and is taken out material, and with liquid nitrogen flash freezer ,-80 DEG C save backup.

(2) high Ficus caricaL (Fig. 2 B): soybean seedling is placed in 200mM by NaCl solution, illumination cultivation takes out material after 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours respectively, and with liquid nitrogen flash freezer ,-80 DEG C save backup.

(3) pyroprocessing (Fig. 2 C): at soybean seedling being placed in 42 DEG C, illumination cultivation is taken out respectively after 30 minutes, 1 hour, 2 hours, 5 hours, 12 hours, 24 hours and used liquid nitrogen flash freezer, and-80 DEG C save backup.

(4) dormin process (Fig. 2 D): dormin (ABA) solution soybean seedling being placed in 100 μMs, illumination cultivation is taken out respectively after 30 minutes, 1 hour, 2 hours, 5 hours, 12 hours, 24 hours and used liquid nitrogen flash freezer, and-80 DEG C save backup.

(5) subzero treatment (Fig. 2 E): soybean seedling is placed in 4 DEG C of incubators, illumination cultivation is taken out after 30 minutes, 1 hour, 2 hours, 5 hours, 12 hours, 24 hours and used liquid nitrogen flash freezer, and-80 DEG C save backup.

(6) hydrogen peroxide process (Fig. 2 F): hydrogen peroxide solution wheat seedling being placed in 20mM, illumination cultivation takes out material after 30 minutes, 1 hour, 2 hours, 5 hours, 12 hours, 24 hours respectively, with after liquid nitrogen flash freezer-80 DEG C save backup.

(7) injury process (Fig. 2 G): soybean seedling scissors is deducted leaf top, and illumination cultivation is taken out respectively after 30 minutes, 1 hour, 2 hours, 5 hours, 12 hours, 24 hours and used liquid nitrogen flash freezer, and-80 DEG C save backup.

(8) treatment with mannitol (Fig. 2 H): N.F,USP MANNITOL (Mannitol) solution soybean seedling being placed in 300mM, illumination cultivation is taken out respectively after 30 minutes, 1 hour, 2 hours, 5 hours, 12 hours, 24 hours and is used liquid nitrogen flash freezer, and-80 DEG C save backup.

(9) process contrasted: directly get without the soybean seedling-80 DEG C of any process frozen in contrast (0 hour).

2, the separation of mRNA

The soybean seedling of above-mentioned each process is adopted Quikprep Micro mRNA Purification Kit(Pharmacia) carry out the separation of mRNA.

3, reverse transcription is cDNA

Be cDNA by the mRNA reverse transcription of purifying.

4, real-time fluorescence quantitative PCR

CDNA is diluted the template that 50 times are used as Q-RT-PCR afterwards.With gene specific primer to (Q-RT-GmNF-YA15F:5'-GTCAATGTACAGTATGCAGCACC-3'; Q-RT-GmNF-YA15R:5'-TGATTTGTCAGCTGATGCCA-3') Q-RT-PCR amplification is carried out to sample, analyzing gene to the response situation of various process, with actin(Q-RT-ActinF:5'-ACATTGTTCTTAGTGGTGGCT-3'; Q-RT-Q-RT-ActinR:5'-CTGTTGGAAGGTGCTGAG-3') internal reference is done.Q-RT-PCR exists 7000 real-time fluorescence quantitative PCR instrument carry out, and 3 repetitions are established in a parallel test.Utilize the method that Livak KJ and Schmittgen TD (2001) reports, namely 2 ^{-Δ Δ CT}calculate relative expression quantity.

ΔΔC _T=（C _T.Target－C _T.Actin） _{Time x}－（C _T.Target－C _T.Actin） _Time0

Time x represents random time point, Time ₀represent that the target gene of 1 times amount after actin corrects is expressed.

The results are shown in Figure 2, under Osmotic treatment, the expression amount of GmNF-YA15 uprises gradually along with the prolongation of dewatering time, 2.3 ± 0.33 times of contrast are reached to processed 2h expression amount, 8.03 ± 0.78 times of contrast are reached to processed 12h expression amount, start again to lower to processed 24h expression amount, see Fig. 2 A.Under high Ficus caricaL, the expression amount of GmNF-YA15 is when processing 2h, and expression amount transient expression is to 8.2 ± 0.33 times of contrast, and expression amount instantaneous downward again afterwards, is shown in Fig. 2 B.

Under pyroprocessing, the expression amount of GmNF-YA15 slowly uprises along with the prolongation of high-temperature time, and reach 2.2 ± 0.30 times of contrast to process 12h expression amount, expression amount starts again slowly to lower afterwards, sees Fig. 2 C.

Under hormone ABA process, the expression amount of GmNF-YA15, without significant variation tendency, is shown in Fig. 2 D.

Under subzero treatment, the expression amount of GmNF-YA15 continues slowly to lower, and sees Fig. 2 E.

Under hydrogen peroxide process, the expression amount of GmNF-YA15 is at 0.5h transient expression to 7.2 ± 0.37 times that contrast, and expression amount instantaneous downward again afterwards, reach 10.4 ± 0.31 times of contrast at 2h expression amount, expression amount is lowered again a little afterwards, sees Fig. 2 F.

Under injury process, the expression amount of GmNF-YA15 is when processing 1h, and expression amount transient expression is to 0.19 ± 0.13 times of contrast, and expression amount slowly raises again afterwards, during to 12h, returns to initial expression level, sees Fig. 2 G.

Under treatment with mannitol, the expression amount of GmNF-YA15 is when processing 5h, and expression amount transient expression is to 4.2 ± 0.23 times of contrast, and when processing 24h, expression amount transient expression is to 6.4 ± 0.36 times of contrast, and expression amount continues slowly to raise trend, sees Fig. 2 H.Two, GmNF-YA15 Subcellular Localization

1, material prepares:

At 9cm culture dish upper berth skim MS substratum, after tearing, internal surface is upward, is laid in MS substratum center, diameter within the scope of 3cm, 25 DEG C of preculture 4h.

2, the process of bronze:

Getting 100mg diameter is that 1.5ml centrifuge tube put into by the bronze of 1.0 μMs, and add 1ml dehydrated alcohol, fully vibrate 3min, with the centrifugal 1min of 12000rpm, removes supernatant, then adds after 1ml sterilized water fully mixes, centrifugal with 12000rpm, repeats above-mentioned steps 3 times.Finally, be suspended in by bronze in 1ml ultrapure water ,-20 DEG C save backup.

3, the structure of Subcellular Localization recombinant vectors

1. according to primers GmNF-YA15-BI and GmNF-YA15-XI of GmNF-YA15 gene, prime end introduces PstI and BamHI restriction enzyme site respectively.

GFP-GmNF-YA15F：5'-AAACTGCAGATGGTTTGGACAGTGT-3'

GFP-GmNF-YA15R：5'-CGCGGATCCGGATGCCCTATCTGAT-3'。

2. cut the pcr amplification product of step 1 recovery with restriction enzyme PstI and BamHI enzyme, reclaim the digestion products of 669bp; Cut expression vector 16318GFP with restriction enzyme PstI and BamHI enzyme, reclaim the carrier framework of about 4kbp;

3. step digestion products is 2. connected with the carrier framework of step;

4. by step connection product freeze-thaw method transform Escherichia coli strain (purchased from Tiangen company) 3., 37 DEG C of incubated overnight, picking positive colony extracts plasmid and checks order;

Sequencing result shows, plasmid is for holding the recombinant vectors obtained between PstI and the BamHI restriction enzyme site of the DNA fragmentation insertion vector 16318GFP shown in the Nucleotide of 150-800 position, called after 16318GFP-GmNF-YA15 from 5' by the sequence 2 of sequence table.

4, particulate bullet is prepared:

3 μ g recombinant plasmid dnas (16318GFP-GmNF-YA15 and 16318GFP empty carrier) individually add the bronze suspension 6 μ l(50mg/ml that diameter is 1.0 μMs), 0.1M spermidine (spermidine) 4 μ l, 2.5M CaCl ₂6 μ l, mixing of bronze, DNA, spermidine and calcium chloride first being vibrated respectively, then after mixing vibration mixing 3min, leaves standstill 15min on ice.The centrifugal 10s(of 12000rpm refers to that rotating speed reaches 10s after 12000rpm), abandon supernatant.Add 140 μ l dehydrated alcohols, the centrifugal 10s of (breaing up bronze) 12000rpm after thick vibration, collect bronze precipitation.20 μ l dehydrated alcohols suspend and precipitate, some film.

5, biolistic bombardment acceptor material:

1. select the split film (this experiment 1100psi) of certain pressure, together with bombardment film, in the alcohol of 70%, soak 1 ~ 2h, taking-up is dried;

2. metal baffle is with alcohol-pickled, sterilizing on spirit lamp, the Bechtop ultraviolet sterilization of particle gun;

3. get the above-mentioned bronze-plasmid complex prepared of 20 μ l, be spread evenly across on the mid-way of bombardment film, be not applied on whole film, size is consistent with the pore diameter range on carrier fixed ring, dries, and is then fixed on carrier fixed ring;

4. above-mentioned carrier fixed ring is installed on launching device;

5. can split film and be installed to gas acceleration tube lower end;

6. onion epidermis culture dish is put into vacuum chamber, take off culture dish lid;

7. pointer is vacuumized to 26In/Hg;

8. put helium in gas acceleration tube, until pressure reaches when can split pressure that film can bear in pipe, film can be split and break;

9. gas is flushed on bombardment film, and carrier moves downward, and is blocked by metal baffle, and bronze-plasmid complex is below through the mesh directive target cell of metal baffle;

10. the onion epidermis cell bombarded is put into 25 DEG C of incubators, observe under laser confocal microscope after light culture 16 ~ 24h.

6, onion epidermis cell microscopy:

By the onion epidermis compressing tablet after biolistic bombardment, light culture 16-24h, then at laser scanning co-focusing microscope (Bio-Rad MicroRadiance) (Laser scanning confocal microscopy, LSMC) observe GFP (green fluorescent protein) fluorescence, and carry out scanning and take pictures.The working parameter of LSCM is: Ex=488nm, Em=525 ± 15nm, Power=10%, Zoom7, medium sweep, Frame512 × 512.Software is TIME-COURSE and PHOTOSHOP5.0.

As shown in Figure 3, A:GmNF-YA15 is positioned in nucleus result; B:16318hGFP empty vector control is positioned in cytolemma and core.

Embodiment 2, GmNF-YA15 are as the application in transcription factor

Prove that with yeast-one-hybrid system the cardinal principle of the activation characteristic of transcription factor is as follows: basic promotor Pmin(minimal promoter CCAAT cis-acting elements and mutant CCAAT cis-acting elements being building up to respectively pHISi-1 carrier and pLacZi carrier) upstream, Pmin promotor downstream connects reporter gene (His3, LacZ and URA3).When being connected with the expression vector YEP-GAP(of goal gene of encoding transcription factors not containing mobilizing function) be transformed into the yeast cell being connected with CCAAT cis-acting elements and mutant CCAAT cis-acting elements respectively after, if the reporter gene be connected with in the yeast cell of mutant CCAAT cis-acting elements can not be expressed, and the reporter gene be connected with in the yeast cell of specific CCAAT cis-acting elements can be expressed, illustrate that this transcription factor can be combined with CCAAT cis-acting elements, and there is mobilizing function, have activated Pmin promotor, reporter gene is impelled to express.Thus demonstrate Binding in vivo specificity and the mobilizing function of object transcription factor.

Expression vector YEP-GAP: Institute of Crop Science, Chinese Academy of Agricultural Science ensures to provide to the public; Reference Liu Q; Kasuga M; Sakuma Y; Abe H; Miura S; Yamaguchi-Shinozaki K; Shinozaki K.Two transcription factors; DREB1and DREB2; with an EREBP/AP2DNA binding domain separate two cellular signal transduction pathways in drought-and low-temperature-responsive gene expression; respectively, in Arabidopsis, Plant Cell1998Aug; 10 (8): 1391-1406.

YPD liquid nutrient medium: microbial culture yeast extract (Bacto-Yeast Extract) 10g/L, microbial culture tryptone (Bacto-Peptone) 20g/L, regulate pH to 5.8,121 DEG C/15min sterilizing, be down to the Glucose that 60 DEG C add 40% later, make its final concentration be 20g/L.

SD/His ^-/ Ura ^-/ Trp ^-selective medium: not containing amino acid whose yeast nitrogen (Yeast nitrogen base) 6.7g/L, auxotroph mixture (drop-out media without His/Ura/Trp) 100ml, agar powder (Bacteriological agar) 20g/L, regulate pH to 5.8,121 DEG C/15min sterilizing, add 40%Glucose after being down to 60 DEG C, make its final concentration be 20g/L.

Auxotroph mixture (Drop-out mix): (10 ×): L-Isoleucine(Isoleucine) 300mg/L, L-Valine(α-amino-isovaleric acid) 1500mg/L, L-Adenine(VITAMIN B4) 200mg/L, L-Arginine(arginine) 200mg/L, L-Histidine Hcl monohydrate(Histidine) 200mg/L, L-Leucine(leucine) 1000mg/L, L-Lysine Hcl(Methionin) 300mg/L, L-Methionine(methionine(Met)) 200mg/L, L-Phenylalanine(phenylalanine) 500mg/L, L-Threonine(Threonine) 2000mg/L, L-Tyrosine(tyrosine) 300mg/L.

1×PEG/LiAc：50％PEG33508ml，10×TE buffer1ml，10×LiAc1ml。

10 × TE Buffer:100mM Tris-Hcl, 10mM EDTA, pH=7.5,121 DEG C of autoclavings, room temperature preservation.

1×TE/LiAc：10×TE buffer1ml，10×LiAc1ml，ddH ₂O8ml。

Z Buffer:Na ₂hPO ₄7H ₂o16.1g/L, NaH ₂pO ₄h ₂o5.5g/L, KCl0.75g/L, MgSO ₄7H ₂o0.246g/L, regulates pH to 7.0,121 DEG C/15min sterilizing, 4 DEG C of preservations.

X-gal storage liquid (X-gal Stock Solution): with N, N-dimethyl-formamide(DMF) dissolve X-gal, make its final concentration be 20mg/ml ,-20 DEG C of storages.

Z buffer damping fluid 100ml(Z buffer with X-gal containing X-gal), matching while using: Z buffer98ml, beta-mercaptoethanol (β-mercaptoethanol) 0.27ml, X-gal storage liquid (X-gal stocksolution) 1.67ml.

10×LiAc：100Mm Tris-Hcl,100mM EDTA,pH=7.5。121 DEG C of autoclavings, room temperature preservation.

One, the structure of recombinant vectors

1, the acquisition of GmNF-YA15 gene

According to primers GmNF-YA15-BI and GmNF-YA15-XI of GmNF-YA15 gene, prime end introduces BamHI and XhoI restriction enzyme site respectively.

GmNF-YA15-BI：5'-CGCGGATCCATGGTTTGGACAGTGT-3'

GmNF-YA15-XI：5'-CCGCTCGAGTTAGGATGCCCTATCT-3'

With the cDNA of the whole plant of rich No. 8 of soybean varieties iron for template, carry out pcr amplification with primer GmNF-YA15-BI and GmNF-YA15 – XI.

Obtain the pcr amplification product of about 672bp.

Reclaim pcr amplification product.

2, the structure of recombinant vectors

1. cut the pcr amplification product of step 1 recovery with restriction enzyme BamHI and XhoI enzyme, reclaim the digestion products of 672bp;

2. expression vector YEP-GAP is cut, the carrier framework of recovery with restriction enzyme BamHI and XhoI enzyme;

3. step digestion products is 1. connected with step carrier framework 2.;

4. by the electroporated JM109 bacterial strain of step connection product 3. (purchased from Clontech company), 37 DEG C of incubated overnight, picking positive colony extracts plasmid and checks order;

Sequencing result shows, plasmid is for holding the recombinant vectors obtained between BamHI and the XhoI restriction enzyme site of the DNA fragmentation insertion vector YEP-GAP shown in the Nucleotide of 150-803 position, called after YEP-GAP-GmNF-YA15 from 5' by the sequence 2 of sequence table.

Two, the Binding in vivo specificity of GmNF-YA15 and the checking of activation characteristic

1, the structure of yeast reporter

(1) structure of normal dual yeast reporter

DNA fragmentation A (containing 4 CCAAT elements):

5’-GAATTC-CCAAT-CCAAT-CCAAT-CCAAT-GTCGAC-3'。

DNA fragmentation A is building up to the Pmin of pHis-1 carrier (MATCHMAKER One-Hybrid System, Clontech company) _hIS3promotor upstream, obtains recombinant vectors pHis-1-CCAAT, with Xho I and Nco I restriction endonuclease, pHis-1-CCAAT carrier is cut into wire.

DNA fragmentation A is building up to pLacZi carrier (MATCHMAKER One-Hybrid System, Clontech company) P _cYCIpromotor upstream, obtains recombinant vectors pLacZi-CCAAT, respectively pLacZi-CCAAT carrier is cut into wire with Xho I and Nco I restriction endonuclease.

First by wire pHis-1-CCAAT vector in yeast cell (YM4271 strain, MATCHMAKER One-Hybrid System, Clontech company), acquisition can at SD/His ^-the yeast transformant (Yeast transformant) of normal growth on substratum.Then with this yeast transformant for host cell, continue to transform the wire pLacZi-CCAAT carrier repeating CCAAT elements containing 4.Lack the SD/His of Histidine and uridylic so at the same time ^-/ Ura ^-on substratum, select to obtain normal dual yeast reporter containing pHis-1-CCAAT and pLacZi-CCAAT.

(2) structure of dual yeast reporter of mutant

DNA fragmentation B (the mutant TTTTA containing 4 CCAAT elements): 5 '-GAATTC-TTTTA-TTTTA-TTTTA-TTTTA-GTCGAC-3'.

Replace DNA fragmentation A with DNA fragmentation B, the same step of method (1), obtain dual yeast reporter of mutant.

2, PEG/LiAc method transformed yeast and interpretation of result

(1) normal dual yeast reporter containing pHis-1-CCAAT and pLacZi-CCAAT and the dual yeast reporter of mutant of inoculating above-mentioned 1 acquisition are respectively sub in 1ml YPD liquid nutrient medium, concuss 2 minutes, after dispersion agglomerate, suspension is gone in the triangular flask containing 50ml YPD liquid nutrient medium, 30 DEG C/250rpm shakes and spends the night, and surveys OD600=1.7-1.8(counting about 4 × 10 ⁷individual/mL);

(2) get 30ml step (1) overnight culture to receive in the fresh YPD substratum of 300ml, 30 DEG C/250rpm cultivates, about 3 hours (to OD600=0.5 ± 0.1), the centrifugal 5min of room temperature 1000g, collects thalline, abandons supernatant, suspend with 1/2 volume 1 × TE, 1000g/5min is centrifugal;

(3) inhale and abandon supernatant, with the freshly prepared 1 × TE/LiAc solution suspension of 1.5ml, vibration mixing is for subsequent use;

(4) take out 0.1ml competent yeast to transform, add following solutions successively: 0.1 μ g is by recombinant vectors YEP-GAP-GmNF-YA15,0.1mg ssDNA(salmon sperm dna of a preparation, SiTaa), 0.6mlPEG/LiAc at a high speed vibration 1 minute, 30 DEG C/200rpm shaking culture 30 minutes;

(5) add 70ul DMSO(siTaa#D8779), be inverted mixing gently, 42 DEG C of heat shocks 30 minutes, vibrate therebetween gently, ice bath 2 minutes, the centrifugal 5min of room temperature 1000g;

(6) inhale and abandon supernatant, add 0.5ml1 × TE buffer suspension cell;

(7) dip suspension with transfering loop, respectively containing 0, the SD/His of 15mmol/L3-AT ^-/ Ura ^-/ Trp ^-on selective medium, setting-out is cultivated.

(8) dull and stereotyped half cultivates normal dual yeast reporter, and second half cultivates dual yeast reporter of mutant, to do check analysis.

(9) be placed upside down in incubator, 30oC cultivates 3-4 days.

(10) found that the SD/His at 0mmol/L3-AT ^-/ Ura ^-/ Trp ^-culture medium flat plate on yeast reporter of normal yeast reporter and sudden change have growth, but the diameter of yeast reporter of sudden change is obviously little; And at the SD/His of 15mmol/L3-AT ^-/ Ura ^-/ Trp ^-culture medium flat plate on normal yeast reporter can normal growth, but yeast reporter of sudden change is by supression not growth.

3, galactosidase activity detects

(1) from the SD/His of 0mmol/L3-AT ^-/ Ura ^-/ Trp ^-culture medium flat plate on the yeast reporter daughter colony of respectively normal yeast reporter of picking and sudden change.Go in YPD liquid nutrient medium, in 30 DEG C of shaking culture, in the logarithmic growth later stage to be grown to, get 1.5ml bacterium liquid, the centrifugal 30s of 3000rpm;

(2) abandon supernatant, liquid in control main, is placed in liquid nitrogen quick-frozen 10min by centrifuge tube, taking-up makes it naturally melt, and adds 50ul Z/X-gal solution, 30 DEG C of incubations, found that normal yeast reporter becomes blue in 6-8h, and the not change in 12h of yeast reporter of sudden change, be still white.

Illustrate that transcription factor GmNF-YA15 can be combined with CCAAT cis-acting elements, and there is mobilizing function, have activated Pmin promotor, impel reporter gene to express.Thus demonstrating Binding in vivo specificity and the mobilizing function of GmNF-YA15, GmNF-YA15 is transcription factor.

Embodiment 3, the GmNF-YA15 application in the resistance of reverse improving plant

One, the acquisition of GmNF-YA15 Arabidopis thaliana is turned

1, the structure of recombinant vectors

1) clone of GmNF-YA15 gene

According to the primers of GmNF-YA15 gene to (GmNF-YA15-121F and GmNF-YA15-121R), prime end introduces Sma I respectively and SacI enzyme cuts recognition site,

GmNF-YA15-121F：5'-TCCCCCGGGATGGTTTGGACAGTGTTACGTG-3'；

GmNF-YA15-121R：5'-GCCGAGCTCTTAGGATGCCCTATCTGATGAAG-3'；

For template, carry out pcr amplification with GmNF-YA15-121F and GmNF-YA15-121R with Glycine soybean (Glycine max L.) (rich No. 8 of iron) cDNA, obtain the PCR primer (GmNF-YA15 gene) that size is about 1Kb.

Reclaim above-mentioned PCR primer.

2), the structure of recombinant vectors

1. cut the PCR primer of step 1 recovery with restriction endonuclease sma I and SacI enzyme, reclaim 672bp digestion products;

2. buy with restriction enzyme sma I and SacI Mei Qie pBI121(Clontech company), reclaim 672bp carrier framework;

3. step digestion products is 1. connected with step carrier framework 2.;

4. by the electroporated TOP10 bacterial strain of step connection product 3. (purchased from Beijing Tian Gen company), 37 DEG C of incubated overnight, picking positive colony extracts plasmid and checks order.

Sequencing result shows, plasmid is for holding the recombinant vectors obtained between the Sma I of the DNA fragmentation insertion vector pBI121 shown in the Nucleotide of 150-803 position and SacI restriction enzyme site, called after pBI121-GmNF-YA15 from 5' by the sequence 2 of sequence table.

3, the acquisition of recombinational agrobacterium

Buy with recombinant plasmid pBI121-GmNF-YA15 gene transformation Agrobacterium C58C1(Beijing Baeyer enlightening biotech company), obtain recombinational agrobacterium C58C1/pBI121-GmNF-YA15(extraction plasmid and send to order-checking, for pBI121-GmNF-YA15, then the recombinant bacterium containing this plasmid is positive).

4, turn GmNF-YA15 Arabidopis thaliana to obtain

1) recombinational agrobacterium obtained 3 is inoculated in LB (containing 50mg/ml Rifampin, 100mg/ml kantlex, 50mg/ml gentamicin) liquid nutrient medium, 28 DEG C, 3000rpm cultivate about 30 hours, obtain bacterium liquid;

2) bacterium liquid is gone in LB (containing 50mg/ml Rifampin, 100mg/ml kantlex, 50mg/ml gentamicin), 28 DEG C, 300rpm cultivates about 14 hours (bacterium liquid OD600 reaches 1.5-3.0);

3) collecting thalline, 4 DEG C, the centrifugal 10min of 4000g, being about 0.8-1.0 with being diluted to OD600 containing 10% sucrose MS liquid nutrient medium (containing 0.02%silwet);

4) by Arabidopis thaliana (Columbia ecotype Col-0, SALK company buys, also referred to as wildtype Arabidopsis thaliana) whole strain tips upside down in the container of the bacterium liquid filling step 4 together with flowerpot, makes flower soak about 50s, after immersion, take out flowerpot, be sidelong in pallet, cover black plastic cloth, plastic cloth is opened after 24hr, upright placing flowerpot, carries out normal illumination cultivation, results T ₁for seed, kantlex screening (concentration is 50 μ g/L kantlex) positive plant is T ₁for regeneration plant, go down to posterity, until obtain T ₃for regeneration plant.

T ₂t is shown in representative ₁the seed produced for selfing and the plant grown up to by it, T ₃t is shown in representative ₂the seed produced for selfing and the plant grown up to by it.

Extract T ₃for the DNA of the whole plant of regeneration plant as template, with primer pair: F:5'-ATGGTTTGGACAGTGTTACGTG; R:5'-TTAGGATGCCCTATCTGATGAAG-3'; Carry out pcr amplification, as shown in Figure 4 A, P is Plasmid, M be Marker, C be Columbia ecotype Arabidopis thaliana Col-0, L1-L3 is T to part sample results ₃for regeneration plant, H is H ₂o; Obtain 654bp for positive.

Extract T ₃for the RNA of the whole plant of regeneration plant, reverse transcription obtains cDNA as template, with primer pair: F:5'-ATGGTTTGGACAGTGTTACGTG; R:5'-TTAGGATGCCCTATCTGATGAAG-3'; Carry out pcr amplification.

Part sample the results are shown in Figure 4B, P is Plasmid, M be Marker, C be Columbia ecotype Arabidopis thaliana Col-0, L1-L3 is T ₃for regeneration plant, H is H ₂o; Obtain size be 915bp fragment for positive.

In above-mentioned DNA and cDNA level, qualification is and positive is T ₃in generation, turns GmNF-YA15 Arabidopis thaliana.

Adopting uses the same method proceeds in wildtype Arabidopsis thaliana by plasmid pBI121, obtains turning empty carrier Arabidopis thaliana, cultivates and obtains T ₃in generation, turns empty carrier Arabidopis thaliana, adopts the qualification that uses the same method, does not obtain 915bp fragment.

Two, the resistance of reverse qualification of transgenic plant

1, Salt-Tolerance Identification

Respectively by T ₃in generation, turns GmNF-YA15 Arabidopis thaliana strain GmNF-YA15-1, GmNF-YA15-2, GmNF-YA15-3, T ₃in generation, turns empty carrier Arabidopis thaliana and wildtype Arabidopsis thaliana (each 60 strains) carries out Salt-Tolerance Identification.Arrange and repeat experiment for three times, results averaged.

For evaluated expression GmNF-YA15 is because of the effect of plant seed Seedling Salt-tolerance.

By T ₃in generation, turns GmNF-YA15 Arabidopis thaliana strain GmNF-YA15-1, GmNF-YA15-2, GmNF-YA15-3, T ₃in generation, turns the empty carrier Arabidopis thaliana seedling (Fig. 5 A) consistent with picking growth conditions after wildtype Arabidopsis thaliana (WT) seed germination 4d, transfers to vertical on the 1/2MS substratum containing 150mM NaCl cultivation 14 days, observes phenotype, takes pictures and add up main root long.

Fig. 5 B is shown in by photo.

The main root of wildtype Arabidopsis thaliana Col-0 is long is 3.62cm;

The main root of GmNF-YA15-1 is long is 5.80cm;

The main root of GmNF-YA15-2 is long is 5.82cm;

The main root of GmNF-YA15-3 is long is 5.85cm.

T ₃in generation, turns empty carrier Arabidopis thaliana and wildtype Arabidopsis thaliana result without significant difference.

Illustrate that gene GmNF-YA15 has salt tolerance.

2, oxidation-resistance qualification

For evaluated expression GmNF-YA15 is because of plant seed oxidation-resistance in seedling stage ground effect.By T ₃in generation, turns GmNF-YA15 Arabidopis thaliana strain GmNF-YA15-1, GmNF-YA15-2, GmNF-YA15-3, T ₃in generation, turns empty carrier Arabidopis thaliana and wildtype Arabidopsis thaliana (WT) seed (each 60 strains) carries out oxidation-resistance qualification.Arrange and repeat experiment for three times, results averaged.

T ₃in generation, turns GmNF-YA15 Arabidopis thaliana strain GmNF-YA15-1, GmNF-YA15-2, GmNF-YA15-3, T ₃in generation, turns the empty carrier Arabidopis thaliana seedling consistent with picking growth conditions after wildtype Arabidopsis thaliana seed germination 4d, transfers to containing 7mM H ₂o ₂1/2MS substratum on vertically cultivate 14d.Observe phenotype, take pictures and add up main root long.

Fig. 5 C is shown in by photo.

The main root of wildtype Arabidopsis thaliana Col-0 is long is 2.91cm;

The main root of GmNF-YA15-1 is long is 5.42cm;

The main root of GmNF-YA15-2 is long is 5.36cm;

The main root of GmNF-YA15-3 is long is 5.38cm.

Illustrate that gene GmNF-YA15 has oxidation-resistance.

3, drought tolerance qualification

For evaluated expression GmNF-YA15 is because of plant seed oxidation-resistance in seedling stage ground effect.To unanimously be processed Arabidopis thaliana adjoining tree and the transfer-gen plants of 3d by 4 DEG C of growths, the seedling that after sprouting 4d, picking growth conditions is consistent, transfers on the 1/2MS substratum containing 4%PEG and vertically cultivates 14d, observes phenotype, takes pictures and add up main root long.Arrange and repeat experiment for three times, results averaged.

Fig. 5 D is shown in by photo.

The main root of wildtype Arabidopsis thaliana Col-0 is long is 2.30cm;

The main root of GmNF-YA15-1 is long is 4.22cm;

The main root of GmNF-YA15-2 is long is 4.22cm;

The main root of GmNF-YA15-3 is long is 4.22cm.

Illustrate that gene GmNF-YA15 has drought tolerance.

Claims

1. a protein is following (a) or (b):

(b) by the aminoacid sequence of sequence 1 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and the protein that by sequence 1 derived relevant to plant stress tolerance and/or oxidation-resistance.

2. the DNA molecular of albumen described in coding claim 1.

3. DNA molecular according to claim 2, is characterized in that: described DNA molecular is following 1)-4) in any one DNA molecular:

1) coding region for shown in sequence in sequence table 2 DNA molecular;

2) coding region is for sequence 2 is from the DNA molecular shown in 5 ' end the 150 to 803 Nucleotide;

3) under strict conditions with 1) or 2) DNA sequence dna that limits hybridizes and the DNA molecular of encode resistance of reverse and/or oxidation-resistance associated protein;

4) with 1) or 2) DNA sequence dna that limits has more than 90% homology, and the DNA molecular of coding resistance of reverse and/or oxidation-resistance associated protein.

4. the recombinant vectors containing DNA molecular described in Claims 2 or 3, expression cassette, transgenic cell line or recombinant bacterium;

Described recombinant vectors is specially and DNA molecular described in Claims 2 or 3 is inserted expression vector, obtains the carrier of expressing protein described in claim 1.

5. the primer pair of DNA molecular or its any fragment described in Claims 2 or 3 of increasing.

6. the DNA molecular described in protein according to claim 1, Claims 2 or 3 or the application in regulating plant resistance of reverse or regulating plant oxidation-resistance of recombinant vectors according to claim 4, expression cassette, transgenic cell line or recombinant bacterium.

7. application according to claim 6, is characterized in that: described resistance of reverse is salt tolerance and/or drought tolerance;

8. cultivate a method for transgenic plant, be that DNA molecular described in Claims 2 or 3 is imported in object plant, obtain transgenic plant; The resistance of reverse of described transgenic plant and/or oxidation-resistance are higher than described object plant.

9. method according to claim 8, is characterized in that: DNA molecular described in Claims 2 or 3 imports described object plant by recombinant vectors described in claim 4;

Described resistance of reverse is drought tolerance and/or salt tolerance;

10. albumen described in claim 1 is as the application of transcription factor.