CN103509094B

CN103509094B - Plant stress tolerance correlative protein GmNF-YC9 and encoding gene thereof and application

Info

Publication number: CN103509094B
Application number: CN201210214142.9A
Authority: CN
Inventors: 马有志; 徐兆师; 郑炜君; 李连城; 陈明
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: 2012-06-25
Filing date: 2012-06-25
Publication date: 2015-09-02
Anticipated expiration: 2032-06-25
Also published as: CN103509094A

Abstract

The invention discloses a kind of plant stress tolerance correlative protein GmNF-YC9 and encoding gene thereof and application.Experiment proves, by the T3 that in sequence 2, shown in the 34th to the 897th nucleotide sequence, the recombinant expression vector pBI121-GmNF-YC9 arabidopsis thaliana transformation of DNA molecular obtains _generationhomozygous transgenic plant, the survival rate in drought tolerance experiment is 90.2%, and WT lines and the survival rate turning empty carrier plant are respectively 27.2% and 28.4%; Germination rate in the experiment of salt tolerant germination rate is 90.4%, and WT lines is respectively 65.8% and 66.2% with the germination rate turning empty carrier plant.GmNF-YC9 albumen provided by the present invention and encoding gene thereof are significant in raising stress resistance of plant.

Description

Plant stress tolerance correlative protein GmNF-YC9 and encoding gene thereof and application

Technical field

The present invention relates to the relevant albumen of a kind of resistance of reverse in biological technical field and encoding gene thereof and application, particularly a kind of plant stress tolerance correlative protein GmNF-YC9 and encoding gene thereof and application, this protein G mNF-YC9 derives from soybean, has the ability improving drought resistance in plants and salt tolerance.

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 bindingprotein) transcription factor family, bZIP(basic region/leucinezipper 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).

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.NF-YB albumen and NF-YC albumen guard territory by HFM each other, adopt connected head-to-tail mode to form heterodimer and make platform mutually, attract NF-YA protein binding to this dimer platform thus form the activated heterotrimer nuclear factor of tool.NF-Y is attached to the CCAAT box of target gene promoters part by the DNA binding domain on NF-YA subunit, performs transcriptional activation or Transcription inhibition function.The conservative territory of three subunits of NF-Y has different protein structure domains respectively, and wherein NF-YA guards territory and has DNA binding domains (DNAbinding domain) and make structural domain (subunit interaction domain) mutually with NF-YB/C heterodimer.NF-YB and NF-YC albumen is guarded territory and is then made up of histone fold motif (Histone-fold motif).Wherein NF-YB and H2B histone fold motif is similar, and NF-YC and H2A histone fold motif is similar, and histone motif is made up of three α spirals and two rings, is responsible for the dimeric formation of H2A/H2B.

Stress tolerance due to plant is the complex character regulated and controled by polygene, relies on importing individual feature protein gene to be difficult to the comprehensive raising realizing stress resistance of plant.Therefore, utilize a key transcription factor to promote the expression of multiple functional gene, strengthen the resistance of plant, become the engineered study hotspot of plant stress-resistance.

Summary of the invention

The object of this invention is to provide a kind of plant stress tolerance correlative protein GmNF-YC9 and encoding gene thereof and application.

Provided by the present invention and plant stress tolerance correlative protein, derives from soybean (Glycine max L.), be a kind of nuclear factor protein name in conjunction with CCAAT-box is GmNF-YC9, and this protein is following protein a) or b):

A) protein be made up of the aminoacid sequence shown in sequence 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 by (a) the derivative protein relevant to following at least one plant stress tolerance: drought tolerance and salt tolerance.

Aminoacid sequence shown in sequence 1 is made up of 287 amino-acid residues, and the aminoacid sequence of the 8th to the 71st is conservative histone fold motif.

In order to make the albumen in above-mentioned (a) be convenient to purifying, label as shown in table 1 can be connected at the N-terminal of the protein be made up of the aminoacid sequence shown in sequence 1 or C-terminal.

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

Albumen in above-mentioned (b) can synthetic, also can first synthesize its encoding gene, then carries out biological expression and obtain.The encoding gene of the albumen in above-mentioned (b) is by the codon by lacking one or several amino-acid residue in the DNA sequence dna shown in 34-897 position in sequence 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 gene of code for said proteins also belongs to protection scope of the present invention.

The encoding gene of described protein is following 1) or 2) or 3) or 4) gene:

1) its nucleotide sequence is the DNA molecular shown in the 34th to the 897th nucleotide sequence in sequence 2;

2) its nucleotide sequence is the DNA molecular shown in sequence 2;

3) with 1) DNA sequence dna that limits at least has 70%, at least have 75%, at least have 80%, at least have 85%, at least have 90%, at least have 95%, at least have 96%, at least have 97%, at least have 98% or at least have the DNA molecular of 99% homology and code for said proteins;

4) under strict conditions with 1) or 2) or 3) DNA sequence dna that limits hybridizes and the DNA molecular of code for said proteins.

Sequence 2 is made up of 938 deoxyribonucleotides, is the full length cDNA sequence of soybean GmNF-YC9 albumen, and wherein the 34th is open reading frame to the 897th.

Described stringent condition can be as follows: 50 DEG C, at 7% sodium lauryl sulphate (SDS), 0.5M Na ₃pO ₄hybridize with in the mixing solutions of 1mM EDTA, at 50 DEG C, rinsing in 2 × SSC, 0.1%SDS; Also can be: 50 DEG C, at 7%SDS, 0.5M Na ₃pO ₄hybridize with in the mixing solutions of 1mM EDTA, at 50 DEG C, rinsing in 1 × SSC, 0.1%SDS; Also can be: 50 DEG C, at 7%SDS, 0.5M Na ₃pO ₄hybridize with in the mixing solutions of 1mM EDTA, at 50 DEG C, rinsing in 0.5 × SSC, 0.1%SDS; Also can be: 50 DEG C, at 7%SDS, 0.5M Na ₃pO ₄hybridize with in the mixing solutions of 1mM EDTA, at 50 DEG C, rinsing in 0.1 × SSC, 0.1%SDS; Also can be: 50 DEG C, at 7%SDS, 0.5M Na ₃pO ₄hybridize with in the mixing solutions of 1mM EDTA, at 65 DEG C, rinsing in 0.1 × SSC, 0.1%SDS; Also can be: in the solution of 6 × SSC, 0.5%SDS, hybridize at 65 DEG C, then use 2 × SSC, 0.1%SDS and 1 × SSC, 0.1%SDS respectively wash film once.

Recombinant vectors containing described gene, expression cassette, transgenic cell line, recombinant bacterium or recombinant virus also belong to protection scope of the present invention.

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.As pROKII, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb(CAMBIA company) etc.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, can add any one enhancement type promotor (ubiquitin promoter (Ubiquitin) as cauliflower mosaic virus (CAMV) 35S promoter, corn), constitutive promoter or organizing specific expression promotor (promotor as seed specific expression) before its transcription initiation Nucleotide, 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 the enzyme of colour-change or the gene (gus gene of luminophor, luciferase genes etc.), antibiotic marker gene is (as given the nptII gene to kantlex and associated antibiotic resistance, give the bar gene to herbicide phosphinothricin resistance, give the hph gene to microbiotic hygromycin resistance, with the dhfr gene given methatrexate resistance, give EPSPS gene to glyphosate) or chemical resistance reagent marker gene etc. (as anti-weedkiller gene), the mannose-6-phosphate isomerase gene of metabolism seminose ability is provided.

Described recombinant vectors containing described gene specifically can be YEP-GAP-GmNF-YC9 or pBI121-GmNF-YC9;

Described YEP-GAP-GmNF-YC9 can be the recombinant expression vector obtaining described gene insertion vector YEP-GAP to express described albumen; Specifically can be the recombinant expression vector will obtained between BamHI and the XhoI restriction enzyme site of the DNA molecular insertion vector YEP-GAP shown in the 34th to the 894th nucleotide sequence in sequence 2;

Described pBI121-GmNF-YC9 can be the recombinant expression vector obtaining described gene insertion vector pBI121 to express described albumen; Specifically can be the recombinant expression vector obtained between the Sma I of the DNA molecular insertion vector pBI121 shown in the 34th to the 897th nucleotide sequence in sequence 2 and SacI restriction enzyme site.

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

The method of cultivation transgenic plant of the present invention is by described channel genes object plant, obtains the transgenic plant of resistance of reverse higher than described object plant.

In the above-mentioned methods, described object plant can be monocotyledons or dicotyledons.

In the above-mentioned methods, described dicotyledons specifically can be Arabidopis thaliana.

In the above-mentioned methods, described resistance of reverse is drought tolerance and/or salt tolerance.

The present invention protects described albumen as the application in transcription factor.

Experiment proves, by the T that in sequence 2, shown in the 34th to the 897th nucleotide sequence, the recombinant expression vector pBI121-GmNF-YC9 arabidopsis thaliana transformation of DNA molecular obtains ₃for homozygous transgenic plant, with the wild-type under the same terms with turn empty carrier plant and compare, in drought tolerance experiment, (seedling normal growth being sprouted 15 days is not watered, until when WT lines is withered, rehydration one week again), WT lines is respectively 27.2% and 28.4% with the survival rate turning empty carrier plant, and the survival rate of transfer-gen plant is 90.2%; (after seed is cultivated 10 days on the MS substratum containing 100mM NaCl, germination rate is added up in the experiment of salt tolerant germination rate, cotyledon is opened completely and is that green seedling counts sprouting), WT lines is respectively 65.8% and 66.2% with the germination rate turning empty carrier plant, and the germination rate of transfer-gen plant is 90.4%.

GmNF-YC9 albumen provided by the present invention and encoding gene thereof are significant in raising stress resistance of plant, for the expression of manual control anti contravariance related gene provides the foundation, will play a significant role in cultivation high resistance to cold and diseases is as strong drought tolerance and strong Salt tolerant plants kind.

Accompanying drawing explanation

Fig. 1 is the expression map of GmNF-YC9 gene under the process of real-time fluorescence quantitative PCR analysis Different stress.Wherein, A-F respectively is the Stress treatment of dormin, arid, low temperature, high temperature, salt marsh and ethene, and X-coordinate is the time of coercing, and ordinate zou is the relative expression quantity of GmNF-YC9 gene.

Fig. 2 is that yeast-one-hybrid system proves transcription factor Binding in vivo specificity and activates the principle schematic of characteristic.

Fig. 3 is the structural representation of recombinant vectors pBI121-GmNF-YC9.

Fig. 4 is that the PCR of transgenic Arabidopsis plants cDNA level identifies electrophorogram.Wherein, swimming lane M is molecular weight standard, is followed successively by 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp from top to bottom, and swimming lane C is Columbia ecotype Arabidopis thaliana Col-0, swimming lane 1-8 is plant to be identified, and what have expection band is transfer-gen plant.

Fig. 5 wild-type and transgenic arabidopsis drought tolerance compare.Wherein, scheming A is wild-type (WT) and transgenic Arabidopsis plants (TL) before Osmotic treatment; Figure B is Osmotic treatment 14 days, the wild-type of rehydration after 3 days (WT) and transfer-gen plant (TL).

Fig. 6 wild-type and transgenic arabidopsis salt tolerance compare.Wherein, scheming A is wild-type (WT) and transgenic arabidopsis (TL) seed germination and growth situation of 10 days on MS substratum; Figure B is wild-type (WT) and transgenic Arabidopsis plants (TL) germination and growth situation of 10 days on the MS substratum containing 100mM NaCl.

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 embodiment, all arranges and repeats experiment for three times, results averaged.

Rich No. 8 of soybean iron (Glycine max L.): Institute of Crop Science, Chinese Academy of Agricultural Science ensures to provide to the public; Reference: Sun Xiao, Dong Jianhui, Chen Ming, Xu Zhaoshi, leaf is made the country prosperous, Li Liancheng, Qu Yanying, the clone of horse strong-willed .2008. soybean adversity gene GmDREB3 promotor and control region piecewise analysis. Acta Agronomica Sinica, 34 (8): 1475-1479

Carrier YEP-GAP: Chinese Academy of Agricultural Sciences's crop science research ensures to provide to the public; Reference: Liu Q; Kasuga M; Sakuma Y; Abe H; Miura S; Yamaguchi-Shinozaki K; Shinozaki K.Twotranscription factors; DREB1 and DREB2; with an EREBP/AP2DNA binding domainseparate two cellular signal transduction pathways in drought-andlow-temperature-responsive gene expression; respectively, in Arabidopsis, Plant Cell1998Aug; 10 (8): 1391-1406.

The clone of embodiment 1, GmNF-YC9 gene

One, the separation of mRNA

By rich No. 8 of the hydroponics growing soybean iron of 10 days (Glycine max L.) four leaf phase seedling Osmotic treatment 2 hours, with liquid nitrogen flash freezer ,-80 DEG C saved backup.Adopt Quikprep Micro mRNA PurificationKit(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.

Two, the acquisition of GmNF-YC9 full length gene sequence

The nuclear factor C race full length gene cDNA sequence of soybean CCAAT-box is obtained by the method for 5 ' RACE and 3 ' RACE, as shown in sequence 2, name is called GmNF-YC9 gene, its open reading frame is 5 ' end the 34th to the 897th Nucleotide from sequence 2, the aminoacid sequence of the Protein G mNF-YC9 of its translation is as shown in sequence 1, be made up of 287 amino-acid residues, the 8th of this albumen is conservative histone fold motif to the 71st amino acids sequence, the aminoacid sequence of this albumen is compared on Genabnk, with the albumin A tNF-YC11(At3g12480 in Arabidopis thaliana) there is higher homology, and in soybean, do not find homologous protein, proof GmNF-YC9 albumen is a new albumen.

Embodiment 2, real-time fluorescence quantitative PCR analyze the expression characterization of GmNF-YC9 gene

One, Stress treatment

Be rich No. 8 seedling of soybean iron of 10 days by potted plant seedling age, carry out following process:

(1) dormin process (Figure 1A): dormin (ABA) solution being placed in 100 μMs, illumination cultivation is taken out respectively after 0.5 hour, 1 hour, 2 hours, 5 hours, 12 hours, 24 hours and used liquid nitrogen flash freezer, and-80 DEG C save backup.

(2) Osmotic treatment (Figure 1B): take out the moisture blotted on root, is placed on dry filter paper, and arid is cultivated after 0.5 hour, 1 hour, 2 hours, 5 hours, 12 hours, 24 hours and taken out material, and with liquid nitrogen flash freezer ,-80 DEG C save backup.

(3) subzero treatment (Fig. 1 C): be placed in 4 DEG C of incubators, illumination cultivation is taken out after 0.5 hour, 1 hour, 2 hours, 5 hours, 12 hours, 24 hours and used liquid nitrogen flash freezer, and-80 DEG C save backup.

(4) pyroprocessing (Fig. 1 D): at being placed in 42 DEG C, illumination cultivation is taken out respectively after 0.5 hour, 1 hour, 2 hours, 5 hours, 12 hours, 24 hours and used liquid nitrogen flash freezer, and-80 DEG C save backup.

(5) salt marsh process (Fig. 1 E): the NaCl solution being placed in 200mM, illumination cultivation takes out material after 0.5 hour, 1 hour, 2 hours, 5 hours, 12 hours, 24 hours respectively, and with liquid nitrogen flash freezer ,-80 DEG C save backup.

(6) ethene (EH) process (Fig. 1 F): the EH solution being placed in 50 μMs, illumination cultivation is taken out respectively after 0.5 hour, 1 hour, 2 hours, 5 hours, 12 hours, 24 hours and used liquid nitrogen flash freezer, and-80 DEG C save backup.The process of contrast: directly get without the soybean seedling-80 DEG C of any process frozen in contrast (0 hour).

Two, the separation of mRNA

Adopt Quikprep Micro mRNA Purification Kit(Pharmacia) material of step one is carried out respectively to the separation and purification of mRNA.

Three, reverse transcription is cDNA

Adopt R103-Quant_Reverse_Transcriptase(TIANGEN Biotech (Beijing) Co., Ltd.) be cDNA by the mRNA reverse transcription of step 2 purifying.

Four, real-time fluorescence quantitative PCR

The cDNA that step 3 obtains is diluted the template that 50 times are used as real-time fluorescence quantitative PCR afterwards.According to the sequence of GmNF-YC9 gene, at its 3 ' end non-coding region design special primer, to sample, real-time fluorescence quantitative PCR amplification is carried out to F and R, analyzing the response situation of the various process of this gene pairs, take actin as reference gene, and primer is actin-F and actin-R.

Real-time fluorescence quantitative PCR is at ABI 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） _Timex－（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 481-499 position of the corresponding sequence 2 of primers F: 5'-CGTGGCCGAGGGCGACCAC-3'()

The 671-690 position of the corresponding sequence 2 of primer R:5'-ATTCAGATCGATGTTCCGGA-3'()

Primer act in-F:5'-CGGTGGTTCTATCTTGGCATC-3';

Primer act in-R:5'-GTCTTTCGCTTCAATAACCCTA-3'

Result as shown in the A-F in Fig. 1, GmNF-YC9 gene pairs above-mentioned 6 kinds coerce in arid (Figure 1B), low temperature (Fig. 1 C), salt marsh (Fig. 1 E) and ethene (Fig. 1 F) coerce and show response.

The activation characteristic of embodiment 3, GmNF-YC9

The cardinal principle of the activation characteristic of transcription factor is proved as shown in Figure 2 with yeast-one-hybrid system, CCAAT cis-acting elements and mutant CCAAT cis-acting elements are building up to respectively the basic promotor Pmin(minimal promoter of 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.

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％PEG3350 8ml，10×TE buffer 1ml，10×LiAc 1ml。

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

1×TE/LiAc：10×TE buffer 1ml，10×LiAc 1ml，ddH ₂O 8ml。

Z Buffer:Na ₂hPO ₄7H ₂o 16.1g/L, NaH ₂pO ₄h ₂o 5.5g/L, KCl 0.75g/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 expression vector

1, the acquisition of GmNF-YC9 gene

According to primers GmNF-YC9-BHI and GmNF-YC9-XI of GmNF-YC9 gene, prime end introduces BamHI and XhoI restriction enzyme site respectively, with the cDNA of rich No. 8 of soybean varieties iron for template, pcr amplification GmNF-YC9 gene, carries out 1.2% agarose gel electrophoresis detection by pcr amplification product.

GmNF-YC9-BHI：5'-TTT GGATCCATGAGGAAGAAGCTCGATAC-3'；

GmNF-YC9-XI：5'-GGT CTCGAGCCCCTCCTCTTCATCATAGTC-3'。

Pcr amplification product carries out 1.2% agarose gel electrophoresis detection.

Agarose Gel DNA PurificationKit Ver.2.0 (TaKaRa company, Code No.DV807A) is adopted to reclaim the PCR primer of purifying 882bp.

2, the structure of recombinant expression vector

1. cut with restriction enzyme BamHI and XhoI enzyme the PCR primer that step 1 reclaims purifying, reclaim digestion products;

2. cut expression vector YEP-GAP with restriction enzyme BamHI and XhoI enzyme, reclaim carrier framework;

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 checks order; Sequencing result shows, obtains recombinant vectors YEP-GAP-GmNF-YC9(and namely between BamHI and the XhoI restriction enzyme site of YEP-GAP, inserts sequence 2 and hold the DNA fragmentation shown in the Nucleotide of 34-894 position from 5').

Two, the Binding in vivo specificity of GmNF-YC9 and the checking of activation characteristic

1, the structure of yeast reporter

(1) structure of normal dual yeast reporter

DNA fragmentation A is (containing 4 CCAAT elements; TTTAA cCAATcAGAAA):

The core sequence of 5 '-GAATTC-CCAAT-CCAAT-CCAAT-CCAAT-GTCGAC-3'(CCAAT: CCAAT).The nucleotide sequence of DNA fragmentation A is shown in the sequence 3 of sequence table.

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, with Xho I and Nco I restriction endonuclease, pLacZi-CCAAT carrier is cut into wire.

First by wire pHis-1-CCAAT vector in yeast cell (YM4271 strain, MATCHMAKEROne-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 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 (containing 4 mCCAAT elements): 5 '-GAATTC-mCCAAT-mCCAAT-mCCAAT-mCCAAT-GTCGAC-3'(MDRE: the core sequence CCAAT of 4 CCAAT elements is mutated into TTTTA).The nucleotide sequence of DNA fragmentation B is shown in the sequence 4 of sequence table.

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) inoculation yeast bacterial strain (YM4271 strain) is in 1ml YPD liquid nutrient medium, concuss 2 minutes, gone to by suspension after dispersion agglomerate 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 YEP-GAP-GmNF-YC9,0.1mg ssDNA(salmon sperm dna, SiTaa), 0.6mlPEG/LiAc vibration 1 minute at a high speed, 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 0.5ml 1 × 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/L 3-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-YC9 can be combined with CCAAT cis-acting elements, and there is mobilizing function, have activated Pmin promotor, impel reporter gene to express.Thus demonstrate Binding in vivo specificity and the mobilizing function of GmNF-YC9.

Embodiment 4, the drought tolerance utilizing GmNF-YC9 gene raising plant and salt tolerance

One, the structure of recombinant expression vector

1, the clone of GmNF-YC9 gene

According to the primers of GmNF-YC9 gene to (GmNF-YC9-121F and GmNF-YC9-121R), prime end introduces Sma I respectively and SacI enzyme cuts recognition site, be template PCR amplifications GmNF-YC9 gene with rich No. 8 of soybean iron (Glycine max L.) cDNA, pcr amplification product is carried out 1.2% agarose gel electrophoresis, Agarose GelDNA Purification Kit Ver.2.0 (TaKaRa company, Code No.:DV807A) is adopted to reclaim the band of purifying about 900bp.

GmNF-YC9-121F：5'-TCC CCCGGGATGAGGAAGAAGCTCGATAC-3'；

GmNF-YC9-121R：5'-C GAGCTCTTACCCCTCCTCTTCATCATAGTC-3'。

2, the structure of recombinant expression vector

1. cut with restriction endonuclease sma I and SacI enzyme the PCR primer that step 1 reclaims purifying, reclaim digestion products;

2. restriction endonuclease sma I and SacI Mei Qie pBI121(Clontech company is used), reclaim 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 checks order; Sequencing result shows, obtain recombinant vectors pBI121-GmNF-YC9(and namely between the Sma I of pBI121 and SacI restriction enzyme site, insert sequence 2 and hold the DNA fragmentation shown in the Nucleotide of 34-897 position from 5', its structural representation as shown in Figure 3).

Two, the acquisition of transgenic plant

1, recombinant plasmid pBI121-GmNF-YC9 transformation Agrobacterium C58C1(Beijing Baeyer enlightening biotech company is bought), obtain recombinational agrobacterium.

2, the recombinational agrobacterium that step 1 obtains is inoculated in LB (containing 50mg/L Rifampin, 100mg/L kantlex, 50mg/L gentamicin) liquid nutrient medium, 28 DEG C, 3000rpm cultivate about 30 hours;

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

4, collecting the thalline of step 3,4 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);

5, by Arabidopis thaliana (Columbia ecotype Col-0, SALK company buys) whole strain tips upside down in the container of the bacterium liquid filling step 4 together with flowerpot, make flower soak about 50s, after immersion, take out flowerpot, be sidelong in pallet, cover black plastic cloth, after 24hr, open plastic cloth, upright placing flowerpot, carry out normal illumination cultivation, individual plant results T ₁for seed.

6, by T ₁after seed disinfection, be laid in (containing 50 μ g/mL kantlex) on MS substratum, 23 DEG C, 16h illumination/8h dark culturing is after 7 days, selects the transgenic Arabidopsis plants (showing as four true leaves for green) of anti-kantlex, by T ₁go to the upper continuation cultivation of MS solid medium (not containing kantlex) for resistant transgenic Arabidopsis plant within 7 days, to move into afterwards in soil, by individual plant results T ₂for seed.Plantation screening T after the same method ₂for seed, transplanting kalamycin resistance segregation ratio is the T of 3:1 ₂for strain 30, and individual plant results T ₂for on individual plant each in strain tie T ₃for seed, get 10 T at random ₃carry out kalamycin resistance screening after the same method for strain seed, obtain 3 T ₃in generation, no longer produces the Transgenic wheat line that kalamycin resistance is separated.The plant of breeding Transgenic wheat line and seed, carry out following PCR qualification and the qualification of further resistance of reverse.

Meanwhile, transform empty carrier pBI121 in the same way as empty vector control, obtain 3 T ₃what generation isozygotied turns empty vector control strain.

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.

Three, the PCR qualification of transfer-gen plant

Get the T that step 2 obtains ₃for Transgenic wheat line plant, carry out PCR qualification in cDNA level, primer pair is 5 '-ATGAGGAAGAAGCTCGATAC-3' and 5'-TTACCCCTCCTCTTCATCATAGTC-3'; Expection band is 864bp; As shown in Figure 4, containing expection band is positive transgenic plant to partial results.

Get the T that step 2 obtains ₃in generation, isozygotys and turns empty vector control strain plant, and the PCR qualification through DNA level is the positive.

Four, the drought tolerance of transgenic plant is identified and Salt-Tolerance Identification

1, drought tolerance qualification

Respectively the PCR that step 3 obtains is identified the T that isozygotys be positive ₃the PCR obtained for transgenic line (TL), step 3 identifies the T that isozygotys be positive ₃in generation, turns empty vector control strain (CK) and each 60 strains of not genetically modified wildtype Arabidopsis thaliana Col-0 (WT), and method in accordance with the following steps carries out drought tolerance qualification (arrange and repeat for three times to test, results averaged) respectively:

The sprouting seedling of 15 days of normal growth is not watered, until WT plant withered (about 2 weeks), then rehydration one week, observes phenotype, take pictures and add up survival results as shown in table 2 and Fig. 5.

The survival rate statistics of table 2. transgenic Arabidopsis plants drought tolerance qualification

Plant	Repeat 1	Repeat 2	Repeat 3	On average
					TL	89.1%	91.2%	90.3%	90.2%
CK	28.4%	29.6%	27.2%	28.4%
					WT	27.3%	28.0%	26.1%	27.2%

Result shows: the survival rate of not genetically modified wildtype Arabidopsis thaliana Col-0 (WT) is 27.2%, and transgenic line survival rate is 90.2% and can normal growth, turns empty vector control strain and is 28.4% and with WT without significant difference; This illustrates that GmNF-YC9 albumen and encoding gene process LAN in Arabidopis thaliana thereof can improve the drought tolerance of plant.

2. Salt-Tolerance Identification

Respectively the PCR that step 3 obtains is identified the T that isozygotys be positive ₃the PCR obtained for transgenic line (TL), step 3 identifies the T that isozygotys be positive ₃in generation, turns each 150 of the seed of empty vector control strain (CK) and not genetically modified wildtype Arabidopsis thaliana Col-0 (WT), and method in accordance with the following steps carries out Salt-Tolerance Identification (arrange and repeat for three times to test, results averaged) respectively:

After seed is sterile-processed, on the MS substratum containing different concns NaCl (0mM, 100mM NaCl) is evenly put with toothpick simple grain, seal with sealed membrane, place after 3 days for 4 DEG C, move into 23 DEG C, 16h illumination/8h is dark, cultivates after 7 days and add up germination rate in the culturing room of 60% relative humidity, cotyledon is opened completely and is that green seedling counts sprouting, and three repetitions are established in each process.Result is as shown in table 3 and Fig. 6.

The germination rate statistics of table 3. transgenic Arabidopsis plants Salt-Tolerance Identification

Result shows: the germination rate of not genetically modified wildtype Arabidopsis thaliana Col-0 (WT) seed is 65.8%, and the germination rate of transgenic line (TL) seed is 90.4%, turns the germination rate of empty vector control (CK) seed and WT without significant difference; This illustrates that GmNF-YC9 albumen and encoding gene process LAN in Arabidopis thaliana thereof can improve the salt tolerance of plant.

Claims

1. cultivate a method for transgenic plant, be by channel genes object plant, obtain the transgenic plant of resistance of reverse higher than described object plant; The protein of the aminoacid sequence composition shown in described gene coded sequence table sequence 1; Described resistance of reverse is drought tolerance and/or salt tolerance; Described plant is dicotyledons.

2. method according to claim 1, is characterized in that: described gene is following 1) or 2) gene:

2) its nucleotide sequence is the DNA molecular shown in sequence 2.

3. method according to claim 1 and 2, is characterized in that: described dicotyledons is Arabidopis thaliana.

4. method according to claim 3, is characterized in that: describedly realized in channel genes object plant by recombinant vectors pBI121-GmNF-YC9; Described pBI121-GmNF-YC9 is that described gene insertion vector pBI121 obtains expressing described recombinant expression of proteins carrier.

5. method according to claim 3, is characterized in that: obtain described recombinant expression vector by between the Sma I of the DNA molecular insertion vector pBI121 shown in the 34th to the 897th nucleotide sequence in sequence 2 and SacI restriction enzyme site.