CN104892739B - Plant stress tolerance correlative protein GmNF-YC6 and its encoding gene and application - Google Patents

Abstract

The invention discloses a kind of plant stress tolerance correlative protein GmNF YC6 and its encoding gene and applications.Protein provided by the invention is as follows（a）Or（b）：（a）The protein being made of the amino acid sequence shown in sequence in sequence table 1；（b）By the amino acid sequence of sequence 1 by one or several amino acid residues substitution and/or lack and or add and with the relevant protein as derived from sequence 1 of plant stress tolerance.The present invention GmNF YC6 expressed under induction arid, with high salt and low temperature, the albumen of coding is navigated in cytoplasm, and can special regulation and control contain CCAAT box cis elements（Core sequence：CCAAT）Gene transcriptional expression, GmNF YC6 of the invention can improve the drought tolerance of plant, be that degeneration-resistant and the correlation gene of resistance to anti-phase expression provides the foundation people in order to control, will play an important role in the plant breeding that resistance and resistance of reverse enhance is cultivated.

Description

Plant stress tolerance correlative protein GmNF-YC6 and its encoding gene and application

Technical field

The present invention relates to biotechnology more particularly to a kind of plant stress tolerance correlative protein GmNF-YC6 and its coding Gene and application.

Background technology

The environment stresses such as arid, with high salt and low temperature seriously restrict the growth of soybean, development.Therefore, soybean is understood to inverse The response of border condition and signal transduction mechanism improve the resistance of soybean varieties, becomes soybean heredity research and breed improvement One of vital task.

A series of responsing reactions can be generated in plant under environment stress, along with many Physiology and biochemistries and developmentally Variation.Reaction mechanism of the plant to adverse circumstance is specified, science argument will be provided for adversity gene engineering research and application.At present, it plants The degeneration-resistant Journal of Sex Research of object is gradually deep into cell, molecular level, and is combined with science of heredity and genetic engineering research, exploration life Object technology improves plant growth characteristics, and the purpose is to improve adaptability of the plant to adverse circumstance.

Under the adverse environmental factor of the environment-stress such as arid, with high salt and low temperature, plant can be in molecule, cell and integral level On make corresponding adjustment, to reduce injury caused by environment and existence to the full extent.Many genes are lured by stress Expression is led, the product of these genes can not only directly participate in the stress response of plant, and can adjust other related genes Expression or participate in signal transduction path, so as to which plant be made to avoid or reduce injury, enhance the resistance to stressful environmental.With stress Relevant gene outcome can be divided into two major class：The product of first kind gene code include ionophorous protein, aquaporin, Osmotic factor（Sucrose, proline and glycine betaine etc.）Synzyme etc. directly participates in the gene outcome of plant stress response；The The product of two genoids coding includes the protein factor for participating in coercing relevant signal transmission and Gene expression and regulation, as albumen swashs Enzyme, transcription factor etc..Wherein, transcription factor plays an important role in the gene expression regulation of plant stress response.

Transcription factor is also referred to as trans-acting factor, is that can be sent out with cis-acting elements in eukaryotic gene promoter region The DNA binding protein of raw specific effect, by the interaction between them and between other GAP-associated protein GAPs, activation or Inhibit transcription.The DNA combined areas of transcription factor determine the specificity that it is combined with cis-acting elements, and transcription regulatory region is determined It is determined and activation or inhibiting effect is risen to gene expression.In addition, the effects that its own activity is also by nuclear location and oligomerization Influence.

Being currently known in plant mainly has with coercing relevant transcription factor：AP2 with AP2 structural domains (APETALA2)/EREBP (element responsive to ethylene binding protein, ethylene responsive element binding Protein) transcription factor family, the bZIP containing basic region and leucine zipper（basic region/leucine zipper motif transcription factors）Class transcription factor, the WRKY containing conservative WRKY amino acid sequences Transcription factor family, with reference to CCAAT-box main nuclear factor CBF（CCAAT binding factor）Class transcription because Son contains basic helix-loop-helix（bHLH）With the MYC families of leucine zipper and with tryptophan cluster（Trp cluster） MYB families.These transcription factor families, in addition to WRKY families are not involved in the water Stress responses of plant, other four families are equal It participates in adjusting environment stress reaction of the plant to arid, with high salt and low temperature etc..Wherein, NF-Y transcription factors are wide in higher plant General presence in recent years, has been reported that this shows that NF-Y transcription factors are generally deposited in higher plant in arabidopsis, corn, rice And play an important roll.

NF-Y（Nuclear factor, Nuclear Factor Y）It is a kind of main nuclear transcription factor of combination CCAAT-box Son, special identification and promoter or the increasing for combining many eucaryote composing types, inductivity and cell cycle dependant gene Conserved sequence CCAAT-box in hadron, and regulate and control the expression of the transcriptional level of these genes.NF-Y is by NF-YA, NF-YB With the tripolymer of tri- different subunit compositions of NF-YC.The histone fold motif of NF-YB and NF-YC（HFM）Interaction is allowed to Dimer is formed, the NF-Y compounds of heterotrimer are formed then in conjunction with NF-YA, so as to reference to CCAAT-box, adjust its target The transcription of gene.At least there are two the combinations that structural domain is used for protein by NF-YA：Structural domain rich in glutamine（Q-rich domain）With the structural domain of a subunit interaction（subunit interaction domain）.Also there are two eggs by NF-YB The structural domain combined in vain：Histone fold motif（histone-fold motif）With TATA binding proteins（TATA-binding protein）Binding structural domain（TBP-binding domain）.There are three the binding structural domains of protein by NF-YC：Histone is rolled over Folded motif, TBP binding structural domains and the structural domain rich in glutamine.The structural amino acid sequence and group of NF-YA and NF-YC Protein folding motif is homologous, and NF-YB is related to H2B histone fold motifs, and NF-YC is related in H2A histones fold motif, The motif is made of three α spirals and two rings, is responsible for the formation of H2A/H2B dimers.

Up to the present, NF-Y genes are cloned into the plants such as arabidopsis, soybean, corn, rice.Functional study table Bright, the AtNF-YB1 for being overexpressed arabidopsis significantly improves the drought resistance of transgenic arabidopsis, intends further study show that being overexpressed The homologous gene ZmNF-YB2 of southern mustard AtNF-YB1, transgenic corns can significantly increase drought resistance under conditions of water shortage.Turn Gene corn is by improving stomatal conductance, reducing leaf temperature, prevent blade from wilting, so as to remain normal under drought condition Photosynthesis improves the yield of corn.In addition, arabidopsis AtNF-YA5 by arid and ABA induced expressions, in vascular bundle and guarantor Specifically expressing in guard cell can reduce moisture content transpiration rate and activation stress response gene by reducing stomatal aperture, Cell normal osmosis gesture is maintained under drought condition, the drought resistance of plant is improved by keeping cell normal physiological function.Due to The stress tolerance of plant is the complex character regulated and controled by polygenes, and plant is difficult to realize by individual feature protein gene is imported The comprehensive of resistance is improved.Therefore, promote the expression of multiple functional genes using a key transcription factor, enhance the anti-of plant Inverse property has become the research hotspot of plant stress-resistance genetic engineering.

Invention content

It is an object of the present invention to provide a kind of plant stress tolerance correlative protein GmNF-YC6 and its encoding genes.

Protein provided by the invention, to combine the nuclear factor albumen of CCAAT-box, entitled GmNF-YC6 comes Derived from Glycine soybean（Glycine max L.）, it is as follows（a）Or（b）：

（a）The protein being made of the amino acid sequence shown in sequence in sequence table 1；

（b）The amino acid sequence of sequence 1 by the substitution of one or several amino acid residues and/or missing and/or is added Add and with the relevant protein as derived from sequence 1 of plant stress tolerance.

The protein that the amino acid residue sequence of sequence 1 is made of 271 amino acid residues in sequence table, wherein from 99 amino acid residues to 172 amino acid residues be conservative histone fold motif.

In order to make a）In GmNF-YC6 convenient for purifying, amino acid sequence that can be in by sequence table shown in sequence 1 forms The amino terminal of protein or the upper label as shown in Table 1 of carboxyl terminal connection.

The sequence of 1. label of table

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

Above-mentioned 1）In GmNF-YC6 can be artificial synthesized, also can first synthesize its encoding gene, then carry out biological expression and obtain. The encoding gene of GmNF-YC6 in above-mentioned 1 can be one or several by will be lacked in the DNA sequence dna shown in sequence in sequence table 2 The codon of amino acid residue and/or carry out the missense mutation of one or several base-pairs and/or at its 5 ' end and/or 3 ' ends The coded sequence for connecting the label shown in table 1 obtains.

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

Above-mentioned DNA molecular is following 1）-4）In any DNA molecular：

1）Code area is the DNA molecular shown in sequence 2 in sequence table；

2）Code area is the DNA molecular shown in sequence 2 from the nucleotide of 5 ' end the 1st to 813；

3）Under strict conditions with 1）Or 2）The DNA sequence dna hybridization of restriction and the DNA molecular of coding stress tolerance correlative protein；

4）With 1）Or 2）The DNA sequence dna of restriction has more than 90% homology, and encodes DNA points of stress tolerance correlative protein Son.

Above-mentioned stringent condition can be in 6 × SSC, the solution of 0.5%SDS, hybridize at 65 DEG C, then with 2 × SSC, It is primary that 0.1%SDS and 1 × SSC, 0.1%SDS respectively wash film.

DNA sequence dna in sequence 2 is made of 816 nucleotide, and the open reading frame of the gene is from 5 ' end 1-816 Position nucleotide.

Recombinant vector, expression cassette, transgenic cell line or recombinant bacterium containing above-mentioned DNA molecular are also that the present invention protects Range.

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

The recombinant expression carrier of the gene can be contained with existing plant expression vector construction.The plant expression vector Carrier including double base agrobacterium vector and available for plant micropellet bombardment etc..The plant expression vector also may include external source base 3 ' end untranslated regions of cause, i.e., comprising polyadenylation signals and any other DNA pieces for participating in mRNA processing or gene expression Section.The bootable polyadenylic acid of polyadenylation signals is added to 3 ' ends of mRNA precursor, as Agrobacterium crown gall nodule induces (Ti) Plasmid gene (such as kermes synzyme Nos genes), plant gene（Such as soybean storage protein genes）The non-translational region of 3 ' end transcriptions It is respectively provided with similar functions.During using the gene constructed recombinant plant expression vector, it can be added before its transcription initiation nucleotide Any enhanced promoter or constitutive promoter, such as cauliflower mosaic virus（CAMV）The ubiquitin of 35S promoter, corn Promoter（Ubiquitin）, they can be used alone or are used in combination with other plant promoters；In addition, use the present invention Gene constructed plant expression vector when, also can be used enhancer, including translational enhancer or transcriptional enhancer, these enhancers Region can be ATG initiation codon or neighboring region initiation codon etc., but must be identical with the reading frame of coded sequence, with Ensure the correct translation of entire sequence.The source of the translation control signal and initiation codon is extensive, can be natural Or synthesis.Translation initiation region can come from transcription initiation region or structural gene.For the ease of to transgenosis Plant cell or plant are identified and are screened, and plant expression vector used can be processed, as add in can in plant table The coding reached can generate the enzyme of color change or the gene of luminophor（Gus gene, luciferase genes etc.）, it is resistant Antibiotic marker（Gentamicin marker, kanamycins marker etc.）Or anti-chemical reagent marker gene（It is removed as anti- Green bristlegrass agent gene）Deng.From the security consideration of genetically modified plants, any selected marker can be not added with, is directly screened with adverse circumstance Transformed plant.

In an embodiment of the present invention, expression vector YEP-GAP, corresponding recombinant vector are YEP-GAP-GmNF- YC6, YEP-GAP-GmNF-YC6 are the recombinant plasmid for obtaining the multiple cloning sites of above-mentioned DNA molecular insertion YEP-GAP, preferably For the DNA fragmentation shown in the sequence 2 of sequence table from the nucleotide of 5 ' end 1-813 to be inserted into the BamHI and PstI of YEP-GAP The recombinant vector obtained between digestion recognition site；

Expression vector is pBI121, and corresponding recombinant vector is pBI121-GmNF-YC6, and pBI121-GmNF-YC6 is will Above-mentioned DNA molecular is inserted into the recombinant plasmid that the multiple cloning sites of pBI121 obtain, preferably by the sequence of sequence table 2 from 5 ' ends DNA fragmentation shown in 1-813 nucleotide is inserted into the recombination obtained between Sma I and SacI the digestion recognition site of pBI121 Carrier.

Expand the primer pair of above-mentioned DNA molecular or its arbitrary segment.

The primer pair is GmNF-YC6-BHI and GmNF-YC6-XI or GmNF-YC6-121F and GmNF-YC6-121R：

GmNF-YC6–BHI：F CGCGGATCC ATGGATAAATCAGAGCAGACTC

GmNF-YC6-PI：R AAACTGCAGTGAGTCTGTTTGTTGATGTTGG.

GmNF-YC6-121F：5'-TCCCCCGGGATGGATAAATCAGAGCAGACTC-3'；

GmNF-YC6-121R：5'-AAAGAGCTCTGAGTCTGTTTGTTGATGTTGG-3'.

Above-mentioned protein, above-mentioned DNA molecular or above-mentioned recombinant vector, expression cassette, transgenic cell line or recombination Application of the bacterium in plant stress tolerance is adjusted is also the scope of protection of the invention.

Above-mentioned adjusting plant stress tolerance is improves plant stress tolerance.

In above application, the resistance of reverse is salt tolerance and/or drought tolerance；

The plant is dicotyledon or monocotyledon；The dicotyledon is specially arabidopsis.

It is a further object to provide a kind of methods for cultivating genetically modified plants.

Method provided by the invention is to import above-mentioned DNA molecular in purpose plant, obtains genetically modified plants；Described turn The resistance of reverse of gene plant is higher than the purpose plant.

In the above method, above-mentioned DNA molecular imports the purpose plant by above-mentioned recombinant vector；

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

The salt tolerance is embodied under NaCl stress, and the survival rate of the genetically modified plants is more than the purpose plant；

The survival rate that the drought tolerance is embodied in the genetically modified plants is more than the purpose plant；

The purpose plant is dicotyledon or monocotyledon；The dicotyledon is specially arabidopsis.

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

The experiment proves that present invention clone from soybean obtains gene GmNF-YC6, arid, with high salt and low Temperature induction under express, the albumen of coding is navigated in cytoplasm, and can special regulation and control contain the cis- members of CCAAT-box Part（Core sequence：CCAAT）Gene transcriptional expression, GmNF-YC6 of the invention can improve the drought tolerance of plant, be people Degeneration-resistant and the correlation gene of resistance to anti-phase expression provides the foundation in order to control, will be in the plant breeding for cultivating resistance and resistance of reverse enhancing In play an important role.

Description of the drawings

Fig. 1 is the sequence analysis result of GmNF-YC6 and arabidopsis amino acid AtNF-YC2 amino acid sequences.

Fig. 2 is the fluorescence real-time quantitative that GmNF-YC6 is expressed by stress-inducing（Real-time）PCR collection of illustrative plates.

Fig. 3 is target gene of the Molecular Detection in transgenic arabidopsis

Fig. 4 is wildtype Arabidopsis thaliana and turns GmNFYA-15 arabidopsis drought resistances and compares

Fig. 5 compares for wild type and transgenic arabidopsis salt tolerance

Specific embodiment

Experimental method used in following embodiments is conventional method unless otherwise specified.

The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.

% in following embodiments is mass percentage unless otherwise specified.Quantitative test in following embodiment, Three repeated experiments are respectively provided with, results are averaged.

The acquisition of embodiment 1, gene GmNF-YC6

First, the acquisition of gene GmNF-YC6

By rich No. 8 of the soybean iron of the growth 10 days or so of water planting（National germplasm resource bank, number ZM242；It is documented in as follows In document：Wang Caijie, Sun Shi, Wu Baomei, Chang Ru town, Chinese large area plantation soybean product since Han Tian richnesses .20 forties in century The pedigree analysis of kind.Chinese oil crops journal.2013,35 (3):246-252, the public can be from Chinese Academy of Agricultural Sciences crops Science Institute obtains）Four leaf stage seedling Osmotic treatment 2 hours, with liquid nitrogen flash freezer, -80 DEG C save backup.Using Quikprep Micro mRNA Purification Kit（Pharmacia）Carry out the separation of mRNA.First chain cDNA synthesis reverse transcriptase XL（AMV）.Ds cDNA are synthesized using SMART methods, PCR product carries out 1.0% agarose gel electrophoresis detection.

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

Unnamed gene in the sequence table shown in sequence 2 is GmNF-YC6, and open reading frame is the sequence from sequence table 5 ' the 1st to 816 nucleotide in end of row 2, the albumen of the gene code are named as GmNF-YC6, and the amino acid sequence of the albumen is Sequence 1 in sequence table, sequence 1 are made of 271 amino acid residues, have conservative histone fold motif.

Above-mentioned sequence 2 can also be by artificial synthesized.

The sequence of GmNF-YC6 is compared on Genabnk, has with the albumin A tNF-YC11 in arabidopsis higher same Source property（Fig. 1）, and not finding homologous protein in soybean, it was demonstrated that GmNF-YC6 albumen is a new albumen.

2nd, the expression characterization of real-time fluorescence quantitative PCR analysis GmNF-YC6

1st, Stress treatment

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

（1）Osmotic treatment（Fig. 2A）：The soybean seedling taking-up of potting is blotted to the moisture on root, is placed in dry filter paper On, arid culture takes out material after 30 minutes, 1 hour, 2 hours, 5 hours, 12 hours, 24 hours, with liquid nitrogen flash freezer, -80 DEG C It saves backup.

（2）Salt marsh processing（Fig. 2 B）：By soybean seedling be placed in 200mM by NaCl solution, illumination cultivation 30 minutes, 1 Hour, 2 hours, 5 hours, 12 hours take out material respectively after 24 hours, and with liquid nitrogen flash freezer, -80 DEG C save backup.

（3）Abscisic acid processing（Fig. 2 C）：Soybean seedling is placed in 100 μM of abscisic acid（ABA）In solution, illumination cultivation 30 Minute, 1 hour, 2 hours, 5 hours, 12 hours, take out respectively after 24 hours and with liquid nitrogen flash freezer, -80 DEG C save backup.

（4）Low-temperature treatment（Fig. 2 D）：Soybean seedling is placed in 4 DEG C of incubators, illumination cultivation 30 minutes, 1 hour, 2 hours, It is taken out and with liquid nitrogen flash freezer after 5 hours, 12 hours, 24 hours, -80 DEG C save backup.

（5）High-temperature process（Fig. 2 E）：Soybean seedling is placed at 42 DEG C, illumination cultivation 30 minutes, 1 hour, 2 hours, it is 5 small When, 12 hours, take out respectively after 24 hours and with liquid nitrogen flash freezer, -80 DEG C save backup.

（6）Ethylene（EH）Processing（Fig. 2 F）：Soybean seedling is placed in 50 μM of GA solution, illumination cultivation 30 minutes, 1 small When, 2 hours, 5 hours, 12 hours, take out respectively after 24 hours and with liquid nitrogen flash freezer, -80 DEG C save backup.

（7）The processing of control：Directly -80 DEG C of the soybean seedling without any processing is taken to freeze as control（0 hour）.

2nd, the separation of mRNA

The above-mentioned soybean seedling respectively handled is used into Quikprep Micro mRNA Purification Kit （Pharmacia）Carry out the separation of mRNA.

3rd, reverse transcription cDNA

It is cDNA by the mRNA reverse transcriptions of purifying.

4th, real-time fluorescence quantitative PCR

CDNA is diluted to the template for being used as Q-RT-PCR after 50 times.The special primer pair of noncoding region is held with gene 3 '（F： 5 ' CGATCTTGGATCCACACAG ' 3 and R：5′GGCATATCAGCTGGAGAAC′3）Q-RT-PCR amplifications are carried out to sample, point The response situation of the various processing of gene pairs is analysed, with actin（F：5 '-GTCTGGTGATGGTGTTAGC-3 ' and R：5′- CCTATCAGCAATTCCAGGAAAC-3′）Do internal reference.Q-RT-PCR existsOn 7000 real-time fluorescence quantitative PCR instrument It carries out, a parallel test sets 3 repetitions.The method reported using Livak KJ and Schmittgen TD (2001), i.e., 2^-ΔΔCTCalculate relative expression quantity.

ΔΔC_T=（C_T.Target- C_T.Actin）_{Time x}（C_T.Target- C_T.Actin）_Time0

Time x represent random time point, and Time0 represents the target gene expression of 1 times of amount after actin is corrected.

As a result see Fig. 2, it can be seen that GmNF-YC6 has response to arid, with high salt, high temperature.

Embodiment 2, GmNF-YC6 are as the application in transcription factor

Prove that the cardinal principle of the activation characteristic of transcription factor is as follows with yeast-one-hybrid system：By CCAAT cis actings Element and mutant CCAAT cis-acting elements are building up to the basic promoter of pHISi-1 carriers and pLacZi carriers respectively Pmin（minimal promoter）Upstream, Pmin promoter downstream connection reporters（His3, LacZ and URA3）.Work as connection There is the expression vector YEP-GAP of the target gene of encoding transcription factors（Without activation function）It is transformed into that be connected with CCAAT suitable respectively After the yeast cells of formula functional element and mutant CCAAT cis-acting elements, if being connected with mutant CCAAT cis actings member Reporter in the yeast cells of part cannot express, and be connected in the yeast cells of specific CCAAT cis-acting elements Reporter can express, and illustrate that the transcription factor can be combined with CCAAT cis-acting elements, and have the function of activation, activation Pmin promoters, promote reporter to express.So as to demonstrate the Binding in vivo specificity of purpose transcription factor and activation work( Energy.

Expression vector YEP-GAP:Institute of Crop Science, Chinese Academy of Agricultural Science ensures to provide to the public；Bibliography 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 fluid nutrient mediums：Bacteria Culture yeast extract（Bacto-Yeast Extract）10g/L, Bacteria Culture Use tryptone（Bacto-Peptone）20g/L, adjusts pH to 5.8, and 121 DEG C/15min sterilizings add in after being down to 60 DEG C 40% Glucose makes its final concentration of 20g/L.

SD/His^-/Ura^-/Trp^-Selective medium：Yeast nitrogen without amino acid（Yeast nitrogen base）6.7g/L, auxotroph mixture（drop-out media without His/Ura/Trp）100ml, agar powder （Bacteriological agar）20g/L, adjusts pH to 5.8, and 121 DEG C/15min sterilizings add in 40% after being down to 60 DEG C Glucose makes its final concentration of 20g/L.

Auxotroph mixture（Drop-out mix）：（10×）：L-Isoleucine（Isoleucine）300mg/L, L-Valine（Valine）1500mg/L, L-Adenine（Adenine）200mg/L, L-Arginine（Arginine）200mg/L, L-Histidine Hcl monohydrate（Histidine）200mg/L, L-Leucine（Leucine）1000mg/L, L-Lysine Hcl（Lysine）300mg/L, L-Methionine（Methionine）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 high pressure sterilizations, room temperature preservation.

1×TE/LiAc：10 × TE buffer1ml, 10 × LiAc1ml, ddH₂O8ml。

Z Buffer：Na₂HPO₄·7H₂O16.1g/L, NaH₂PO₄·H₂O5.5g/L, KCl0.75g/L, MgSO₄· 7H₂O0.246g/L adjusts pH to 7.0,121 DEG C/15min sterilizings, 4 DEG C of preservations.

X-gal storing liquids（X-gal Stock Solution）：With N, N-dimethyl-formamide（DMF）Dissolve X- Gal makes its final concentration of 20mg/ml, -20 DEG C of storages.

Z buffer buffer solutions 100ml containing X-gal（Z buffer with X-gal）, matching while using：Z Buffer98ml, beta -mercaptoethanol（β-mercaptoethanol）0.27ml, X-gal storing liquid（X-gal stock solution）1.67ml.

10×LiAc：100Mm Tris-Hcl,100mM EDTA,pH=7.5.121 DEG C of high pressure sterilizations, room temperature preservations.

First, the structure of recombinant vector

1st, the acquisition of GmNF-YC6 genes

According to the primers GmNF-YC6-BHI and GmNF-YC6-XI of GmNF-YC6 genes, prime end difference Introduce BamHI and PstI restriction enzyme sites.

GmNF-YC6–BHI：F CGCGGATCC ATGGATAAATCAGAGCAGACTC

GmNF-YC6-PI：R AAACTGCAGTGAGTCTGTTTGTTGATGTTGG.

Using the cDNA of whole rich No. 8 of strain soybean iron as template, PCR is carried out with primer GmNF-YC6-BHI and GmNF-YC6-XI Amplification.

Obtain the pcr amplification product of 816bp or so.

Recycle pcr amplification product.

2nd, the structure of recombinant vector

1. the pcr amplification product recycled with restriction enzyme BamHI and PstI digestion step 1 recycles the digestion of 816bp Product；

2. with restriction enzyme BamHI and PstI digestion expression vector YEP-GAP, carrier framework is recycled；

3. the digestion products of step 1. are connected with the carrier framework of step 2.；

4. by the electroporated JM109 bacterial strains of the connection product of step 3.（Purchased from Clontech companies）, 37 DEG C are incubated overnight, Picking positive colony extraction plasmid is sequenced；

Sequencing result shows plasmid for the DNA pieces shown in by the sequence 2 of sequence table from the nucleotide of 5 ' end 1-813 Section is inserted into the recombinant vector obtained between BamHI the and PstI restriction enzyme sites of carrier YEP-GAP, is named as YEP-GAP-GmNF- YC6。

2nd, the verification of the Binding in vivo specificity and activation characteristic of GmNF-YC6

1st, the structure of yeast reporter

（1）The structure of normal dual yeast reporter

DNA fragmentation A (contains 4 CCAAT elements):

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

DNA fragmentation A is building up to pHis-1 carriers（MATCHMAKER One-Hybrid System, Clontech companies） Pmin_HIS3Promoter upstream obtains recombinant vector pHis-1-CCAAT, with Xho I and I restriction endonucleases of Nco by pHis-1-CCAAT Carrier is cut into threadiness.

DNA fragmentation A is building up to pLacZi carriers（MATCHMAKER One-Hybrid System, Clontech companies） P_CYCIPromoter upstream obtains recombinant vector pLacZi-CCAAT, with Xho I and I restriction endonucleases of Nco respectively by pLacZi-CCAAT Carrier is cut into threadiness.

Linear pHis-1-CCAAT carriers are first transformed into yeast cells（YM4271 strains, MATCHMAKER One- Hybrid System, Clontech companies）It is interior, obtain can on SD/His- culture mediums normal growth yeast transformant （Yeast transformant）.Then using this yeast transformant as host cell, continue conversion and contain 4 repetition CCAAT The linear pLacZi-CCAAT carriers of element.Lack the SD/His of histidine and uracil at the same time in this way^-/Ura^-On culture medium, Selection obtains normal dual yeast reporter containing pHis-1-CCAAT and pLacZi-CCAAT.

（2）The 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'。

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

2nd, PEG/LiAc methods transformed yeast and interpretation of result

(1) the above-mentioned 1 normal dual yeast reporter containing pHis-1-CCAAT and pLacZi-CCAAT obtained is inoculated with respectively In son and mutant dual yeast reporter to 1ml YPD fluid nutrient mediums, acutely concussion 2 minutes, will suspend after disperseing agglomerate Liquid is gone in the triangular flask containing 50ml YPD fluid nutrient mediums, and 30 DEG C/250rpm shakes overnight, surveys OD600=1.7-1.8（It counts About 4 × 10⁷A/mL）；

(2) 30ml steps are taken（1）Overnight culture is connected in the fresh YPD culture mediums of 300ml, 30 DEG C/250rpm cultures, About 3 hours（To OD600=0.5 ± 0.1）, room temperature 1000g centrifugation 5min, collect thalline, abandon supernatant, hanged with 1/2 1 × TE of volume It is floating, 1000g/5min centrifugations；

(3) inhale and abandon supernatant, with 1 × TE/LiAc solution suspensions of 1.5ml Fresh, oscillation mixing is spare；

(4) take out 0.1ml competent yeasts to be converted, add following solutions successively：The recombinant vector that 0.1 μ g are prepared by one YEP-GAP-GmNF-YC6、0.1mg ssDNA（Salmon sperm dna, SiTaa）, 0.6mlPEG/LiAc at a high speed oscillation 1 minute, 30 DEG C/200rpm shaken cultivations 30 minutes；

(5) 70ul DMSO are added in（siTaa#D8779）, gently it is inverted mixing, 42 DEG C of heat shocks 30 minutes are gently shaken therebetween It swings, ice bath 2 minutes, room temperature 1000g centrifugations 5min；

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

(7) suspension is dipped with oese, respectively in the SD/His containing 0,15mmol/L3-AT^-/Ura^-/Trp^-Selectivity Setting-out culture on culture medium.

(8) normally dual yeast reporter is sub for the half culture of tablet, dual yeast reporter of the other half culture mutant, with Just check analysis is done.

(9) it is placed upside down in incubator, 30 DEG C are cultivated 3-4 days.

As a result, it has been found that 0mmol/L3-AT SD/His^-/Ura^-/Trp^-Culture medium flat plate on normal yeast reporter Son and yeast reporter of mutation have growth, but the diameter for yeast reporter being mutated is significantly small；And in 15mmol/L3-AT SD/His^-/Ura^-/Trp^-Culture medium flat plate on normal yeast reporter energy normal growth, but yeast reporter being mutated It is suppressed and does not grow.

3rd, galactosidase activity detects

(1) from the SD/His of 0mmol/L3-AT^-/Ura^-/Trp^-Culture medium flat plate on respectively the normal yeast reporter of picking Son and the yeast reporter daughter colony of mutation.It goes in YPD fluid nutrient mediums, in 30 DEG C of shaken cultivations, after length to logarithmic growth Phase takes 1.5ml bacterium solutions, 3000rpm centrifugations 30s；

(2) supernatant is abandoned, drains liquid in pipe, centrifuge tube is placed in quick-frozen 10min in liquid nitrogen, taking-up makes it melt naturally, adds 50ul Z/X-gal solution, 30 DEG C of incubations, as a result, it has been found that normal yeast reporter becomes yeast report that is blue, and being mutated in 6-8h Line does not change in 12h, is still white.

Illustrate that transcription factor GmNF-YC6 can be combined with CCAAT cis-acting elements, and there is activation, have activated Pmin promoters, promote reporter to express.So as to demonstrate the Binding in vivo specificity of GmNF-YC6 and activation function, to turn Record the factor.

The application of embodiment 3, GmNF-YC6 in the resistance of reverse for improving plant

First, turn the acquisition of GmNF-YC6 arabidopsis

1st, the structure of recombinant vector

1）The clone of GmNF-YC6 genes

According to the primers pair of GmNF-YC6 genes（GmNF-YC6-121F and GmNF-YC6-121R）, primer end End introduces Sma I and SacI digestion recognition sites respectively,

GmNF-YC6-121F：5'-TCCCCCGGGATGGATAAATCAGAGCAGACTC-3'；

GmNF-YC6-121R：5'-AAAGAGCTCTGAGTCTGTTTGTTGATGTTGG-3'.

With Glycine soybean（Glycine max L.）Rich No. 8 whole strain cDNA of iron are template, with GmNF-YC6-121F and GmNF-YC6-121R carries out PCR amplification, obtains the PCR product of size about 1Kb（GmNF-YC6 genes）.

Recycle above-mentioned PCR product.

2）, recombinant vector structure

1. the PCR product recycled with restriction endonuclease sma I and SacI digestions step 1, recycles 816bp digestion products；

2. with restriction enzyme sma I and SacI digestions pBI121（Clontech companies buy）, recycling 14000bp loads Body skeleton；

3. the digestion products of step 1. are connected with the carrier framework of step 2.；

4. by the electroporated TOP10 bacterial strains of the connection product of step 3.（Purchased from Beijing Tiangeng company）, 37 DEG C are incubated overnight, Picking positive colony extraction plasmid is sequenced.

Sequencing result shows plasmid for the DNA fragmentation shown in by the sequence 2 of sequence table from 1-813,5' ends nucleotide The recombinant vector obtained between the Sma I of carrier pBI121 and SacI restriction enzyme sites is inserted into, is named as pBI121-GmNF-YC6.

3rd, the acquisition of recombinational agrobacterium

With recombinant plasmid pBI121-GmNF-YC6 genetic transformation Agrobacteriums C58C1（Beijing Baeyer enlightening biotech company purchases It buys）, obtain recombinational agrobacterium C58C1/pBI121-GmNF-YC6（Extraction plasmid sends to sequencing, is pBI121-GmNF-YC6, then Recombinant bacterium containing the plasmid is the positive）.

4th, turn the acquisition of GmNF-YC6 arabidopsis

1) recombinational agrobacterium obtained 3 is inoculated in LB (rifampin containing 50mg/ml, 100mg/ml kanamycins, 50mg/ Ml gentamicins) in fluid nutrient medium, 28 DEG C, 3000rpm cultivate about 30 hours, obtain bacterium solution；

2) bacterium solution is gone in LB (rifampin containing 50mg/ml, 100mg/ml kanamycins, 50mg/ml gentamicins), 28 DEG C, 300rpm cultivate about 14 hours（Bacterium solution OD600 reaches 1.5-3.0）；

3) thalline, 4 DEG C, 4000g centrifugation 10min, with containing 10% sucrose MS fluid nutrient mediums are collected（Containing 0.02%silwet） It is about 0.8-1.0 to be diluted to OD600；

4) by arabidopsis（Columbia ecotype Col-0, SALK companies buy, also referred to as wildtype Arabidopsis thaliana）Whole strain with Flowerpot is tipped upside down on together in the container for the bacterium solution for filling step 4, and flower is made to impregnate 50s or so, after immersion, takes out flowerpot, side It is put in pallet, covers black plastic cloth, open plastic cloth after r for 24 hours, uprightly place flowerpot, carry out normal illumination cultivation, receive Obtain T₁For seed, kanamycins screening（A concentration of 50 μ g/L kanamycins）Positive plant is T₁For regeneration plant, pass on, until Obtain T₃For regeneration plant.

T₂T is shown in representative₁The generation selfing seed generated and the plant grown up to by it, T₃T is shown in representative₂The kind that generation selfing generates Son and the plant grown up to by it.

Extract T₃For the RNA of the blade of regeneration plant, reverse transcription obtains cDNA as template, uses primer pair：F:5'- ATGGATAAATCAGAGCAGACTC-3'；R:5'-TGAGTCTGTTTGTTGATGTTGG-3'；Carry out PCR amplification.

The result of part sample is shown in Fig. 3, and M Marker, col are Columbia ecotype arabidopsis, L1-L8 T₃In generation, is again Raw plant.

It is 816bp segments is the positive that L1-L2, L4-L8, which obtain size, as T₃In generation, turns GmNF-YC6 arabidopsis.

Plasmid pBI121 is transferred in wildtype Arabidopsis thaliana using same method, obtains turning empty carrier arabidopsis, is cultivated Obtain T₃In generation, turns empty carrier arabidopsis, is identified using same method, does not obtain 915bp segments.

2nd, the resistance of reverse identification of genetically modified plants

1st, drought tolerance is identified

By T₃In generation, turns GmNF-YC6 arabidopsis strains NFYC6-2, NFYC6-8, NFYC6-10, T₃In generation, turns empty carrier arabidopsis And wildtype Arabidopsis thaliana（WT）The seedling that seed is sprouted 15 days is not watered, until WT lines are withered（When not watering 2 weeks）, so Rehydration one week afterwards, observation phenotype take pictures and count survival rate.Three repeated experiments are set, and results are averaged.

Photo is shown in Fig. 4, A：Wild type and transfer-gen plant before Osmotic treatment；B:After Osmotic treatment 14 days, rehydration is after 3 days Wild type and transfer-gen plant, the survival rate after statistical disposition are as follows：

Wildtype Arabidopsis thaliana（WT）Survival rate be 27%；

The survival rate of NFYC6--2 is 100%；

The survival rate of NFYC6--8 is 100%；

The survival rate of NFYC6--10 is 83%；

T₃In generation, turns empty carrier arabidopsis and wildtype Arabidopsis thaliana result without significant difference.

After Osmotic treatment, transfer-gen plant is higher than WT lines survival rate, illustrates that gene GmNF-YC6 has drought tolerance.

2nd, Salt-Tolerance Identification

Respectively by T₃In generation, turns GmNF-YC6 arabidopsis strains NFYC6--2, NFYC6-8, NFYC6-10, T₃In generation, turns empty carrier plan Southern mustard and wildtype Arabidopsis thaliana（Each 60 plants）Carry out Salt-Tolerance Identification.Three repeated experiments are set, and results are averaged.

By T₃In generation, turns GmNF-YC6 arabidopsis strains GmNF-YC6-1, GmNF-YC6-2, GmNF-YC6-3, T₃In generation, turns zero load Body arabidopsis and wildtype Arabidopsis thaliana（WT）The seedling that seed is sprouted 15 days pours 100mM NaCl solutions, until WT lines Wilting, which occurs, in blade to turn yellow, then rehydration one week, observes phenotype, takes pictures and counts survival rate.

Photo is shown in Fig. 5, upper figure：The sprouting situation of MS culture mediums wild type and transgenic seed；Figure below:Transgenosis and open country Raw type germination and growth 7 days on the MS culture mediums of the NaCl containing 100mM.

Count treated survival rate, wildtype Arabidopsis thaliana with high salt（WT）Survival rate be 10%；

The survival rate of GmNF-YC6-2 is 81%；

The survival rate of GmNF-YC6-8 is 60%；

The survival rate of GmNF-YC6-10 is 40%；

T₃In generation, turns empty carrier arabidopsis and wildtype Arabidopsis thaliana result without significant difference.

After high salt treatment, transfer-gen plant is higher than WT lines survival rate, illustrates that gene GmNF-YC6 has salt tolerance.

Claims (7)

1. a kind of application of protein or its encoding gene in plant stress tolerance is adjusted；

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

The protein is made of the amino acid sequence shown in sequence in sequence table 1.

2. application according to claim 1, it is characterised in that：The encoding gene for it is following 1) or 2)：

1) DNA molecular in sequence table shown in sequence 2；

2) DNA molecular shown in sequence 2 from the nucleotide of 5 ' end the 1st to 813.

3. application according to claim 1 or 2, it is characterised in that：The plant is dicotyledon or monocotyledon.

4. application according to claim 3, it is characterised in that：The dicotyledon is arabidopsis.

5. a kind of method for cultivating genetically modified plants is to import a kind of DNA molecular in purpose plant, obtains genetically modified plants； The resistance of reverse of the genetically modified plants is higher than the purpose plant；

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

The DNA molecular for it is following 1) or 2)：

1) DNA molecular in sequence table shown in sequence 2；

2) DNA molecular shown in sequence 2 from the nucleotide of 5 ' end the 1st to 813.

6. according to the method described in claim 5, it is characterized in that：The DNA molecular imports the purpose by recombinant vector Plant；

The recombinant vector is that the DNA molecular shown in sequence in sequence table 2 is inserted into expression vector, obtains sequence in expressed sequence table The carrier of the protein of amino acid sequence composition shown in row 1；

The purpose plant is dicotyledon or monocotyledon.

7. according to the method described in claim 6, it is characterized in that：The dicotyledon is arabidopsis.