CN106893738A - The application of OsSGT1 albumen and its encoding gene in plant salt tolerance resistance is regulated and controled - Google Patents

Abstract

The invention discloses a kind of application of OsSGT1 albumen and its encoding gene in plant salt tolerance resistance is regulated and controled.The present invention provides a kind of method for cultivating genetically modified plants, comprises the following steps：The channel genes for encoding OsSGT1 albumen are set out plant, the genetically modified plants of salt tolerance reduction are obtained.The OsSGT1 albumen, is following (a1) or (a2)：(a1) protein that the amino acid sequence shown in sequence in sequence table 2 is constituted；(a2) by the amino acid sequence of sequence 2 by the substitution of one or several amino acid residues and/or missing and/or addition and as sequence 2 derived from the protein related to plant salt endurance.The present invention understands the genesis mechanism of salt stress for excavating plant salt tolerance gene, improves plant salt endurance, cultivates the strong plant variety of salt tolerance, is worth with important theory directive significance and production application.

Description

The application of OsSGT1 albumen and its encoding gene in plant salt tolerance resistance is regulated and controled

Technical field

The present invention relates to a kind of application of OsSGT1 albumen and its encoding gene in plant salt tolerance resistance is regulated and controled.

Background technology

Abiotic stress is for example saline and alkaline, arid and low temperature severely impacts the high yield and stable yields of crop, is to threaten world food Safety the principal element soil salinization be limit crop growth, cause grain drop in production the main abiotic stress factor it One.In recent years, soil secondary salinization area is increasing year by year, and salt stress has turned into influence biological husbantry production in world wide One of most important environmental stress factor.Salt stress is mainly shown as osmotic stress, influence plant pair Na⁺And K⁺Absorption and row Reprimand, and the normal ion of cell distribution and dynamic equilibrium.Paddy rice is grass family model plant, is also a kind of to salt medium sensitivity Crop, salt stress turned into the main restricting factor of Chinese saline and alkaline rice region paddy rice steady production.Salt stress patience related gene Including participating in the signal transduction gene related to transcriptional modulatory gene, osmotic adjustment metabolism and transporter gene etc..Water The Study on Molecular Mechanism of rice salt stress response achieves some substantial progresses, is mainly manifested in multiple related to salt stress tolerance Gene and regulatory factor by successful identification, the biological function of portion gene also illustrates clearer.Though identify at present The related molecular labeling of some salt stresses and gene (or QTL) site, because salt stress patience is a quantitative character, by many Individual gene regulation.Therefore also need to increase the excavation and utilization of Rice Salt germ plasm resource, strengthen Rice Salt molecular labeling Position Research, clones resistant gene of salt, and study its mechanism of action.Plant salt tolerance gene is excavated, the genesis mechanism of salt stress is understood, Looking for plant responds the signal pathway node of salt stress, improves the disease resistance and salt tolerance of paddy rice, cultivates the strong paddy rice of salt tolerance Kind, is worth with important theory significance and production application.

The content of the invention

It is an object of the invention to provide a kind of OsSGT1 albumen and its encoding gene answering in plant salt tolerance resistance is regulated and controled With.

The invention provides a kind of method for cultivating genetically modified plants, comprise the following steps：OsSGT1 albumen will be encoded Channel genes set out plant, obtain the genetically modified plants of salt tolerance reduction.

The OsSGT1 albumen, is following (a1) or (a2) available from paddy rice：

(a1) protein that the amino acid sequence shown in sequence in sequence table 2 is constituted；

(a2) by the amino acid sequence of sequence 2 is by the substitution of one or several amino acid residues and/or missing and/or adds Plus and as sequence 2 derived from the protein related to plant salt endurance.

In order that the OsSGT1 albumen in (a1) is easy to purify and is detected, amino that can be in as sequence table shown in sequence 2 The amino terminal or the upper label as shown in table 1 of carboxyl terminal connection of the protein of acid sequence composition.

The sequence of the label of table 1

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

OsSGT1 albumen in above-mentioned (a2) can be artificial synthesized, also can first synthesize its encoding gene, then carry out biological expression Obtain.The encoding gene of the OsSGT1 albumen in above-mentioned (a2) can be by will lack in the DNA sequence dna shown in sequence in sequence table 1 The codon of one or several amino acid residues, and/or carry out the missense mutation of one or several base-pairs, and/or its 5 ' The coded sequence that end and/or 3 ' ends connect the label shown in table 1 is obtained.

" gene (abbreviation OsSGT1 genes) of coding OsSGT1 albumen " is following (b1) or (b2) or (b3)：

(b1) DNA molecular of the code area as shown in the sequence 1 from the nucleotides of 5 ' end 1-1104 of sequence table；

(b2) the DNA sequence dna hybridization for being limited to (b1) under strict conditions and the coding protein related with plant salt endurance DNA molecular；

(b3) there is more than 90% homology to the DNA sequence dna that (b1) or (b2) is limited and encodes related with plant salt endurance Protein DNA molecule.

Above-mentioned stringent condition can be that with 0.1 × SSPE (or 0.1 × SSC), the solution of 0.1%SDS is miscellaneous in DNA or RNA Hand over and hybridize at 65 DEG C in testing and wash film.

In methods described, the OsSGT1 genes can import the plant that sets out by recombinant expression carrier.The restructuring table Can be by Ti-plasmids, Ri plasmids, plant viral vector, directly delivered DNA, microinjection, conductance, agriculture bacillus mediated etc. up to carrier Conventional biology methods are transformed into plant cell or tissue.

The recombinant expression carrier of the gene can be contained with existing plant expression vector construction.The plant expression vector Including double base agrobacterium vector with the carrier that can be used for plant micropellet bombardment etc..Use the gene constructed recombinant expression carrier When, any enhanced, composing type, organizing specific type or inducible promoter can be added before its transcription initiation nucleotides, They can be used alone or are used in combination with other plant promoters；Additionally, using the gene constructed recombinant expression carrier When, enhancer, including translational enhancer or transcriptional enhancer are it is also possible to use, these enhancer regions can be ATG initiation codons Son or neighboring region initiation codon etc., but must be identical with the reading frame of coded sequence, to ensure that the correct of whole sequence turns over Translate.The source of the translation control signal and initiation codon is extensive, can be natural, or synthesis.Turn over Translate initiation region and can come from transcription initiation region or structural gene.For the ease of being reflected to transgenic plant cells or plant It is fixed and screen, plant expression vector used can be processed, be such as added in expression in plant can produce color change enzyme or The gene of luminophor, resistant antibiotic marker or anti-chemical reagent marker gene etc..

The recombinant expression carrier will concretely insert the double-strand shown in the sequence 1 of sequence table in carrier pGWB18 The recombinant plasmid that DNA molecular is obtained.

The plant that set out described in any of the above is monocotyledon or dicotyledon.The monocotyledon can be Poales Plant.The Poales plant can be grass.The grass can be oryza plant.The oryza plant is specific Can be paddy rice, such as Nipponbare paddy rice.

The salt tolerance reduction is embodied as at least one in following (d1)-(d3)：

(d1) under high salt conditions, survival rate is less than the plant that sets out；

(d2) under high salt conditions, peroxidase activity is less than the plant that sets out；

(d3) under high salt conditions, mda content is higher than the plant that sets out.

The high salt conditions concretely 100mM NaCl treatment.

The present invention also protects the application of OsSGT1 albumen, is following (c1) or (c2)：

(c1) plant salt endurance is regulated and controled；

(c2) plant salt endurance is reduced.

The present invention also application of the protection any of the above methods described in plant breeding.

The purpose of the breeding is the low plant of seed selection salt tolerance.

The present invention also protects a kind of plant breeding method, comprises the following steps：Increase the expression of OsSGT1 albumen in plant Amount and/or activity, obtain the paddy rice of salt tolerance reduction.

The present invention also protects a kind of method for reducing plant salt endurance, comprises the following steps：Increase OsSGT1 eggs in plant White expression quantity and/or activity, so as to reduce plant salt endurance.

Plant described in any of the above is monocotyledon or dicotyledon.The monocotyledon can be planted for Poales Thing.The Poales plant can be grass.The grass can be oryza plant.The oryza plant specifically may be used It is paddy rice, such as Nipponbare paddy rice.

The present invention has found that OsSGT1 albumen and its encoding gene have the function of regulation and control plant salt endurance by studying.Raise The expression of OsSGT1 genes can significantly reduce the salt tolerance of plant in plant.The present invention for excavate plant salt tolerance gene, The genesis mechanism of salt stress is solved, plant salt endurance is improved, the strong plant variety of salt tolerance is cultivated, anticipated with important theoretical direction Justice and production application value.

Brief description of the drawings

Fig. 1 is the PCR qualification results of partial transgenic plant.

Fig. 2 is the Western blot results of transfer-gen plant.

Fig. 3 is WT lines and transfer-gen plant salt-tolerant phenotype observation result.

Fig. 4 is WT lines and transfer-gen plant survival rate statistics.

Fig. 5 is WT lines and transfer-gen plant MDA (MDA) content statistics.

Fig. 6 is WT lines and transfer-gen plant POD (peroxidase) activity statisticses.

Specific embodiment

Following embodiment facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments Method, unless otherwise specified, is conventional method.Test material used in following embodiments, unless otherwise specified, is certainly What routine biochemistry reagent shop was commercially available.Quantitative test in following examples, is respectively provided with three repetitions and tests, and as a result makes even Average.

The coding region sequence of OsSGT1 genes as shown in the sequence 1 of sequence table, the albumen shown in the sequence 2 of polynucleotide Matter (OsSGT1 albumen).

Rice strain Nipponbare：Bibliography：Li Lei, Xue's fragrance a, left side shows quick, waits the most suitable blade of rice varieties Nipponbare to roll up Curvature research [J] Yangzhou Universitys journal agricultural and life science version, 2013 (2)：47-51.；The public can be from Chinese agriculture section Crop science research institute of institute obtains.

Carrier pDONR201：Invitrogen companies, article No.：11798-014.

Carrier pGWB18：BioVectorNTCC Type Tissue Collections.

Inducing culture：CaCl₂·2H₂O440mg, KH₂PO₄170mg, MgSO₄·7H₂O370mg, NH₄NO₃1650mg, KNO₃1900mg, KI 0.83mg, CoCl₂·6H₂O0.025mg, H₃BO₃6.2mg, Na₂MoO₄·2H₂O0.25mg, MnSO₄· 4H₂O22.3mg, CuSO₄·5H₂O0.025mg, ZnSO₄·7H₂O8.6mg, Na₂-EDTA·2H₂O37.3mg, FeSO₄· 7H₂O27.8mg, VB10.1mg, VB60.5mg, nicotinic acid 0.5mg, inositol 100mg, glycine 2mg, 2,4-D 2mg hydrolyze junket egg White 2g, maltose 30g, agar 3g, deionized water adds to 1L.

Infect culture medium：Compound method is referring to bibliography：Hiei Y, Ohta S, Komari T, et Al.Efficient transformation of rice (Oryza sativa, L.) mediated by Agrobacterium, and sequence analysis of the boundaries of the T-DNA [J] .Plant Journal, 1994,6 (2)：271-282..The concentration of acetosyringone in bibliography is replaced with 200 μM.

Co-culture culture medium：Acetosyringone and glucose are added in inducing culture, makes acetosyringone in culture Final concentration of 200 μM in base, glucose final concentration of 10g/L in the medium.

Micro-organisms base：The cephalosporin in inducing culture, makes cephalosporin in the medium final concentration of 500mg/L。

Screening and culturing medium：Hygromycin and cephalosporin are added in inducing culture, makes hygromycin end in the medium Concentration is 65mg/L, cephalosporin final concentration of 500mg/L in the medium.

Pre- regeneration culture medium：CaCl₂·2H₂O440mg, KH₂PO₄170mg, MgSO₄·7H₂O370mg, NH₄NO₃1650mg, KNO₃1900mg, KI 0.83mg, CoCl₂·6H₂O0.025mg, H₃BO₃6.2mg, Na₂MoO₄·2H₂O0.25mg, MnSO₄· 4H₂O22.3mg, CuSO₄·5H₂O0.025mg, ZnSO₄·7H₂O8.6mg, Na₂-EDTA·2H₂O37.3mg, FeSO₄· 7H₂O27.8mg, VB1 0.1mg, VB60.5mg, nicotinic acid 0.5mg, inositol 100mg, glycine 2mg, caseinhydrolysate 2g, malt Sugared 30g, agar 3g, kinetin 2mg, methyl α-naphthyl acetate 1mg, deionized water adds to 1L；Hygromycin is added before being down flat plate and makes its dense It is 50mg/L to spend.

Regeneration culture medium：CaCl₂·2H₂O440mg, KH₂PO₄170mg, MgSO₄·7H₂O370mg, NH₄NO₃1650mg, KNO₃1900mg, KI0.83mg, CoCl₂·6H₂O0.025mg, H₃BO₃6.2mg, Na₂MoO₄·2H₂O0.25mg, MnSO₄· 4H₂O22.3mg, CuSO₄·5H₂O0.025mg, ZnSO₄·7H₂O8.6mg, Na₂-EDTA·2H₂O37.3mg, FeSO₄· 7H₂O27.8mg, VB1 0.1mg, VB60.5mg, nicotinic acid 0.5mg, inositol 100mg, glycine 2mg, caseinhydrolysate 2g, malt Sugared 30g, agar 6g, kinetin 2mg, methyl α-naphthyl acetate 1mg, deionized water adds to 1L；Hygromycin is added before being down flat plate and makes its dense It is 50mg/L to spend.

POD reaction solutions：The μ L of guaiacol 28, magnetic stirring apparatus are added in 50ml 0.1M phosphate buffers (pH 6.0) Heating stirring is allowed to be completely dissolved, and 30% (percent by volume) H is added after cooling₂O₂The μ L of the aqueous solution 19 mix.

MDA reaction solutions：0.6gTBA (thiobarbituricacidα-), first with a small amount of 1M NaOH aqueous dissolutions, then with 10% (percent by volume) TCA (trichloroacetic acid) aqueous solution is settled to 100 milliliters.

The functional analysis of embodiment 1, OsSGT1 albumen and its encoding gene

First, the structure of OsSGT1 gene overexpressions carrier

1st, the total serum IgE of Nipponbare rice leaf is extracted, reverse transcription is cDNA.

2nd, cDNA as template is obtained with step 1, entering performing PCR using primer attB-F1 and primer attB-R1 expands, and obtains Amplified production.

attB-F1：5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTGCATGGCAACCGCCGCC-3′；

attB-R1：5′-GGGGACCACTTTGTACAAGAAAGCTGGGTGAGTACTCCCATTTCTTAAGCTCC-3′。

3rd, reacted by BP, the amplified production that step 2 is obtained imports carrier pDONR201, obtains containing sequence The positive Entry clone plasmids pDONR201-OsSGT1 of the double chain DNA molecule shown in 1.

BP reaction systems：Amplified production 2.7 μ L (50-100ng), carrier pDONR201 1.0 μ L (30-50ng), 5 × BP The μ L of 1.0 μ L, BP Enzymemix of ReactionBuffer 0.3.

BP reaction conditions：25 DEG C of warm bath 1h.

4th, the positive Entry clone plasmids pDONR201-OsSGT1 that step 3 is obtained is taken, it is anti-to carry out LR with carrier pGWB18 Should, obtain the 35S containing the double chain DNA molecule shown in sequence 1：：MYC-OsSGT1 expression vectors (have been sequenced and have tested Card).

LR reaction systems：Entry clone plasmids pDONR201-OsSGT1 1 μ L (50-100ng), carrier pGWB18 1 μ L (50-100ng), the μ L of LR enzymemix 0.5.

LR reaction conditions：25 DEG C of warm bath 1h.

2nd, the acquisition of overexpression transgenic paddy rice

1st, the mature seed of Nipponbare paddy rice is taken, mechanical dejacketing, the full bright and clean seed without bacterial plaque of picking carries out disinfection.

2nd, the seed after step 1 is sterilized is inoculated into 28 DEG C of inducing culture, light culture 14 days or so, chooses outward appearance good It is good, the good callus of growing power.

3rd, the expression vector 35S of step one structure is taken：：MYC-OsSGT1, imports Agrobacterium tumefaciems EHA105, is recombinated Bacterium EHA105/35S：：MYC-OsSGT1.

4th, the recombinant bacterium EHA105/35S that step 3 is obtained is taken：：MYC-OsSGT1, with the resuspended thalline of culture medium is infected, obtains Bacteria suspension (OD_600nm=1.0).

5th, the callus for completing step 2 is soaked in bacteria suspension prepared by step 4, infects 20min.By bacterium after infecting Suspension is outwelled, and takes callus, uses aseptic filter paper suck dry moisture, is subsequently placed on co-cultivation culture medium, cultivates 28 DEG C of light cultures 50-55h。

6th, after completing step 5, select during surface do not have the callus of obvious Agrobacterium to move to micro-organisms base, 28 DEG C dark Culture 3-4 days.

7th, after completing step 6, callus is moved on screening and culturing medium 28 DEG C of light culture cultures 30 days, every 10 days subcultures Once.

8th, after completing step 7, the callus of fresh hygromycin resistance is taken, is connected in pre- regeneration culture medium, 28 DEG C of dark trainings Support 7 days, (12h illumination/12h is dark) continues to be transferred on regeneration culture medium after cultivating 7 days between being subsequently placed in illumination cultivation, continues light According to culture, until growing regeneration plant, T is obtained₀For plant.T₀For plant selfing, T is obtained₁For plant.T₁For plant selfing, Obtain T₂For plant.T₂For plant selfing, T is obtained₃For plant.

3rd, the identification of overexpression transgenic paddy rice

1st, the T of each strain of step 2 acquisition is taken₂For plant, the total serum IgE and reverse transcription for extracting plant leaf are cDNA. With cDNA as template, performing PCR is entered using primer Hyg-F and Hyg-R and is identified.Using over-express vector 35S：：MYC-OsSGT1 makees It is positive control, using the cDNA of Nipponbare paddy rice as negative control.

Hyg-F：5’-CTATTTCTTTGCCCTCGGAC-3’；

Hyg-R：5’-CCTGACCTATTGCATCTCCC-3’.

If for a certain T₀For plant, the T of its sampling Detection₂PCR qualification results for plant are the positive, the T₀Generation Plant and its self progeny are a transgenic line excessively for homozygosis.

The PCR qualification results of plant part are as shown in Figure 1.In Fig. 1, M is DNA Maker ,+it is positive control ,-it is feminine gender Control, swimming lane 1-17 is corresponding in turn to 17 T of different strains₂For plant.Result shows that 17 plant of strain can amplify The specific band consistent with plasmid vector hygromycin gene, 17 strains are the transgenic line excessively of homozygosis.

2nd, selecting step 1 identify correct 6 transgenic lines (be named as SA11, SA22, SA24, SA28, SA35 and SA36), each strain T is analyzed using western blot₂For OsSGT1 expressing quantities in plant (MYC antibody), identification turns base Because of the overexpression efficiency of strain.

Testing result is as shown in Figure 2.Result shows：Purpose band is detected in 6 transgenic lines, on total protein On the premise of sample amount is consistent, the protein accumulation amount of OsSGT1 is significantly higher than transgenosis in transgenic line SA22, SA24 and SA28 The protein content of OsSGT1 in strain SA11, SA35 and SA36.

4th, the acquisition of empty carrier strain is turned

Expression vector 35S is substituted using carrier pGWB18：：MYC-OsSGT1 is operated according to the 1-8 of step 2, is obtained Turn empty carrier strain.

5th, the Salt-Tolerance Identification of overexpression transgenic line

Plant to be measured is：Nipponbare paddy rice (wild type), the T of overexpression transgenic line SA24₃Turn for plant, overexpression The T of gene strain SA28₃For plant, turn the T of empty carrier strain₃For plant.

1st, the seed of plant to be measured is put in 50 DEG C of baking ovens and is dried 3 days, it is water-soluble with 0.5% (percent by volume) sodium hypochlorite Immersion bubble surface sterilization was carried out to seed in 30 minutes, then in 37 DEG C of incubators seed soaking, vernalization to showing money or valuables one carries unintentionally, period, about 12h was changed Water.

2nd, select the step 1 consistent seed that germinates to be sowed, cultivated one week with distilled water (pH=5.5), Zhi Houyong (compound method sees reference document Yoshida nutrient solutions：Yoshida S, Forno D A, Cock J H.Laboratory manual for physiological studies of rice.Laboratory manual for physiological Studies of rice.International Rice Research Institute, 1976.) 28 DEG C continue to cultivate to three Ye Qi, is then transferred to plant 28 DEG C of the Yoshida nutrient solutions containing 100mM NaCl and continues to cultivate (salt treatment), exists respectively The 3rd day, the 4th day, the 5th day, the 6th day, the 7th day, the 8th day and the 10th day of salt treatment counts its survival rate.

Result is as shown in Figure 3 and Figure 4.Fig. 3 is the Phenotypic Observation result of the 10th day.In Fig. 3, upper left and upper right are wild type (CK) Phenotypic Observation result, lower-left is the Phenotypic Observation result of transfer-gen plant SA28, and bottom right is transfer-gen plant SA24's Phenotypic Observation result.Fig. 4 is survival rate statistics.Result shows that 100mM NaCl are processed the 4th day and started, WT lines (CK) survival rate is higher than transfer-gen plant SA24 and SA28, and the 10th day, the survival rate of WT lines was respectively 60% He 80%, and the survival rate of transfer-gen plant SA24 is 38% for the survival rate of 13.3%, SA28, i.e., transfer-gen plant is to salt stress Sensitiveness be significantly higher than wild type.There was no significant difference with wild type to turn the survival rate of empty carrier plant.

3rd, take step 2 using 100mM NaCl process the 7th day Leaf Age bit length gesture it is homogeneous as plant leaf blade, weigh 0.5g, shreds and is placed in the mortar of precooling, adds the PBS (NaH of 21.25ml 0.2M of 3ml precoolings₂PO₄The aqueous solution with The Na of 228.25ml 0.2M₂HPO₄The aqueous solution is mixed, and distilled water is settled to 1000ml, pH=7.8) it is fully ground, by lapping liquid Pour into centrifuge tube, then mortar is washed with the PBS of 2ml precoolings, and pour into centrifuge tube, 4 DEG C, 6,000rpm centrifugation 30min inhale 5ml supernatants (i.e. zyme extract) are taken, the measure of POD (peroxidase) activity and MDA (MDA) content is carried out.

(1) POD (peroxidase) determination of activity：During 20 μ L zyme extracts and 3ml POD reaction solutions are added into cuvette, It is quick to mix and determine the light absorption value under 470nm immediately, every 1 minute reading 1 time, read 3 times altogether, with absorbance per minute change Change value (Δ A470/mingFW) represents the size of enzyme activity.

Set and replace the blank of zyme extract using 20 μ L PBS, zeroing.

POD activity (Δ A470/mingFW)=Δs A470 × V × Va × W=Δs A470 × 5 × 0.02 × 0.5 =Δ A470 × 500

ΔA470：Differences of the A470 in 0-3min；V：Extract solution cumulative volume (mL)；Va：Determine zyme extract volume (mL)； W：Leaf dry weight (g)；

(2) MDA is determined：1ml zyme extracts and 2ml MDA reaction solutions are added in centrifuge tube, boiling water bath is sealed 15min, after rapid cooling, 12,000rpm centrifugation 10min, Aspirate supernatant is simultaneously determined under 600,532, tri- wavelength of 450nm Its light absorption value.

MDA(μmol·g^-1·F^-1W^-1)=(6.45 × (D532-D600) -0.56 × D450) × V/Va/W

V：Extract solution cumulative volume (mL)；Va：Determine zyme extract volume (mL)；W：Leaf dry weight (g)；

Result is as shown in Figure 5 and Figure 6.Fig. 5 is MDA content statisticses, and Fig. 6 is POD activity statisticses.As a result table Bright, under condition of salt stress, the content of MDA is significantly higher than wild type (CK) in rotaring gene plant blade, and cell membrane is by situation than wild Raw type is serious, and POD enzymatic activitys are substantially less than wild type, show effectively remove internal oxygen in rotaring gene plant blade Free radical, reduces the peroxidative damage that the oxygen radical of salt stress generation is caused to cell membrane.Turn the statistics knot of empty carrier plant There was no significant difference with wild type for fruit.

<110>Institute of Crop Science, Chinese Academy of Agricultural Science

Shenzhen Biology Breeding innovation research institute of the Chinese Academy of Agricultural Sciences

<120>The application of OsSGT1 albumen and its encoding gene in plant salt tolerance resistance is regulated and controled

<160> 2

<210> 1

<211> 1104

<212> DNA

<213>Paddy rice（Oryza sativa）

<400> 1

atggcaaccg ccgccgcgtc ggatctggag agcaaggcca aggcggcctt cgtcgacgac 60

gacttcgagc tcgccgccga gctctacacg caggcaatcg aggccagccc cgccaccgcc 120

gagctctacg ccgaccgcgc ccaggcccat atcaagctag gcaactacac tgaggctgta 180

gctgatgcta acaaggccat tgaacttgac ccatcaatgc acaaggctta tcttcgtaaa 240

ggcgctgcat gtatacgact ggaggagtat caaactgcaa aagcagctct tgaattgggt 300

tactcgttcg catctggtga ctcaaggttt actcgcctaa tgaaggagtg tgatgagcgc 360

attgctgagg agcttagtga agtccctgtt aagaaggctg aagatggagc agctgccccc 420

tctgttgctt cttttgttga ggaaaaggat gatgctgcaa acatggataa tacaccacca 480

atggtagaag tgaagccaaa atacaggcac gacttctaca acagtgctac agaagttgta 540

ttgacaattt ttgcaaaggg tgttcctgct gagaatgttg ttgttgattt tggtgaacaa 600

atgttaagtg tgtcgattga agtccctgga gaggagccgt accattttca gcctcgtctg 660

ttttctaaga tcatccctga gaaaagcaga taccaagtgc tatccacgaa ggttgaaata 720

agactggcta aagctgaaca gattacatgg acctcacttg attatgataa aaaaccaaag 780

gctgttccac aaaagataat ccctccagct gaatcggccc agaggccatc atatccttcc 840

tcaaaatcca agaaagactg ggataaactg gaagctgaag ttaaaaagga ggagaaggag 900

gagaagcttg aaggcgatgc tgcattgaac aaatttttcc gtgacatcta cagtgatgct 960

gatgaagaca tgcgacgagc aatgatgaaa tcttttgttg aatctaacgg tactgttctg 1020

tcgaccaatt ggaaagatgt tggctcgaag aaggtagagg gaagcccacc tgatgggatg 1080

gagcttaaga aatgggagta ctaa 1104

<210> 2

<211> 367

<212> PRT

<213>Paddy rice（Oryza sativa）

<400> 2

Met Ala Thr Ala Ala Ala Ser Asp Leu Glu Ser Lys Ala Lys Ala Ala

1 5 10 15

Phe Val Asp Asp Asp Phe Glu Leu Ala Ala Glu Leu Tyr Thr Gln Ala

20 25 30

Ile Glu Ala Ser Pro Ala Thr Ala Glu Leu Tyr Ala Asp Arg Ala Gln

35 40 45

Ala His Ile Lys Leu Gly Asn Tyr Thr Glu Ala Val Ala Asp Ala Asn

50 55 60

Lys Ala Ile Glu Leu Asp Pro Ser Met His Lys Ala Tyr Leu Arg Lys

65 70 75 80

Gly Ala Ala Cys Ile Arg Leu Glu Glu Tyr Gln Thr Ala Lys Ala Ala

85 90 95

Leu Glu Leu Gly Tyr Ser Phe Ala Ser Gly Asp Ser Arg Phe Thr Arg

100 105 110

Leu Met Lys Glu Cys Asp Glu Arg Ile Ala Glu Glu Leu Ser Glu Val

115 120 125

Pro Val Lys Lys Ala Glu Asp Gly Ala Ala Ala Pro Ser Val Ala Ser

130 135 140

Phe Val Glu Glu Lys Asp Asp Ala Ala Asn Met Asp Asn Thr Pro Pro

145 150 155 160

Met Val Glu Val Lys Pro Lys Tyr Arg His Asp Phe Tyr Asn Ser Ala

165 170 175

Thr Glu Val Val Leu Thr Ile Phe Ala Lys Gly Val Pro Ala Glu Asn

180 185 190

Val Val Val Asp Phe Gly Glu Gln Met Leu Ser Val Ser Ile Glu Val

195 200 205

Pro Gly Glu Glu Pro Tyr His Phe Gln Pro Arg Leu Phe Ser Lys Ile

210 215 220

Ile Pro Glu Lys Ser Arg Tyr Gln Val Leu Ser Thr Lys Val Glu Ile

225 230 235 240

Arg Leu Ala Lys Ala Glu Gln Ile Thr Trp Thr Ser Leu Asp Tyr Asp

245 250 255

Lys Lys Pro Lys Ala Val Pro Gln Lys Ile Ile Pro Pro Ala Glu Ser

260 265 270

Ala Gln Arg Pro Ser Tyr Pro Ser Ser Lys Ser Lys Lys Asp Trp Asp

275 280 285

Lys Leu Glu Ala Glu Val Lys Lys Glu Glu Lys Glu Glu Lys Leu Glu

290 295 300

Gly Asp Ala Ala Leu Asn Lys Phe Phe Arg Asp Ile Tyr Ser Asp Ala

305 310 315 320

Asp Glu Asp Met Arg Arg Ala Met Met Lys Ser Phe Val Glu Ser Asn

325 330 335

Gly Thr Val Leu Ser Thr Asn Trp Lys Asp Val Gly Ser Lys Lys Val

340 345 350

Glu Gly Ser Pro Pro Asp Gly Met Glu Leu Lys Lys Trp Glu Tyr

355 360 365

Claims (10)

1. a kind of method for cultivating genetically modified plants, comprises the following steps：The channel genes for encoding OsSGT1 albumen are set out plant Thing, obtains the genetically modified plants of salt tolerance reduction.

2. the method for claim 1, it is characterised in that：The OsSGT1 albumen, is following (a1) or (a2)：

(a1) protein that the amino acid sequence shown in sequence in sequence table 2 is constituted；

(a2) by the amino acid sequence of sequence 2 by the substitution of one or several amino acid residues and/or missing and/or addition and As sequence 2 derived from the protein related to plant salt endurance.

3. method as claimed in claim 1 or 2, it is characterised in that：" gene of coding OsSGT1 albumen " is as follows Or (b2) or (b3) (b1)：

(b1) DNA molecular of the code area as shown in the sequence 1 from the nucleotides of 5 ' end 1-1104 of sequence table；

(b2) the DNA sequence dna hybridization that is limited to (b1) under strict conditions and the protein related with plant salt endurance is encoded DNA molecular；

(b3) there is more than 90% homology to the DNA sequence dna that (b1) or (b2) is limited and encodes the egg related with plant salt endurance The DNA molecular of white matter.

4. the method as described in claim 1-3 is any, it is characterised in that：The plant that sets out is for monocotyledon or dicotyledonous Plant.

The application of 5.OsSGT1 albumen, is following (c1) or (c2)：

(c1) plant salt endurance is regulated and controled；

(c2) plant salt endurance is reduced.

6. application as claimed in claim 5, it is characterised in that：The OsSGT1 albumen, is following (a1) or (a2)：

(a1) protein that the amino acid sequence shown in sequence in sequence table 2 is constituted；

(a2) by the amino acid sequence of sequence 2 by the substitution of one or several amino acid residues and/or missing and/or addition and As sequence 2 derived from the protein related to plant salt endurance.

7. application of any described methods of claim 1-4 in plant breeding.

8. a kind of plant breeding method, comprises the following steps：Increase the expression quantity and/or activity of OsSGT1 albumen in plant, obtain To the paddy rice of salt tolerance reduction.

9. a kind of method for reducing plant salt endurance, comprises the following steps：Increase plant in OsSGT1 albumen expression quantity and/or Activity, so as to reduce plant salt endurance.

10. method as claimed in claim 8 or 9, it is characterised in that：The OsSGT1 albumen, is following (a1) or (a2)：

(a1) protein that the amino acid sequence shown in sequence in sequence table 2 is constituted；

(a2) by the amino acid sequence of sequence 2 by the substitution of one or several amino acid residues and/or missing and/or addition and As sequence 2 derived from the protein related to plant salt endurance.