CN102146129A - Protein SeVP1 related to stress tolerance of plants and encoding gene and application thereof - Google Patents

Abstract

The invention discloses a protein SeVP1 related to stress tolerance of plants and an encoding gene and application thereof. The protein is a protein in the following (a) and (b): (a) a protein consisting of an amino acid sequence shown in SEQ ID NO:1; and (b) a protein which is obtained by carrying out substitution and/or deletion and/or addition of one or more amino acid residues on the amino acid sequence in SEQ ID NO:1, is related to the stress tolerance of the plants and is derivative from (a). Experiments prove that by the protein and the encoding gene thereof, the salt tolerance and the low phosphorous resistance of the plants can be obviously improved; a theoretical principle and a gene resource are provided for genetic improvement of salt resistance and low phosphorous resistance crops; and the protein and the encoding gene thereof have wide application prospect in the field of genetic breeding of the plants.

Description

Plant stress tolerance correlative protein SeVP1 and encoding gene thereof and application

Technical field

The present invention relates to a kind of plant stress tolerance correlative protein SeVP1 and encoding gene and application.

Background technology

Salt stress is one of main abiotic stress of occurring in nature, soil middle and high concentration Na ⁺G and D to many plants causes very big injury.Salt stress mainly contains to the injury of plant that the factor of two aspects causes, and the one, ion is coerced, and the 2nd, osmotic stress.Since the toxic action that ion is coerced, a large amount of Na ⁺Enter in the plant, not only broken the intravital ionic equilibrium of plant, and excessive N a in the vegetable cell ⁺Can influence the biochemical metabolism of vegetable cell, intracellular reactive oxygen species is risen to be increased, and the peroxidation aggravation causes the damage of film fat or membranin, and membrane permeability increases, and soluble substance exosmoses in the born of the same parents.Salinity also makes the flow of water in the soil reduce, thereby causes osmotic stress, causes that plant materials moisture lacks.Along with plant genetics and development of molecular biology, people have had comparatively deep understanding to plant to the molecular mechanism that salt stress reacts.At present, many plant salt tolerance genes involveds are cloned in succession, and the relation of these genes and plant salt tolerance proterties is also tentatively confirmed.

Phosphorus is as one of necessary macroelement of growth and development of plants, and it has almost participated in all vital movement processes of plant, and its shortage must have a strong impact on growth and development of plant.Plant is mainly satisfied its phosphorus nutrition demand by the soluble phosphate that root system absorbs in the soil.The further investigation plant efficient absorbs the molecular biology basis of soil phosphorus, and the efficient phosphorus nutrition genetic resources of digging utilization plant and even other biological is cultivated the phosphorus efficiency crop varieties, has become the focus of plant biology area research.The variation of plant root species form is undoubtedly the most significant adaptation mechanism of plant to low-phosphorus stress.Studies show that in a large number low-phosphorus stress causes plant root species form generation noticeable change, comprise length and the number and the root absorbing area increase of root/shoot ratio, total root length, lateral root, root average diameter reduction etc., to enlarge the contact area of root system and soil, improve the efficient that absorbs of soil phosphorus.In recent years, the generation of root system of plant and the research of change mechanism there have been very big breakthrough, have mainly concentrated on hormone such as growth hormone and form effect during especially lateral root takes place at root system.Growth hormone is considered to play an important role in root system configuration and lateral root generating process.

H ⁺-PPase is a kind of H that is positioned on the vegetation water vacuolar membrane ⁺Translocator, it can be the free energy and the H of PPi hydrolysis generation ⁺Transmembrane transport phase coupling connection is when being hydrolyzed to PPi 2 Pi, also with the H in the tenuigenin ⁺Pump in the vacuole, play proton pump.H ⁺-PPase and vacuole skin H ⁺-ATPase sets up H together ⁺Stride the vacuole skin electrochemical gradient, the secondary active transport that one side is striden vacuole skin for various solutes (as positively charged ion, negatively charged ion, amino acid and carbohydrate etc.) molecule provides motivating force, cell plasma balance and osmotic equilibrium can be kept, some mineral ions can be alleviated again (as Na ⁺And Cl ^-) murder by poisoning of pair cell matter; Can make the alkalization of vacuole acidifying and tenuigenin on the other hand, help carrying out smoothly of biochemical reactions in the tenuigenin.

Salicornia europaeal (Salicornia europaea) is the true halophytes of a kind of carnification that belongs to Chenopodiaceae, extensively is distributed near coastal and the inland brine lake, can accumulate high NaCl to dry weight 50%, is considered in the world a kind of higher plant of salt tolerant.

Summary of the invention

An object of the present invention is to provide a kind of albumen and encoding gene thereof.

Albumen provided by the present invention, be following a) or b) protein:

A) protein of forming by the aminoacid sequence shown in the SEQ ID NO:1;

B) with the aminoacid sequence shown in the SEQ ID NO:1 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and relevant with plant stress tolerance by a) deutero-protein.

Described encoding gene is following 1) or 2) or 3) or 4) shown in:

1) its nucleotide sequence is from dna molecular shown in 5 ' terminal 197-2491 or the 168-2616 position Nucleotide among the SEQ ID NO:2;

2) its nucleotide sequence is a dna molecular shown in the SEQ ID NO:2;

3) under stringent condition with 1) or 2) the dna sequence dna hybridization that limits and the dna molecular of encoding said proteins; Described stringent condition can be 0.1 * SSPE (or 0.1 * SSC), in the solution of 0.1%SDS,, and wash film with this solution 65 ℃ of hybridization down.

4) with 1) or 2) dna sequence dna that limits has the homology more than 90% and the dna molecular of encoding said proteins.

Albumen in above-mentioned in order to make (a) is convenient to purifying, can connect label as shown in table 1 at proteinic N-terminal of being made up of the aminoacid sequence shown in the SEQ ID NO:1 or C-terminal.

The sequence of table 1 label

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

Above-mentioned (a) but or the albumen synthetic (b), also can synthesize its encoding gene earlier, carry out biology again and express and to obtain.Proteic encoding gene in above-mentioned (b) can be by the codon with one or several amino-acid residue of disappearance in the dna sequence dna shown in the SEQ ID NO:2, and/or carry out the missense mutation of one or several base pair, and/or obtain at the encoding sequence that its 5 ' end and/or 3 ' end connects the label shown in the table 1.

Increase above-mentioned arbitrary described encoding gene total length or its any segmental primer to also belonging to protection scope of the present invention; Described primer is to being specially dna molecular shown in dna molecular shown in the SEQ ID NO:3 and the SEQ ID NO:4.

The recombinant vectors, reorganization bacterium, transgenic cell line, recombinant virus or the expression cassette that contain above-mentioned arbitrary described encoding gene also belong to protection scope of the present invention.

Described recombinant vectors is for to insert the recombinant expression vector that described encoding gene obtains in the multiple clone site of expression vector pCAMBIA3300-ubiquitin;

Described pCAMBIA3300-ubiquitin makes up as follows: insert the ubiquitin promoter sequence between the HindIII of pCAMBIA3300 and BamHI; Described ubiquitin promoter sequence is shown in SEQ ID NO:5.

Above-mentioned arbitrary described albumen, described encoding gene, the application of recombinant vectors in the resistance of reverse that improves plant also belong to protection scope of the present invention.

In the above-mentioned application, described plant is monocotyledons or dicotyledons; Described dicotyledons is specially Arabidopis thaliana.

In the above-mentioned application, described resistance of reverse is a salt tolerant and/or anti-low-phosphorous.

Another object of the present invention provides a kind of method of cultivating the transgenic plant of resistance of reverse raising.

The method of the transgenic plant that cultivation resistance of reverse provided by the present invention improves comprises the steps: to import above-mentioned arbitrary described proteic encoding gene in the plant that sets out, and obtains the purpose transgenic plant that resistance of reverse is higher than the described plant that sets out.

In the said process, described encoding gene imports by above-mentioned arbitrary described recombinant vectors.

In the said process, described plant is monocotyledons or dicotyledons; Described dicotyledons is specially Arabidopis thaliana.

In the said process, described resistance of reverse is a salt tolerant and/or anti-low-phosphorous.

Experimental results show that, albumen of the present invention and encoding gene thereof can significantly improve salt tolerance and the anti-low-phosphorous property of plant, the present invention provides theoretical basis and genetic resources for anti-salt and anti-low-phosphorous crop genetic improvement, has broad application prospects in the genetic breeding field of plant.

Description of drawings

Fig. 1 is under NaCl and the low-phosphorus stress, and SeVP1 is at salicornia europaeal root and the relative accumulation volume of transcript in the over-ground part.(a) 200 and 800mM NaCl handle; (b) low-phosphorous (10 μ M Pi) handles.

Fig. 2 is the transcript expression of SeVP1 in each Arabidopis thaliana strain system.WT, wild-type; Avp1, the afunction mutant; SeVP1-1 ,-2 ,-3 ,-4 ,-5, cross the transgenic line of expressing SeVP1; Avp1:SeVP1-1 ,-2 ,-3, the strain that has complementary functions of expressing SeVP1 is.

Fig. 3 handles the germination rate of each transgenic arabidopsis seed down for 150mM NaCl.(a) 5 days growing state behind the seed germination; (b) statistics of the germination rate in the seed germination 7 days.WT is a wild-type; L1-L5 is that 5 SeVP1 cross expression strain system.

Fig. 4 is the growing state of Arabidopis thaliana seedling under normal growth condition and condition of salt stress.WT, wild-type; Avp1, the afunction mutant; SeVP1-1 ,-2 ,-3 ,-4 ,-5, cross the transgenic line of expressing SeVP1; Avp1:SeVP1-1 ,-2 ,-3, the strain that has complementary functions of expressing SeVP1 is.

Fig. 5 is that the Arabidopis thaliana seedling is at the main root of salt stress in the time of 10 days long (a), fresh weight (b) and chlorophyll content (c).WT, wild-type; Avp1, the afunction mutant; SeVP1-1 ,-2 ,-3 ,-4 ,-5, cross the transgenic line of expressing SeVP1; Avp1:SeVP1-1 ,-2 ,-3, the strain that has complementary functions of expressing SeVP1 is.* be illustrated under the same treatment condition with * *, compare with wild-type, this value reaches significant difference respectively on P＜0.0 and P＜0.01 level.

Fig. 6 is the germination rate of each transgenic arabidopsis seed under the low-phosphorous processing.WT is a wild-type; L1-L5 is that 5 SeVP1 cross expression strain system.

Fig. 7 is the growing state of Arabidopis thaliana seedling under normal growth condition and scarce phosphorus stress conditions.WT, wild-type; Avp1, the afunction mutant; SeVP1-1 ,-2 ,-3 ,-4 ,-5, cross the transgenic line of expressing SeVP1; Avp1:SeVP1-1 ,-2 ,-3, the strain that has complementary functions of expressing SeVP1 is.

Fig. 8 is that the Arabidopis thaliana seedling is lacking the main root of phosphorus processing in the time of 10 days long (a), lateral root number (b) and fresh weight (c).WT, wild-type; Avp1, the afunction mutant; SeVP1-1 ,-2 ,-3 ,-4 ,-5, cross the transgenic line of expressing SeVP1; Avp1:SeVP1-1 ,-2 ,-3, the strain that has complementary functions of expressing SeVP1 is.* be illustrated under the same treatment condition with * *, compare with wild-type, this value reaches significant difference respectively on P＜0.0 and P＜0.01 level.

Embodiment

Employed experimental technique is ordinary method if no special instructions among the following embodiment.

Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.

PCAMBIA3300-ubiquitin makes up as follows: insert the ubiquitin promoter sequence between the HindIII of pCAMBIA3300 and BamHI; Described ubiquitin promoter sequence is shown in SEQ ID NO:5.

PCAMBIA3300 is available from the Beijing DingGuo ChangSheng Biology Technology Co., Ltd.

Agrobacterium C58 bacterial strain disclosed in document " Overexpression of Organellar and Cytosolic AtHSP90in Arabidopsis thaliana Impairs Plant Tolerance to Oxidative Stress.HongmiaoSong; Pengxiang Fan; Yinxin Li.Plant Mol Biol Rep (2009) 27:342-349. ", and the public can obtain from Institute of Botany, Chinese Academy of Sciences.

(Arabidopsis biologicalresource center, ABRC), catalog number is Salk_046492C to Arabidopis thaliana afunction mutant avp1 available from Arabidopis thaliana Biological resources center.

Salicornia europaeal (S.europeae L.) seed is available from the brilliant grand marine industries Development Co., Ltd in Dafeng City, Jiangsu Province.

The salicornia europaeal seed is evenly sowed in vermiculite, treated seed germination, with the pouring of l/2Hoagland (Hoagland, 1935) nutritive medium.Seedling is cultivated in plant institute of Chinese Academy of Sciences greenhouse, and day temperature maintains 25～30 ℃, and nocturnal temperature is at 18～20 ℃, and relative humidity maintains 60～80%, and illumination condition is 16h illumination/8h dark.

The l/2Hoagland nutrient solution prescription is as follows:

The preparation of embodiment 1, gene

One, the clone of gene

The acquisition of (1) 3 ' RACE gene fragment

Utilize the Trizol total RNA of extraction method extraction salicornia europaeal blade in a small amount, utilize the golden reverse transcription of full formula system to obtain the first chain cDNA.Use primer VPP3 and 3 ' oligadT to carry out the amplification of cDNA3 ' end.Concrete PCR reaction conditions is as follows: 5cycles:94 ℃ of 30sec, 72 ℃ of 3min; 5cycles:94 ℃ of 30sec, 70 ℃ 30sec dT=-1 ℃, 72 ℃ of 3min; 27cycles:94 ℃ of 30sec, 68 ℃ of 30sec, 72 ℃ of 3min.The RACE product that obtains is carried out agarose gel electrophoresis and required dna fragmentation is cut glue reclaim, check order after being connected into the T carrier

Upstream primer VPP3 (5 '-GGTTGATGGAAGGAACTGCCA-3 ').

(2) acquisition of intermediate segment

Design upstream degenerate primer ZP1 (5 '-GGTTAA (AG) CTTTCCCCTGATTCTGG-3 '), according to 3 ' RACE gene order, design downstream special primer PR1 (5 '-CACCTCGAGCTTATCCTTCTGTTGCC-3 ').With salicornia europaeal cDNA is masterplate, and amplification obtains intermediate segment.The PCR product that obtains is carried out agarose gel electrophoresis and required dna fragmentation is cut glue reclaim, check order after being connected into the T carrier.

The experimental procedure of (3) 5 ' RACE

The primer GSP5 of use 5 ' RACE anchor primer (UPM) and gene specific (5 '-ATATGCCTCCGCCAACTCTACCGAAA-3 '), with warm start Hot

Taq archaeal dna polymerase (available from Takara company) is according to Clontech SMART ^TMThe method of RACE cDNA Amplification Kit and step are carried out the amplification of cDNA5 ' end.Concrete PCR reaction conditions is as follows: 94 ℃ of 5min; 5cycles:94 ℃ of 30sec, 68 ℃ of 30sec, 72 ℃ of 2min; 30cycles:94 ℃ of 30sec, 65 ℃ of 30sec, 72 ℃ of 2min; 72 ℃ of 10min.

Amplified production carries out agarose gel electrophoresis and required dna fragmentation is cut glue reclaim, be connected into the T carrier after, check order.

(4) acquisition of full length cDNA sequence and bioinformatic analysis

Use information biology software Genetool to 5 ' and the sequence information of 3 ' RACE carry out sequence assembling, result according to sequence assembling, (SEQ ID NO:3 is complementary with the 168-190 base of SEQ ID NO:2 at gene ORF (Open Reading Frame) both sides designs primer PF168 (5 '--ACCGAAAAAAAGGAAATTCACAC-3 ').) and PR4 (5 '--ACAGCCTTGTCATCTAACCGAGT-3 ') (SEQ IDNO:4 is complementary with the 2594-2616 base of SEQ ID NO:2.), be template with the high-fidelity Taq polysaccharase full-length cDNA that increases with salicornia europaeal children stem cDNA then.The full-length cDNA that obtains is connected into the T carrier.To the affirmation of checking order of this carrier.Then this sequence is carried out ORF and analyze, confirm the opening code-reading frame of this gene.

Sequencing result shows, the cDNA sequence of amplification shown in SEQ ID NO:2, this cDNA sequence total length 2903bp, we are with its called after SeVP1.This gene order comprises 5 ' non-translational region (5 ' UTR) and 3 ' non-translational region (opening code-reading frame of 3 ' UTR) and 2292bp (197-2491 position Nucleotide is opening code-reading frame among the SEQ ID NO:2) of 412bp of 196bp.This reading frame 764 amino acid of having encoded, aminoacid sequence is shown in SEQID NO:1.

Two, gene expression analysis under salt and the low-phosphorous condition

Salicornia europaeal is sprouted in vermiculite, change the Hoagland nutritive medium after 4 weeks over to and cultivate, 4 week the back in nutritive medium, add 200 or the NaCl of 800mM, handling 0, when 6h, 12h, 24h, 48h, getting over-ground part and root, be used for genetic expression and detect respectively.

Because the NaCl of the suitableeest growth needs 200-400mM of salicornia europaeal, so low-phosphorous processing experiment is a unfolded on the basis of the Hoagland nutritive medium that contains 200mM NaCl.Sprout 4 all backs salicornia europaeals in the vermiculite, change the Hoagland nutritive medium that contains 200mM NaCl over to and cultivated for 4 weeks, then with containing 10 μ M KH ₂PO ₄The Hoagland nutritive medium handle.When processing 0,6h, 12h, 24h, 48h, get over-ground part and root respectively, be used for genetic expression and detect.

Use Trizol reagent to extract total RNA, total RNA of extraction digests through DNase, and after the reverse transcription, (Toyobo Japan) carries out fluorescence quantitative RT-RCR and detects to utilize SYBR GreenRealtime PCR Master Mix.With salicornia europaeal actin gene as confidential reference items, by 2 ^{-Δ Δ Ct}Method relative quantification is carried out in genetic expression.

The primer sequence is as follows in the fluorescence quantitative RT-RCR:

Mark actin primer in the salicornia europaeal:

SeActup1：5′-TGAGAGATTCCGTTGCCCAG-3′

SeActdn1：5′-CCACCACTGAGCACGATGTTAC-3′

Salicornia europaeal SeVP1 gene primer:

P1-1：5′-GAGGTGTTTTCTGCCCTTATGTC-3′

P1-2：5′--ACAGCCTTGTCATCTAACCGAGT-3′

The fluorescence quantitative RT-RCR detected result shows, 200 and 800mM NaCl condition under, SeVP1 the expression amount of the transcriptional level of root and over-ground part all present the variation tendency that afterwards reduces of raising earlier (Fig. 1, a).200mM NaCl handled 12 hours, and it is the highest that expression amount reaches, and wherein the performance in root is more obvious.Compare with the NaCl processing of low concentration, 800mM NaCl induces degree bigger to what SeVP1 expressed.When 800mM NaCl handled 24h, the SeVP1 expression level of part on the ground was 5 times of untreated control approximately.

Under low-phosphorus stress, SeVP1 also presents at the relative expression quantity of root and over-ground part and raises earlier that (Fig. 1, b), wherein when processing 12h, expression level is the highest for the variation tendency that afterwards reduces.The inducing action that low-phosphorus stress is expressed SeVP1 shows particularly evident in root, and when handling 12h, this expression of gene level has improved about 50 times.

The application of embodiment 2, gene

1, the structure of plant expression vector

Used plant expression vector is pCAMBIA3300-ubiquitin, contains ubiquitin promotor and weedicide Basta selectable marker gene bar.Utilize the SmaI and the SacI of this carrier multiple clone site, the SeVP1 gene is inserted into pCAMBIA3300-ubiquitin, made up the pCAMBIA3300-ubiquitin-SeVP1 carrier.

The pCAMBIA3300-ubiquitin-SeVP1 carrier is carried out sequence verification, the result is between the SmaI and SacI site of pCAMBIA3300-ubiquitin-SeVP1 carrier, along the direction from SmaI to SacI, the sequence of gene is shown in 197-2491 position Nucleotide among the SEQ ID NO:2.

2, reorganization bacterium

Method with heat shock transforms Agrobacterium C58 bacterial strain with the plant expression vector pCAMBIA3300-ubiquitin-SeVP1 that makes up, and obtains positive reorganization bacterium.

3, transform

With the Agrobacterium C58 bacterial strain that carries plant expression vector pCAMBIA3300-ubiquitin-SeVP1, utilize agriculture bacillus mediated flower-dipping method to transform wild-type Arabidopis thaliana (Col-0) and Arabidopis thaliana afunction mutant avp1 respectively, to obtain expression strain system and the strain system that has complementary functions.Seed (the T of the Arabidopis thaliana of results ₀Generation) through broadcasting sowing equably behind the surface sterilization on the 1/2MS solid medium that contains 0.02%Basta, at first placed 2 days down in 4 ℃ the dark place, place then and cultivate after 5-10 days under 22 ℃ of illumination, the seedling of will surviving changes over to and does not contain on the antibiotic 1/2MS substratum, grew about 3 days, and moved to and grow to seed maturity in the compost.Repeat this step, be sheerly for transgenosis up to obtaining T2 or T3.

Establish following contrast simultaneously:

Change empty carrier contrast 1: in wild-type Arabidopis thaliana (Col-0), change pCAMBIA3300-ubiquitin over to, the commentaries on classics empty carrier Arabidopis thaliana that obtains.

Change empty carrier contrast 2: in Arabidopis thaliana afunction mutant avp1, change pCAMBIA3300-ubiquitin over to, the commentaries on classics empty carrier Arabidopis thaliana that obtains.

4, the evaluation of transgenic line

After the seed-coat sterilization from wild-type, mutant and each transgenic line, be seeded on the 1/2MS substratum, 4 ℃ of dark down cultivations 2 days, after 22 ℃ of illumination cultivated for two weeks down, respectively get 10 strains (about 100ng), extract total RNA and digest, after the reverse transcription through DNase, (Toyobo Japan) carries out fluorescence quantitative RT-RCR and detects to utilize SYBR Green Realtime PCR Master Mix.With Arabidopis thaliana Actin gene as confidential reference items, by 2 ^{-Δ Δ Ct}Method relative quantification is carried out in genetic expression.

The primer sequence is as follows in the fluorescence quantitative RT-RCR:

Mark Actin gene primer in the Arabidopis thaliana:

AtAct1：5′-CCCGCTATGTATGTCGCCA-3′

AtAct2：5′-AACCCTCGTAGATTGGCACA-3′

Salicornia europaeal SeVP1 gene specific primer:

Vp1up：5′-TTCCCCTGATTCTGGTCTCG-3′

Vp1down：5′-GGTTGCTCCTTCAGAAATGGC-3′

As a result, obtain 8 altogether and crossed expression strain system and 5 strain systems that have complementary functions.Getting 5 mistake expression strain systems and 3 complementary strains is that design SeVP1 special primer carries out the genetic expression component analysis.As shown in Figure 2, cross the transcript degree of expressing SeVP1 in the strain system and improved 1-3 doubly than wild-type.This this level of gene transcription significantly is lower than wild-type among the mutant avp1.Except avp1:SeVP1-2, SeVP1 is higher than wild-type at the expression amount that other two complementary strains are.These results show that SeVP1 has changed Arabidopis thaliana over to, and can stably express.It is consistent with wild-type to change empty carrier contrast 1, and it is consistent with mutant to change empty carrier contrast 2.

5, the salt resistance of transgenic arabidopsis detects

(1) seed germination experiment

Cross the Arabidopis thaliana seeds of expression strains systems (L1, L2, L3, L4, L5) from wild-type (WT) and 5, behind surface sterilization, respectively sowing and 1/2MS with contain on the 1/2MS substratum of 150mM NaCl.4 ℃ of dark down cultivations 2 days, cultivate down in 22 ℃ of illumination then, add up seed germination rate every day, second day note of 22 ℃ of illumination cultivation done the 1st day.

Result such as table 1 and shown in Figure 3.

Under the normal growth condition, from the sprouting speed no significant difference of the seed of transgenic line and wild-type, in the time of the 3rd day, the seed germination rate of all strains systems has nearly all reached 100%.150mM NaCl coerces, and has significantly suppressed the sprouting of Arabidopis thaliana seed, and the seed that nearly all strain is just began to sprout in the time of the 4th day.Yet, to compare with wild-type, seed germination rate that 5 mistakes are expressed strain system all is significantly higher than wild-type, and wherein in the time of the 5th and the 6th day, this species diversity is the most obvious.In the time of the 7th day, the germination rate of wild type seeds has only about 70%, and transfer-gen plant all more than 90%.These results show that under the salt stress, the seed germination rate that has significantly improved Arabidopis thaliana is expressed in crossing of SeVP1 gene.

Table 1, seed germination rate

"-" expression does not add NaCl, and 150mM NaCl is added in "+" expression.

(2) seedling stage, salt resistance detected

(culture condition: temperature is 22 ℃ after 3 days in growth on the 1/2MS substratum to express the Arabidopis thaliana seed of strain system and 3 strain systems that have complementary functions from wild-type, avp1 mutant, 5 mistakes, 16h illumination/8h dark)), the consistent seedling of picking growth forwards 1/2MS respectively to and contains on the 1/2MS substratum of 150mM NaCl and vertically cultivate (vertical culture condition: culture dish is vertically placed, 60 degree that tilt are cultivated, temperature is 22 ℃, 16h illumination/8h dark.)。Handle after 10 days, measure plant main root length, fresh weight and chlorophyll content.

The measuring method of chlorophyll content: round the over-ground part of strain Arabidopis thaliana, extract chlorophyll with 80% acetone extraction method, spectrophotometer method calculates chlorophyll content: C as follows _T=20.29D ₆₄₅+ 8.05D ₆₆₃

The result is as follows:

Long (em): the WT:1.62 of main root ± 0.36, SeVP1-1:3.24 ± 0.53, SeVP1-2:3.17 ± 0.54, SeVP1-3:2.08 ± 0.57, SeVP1-4:2.48 ± 0.48, SeVP1-5:2.56 ± 0.39, avp1:1.07 ± 0.24, avp1:SeVP1-1:1.48 ± 0.29, avp1:SeVP1-2:1.69 ± 0.49, avp1:SeVP1-3:1.89 ± 0.55.

Fresh weight (g/3 strain): WT:0.019 ± 0.003, SeVP1-1:0.031 ± 0.005, SeVP1-2:0.031 ± 0.007, SeVP1-3:0.025 ± 0.002, SeVP1-4:0.029 ± 0.004, SeVP1-5:0.029 ± 0.003, avp1:0.007 ± 0.003, avp1:SeVP1-1:0.021 ± 0.002, avp1:SeVP1-2:0.014 ± 0.007, avp1:SeVP1-3:0.026 ± 0.002.

Chlorophyll content (mg/g fresh weight): WT:0.21 ± 0.04, SeVP1-1:0.36 ± 0.08, SeVP1-2:0.39 ± 0.08, SeVP1-3:0.36 ± 0.04, SeVP1-4:0.36 ± 0.08, SeVP1-5:0.24 ± 0.03, avp1:0.17 ± 0.07, avp1:SeVP1-1:0.27 ± 0.06, avp1:SeVP1-2:0.30 ± 0.05, avp1:SeVP1-3:0.36 ± 0.07.

As shown in Figure 4, under the normal growth condition, transgenic line and wild-type do not have significant difference.Coerce processing in the time of 10 days at 150mMNaCl, long-living apparent being suppressed kept burning day and night of the main root of Arabidopis thaliana, the blade flavescence, mutant avp1 major part is dead.The statistics of physical signs shows, what when salt was handled 10 days, SeVP1 crossed that the main root of expressing strain system is long, fresh weight and chlorophyll content all are significantly higher than wild-type; And the fresh weight of mutant and chlorophyll content all significantly are lower than wild-type, and each index of the strain that has complementary functions system all is higher than mutant, have in addition be higher than wild-type (Fig. 5).Under the condition of salt stress, the fresh weight of expressing plant illustrates that than the high 25-30% of wild-type the expression of crossing of SeVP1 has improved the salt resistance in Arabidopis thaliana seedling stage excessively.

Among Fig. 4, WT, wild-type; Avp1, the afunction mutant; SeVP1-1 ,-2 ,-3 ,-4 ,-5, cross the transgenic line of expressing SeVP1; Avp1:SeVP1-1 ,-2 ,-3, the strain that has complementary functions of expressing SeVP1 is.

Among Fig. 5, WT, wild-type; Avp1, the afunction mutant; SeVP1-1 ,-2 ,-3 ,-4 ,-5, cross the transgenic line of expressing SeVP1; Avp1:SeVP1-1 ,-2 ,-3, the strain that has complementary functions of expressing SeVP1 is.* be illustrated under the same treatment condition with * *, compare with wild-type, this value reaches significant difference respectively on P＜0.0 and P＜0.01 level.

6, the anti-low-phosphorous property mensuration of transgenic arabidopsis

(1) seed germination experiment

Express the Arabidopis thaliana seed that strain is from wild-type and 5 mistakes, behind surface sterilization, respectively on sowing and 1/2MS and the scarce phosphorus 1/2MS substratum.4 ℃ of dark down cultivations 2 days, cultivate down in 22 ℃ of illumination then, add up seed germination rate every day, second day note of 22 ℃ of illumination cultivation done the 1st day.

Lacking phosphorus 1/2MS substratum forms and each concentration of component:

Lack the preparation and the preservation of phosphorus 1/2MS substratum mother liquor

More than in the various mother liquors, the macroelement mother liquor is 20 times of concentrated solutions, micro-mother liquor and mother liquid of iron salt are 200 times of concentrated solutions, the organic composition mother liquor is 100 times of concentrated solutions.When being used to cultivate Arabidopis thaliana, be diluted with water to 1/2MS, add 1% sucrose and 0.488g/L MES, transfer pH=5.8～6.0, add 8% agar powder at last, autoclaving with the NaOH solution of 1M.

With different under the salt stress, low-phosphorous processing has just reduced seed germination speed to the not obviously influence of final germination rate of Arabidopis thaliana seed.Under the normal condition, Arabidopis thaliana seed germination rate in the time of the 3rd day has just almost reached 100%, and under low-phosphorous condition, the germination rate of wild type seeds had only about 30% in the time of the 3rd day, just reached more than 90% during by the 4th day.The performance in crossing expression strain system of this sprouting delay phenomenon is not obvious, and in the time of the 3rd day, the seed germination rate of each transgenic line has still reached more than 90%, is significantly higher than (Fig. 6) of wild-type.

(2) seedling stage, anti-low-phosphorous ability was measured

(culture condition: temperature is 22 ℃ after 3 days in growth on the 1/2MS substratum to express the Arabidopis thaliana seed of strain system and 3 strain systems that have complementary functions from wild-type, avp1 mutant, 5 mistakes, 16h illumination/8h dark), the consistent seedling of picking growth forwards on the 1/2MS substratum of 1/2MS and scarce phosphorus and vertically cultivates (vertically culture condition: with experiment 5) respectively to.Handle after 10 days, measure plant main root length, lateral root number and fresh weight.

The result is as follows:

Long (cm): the WT:4.03 ± 0.68cm of main root, SeVP1-1:4.23 ± 0.5, SeVP1-2:4.6 ± 0.49, SeVP1-3:4.26 ± 0.7, SeVP1-4:3.54 ± 0.47, SeVP1-5:4.06 ± 0.55, avp1:2.04 ± 1.31, avp1:SeVP1-1:2.27 ± 0.82, avp1:SeVP1-2:2.71 ± 0.83, avp1:SeVP1-3:3.62 ± 1.26.

Fresh weight (g/3 strain): WT:0.008 ± 0.001g, SeVP1-1:0.01 ± 0.002, SeVP1-2:0.01 ± 0.001, SeVP1-3:0.008 ± 0.001, SeVP1-4:0.008 ± 0.005, SeVP1-5:0.011 ± 0.001, avp1:0.009 ± 0.004, avp1:SeVP1-1:0.009 ± 0.002, avp1:SeVP1-2:0.008 ± 0.002, avp1:SeVP1-3:0.01 ± 0.003.

Lateral root number: WT:10.8 ± 2.4, SeVP1-1:18.4 ± 1.3, SeVP1-2:21.6 ± 1.9, SeVP1-3:16 ± 1.9, SeVP1-4:14.7 ± 1.7, SeVP1-5:16.2 ± 3.2, avp1:8.6 ± 3.8, avp1:SeVP1-1:11.6 ± 4.0, avp1:SeVP1-2:12.7 ± 4.3, avp1:SeVP1-3:13.8 ± 3.7.

As shown in Figure 7, under the normal growth condition, transgenic line and wild-type do not have significant difference.When scarce phosphorus is handled 10 days, long-living apparent being suppressed kept burning day and night of the main root of Arabidopis thaliana, and lateral root number showed increased, blade reddens.The statistical result showed of physical signs (Fig. 8), when lacking phosphorus and handling 10 days, the plant main root of SeVP1-1 and SeVP1-2 significantly is longer than wild-type, and the avp1 mutant significantly be shorter than wild-type.5 mistakes are expressed lateral root number that strain system and complementary strain be all obviously more than wild-type.And aspect the plant fresh weight, except SeVP1-5 was significantly higher than wild-type, all the other strain systems and wild-type did not have significant difference.

In above-mentioned salt resistance and the anti-low-phosphorous property experiment, the result who changes empty carrier control group 1 is all consistent with wild-type.The result who changes empty carrier control group 2 is all consistent with mutant.

Claims (10)

1. albumen, be following a) or b) protein:

A) protein of forming by the aminoacid sequence shown in the SEQ ID NO:1;

B) with the aminoacid sequence shown in the SEQ ID NO:1 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and relevant with plant stress tolerance by a) deutero-protein.

2. the described proteic encoding gene of claim 1.

3. encoding gene according to claim 2 is characterized in that: described encoding gene is following 1) or 2) or 3) or 4) shown in:

1) its nucleotide sequence is from dna molecular shown in 5 ' terminal 197-2491 or the 168-2616 position Nucleotide among the SEQ ID NO:2;

2) its nucleotide sequence is a dna molecular shown in the SEQ ID NO:2;

3) under stringent condition with 1) or 2) the dna sequence dna hybridization that limits and the dna molecular of encoding said proteins;

4) with 1) or 2) dna sequence dna that limits has the homology more than 90% and the dna molecular of encoding said proteins.

4. amplification claim 2 or 3 described encoding gene total lengths or its any segmental primer are right; Described primer is to being specially dna molecular shown in dna molecular shown in the SEQ ID NO:3 and the SEQ ID NO:4.

5. the recombinant vectors, reorganization bacterium, transgenic cell line, recombinant virus or the expression cassette that contain claim 2 or 3 described encoding genes.

6. recombinant vectors according to claim 5 is characterized in that: described recombinant vectors is for to insert the recombinant expression vector that described encoding gene obtains in the multiple clone site of expression vector pCAMBIA3300-ubiquitin;

Described pCAMBIA3300-ubiquitin makes up as follows: insert the ubiquitin promoter sequence between the HindIII of pCAMBIA3300 and BamHI; Described ubiquitin promoter sequence is shown in SEQ ID NO:5.

7. the described albumen of claim 1, claim 2 or 3 described encoding genes, claim 6 or the 7 described recombinant vectorss application in the resistance of reverse that improves plant.

8. a method of cultivating the transgenic plant of resistance of reverse raising comprises the steps: to import the described proteic encoding gene of claim 1 in the plant that sets out, and obtains the purpose transgenic plant that resistance of reverse is higher than the described plant that sets out.

9. method according to claim 8 is characterized in that: described encoding gene imports by claim 5 or 6 described recombinant vectorss.

10. application according to claim 7 or claim 8 or 9 described methods is characterized in that: described plant is monocotyledons or dicotyledons;

Described resistance of reverse is a salt tolerant and/or anti-low-phosphorous.

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