CN102250227B

CN102250227B - Plant sucrose transport protein, and coding gene and application thereof

Info

Publication number: CN102250227B
Application number: CN2010101786121A
Authority: CN
Inventors: 刘公社; 苏蔓; 李晓峰; 齐冬梅; 陈双燕; 刘辉
Original assignee: Institute of Botany of CAS
Current assignee: Institute of Botany of CAS
Priority date: 2010-05-17
Filing date: 2010-05-17
Publication date: 2013-05-29
Anticipated expiration: 2030-05-17
Also published as: CN102250227A

Abstract

The invention discloses a plant sucrose transport protein, and a coding gene and application thereof. The protein disclosed by the invention is (a) a protein composed of amino acid sequences shown in sequence 1 in a sequence list, or (b) a protein derived from sequence 1. The protein and the coding gene thereof can regulate and control the growth and development of plants, can be induced by multiple stresses and can participate in the response of guinea grass to the multiple stresses. When the gene is transduced into plants, the stress resistance of the plants can be improved. The protein and the coding gene thereof have important practical values in the aspect of cultivating guinea grass having improved stress resistance and other new plant varieties. The invention is beneficial to learning the action mechanism of potential internal/external factors in the plant growth and development process on the regulation and control of the transport protein, thereby showing that the sucrose transport mechanism has a function of regulating the growth and development and the stress resistance of plants, and providing a genetic basis for the gene engineering research on increasing the crop yield and improving the quality.

Description

Sucrose Transporters in Plants and encoding gene thereof and application

Technical field

The present invention relates to a kind of Sucrose Transporters in Plants and encoding gene thereof and application.

Background technology

The assimilate that the photosynthesis of most plants blade produces all is transported to non-photosynthetic organs by sucrose as principal mode.Sucrose is main even unique form of carbohydrate long-distance transportation in the higher plant body.It is that the major cause of transhipment material is because the solubleness of sucrose is high, viscosity is low that sucrose is taked in transportation from the source to the storehouse, good stability and form very high osmotic potential, and this is that long-distance transportation is necessary.

Sucrose transporter (sucrose transporter, be abbreviated as SUT or SUC) be 12 cross-film embrane-associated proteins of a class, extensively be present in the various tissues and organ of higher plant, be responsible for the cross-film transportation of sucrose, in the links such as storehouse transhipment, played a significant role by the source at sucrose.Understand the mechanism of action of Sucrose Transporters in Plants regulation and control, the relation that discloses sucrose transporter and environment stress will help to disclose the sucrose transporting mechanism to the regulatory function of growth and development of plants and opposing adverse circumstance, provide the genetics foundation for improving crop yield with the genetically engineered research that improves quality.

Summary of the invention

The purpose of this invention is to provide a kind of Sucrose Transporters in Plants and encoding gene thereof and application.

Albumen provided by the invention (LcSUT1) is a kind of sucrose transporter, derives from sheep's hay (Leymus chinensis (Trin.) Tzvel.), is following (a) or (b):

(a) protein that is formed by the aminoacid sequence shown in the sequence in the sequence table 1;

(b) with the aminoacid sequence of sequence 1 through replacement and/or disappearance and/or the interpolation of one or several amino-acid residue and participate in the protein of being derived by sequence 1 of sucrose transhipment.

Sequence 1 is comprised of 523 amino-acid residues in the sequence table, being the conserved domain of GPH-Sucrose super family from the 34th the-the 513rd amino acids residue of N-terminal, is MelB domain sequence from the 26th the-the 523rd amino acids residue of N-terminal.

For the ease of the purifying of LcSUT1, label as shown in table 1 on the aminoterminal of the protein that can form at the amino acid residue sequence by sequence 1 or carboxyl terminal connect.

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 (b) but in the LcSUT1 synthetic, also can synthesize first its encoding gene, carry out again biological expression and obtain.The encoding gene of LcSUT1 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 sequence in the sequence table 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.

The gene (LcSUT1) of above-mentioned sucrose transporter of encoding also belongs to protection scope of the present invention.

Described gene can be following 1) or 2) or 3) or 4) dna molecular:

1) in the sequence table sequence 2 from the dna molecular shown in the Nucleotide of 5 ' terminal 118-1689 position;

2) dna molecular shown in the sequence in the sequence table 2;

3) can be with 1 under stringent condition) or 2) gene recombination that limits and the gene of encoding said proteins;

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

Above-mentioned stringent condition can be 0.1 * SSPE (or in the solution of 0.1 * SSC), 0.1%SDS, hybridization and wash film under 65 ℃ of conditions.

Sequence 2 in the sequence table is comprised of 1842 Nucleotide, and its open reading frame (ORF) is from 5 ' terminal 118-1689 position Nucleotide.

The recombinant vectors that contains above arbitrary described gene also belongs to protection scope of the present invention, such as recombinant expression vector.

Available existing plant expression vector construction contains the recombinant expression vector of described gene.

Described plant expression vector comprises double base agrobacterium vector (such as pBI121, pBin19, pCAMBIA2301, pCAMBIA3301, pCAMBIA1301-UbiN, pCAMBIA1300 etc.) and can be used for carrier of plant micropellet bombardment etc.Described plant expression vector also can comprise 3 ' end untranslated zone of foreign gene, namely comprises the dna fragmentation of polyadenylic acid signal and any other participation mRNA processing or genetic expression.The bootable polyadenylic acid of described polyadenylic acid signal joins 3 ' end of mRNA precursor, and the non-translational region of inducing (Ti) plasmid gene (such as kermes synthetic enzyme Nos gene), plant gene (storing protein gene such as soybean) 3 ' end to transcribe such as the Agrobacterium crown-gall nodule all has similar functions.

When using described gene constructed recombinant plant expression vector, before its transcription initiation Nucleotide, can add any enhancement type promotor, constitutive promoter or inducible promoter, such as the ubiquitin promoter (Ubiquitin) of cauliflower mosaic virus (CAMV) 35S promoter, corn, stress induced promoter Rd29A etc., they can use separately or be combined with other plant promoter; In addition, when using gene constructed plant expression vector of the present invention, also can use enhanser, comprise translational enhancer or transcriptional enhancer, these enhanser zones can be ATG initiator codon or neighboring region initiator codon etc., but must be identical with the reading frame of encoding sequence, to guarantee the correct translation of whole sequence.The source of described translation control signal and initiator codon is widely, can be natural, also can synthesize.Translation initiation region can be from transcription initiation zone or structure gene.

For the ease of transgenic plant cells or plant being identified and screening, can process used plant expression vector, can produce the enzyme of colour-change or the gene of luminophor (gus gene, luciferase genes etc.) as adding the coding that in plant, to express, have the antibiotic marker thing (gentamicin marker, kantlex marker etc.) of resistance or anti-chemical reagent marker gene (such as anti-weedkiller gene) etc.From the security consideration of transgenic plant, can not add any selected marker, directly with adverse circumstance screening transformed plant.

Described recombinant expression vector can be described gene is inserted the recombinant plasmid that the multiple clone site of Yeast expression carrier PDR196 obtains.Insert the recombinant plasmid that described gene obtains between the EcorI that described recombinant expression vector specifically can be at PDR196 and the SalI restriction enzyme site.

The expression cassette, transgenic cell line and the recombinant bacterium that contain above arbitrary described gene (LcSUT1) all belong to protection scope of the present invention.

The total length of amplification said gene or the primer of its arbitrary fragment are to also belonging within protection scope of the present invention.

In described albumen, described gene, described recombinant expression vector, expression cassette, transgenic cell line or the recombinant bacterium any one all can be applicable to cultivate plant with adverse resistance.

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

The method of cultivation transgenic plant provided by the present invention can import the gene of the described plant stress tolerance correlative protein of coding in the purpose plant, obtains the transgenic plant that resistance of reverse is higher than described purpose plant.

Utilize any carrier that can guide foreign gene in plant, to express, with the gene transfered plant cell of encoding said proteins, can obtain transgenic cell line and transfer-gen plant that resistance of reverse strengthens.Carry described gene expression vector can Ti-plasmids, Ri plasmid, plant viral vector, directly delivered DNA, microinjection, electricity be led, conventional biological method transformed plant cells or the tissue such as agriculture bacillus mediated by using, and the plant tissue that transforms is cultivated into plant.The plant host that is converted both can be monocotyledons, also can be dicotyledons, such as Arabidopis thaliana, tobacco, Root or stem of Littleleaf Indianmulberry, paddy rice, wheat, corn, cucumber, tomato, willow, turfgrass, lucerne place, soybean or cotton.

The present invention also protects a kind of method of cultivating transgenic yeast, is that described gene is changed in the purpose yeast, obtains transgenic yeast, and the sucrose of described transgenic yeast utilizes ability to be higher than described purpose yeast.Described gene specifically can import in the described purpose yeast by described recombinant vectors; Described purpose yeast specifically can be yeast mutants SUSY7 strain.

Albumen provided by the invention and encoding gene thereof can regulating plant growth and growths, induced by various abiotic stress, participate in sheep's hay to the response of various abiotic stress.With gene transfered plant of the present invention, can improve the resistance of plant.Particularly cradle (cut) and can induce this protein expression, improve the conveying efficiency of sugar in plant materials, improve crop sucrose transporter expression amount by the transgenosis means, can improve overcompensate growth and anti-herding property after the cradling of careless class, help to realize the healthy and sustainable development of grassland ecosystem.Albumen provided by the invention and encoding gene thereof have important practical value for the sheep's hay of cultivating the resistance raising and other neies variety of plant.The present invention helps to understand internal and external factors potential in the plant growth and development process to the mechanism of action of sucrose transporter regulation and control, provides the genetics foundation for disclosing the sucrose transporting mechanism to the regulatory function of growth and development of plants and resistance, for the genetically engineered research that improves crop yield and improvement quality.

Description of drawings

Fig. 1 is the agarose gel electrophoresis detected result of the total RNA of chinense seedlings;

Fig. 2 is the agarose gel electrophoresis detected result of sheep's hay sucrose transporter fragment product;

Fig. 3 is the agarose gel electrophoresis detected result of 5 ' RACE product;

Fig. 4 is the agarose gel electrophoresis detected result of 3 ' RACE product;

Fig. 5 is the agarose gel electrophoresis detected result of pcr amplification LcSUT1 full-length cDNA;

Fig. 6 is the structural domain synoptic diagram of LcSUT1;

Fig. 7 is the close and distant water prediction of LcSUT1;

Fig. 8 is the secondary structure prediction of LcSUT1;

Fig. 9 is the systematic evolution tree of LcSUT1;

Figure 10 is the response modes (sxemiquantitative RT-PCR) of LcSUT1 gene under various treatment condition;

Figure 11 is that sucrose is the growing state of transgenosis and contrast yeast strain in the substratum of sole carbon source;

Embodiment

Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment if no special instructions, is ordinary method.Used test materials among the following embodiment if no special instructions, is and purchases available from routine biochemistry reagent shop.% among the following embodiment if no special instructions, is the quality percentage composition.Quantitative test in following examples all arranges repeated experiments three times, results averaged.

Sheep's hay (lucky giving birth to No. one): in October, 2005 is available from Jisheng Improved Variety Station of Aneurolepidium Chinense, Jilin Province.

Yeast expression carrier PDR196: Institute of Botany, Chinese Academy of Sciences's carrier guarantees to provide to the public.

Yeast mutants SUSY7/ura3 strain: Institute of Botany, Chinese Academy of Sciences's carrier guarantees to provide to the public.

The reference of Yeast expression carrier PDR196 and yeast mutants SUSY7/ura3 strain: Rlesmeier JW, Willmitzer L, Frommer WB (1992) .Isolation and characterization of a sucrose carriercDNA from spinach by functional expression in yeast.EMBO J.11:4705-4713.

Plasmid PDR196-StSUT: Institute of Botany, Chinese Academy of Sciences's carrier guarantees to provide to the public; Reference: K ü hnC, Quick WP, Schulz A, Riesmeler TW, Sonnewald U, Frommer WB (1996) .Companioncel l-specific inhibition of the potato sucrose transporter SUT 1.Plant Cell Environ.19:1115-1123.

The acquisition of embodiment 1, sheep's hay sucrose transporter gene

One, the clone of sheep's hay sucrose transporter gene LcSUT1 fragment

1, the extraction of vegetable material processing and total RNA

Take chinense seedlings as material, extract total RNA, carry out 1.2% agarose gel electrophoresis and detect, the result is as shown in Figure 1.The RNA that extracts has two obvious electrophoretic bands, is followed successively by from top to bottom 28S RNA and 18S RNA, shows to have obtained higher, the more completely total RNA of purity.

2, the clone of sheep's hay sucrose transporter intermediate sequence

Amino acid residue sequence according to published Sucrose Transporters in Plants is sought conservative region, and according to the conservative region primers, concrete primer sequence is as follows:

S 15′-TGTGGCTATGCGGTCCTA-3′；

S25′-TTCGTGCTTGCGGTGATG-3′。

The total RNA that extracts chinense seedlings take step 1 uses PrimeScript as template ^TM1st Strand cDNASynthesized Kit test kit (Takara company) and reference reagent box specification sheets, synthetic the first chain cDNA of counter-rotating.Reaction system and reaction conditions are as follows: Oligo-dT (10pmol/ μ l) 1.0 μ l, dNTP Mixture (10mmol/l each) 1.0 μ l, Total RNA (≤1 μ g) 1.0 μ l, RNase-free water7.0 μ l, 65 ℃ of 5min; Then add 5 * Buffer4.0 μ l, RNase Inhibitor (40U/ μ l) 0.5 μ l, PrimeScript RTase (200U/ μ l) 0.5 μ l, 42 ℃ of 45min, 70 ℃ of 15min, with the first synthetic chain cDNA be stored in-20 ℃ for subsequent use.

Take the first chain cDNA of obtaining as template, with the primer of S1 and S2 composition to carrying out pcr amplification; The PCR reaction system is: each 1 μ l of cDNA template, S1 and S2,10 * Buffer, 2.5 μ l, dNTP Mixture (10mmol/leach) 2 μ l, Taq enzyme 0.25 μ l, ddH2O 12.25 μ l; Reaction conditions is: 94 ℃ of denaturation 5min of elder generation; Then 94 ℃ of 30s, 52 ℃ of 30s, 72 ℃ of 60s, totally 35 circulations; Last 72 ℃ are extended 10min.

After reaction finishes, pcr amplification product is carried out 1.2% agarose gel electrophoresis detect, the result as shown in Figure 2.Among Fig. 2, swimming lane M is DL2000DNA molecular weight standard (Beijing Quanshijin Biotechnology Co., Ltd), and swimming lane 1 is pcr amplification product.The result shows, has obtained the purpose fragment of the about 1000bp of length through pcr amplification.Reclaim and purified pcr product, be connected on the PMD-18T carrier (Takara company), connect product and transform bacillus coli DH 5 alpha competent cell (Beijing Quanshijin Biotechnology Co., Ltd), screening positive clone carries out bacterium liquid PCR to be identified, the plasmid that extracts positive colony checks order, and sequencing result is carried out BLAST analyze.The result shows that the length of this fragment is 1005bp, and its deoxyribonucleotide sequence has higher homology with the sequence of SUT genoid known in the plant shown in sequence in the sequence table 3, show that this fragment may be the fragment of sheep's hay LcSUT1.

Two, the clone of sheep's hay sucrose transporter gene LcSUT15 ' end and 3 ' terminal sequence

1, the clone of sheep's hay sucrose transporter gene LcSUT15 ' terminal sequence

LcSUT1 gene fragment cDNA sequences Design RACE5 ' nested primer according to the step 1 acquisition:

SUTa-n：：5′-CCACTAAGCCAGCAATAGGACCGCATA-3′；

SUTa-w 5′-GAGGATGAACGGGAACCACGAGAGC-3′。

Total RNA of the chinense seedlings of extracting take step 1 adopts the SMARTer of Promega company as template ^TMRACEcDNAAmplification Kit and reference 5 ' process specifications, its first chain cDNA is synthesized in reverse transcription.Reaction system and condition are as follows: 1 μ l RNA, 1 μ l, 5 '-CDS primerA, 1 μ l SMART II A oligo, 1 μ l DTT (20mM), 1 μ l dNTP Mix (10mM), 1 μ l MMLV ReverseTranscriptase, 2 μ l 5X First-StrandBuffer, 2 μ l sterile H ₂O; 70 ℃ of 2min, 2min on ice, 42 ℃ of 1.5h, 72 ℃ of 7min.

Take the first chain cDNA of obtaining as template, primer SUTa-w and primer UPM (Promega company: Long (0.4 μ M): 5 '-CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-3 ', Short (2 μ M): 5 '-CTAATACGACTCACTATAGGGC-3 ') pcr amplification is carried out in pairing; The PCR reaction system is: 1 μ l 50X Advantage 2Polymerase Mix, 34.5 μ l PCR-Grade Water, 5 μ l10XAdvantage 2PCR Buffer, 1 μ l dNTP Mix (10mM), 1 μ l 50X Advantage 2Polymerase Mix, 5 μ lUPM, 1 μ l primer SUTa-n:, 2.5 μ lcDNA templates; Reaction conditions is: first 94 ℃ of 30s, 68 ℃ of 30s then, 70 ℃ of 60s, totally 35 circulations; Last 70 ℃ are extended 10min.

After reaction finishes, getting PCR product 1 μ l adds PCR-Grade Water and is diluted to 100 μ l, get 1 μ l and work as template, be nest PCR with Nested Universal primer A (NUP) 5 '-AAGCAGTGGTATCAACGCAGAGT-3 ' and SUTa-n, reaction system is 1 μ l 50X Advantage 2Polymerase Mix, 38.5 μ l PCR-GradeWater, 5 μ l 10XAdvantage 2PCRBuffer, 1 μ l dNTP Mix (10mM), 1 μ l 50XAdvantage2Polymerase Mix, 1 μ lNUP, 1 μ l primer SUTa-n.

After reaction finishes, pcr amplification product is carried out 1.2% agarose gel electrophoresis detect, the result as shown in Figure 3.Among Fig. 3, swimming lane M is DL2000DNA molecular weight standard (Beijing Quanshijin Biotechnology Co., Ltd), and swimming lane 1 is 5 ' RACE pcr amplification product.The result shows, obtained the purpose fragment that length is about 500bp through pcr amplification.Reclaim and purifying 5 ' RACE product, be connected on the PMD-18T carrier, connect product and transform the bacillus coli DH 5 alpha competent cell, screening positive clone carries out bacterium liquid PCR to be identified, the plasmid that extracts positive colony checks order, and sequencing result is carried out BLAST analyze.The result shows, the length of this fragment is 503bp, its deoxyribonucleotide sequence has higher homology with 5 ' terminal sequence of SUT genoid known in the plant shown in sequence in the sequence table 4, show that this fragment may be 5 ' terminal sequence of sheep's hay SUT encoding gene.

2, the clone of sheep's hay sucrose transporter gene LcSUT13 ' terminal sequence

LcSUT1 gene fragment cDNA sequences Design RACE3 ' primer according to the step 1 acquisition: SUTs:5 '-TTCGGGCTGCTTCTCAAC-3 '.Total RNA of the chinense seedlings of extracting take step 1 adopts the SMARTer of Promega company as template ^TMRACE cDNA Amplification Kit and reference 3 ' process specifications, its first chain cDNA is synthesized in reverse transcription.Reaction system and condition are as follows: 1 μ l RNA, 1 μ l, 3 '-CDS primer A, 1 μ l DTT (20mM), 1 μ l dNTP Mix (10mM), 1 μ l MMLV ReverseTranscriptase, 2 μ l 5XFirst-Strand Buffer, 3 μ l sterile H ₂O; 70 ℃ of 2min, 2min on ice, 42 ℃ of 1.5h, 72 ℃ of 7min.The PCR reaction adds 1 μ l 50X Advantage 2Polymerase Mix, 34.5 μ l PCR-Grade Water, 5 μ l10X Advantage 2PCR Buffer, 1 μ l dNTP Mix (10mM), 1 μ l 50X Advantage 2PolymeraseMix, 5 μ lUPM, 1 μ l primer SUTs, 2.5 μ l cDNA templates; Reaction conditions is: first 94 ℃ of 30s, 58 ℃ of 30s then, 70 ℃ of 60s, totally 35 circulations; Last 70 ℃ are extended 10min.

After reaction finishes, pcr amplification product is carried out 1.2% agarose gel electrophoresis detect, the result as shown in Figure 4.Among Fig. 4, swimming lane M is DL2000DNA molecular weight standard (Beijing Quanshijin Biotechnology Co., Ltd), and swimming lane 1 is 3 ' RACE pcr amplification product.The result shows, obtained the purpose fragment that length is about 900bp through pcr amplification.Reclaim and purifying 3 ' RACE product, be connected on the PMD-18T carrier, connect product and transform the bacillus coli DH 5 alpha competent cell, screening positive clone carries out bacterium liquid PCR to be identified, the plasmid that extracts positive colony checks order, and sequencing result is carried out BLAST analyze.The result shows, the length of this fragment is 927bp, its deoxyribonucleotide sequence has higher homology with 3 ' terminal sequence of SUT genoid known in the plant shown in sequence in the sequence table 5, show that this fragment may be 3 ' terminal sequence of sheep's hay SUT encoding gene.

Three, the acquisition of sheep's hay sucrose transporter gene LcSUT1 full length cDNA sequence and PCR detect

5 ' and 3 ' the RACE sequence of utilizing step 2 to obtain, design comprise the full-length cDNA primer sequence (S1 in sheep's hay translation district; A1).

S1：5′-TGCGTTGATTCGATCAACGATCTTTTCC-3′；

A1：5′-CGGTTCCTCACATTCTTATTCTGC-3′。

Extract total RNA of chinense seedlings, reverse transcription is cDNA, take cDNA as template, with primer (S1 and A1) is carried out pcr amplification.Pcr amplification product is carried out 1.2% agarose gel electrophoresis detect, the result as shown in Figure 5.Among Fig. 5, swimming lane M is DL2000DNA molecular weight standard (Beijing Quanshijin Biotechnology Co., Ltd), and swimming lane 1 is pcr amplification product.The result shows, obtained the fragment that length is about 1800bp through pcr amplification.Reclaim and this product of purifying, be connected on the PMD-18T carrier, connect product and transform the bacillus coli DH 5 alpha competent cell, screening positive clone carries out bacterium liquid PCR identifies that the plasmid that extracts positive colony checks order.Sequencing result shows shown in sequence 2 by analysis, this sheet segment length 1842bp, the protein shown in the sequence 1 of code sequence tabulation.With the protein called after LcSUT1 shown in the sequence 1 (being formed by 523 amino-acid residues).With the encoding gene called after LcSUT1 of LcSUT1, its full-length cDNA is shown in the sequence 2 of sequence table, and open reading frame (ORF) is from 5 ' terminal 118-1689 position Nucleotide.

The bioinformatic analysis of embodiment 2, LcSUT1 and LcSUT1

One, the structure function of the sequential analysis of LcSUT1 gene and proteins encoded thereof prediction

Utilize DNAMAN and OMIGA software that the full length cDNA sequence of LcSUT1 is carried out bioinformatic analysis, this sequence total length 1842bp is ORF from 5 ' the 118th the-the 1689th at end.The structural representation of LcSUT1 as shown in Figure 6.Infer that its molecular weight is 55.358kDa, iso-electric point pI value 8.735.With the structural domain of online BlAST instrument (http://blast.ncbi.nlm.nih.gov/Blast.cgi) analysis LcSUT1, the result shows that this albumen belongs to GPH-sucrose superfamily, shows that this albumen is a member in the SUT family.With membranin on-line prediction instrument TopPred2 prediction, this albumen all has wetting ability (Fig. 7), is the albumen (Fig. 8) of 12 cross-films, meets the sucrose transporter family structure, illustrates that this albumen belongs to sucrose transporter family.

Two, homology and the systematic evolution tree analysis of LcSUT1 and other SUT proteinoid sequence

(the GeneBank number of logging in is: TaSUT1A to other SUT aminoacid sequence of having cloned in LcSUT1 and the plant to utilize online Blast instrument, TaSUT1B, TaSUT1D is respectively AF408842, AF408843, AF408845, HvSUT1AJ272309, SbSUT1GU126430, OsSUT D87819, OsSUT2AB091672, ZmSUT1NM001111370, ZmSUT4NM001144014, ZmSUT2NM001153179, SbSUT4GU045300, HvSUT2AJ272308, NtSUT FM164638, PmSUC1X84379, AgSUTAF063400, AgSUT2A AF167415, AtSUT8NM127031, AtSUT6NM123727) carry out the homology analysis comparison, see Fig. 9.

The result shows that the homology of LcSUT1 and monocotyledons barley, wheat, corn and Chinese sorghum SUTgroup1 (SUT1) is respectively 94%, 90%, 82% and 83%; And lower with dicots SUT group1 homology with other group of unifacial leaf SUT shows that the homology of LcSUT and monocotyledons SUT group1 is higher, and lower with the homology of other group of SUT and dicots SUT group1.Phylogenetic analysis shows that larger difference has appearred in SUT proteinoid during evolution, but corresponding this albumen of group is relatively conservative in plant.

Embodiment 3, the LcSUT1 expression pattern analysis under the various abiotic stress condition

The chinense seedlings in 8 weeks of normal growth is carried out different treatment.

First group (NaCl): soak root with the 250mMNaCl aqueous solution, incubated at room temperature;

Second group (PEG): soak root, incubated at room temperature with 20% (quality percentage composition) PEG aqueous solution;

The 3rd group (ABA): soak root, incubated at room temperature with the 100 μ M dormin aqueous solution;

The 4th group (MJ): soak root, incubated at room temperature with the 100 μ M methyl jasmonate aqueous solution;

The 5th group (cut): cradle;

The 6th group (Cold): cultivate under 4 ℃ of conditions.

The 7th group (CK): room temperature is normally cultivated.

Process after 2 hours, extract respectively total RNA of careless seedling, carry out RT-PCR.

LcSUT1 primer: 1:5 '-GCTGGCTTAGTGGTTCAA-3 ';

2：5′-TCCAGTCGGTGTCGTAGA-3′；

Confidential reference items Actin primer: 1:5 '-TGGACTCTGGTGATGGTGTGAG-3 ';

2：5′-GTGCTAAGGGAGGCAAGGATG-3′。

Reaction conditions: 94 ℃ of 4min; 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 30s, 25 circulations.

The results are shown in Figure 10.The result shows that the LcSUT1 transcriptional level obviously is subjected to stress-inducing, and under inductive condition, the relative expression quantity of LcSUT1 gene increases sharply, particularly ABA and cradle (cut) to process the expression amount increasing amount obvious.

The functional verification of embodiment 4, LcSUT1

One, LcSUT1 gene cloning

Extract total RNA of chinense seedlings, reverse transcription is cDNA; Take cDNA as template, with primer (S1 and A1) carried out pcr amplification.

S1：5′-TGCGTTGATTCGATCAACGATCTTTTCC-3′；

A1：5′-CGGTTCCTCACATTCTTATTCTGC-3′。

The 1.7kb left and right sides PCR product that obtains.

Two, LcSUT1 changes yeast over to

1, take the PCR product of step 1 as template, with following primer to carrying out pcr amplification:

5′-CCGGAATTCCATCGTCCACCATGGCGCG-3′；

5′-CGCGTCGACGTGACCGCCGGTGCTGAC-3′。

Introduce EcorI and SalI restriction enzyme site at the upstream and downstream of open reading frame respectively.

Obtain the PCR product about 1.5kb.

2, reclaim PCR product and purifying, be connected on pMD-19T simple (TaKaRa Code:D104A) carrier, connect product and transform bacillus coli DH 5 alpha (Beijing Quanshijin Biotechnology Co., Ltd) competent cell, screening positive clone carries out bacterium liquid PCR identifies that the plasmid that extracts positive colony checks order.Sequencing result shows that the fragment that amplifies has the sequence 2 of sequence table from 5 ' terminal 118-1679 position Nucleotide.

3, the pcr amplification product of step 1 is cut with EcorI and SalI enzyme, reclaimed LcSUT1 gene (the PCR product about 1.5kb); Yeast expression carrier PDR196 is cut with EcorI and SalI enzyme, reclaim skeleton carrier; The LcSUT1 gene that reclaims is connected skeleton carrier to be connected with recovery, obtain recombinant plasmid, sequencing result shows, obtained purpose plasmid PDR196-LcSUT1 (the EcorI of PDR196 and SalI enzyme cut inserted sequence table between the recognition site sequence 2 from the DNA shown in 5 ' the terminal 118-1686 position Nucleotide).

4, plasmid PDR196-LcSUT1 is imported in the yeast mutants SUSY7/ura3 strain of SUT gene function disappearance, obtain recombination microzyme A.Plasmid PDR196-StSUT is imported in the yeast mutants SUSY7/ura3 strain of SUT gene function disappearance, obtain recombination microzyme B (positive control).The PDR196 empty carrier is imported in the yeast mutants SUSY7/ura3 strain of SUT gene function disappearance, obtain recombination microzyme C (negative contrast).

It is that (1.7g/L is without the amino acid yeast nitrogen for the MD substratum of sole carbon source that recombination microzyme A, recombination yeast B, recombination microzyme C are coated in respectively sucrose, 2% sucrose, 5g/L ammonium sulfate, 20mg/L tryptophane and 1.5% agar are adjusted PH=5.0) carried out 30 ℃ of cultured continuously 5 days on the flat board.Observe the yeast colony growing state.

The result as shown in figure 11.Among Figure 11, a represents recombination microzyme A, and b represents recombination microzyme B, and c represents recombination microzyme C.The result shows, is that the growth conditions of a is identical with b in the MD substratum of sole carbon source at sucrose, and c then can not normal growth.Can the encode LCSUT1 albumen of function of presentation of results LcSUT1, yeast mutants strain that can complementary sucrose transporter afunction makes its normal growth.

Sequence table

＜110〉Institute of Botany, Chinese Academy of Sciences

＜120〉Sucrose Transporters in Plants and encoding gene thereof and application

<130>CGGNARY102311

<160>5

<210>1

<211>523

<212>PRT

＜213〉sheep's hay (Leymus chinensis (Trin.) Tzvel.)

<400>1

Met Ala Arg Gly Gly Gly Asn Gly Glu Val Glu Leu Ser Val Gly Val

1 5 10 15

Gly Gly Gly Gly Gly Gly Ala Ala Gly Gly Ala Val Asp Pro Pro Val

20 25 30

Arg Ile Ser Leu Gly Arg Leu Ile Leu Ala Gly Met Val Ala Gly Gly

35 40 45

Val Gln Tyr Gly Trp Ala Leu Gln Leu Ser Leu Leu Thr Pro Tyr Val

50 55 60

Gln Thr Leu Gly Leu Ser His Ala Leu Thr Ser Phe Met Trp Leu Cys

65 70 75 80

Gly Pro Ile Ala Gly Leu Val Val Gln Pro Cys Val Gly Leu Tyr Ser

85 90 95

Asp Lys Cys Thr Ser Arg Trp Gly Arg Arg Arg Pro Phe Ile Leu Thr

100 105 110

Gly Cys Leu Leu Ile Cys Leu Ala Val Ile Ile Val Gly Phe Ser Ala

115 120 125

Asp Ile Gly Ala Val Leu Gly Asp Ser Lys Gly Glu Cys Ser Leu Tyr

130 135 140

His Gly Pro Arg Trp His Ala Ala Ile Val Tyr Val Leu Gly Phe Trp

145 150 155 160

Leu Leu Asp Phe Ser Asn Asn Thr Val Gln Gly Pro Ala Arg Ala Leu

165 170 175

Met Ala Asp Leu Ser Ala Gln His Gly Pro Ser Ala Ala Asn Ser Ile

180 185 190

Phe Cys Ser Trp Met Ala Leu Gly Asn Ile Leu Gly Tyr Ser Ser Gly

195 200 205

Ser Thr Asn Asn Trp His Lys Trp Phe Pro Phe Leu Arg Thr Arg Ala

210 215 220

Cys Cys Glu Ala Arg Ala Asn Leu Lys Gly Ala Phe Leu Val Ala Val

225 230 235 240

Leu Phe Leu Ser Phe Cys Leu Val Ile Thr Leu Ile Phe Ala Lys Glu

245 250 255

Val Pro Tyr Lys Ala Ile Ala Pro Leu Pro Thr Lys Ala Asn Gly Gln

260 265 270

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

275 280 285

Asn Leu Pro Pro Gly Met Pro Ser Val Leu Leu Val Thr Gly Leu Thr

290 295 300

Trp Leu Ser Trp Phe Pro Phe Ile Leu Tyr Asp Thr Asp Trp Met Gly

305 310 315 320

Arg Glu Ile Tyr His Gly Asp Pro Lys Gly Thr Pro Glu Glu Thr Asn

325 330 335

Ala Phe Gln Glu Gly Val Arg Ala Gly Ala Phe Gly Leu Leu Leu Asn

340 345 350

Ser Ile Val Leu Gly Phe Ser Ser Phe Leu Ile Glu Pro Leu Cys Lys

355 360 365

Arg Leu Gly Pro Arg Val Val Trp Val Ser Ser Asn Phe Leu Val Cys

370 375 380

Leu Ser Met Ala Ala Ile Cys Ile Ile Ser Trp Trp Ala Thr Lys Asp

385 390 395 400

Leu His Gly Tyr Ile Gln His Ala Ile Thr Ala Ser Lys Asp Val Lys

405 410 415

Ala Val Ser Leu Val Leu Phe Ala Phe Leu Gly Val Pro Leu Ala Ile

420 425 430

Leu Tyr Ser Val Pro Phe Ala Val Thr Ala Gln Leu Ala Ala Asn Lys

435 440 445

Gly Gly Gly Gln Gly Leu Cys Thr Gly Val Gln Asn Ile Ala Ile Val

450 455 460

Ile Pro Gln Val Ile Ile Ala Val Gly Ala Gly Pro Trp Asp Glu Leu

465 470 475 480

Phe Gly Lys Gly Asn Ile Pro Ala Phe Gly Met Ala Ser Gly Phe Ala

485 490 495

Leu Ile Gly Gly Ile Ala Gly Ile Phe Leu Leu Pro Lys Ile Ser Arg

500 505 510

Arg Gln Phe Arg Ala Val Ser Thr Gly Gly His

515 520

<210>2

<211>1842

<212>DNA

＜213〉sheep's hay (Leymus chinensis (Trin.) Tzvel.)

<400>2

tgcgttgatt cgatcaacgt acttttcctg tcccgagatc cttggccggg cacagacaca 60

ccgtagaatt gataggcgaa cgaacgaggt ggtgatcgcc cgggcggcct ccctgcgatg 120

gcgcgcggcg gcggcaacgg cgaggtggag ctctcggtgg gggtcggtgg cggaggcggc 180

ggcgccgccg gcggggcggt ggaccccccc gtgcggatca gcctcggcag gctcatcctc 240

gccggcatgg tcgccggcgg cgtgcagtac ggatgggcgc tccagctctc cctgctcacc 300

ccctacgtcc agactctggg actttcacat gctctgactt cattcatgtg gctctgcggc 360

cctattgctg gtttggtggt tcaaccatgc gttgggctct acagtgacaa gtgcacttcc 420

agatggggaa gacgcaggcc gtttattctg acaggatgtc tgctcatctg ccttgctgtc 480

atcatcgtcg gcttctcggc tgacattgga gctgttctgg gcgatagcaa gggagagtgc 540

agtctctatc atggacctcg ttggcacgct gcaattgtgt atgttcttgg gttctggctt 600

cttgacttct ccaacaacac tgtgcaaggt ccagcgcgtg ctctgatggc tgatttatca 660

gcgcaacatg gacccagtgc tgcaaattca atcttctgtt cttggatggc actaggaaat 720

atcctaggat attcctctgg ttccacgaat aactggcaca agtggtttcc gttcctccgg 780

acaagagctt gctgcgaagc ccgcgcaaat ctgaaaggcg catttctggt ggcagtgctg 840

ttcctgtcct tctgtttggt gataactctg atcttcgcca aggaggtgcc ctacaaggcg 900

attgcacccc tcccaacaaa ggccaatggc caggttgaag tcgagcctac cgggccgctt 960

gccgtgttca aaggcttcaa gaacttgccc cctggaatgc catcagtgct cctcgtgact 1020

ggtctcacct ggctgtcctg gttcccgttc atcctgtacg acaccgactg gatgggtcgc 1080

gagatctacc acggtgaccc caagggaacc cccgaggaga ccaatgcgtt ccaggaaggt 1140

gtcagggctg gggcgttcgg tctgctactc aactcgatcg ttctcgggtt cagctcgttc 1200

ctgatcgagc cgctgtgcaa gaggctaggc ccgagggtgg tgtgggtgtc tagcaacttc 1260

ctcgtctgcc tttccatggc ggccatttgc atcataagct ggtgggctac taaggactta 1320

catgggtaca tccagcacgc catcaccgcc agcaaggacg tcaaggccgt ctccctcgtc 1380

ctcttcgcct tcctcggagt ccctctcgcc attctgtaca gtgtcccttt cgcggtgacg 1440

gcgcagctgg cggcgaacaa agggggtgga caagggctgt gcacgggcgt gcagaacatc 1500

gccatcgtga taccccaggt gatcatcgcg gtgggggcgg ggccgtggga cgagctgttc 1560

ggcaagggca acatcccggc gttcggcatg gcgtccggct tcgcgctcat cggcggcatc 1620

gccggcatat tcctgctgcc caagatctcc aggcgccagt tccgggccgt cagcaccggc 1680

ggtcactgac cgcgccgcgc ttcggtcggc ctgagcatgg cgaaggccga tcgcgccggc 1740

ctggaggtcc cagctcgcaa tttaccagat tttcgcatag gcttaactag gtggctctcg 1800

cttaaggaca ccggtagagc agaataagaa tgtgaggaac cg 1842

<210>3

<211>1005

<212>DNA

＜213〉sheep's hay (Leymus chinensis (Trin.) Tzvel.)

<400>3

tgtggctatg cgtcctatgc tggcttagtg gtcaaccgtt ggttgctgta cagtgatagg 60

tgtacagcaa gatgggaaga cggagaccat tcattctgac aggatgtgtg ctcatctgca 120

ttgctgtcat tgttgttggc ttttcgtcag acatcggagc tgcgctaggg gacacaaagg 180

aacattgcag tctctatcat ggccctcgct ggcatgctgc gattgtatat gttctggggt 240

tttggctcct tgacttctcc aacaatactg tgcaaggtcc agcacgtgct atgatggctg 300

ttttgtgcgg tcatcatggg cctagtgcag ctagttcaat cttctgttct tggatgcgct 360

gggaaacatc ctaggttatt cctctggttc cacaaacaat cggcacaagt ggtttcccct 420

cctcaaaaca aatgcctgtt gtgaagcctg tgcaaacctg aaaggtgcat ttctggtggc 480

tgtggtgttc ctaatcatat gcttggctat aaccctcgtc ttcgccaagg aagtaccata 540

cggaggaaac gagaacctcc caacaaaagc aaacggcgag gttgaagctg aacctaccgg 600

gccacttgct gtgcccaagg gcttcaagaa cttgccccgc gggatgccat ccgttcttct 660

cgtaactggc ctcacctggc tctcgtggtt cccgttcatc ctctacgaca cggactggat 720

gggccgtgag atctaccacg gcgacccaag gggcaccaat gcccagatct cggcattcaa 780

cgaaggtgtc agaataggcg caatcgggca gcttctcaac tcgattgttc taggattcag 840

ctcgttcctg atcgagccca tgtgccggaa ggtcgggccg agggttgtgt gggtgacgag 900

caacttcatg gtgtgcatcg ccatggcggc caccgcgctg atcagcttct ggtcgctcaa 960

ggactgccac ggatacgtgc agaacgccat caccgcaagc acgaa 1005

<210>4

<211>927

<212>DNA

＜213〉sheep's hay (Leymus chinensis (Trin.) Tzvel.)

<400>4

ttcgggctgc ttctcaactc gatcatcctg gggttcagct cgttcctgat cgagccgatg 60

tgcaagcggc tgggcccgag tgtgggtgtc gagcaacttc ctcgtctgca tcgccatggc 120

cgccaccgcc atcatcagct ggtggtctac caaggagttc catgagtacg tacagcatgc 180

catcaccgcc agcaaggaca tcaagatcgt atgcatggtc ctcttcgcat tcctcggagt 240

gcctctcgcg attttgtaca gcgttccctt tgcggtgacg gcgcagttgg cggcaagcaa 300

aggaggcggc caagggctgt gcaccggcgt gctgaacatc tccatcgtca tcccacaggt 360

gatcatcgcg ctgggggcgg ggccgtggga ccagctgttc gggaagggca acatcccggc 420

cttcgccgcg gcctctgcct tcgcgctcat cggcggcatc gtcggcatat tcctgctgcc 480

caagatatcc aggcactcgt tccgggccgt cagcgccggc ggtcactgac cgcgccgggc 540

gcctgcctca ccacgggcga aagctcgatg gtgcaggctg ggcggttcca gctcgcatgt 600

gccaattttt acataggctt aaaaataggt ggctctcgct tcaagactcc gtacagcaga 660

ataagaatgt gaggaaccgt atgtttgtgt atgtgtacta gtgtgtgtaa cagaacggtg 720

agaggggaat gtggtcatcc attaccggct aggtggtctg tgaaggctat gtggtcgtcg 780

gatttggatc ggagcgcctt tactgtggtc aggtgtaatc cttgtgttgt gacttgtgtg 840

tagcaaacca ggttaagcta ctaatgagaa gacgaatgga cggtggcttt ttcagcaccc 900

caaaaaaaaa aaaaaaaaaa aaaaaaa 927

<210>5

<211>503

<212>DNA

＜213〉sheep's hay (Leymus chinensis (Trin.) Tzvel.)

<400>5

ggaacacaaa cccacaccac ccctctcctc actccacgct ccctccctcc ccccctctct 60

tccactcgca ctttccgccc tcgtctcctc ctcttcttcc tcccgtcagc cccgttcctg 120

gcgccacctt cttcttcctc gcatgcgttg attcgatcaa cgtacttttc ccctctctag 180

atccttggcc gaagaattga taggcgaacg aggtgatcat cgttcgcacg acgtcccggc 240

catgggtgcc ggcggcggca acggcgaggt ggagctctcg gtgggggtcg gtggcggagg 300

cggcggcgcc gccggcgggg cggtggaccc ccccgtgcgg atcagcctcg gcaggctcat 360

cctcgccggc atggtcgccg gcggcgtgca gtatggatgg gccctccagc tctccctgct 420

caccccctac gtccagactc tgggactttc acatgccctg acttcattca tgtggctatg 480

cggtcctatt gctggcttag tgg 503

Claims

1. protein, the protein that is formed by the aminoacid sequence shown in the sequence in the sequence table 1.

2. the gene of coding claim 1 described albumen.

3. gene according to claim 2, it is characterized in that: described gene is following 1) or 2) dna molecular:

2) dna molecular shown in the sequence in the sequence table 2.

4. the recombinant bacterium that contains the described gene of claim 2.

5. the recombinant bacterium that contains the described gene of claim 3.

6. the expression cassette that contains the described gene of claim 2.

7. the expression cassette that contains the described gene of claim 3.

8. the recombinant expression vector that contains the described gene of claim 2.

9. recombinant expression vector according to claim 8 is characterized in that: described recombinant expression vector is that the described gene of claim 2 is inserted the recombinant plasmid that the multiple clone site of PDR196 obtains.

10. the recombinant expression vector that contains the described gene of claim 3, described recombinant expression vector are that the described gene of claim 3 is inserted the recombinant plasmid that the multiple clone site of PDR196 obtains.

11. the application of the described albumen of claim 1 in cultivating plant with adverse resistance; Described plant is sheep's hay.

12. the application of the described gene of claim 2 in cultivating plant with adverse resistance; Described plant is sheep's hay.

13. the application of the described gene of claim 3 in cultivating plant with adverse resistance; Described plant is sheep's hay.

14. a method of cultivating transgenic plant is that the described gene of claim 2 is imported in the purpose plant, obtains the transgenic plant that resistance of reverse is higher than described purpose plant; Described purpose plant is sheep's hay.

15. a method of cultivating transgenic plant is that the described gene of claim 3 is imported in the purpose plant, obtains the transgenic plant that resistance of reverse is higher than described purpose plant; Described purpose plant is sheep's hay.

16. a method of cultivating transgenic yeast is that gene claimed in claim 2 is changed in the purpose yeast, obtains transgenic yeast, the sucrose of described transgenic yeast utilizes ability to be higher than described purpose yeast.

17. method according to claim 16 is characterized in that: gene claimed in claim 2 imports in the described purpose yeast by recombinant vectors claimed in claim 9; Described purpose yeast is yeast mutants SUSY7/ura3 strain.

18. a method of cultivating transgenic yeast is that gene claimed in claim 3 is changed in the purpose yeast, obtains transgenic yeast, the sucrose of described transgenic yeast utilizes ability to be higher than described purpose yeast.

19. method according to claim 18 is characterized in that: gene claimed in claim 3 imports in the described purpose yeast by recombinant vectors claimed in claim 10; Described purpose yeast is yeast mutants SUSY7/ura3 strain.