CN102250227A

CN102250227A - Plant sucrose transport protein, and coding gene and application thereof

Info

Publication number: CN102250227A
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: 2011-11-23
Anticipated expiration: 2030-05-17
Also published as: CN102250227B

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

Plant sucrose transporter and encoding gene thereof and application

Technical field

The present invention relates to a kind of plant sucrose transporter and encoding gene and application.

Background technology

The assimilate that the photosynthesis of most of plant leafs produces all is transported to non-photosynthetic organ 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 solubleness height, the viscosity of sucrose 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 that a class is striden the film embrane-associated protein 12 times, extensively be present in the various tissues and organ of higher plant, the film of striding of being responsible for sucrose transports, and is waited in each link to the storehouse transhipment by the source at sucrose to play a significant role.Understand the mechanism of action of plant sucrose transporter regulation and control, the relation that discloses sucrose transporter and environment stress will help to disclose the regulatory function of sucrose transporting mechanism to 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 plant sucrose transporter and encoding gene 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 of forming by the aminoacid sequence shown in the sequence in the sequence table 1;

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

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

For the ease of the purifying of LcSUT1, can connect label as shown in table 1 at proteinic aminoterminal of forming by the amino acid residue sequence of sequence 1 or carboxyl 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 (b) but in the LcSUT1 synthetic, also can synthesize its encoding gene earlier, carry out biology again and express and to 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 the homology more than 90% and the gene of encoding said proteins.

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

Sequence 2 in the sequence table is made up 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, 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 (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, promptly 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 (as kermes synthetic enzyme Nos gene), plant gene (as soybean storage protein gene) 3 ' end to transcribe 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, as the ubiquitin promoter (Ubiquitin) of cauliflower mosaic virus (CAMV) 35S promoter, corn, stress induced promoter Rd29A etc., they can use separately or be used in combination 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 be synthetic.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 (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 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 reorganization 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 its arbitrary segmental primer 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 reorganization 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,, can obtain resistance of reverse enhanced transgenic cell line and transfer-gen plant the gene transfered plant cell of encoding said proteins.Carry that described expression carrier can Ti-plasmids, Ri plasmid, plant viral vector, directly DNA conversion, microinjection, electricity be led, conventional biological method transformed plant cells or tissue such as agriculture bacillus mediated by using, and the plant transformed tissue cultivating is become plant.By the plant transformed host both can be monocotyledons, also can be dicotyledons, 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 system.

Albumen provided by the invention and encoding gene thereof can regulating plant growth and growths, are subjected to multiple stress-inducing, participate in sheep's hay to multiple response of coercing.With gene transfered plant of the present invention, can improve the resistance of plant.Particularly cradle (cut) and can induce this proteic expression, improve sugar in the intravital conveying efficiency of plant, 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 the mechanism of action to the sucrose transporter regulation and control of potential inside and outside factor in the plant growth and development process, 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 the growing state of transgenosis and contrast yeast strains system in the substratum of sole carbon source for sucrose;

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 to buy from routine biochemistry reagent shop and obtains.% among the following embodiment if no special instructions, is the quality percentage composition.Quantitative test in following examples all is provided with 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 system: 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 system: 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 segmental clone of sheep's hay sucrose transporter gene LcSUT1

1, the extraction of vegetable material processing and total RNA

With the chinense seedlings is material, extracts total RNA, carries out 1.2% agarose gel electrophoresis and detects, and the result as shown in Figure 1.The RNA that is extracted has two tangible electrophoretic bands, is followed successively by 28S RNA and 18S RNA from top to bottom, shows to have obtained higher, the more complete total RNA of purity.

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

Amino acid residue sequence according to disclosed plant sucrose transporter is sought conservative region, and according to conservative region sequences Design primer, concrete primer sequence is as follows:

S?15′-TGTGGCTATGCGGTCCTA-3′；

S25′-TTCGTGCTTGCGGTGATG-3′。

The total RNA that extracts chinense seedlings with step 1 is a template, uses PrimeScript ^TM1st Strand cDNASynthesized Kit test kit (Takara company) and reference reagent box specification sheets, the synthetic 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; Add 5 * Buffer4.0 μ l, RNase Inhibitor (40U/ μ l) 0.5 μ l, PrimeScript RTase (200U/ μ l) 0.5 μ l then, 42 ℃ of 45min, 70 ℃ of 15min, with the synthetic first chain cDNA be stored in-20 ℃ standby.

With the first chain cDNA that obtains is 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 pre-sex change 5min of elder generation; 94 ℃ of 30s then, 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 a 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 transformed into escherichia coli DH5 α 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 this segmental length is 1005bp, and its deoxyribonucleotide sequence has higher homology with the sequence of known SUT genoid 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 with step 1 is a template, adopts the SMARTer of Promega company ^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.

The first chain cDNA with acquisition is a 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 transformed into escherichia coli DH5 α 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, this segmental length is 503bp, its deoxyribonucleotide sequence has higher homology with 5 ' terminal sequence of known SUT genoid 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 with step 1 is a template, adopts the SMARTer of Promega company ^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 transformed into escherichia coli DH5 α 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, this segmental length is 927bp, its deoxyribonucleotide sequence has higher homology with 3 ' terminal sequence of known SUT genoid 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, is template with cDNA, 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 a 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 transformed into escherichia coli DH5 α 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 (forming) 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 LcSUT1 Gene Sequence Analysis 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 an albumen (Fig. 8) of striding film for 12 times, meets the sucrose transporter family structure, illustrates that this albumen belongs to sucrose transporter family.

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

(the GeneBank number of landing 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 it is 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.The evolutionary tree analysis revealed, bigger 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 multiple stress conditions

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.

Handle after 2 hours, extract total RNA of careless seedling respectively, 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 handle the expression amount increasing amount obvious.

The functional verification of embodiment 4, LcSUT1

One, the clone of LcSUT1 gene

Extract total RNA of chinense seedlings, reverse transcription is cDNA; With cDNA is template, with primer (S1 and A1) is 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, the PCR product with step 1 is a 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 transformed into escherichia coli DH5 α (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 of recovery and the skeleton carrier of recovery are connected, 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 system of SUT gene function disappearance, obtain recombination microzyme A.Plasmid PDR196-StSUT is imported in the yeast mutants SUSY7/ura3 strain system of SUT gene function disappearance, obtain recombination microzyme B (over against shining).The PDR196 empty carrier is imported in the yeast mutants SUSY7/ura3 strain system of SUT gene function disappearance, obtain recombination microzyme C (negative contrast).

It is that (1.7g/L does not have 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 sucrose respectively, 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 system that can complementary sucrose transporter afunction makes its normal growth.

Sequence table

＜110〉Institute of Botany, Chinese Academy of Sciences

＜120〉plant sucrose transporter 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 is following (a) or (b):

2. coding claim 1 described proteic gene.

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

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

3) under stringent condition with 1) or 2) the dna sequence dna hybridization that limits and the dna molecular of encoding sucrose transfer related protein;

4) with 1) or 2) or 3) dna sequence dna that limits has 90% above homology, and the dna molecular of encoding sucrose transfer related protein.

4. the recombinant expression vector, expression cassette, transgenic cell line or the reorganization bacterium that contain claim 2 or 3 described genes.

5. recombinant expression vector according to claim 4 is characterized in that: described recombinant expression vector is that claim 2 or 3 described genes are inserted the recombinant plasmid that the multiple clone site of PDR196 obtains.

6. total length or its any segmental primer of amplification claim 2 or 3 described genes are right.

7. any one application in cultivating plant with adverse resistance in the described albumen of claim 1, claim 2 or 3 described genes, claim 4 or 5 described recombinant expression vectors, expression cassette, transgenic cell line or the reorganization bacterium.

8. a method of cultivating transgenic plant is that claim 2 or 3 described genes are imported in the purpose plant, obtains the transgenic plant that resistance of reverse is higher than described purpose plant; Described purpose plant is Arabidopis thaliana, tobacco, Root or stem of Littleleaf Indianmulberry, paddy rice, wheat, corn, cucumber, tomato, willow, turfgrass, lucerne place, soybean or cotton.

9. a method of cultivating transgenic yeast is that claim 2 or 3 described genes are changed in the purpose yeast, obtains transgenic yeast, and the sucrose of described transgenic yeast utilizes ability to be higher than described purpose yeast.

10. method according to claim 9 is characterized in that: claim 2 or 3 described genes import in the described purpose yeast by claim 4 or 5 described recombinant vectorss; Described purpose yeast is a yeast mutants SUSY7/ura3 strain system.