CN107723295A

CN107723295A - A kind of sugarcane saccharide transporter ShSWEET1 genes and its application

Info

Publication number: CN107723295A
Application number: CN201710152152.7A
Authority: CN
Inventors: 王俊刚; 赵婷婷; 张树珍; 王文治; 杨本鹏; 冯翠莲; 冯小艳; 曾军; 蔡文伟; 熊国如; 伍苏然
Original assignee: Institute of Tropical Bioscience and Biotechnology Chinese Academy of Tropical Agricultural Sciences
Current assignee: Institute of Tropical Bioscience and Biotechnology Chinese Academy of Tropical Agricultural Sciences
Priority date: 2017-03-15
Filing date: 2017-03-15
Publication date: 2018-02-23
Anticipated expiration: 2037-03-15
Also published as: CN107723295B

Abstract

The invention provides a kind of sugarcane saccharide transporter ShSWEET1 genes, its nucleotide sequence such as SEQ ID NO:Shown in 1.Present invention also offers the application of the albumen of the ShSWEET1 gene codes and the ShSWEET1 genes.Clone obtains ShSWEET1 genes to the present invention from sugarcane first, the gene has the transport function of saccharide transporter, it can be located in the cell membrane of cell, cold resistance of plant stress ability can be improved, be advantageous to the plants such as sugarcane to grow under adverse condition, to disclose regulatory function of the sucrose transporting mechanism to growth and development of plants and quality-improving, science of heredity foundation is provided to improve genetic engineering research of the crop yield with improveing quality.

Description

A kind of sugarcane saccharide transporter ShSWEET1 genes and its application

Technical field

Biological technical field of the present invention, it is related to a kind of sugarcane saccharide transporter ShSWEET1 genes and its application.

Background technology

Sugarcane (Saccharum officinarum L.) belongs to grass family, chinese sorghum race, Saccharinae, saccharum, 1 year Raw or perennial herb plant, it is homologous with corn and sorghum, with originating in the torrid zone, the subtropical zone of New Guinea or India Area, sugarcane are to convert solar energy into one of maximally effective plant of chemical energy as C4 plants.Sugarcane plant light saturation point is high, and two Carbonoxide crystallized ability is strong, and photosynthetic rate is high, and rate of photosynthisis is big, and biomass is high, and sugar content very abundant simultaneously can also conduct Bio-fuel.Past more than 30 years, because sugarcane can obtain the whole world as a kind of superior potential substituting regenerative resource Pay attention to.Carbon distribution is photosynthesis of plant energy and the critical process of dispensed materials.Generally, C4 photosynthesis of plant and Chloroplaset and vascular bundle sheath cell in mesophyll cell occurs for carbon assimilation.The carbon that photosynthesis is fixed is converted into by photosynthetic source cell The derivative of sugar or sugar is consumed and stored into distal end storehouse cell and in the cell of storehouse then along bast long-distance transportation (Poorterand Villar,1997).Living cells consumption 35%-40% sugar is used to provide energy for cell under normal circumstances Grow (HallandRao, 1999), including cell amplification, division, differentiation, the development of plants of Nutrient Absorption and maintenance.Some are made For the Metabolic Intermediate of cell, such as monose, amino acid, organic acid etc., unnecessary sugar is stored in vacuole or recycling more On polymers (such as amyloplaste), or form biological structure material (such as cellulose, hemicellulose and lignin).

The high-energy transfer capability of sugarcane becomes a kind of high-yield feed crop having good prospects；Cane sugar manufacture mistake simultaneously Caused molasses are even more the animal feeds such as high-quality pig, ox in journey, and bagasse is also used as paper making raw material and edible fungus culturing Raw material；Sugarcane is also the important source material of China's light industry, is raw material and the product of auxiliary material up to 56 classes kind more than 2300 using sucrose (Qin Wenxin etc., 2006)；Fruit sugarcane and juice from sugar cane in sugarcane are also first-class health food, and sugarcane is cool in nature, there is heat-clearing, solution Wine, diuresis, the effect for helping spleen stomach invigorating, nourishing.Sugarcane is the C4 plants of specular removal, and biological yield is very high, flat in sugarcane high-yield area Annual per hectare sugarcane can about produce 7000L alcohol (Matsuoka et al., 2009), and 1-17 is higher by more than other crops Times, as corn (4000L/hm2) and sugar grass (3000L/hm2) (Teetor et al., 2011；Hill et al.,2006). Enjoying Brazil of " state of green energy resource " good reputation, sugarcane year output nearly 10,000,000 tons of alcohol, utilize alcohol and gasoline mixing New energy sources for automobile, enough Brazil the country three into automobile use completely.In addition, using the byproduct of sugarcane and sugarcane, lead to Certain technological means is crossed, can also be generated electricity so as to promote the well-being of mankind, moreover, in Brazil, is generated electricity with cane kind raw material The summation of water generating is alreadyd exceed, becomes the important energy sources of Brazil.

At present, domestic sugarcane plantation and management still suffer from problems, improve cultivation of sugar cane and way to manage lifting be sweet Much room also be present in sugarcane biological yield.Meanwhile the maximum bottleneck that faces of China's cane planting be kind unification trend it is tight Weight, there is an urgent need to cultivate new wide suitable, high yield, high sugar kind to be used to produce in production, due to the chromosome quantitative of sugarcane is more, Genome structure complexity produces the narrow of the hereditary basis of use kind in addition so that is grown the time required to conventional herd breeding kind and is difficult to carry High total sugar content, gene engineering method also simply change the distribution of carbonizable substance, do not improve sugarcane sugared content on the whole, because This is also far from the needs (Tingting ZHAO, 2012) for meeting production and living.Sugar accumulation and distribution mechanism in sugarcane body are studied, finds out sugar The CCP divided in cumulative process, it is to realize sugarcane high-yield, a high sugared important approach then to be improved.To be to carry High sugarcane quality, realize that sugarcane high-yield, high sugar and high anti-breeding objective provide science reference.

The content of the invention

It is an object of the invention to overcome deficiency of the prior art, there is provided a kind of sugarcane saccharide transporter ShSWEET1 and The application of its encoding gene and the gene.It is a further object to provide sugarcane saccharide transporter ShSWEET1 genes Cloning process and used primer pair.

The first aspect of the invention is to provide a kind of sugarcane saccharide transporter ShSWEET1 genes, and its nucleotide sequence is such as SEQ ID NO:Shown in 1.

The second aspect of the invention is to provide a kind of sugarcane saccharide transporter ShSWEET1, and it is first side of the invention The protein of sugarcane saccharide transporter ShSWEET1 gene codes described in face, its nucleotide sequence such as SEQ ID NO:Shown in 8.

The third aspect of the invention is to provide the sugarcane saccharide transporter described in a kind of one side of the invention The cloning process of ShSWEET1 genes, comprises the following steps：(1) total serum IgE is extracted from the root, stem and/or leaf of ripe sugarcane； (2) reverse transcription is carried out as template using total serum IgE and obtains cDNA；(3) ShSWEET1 full length gene sequence amplification primers pair are designed, are carried out PCR is expanded, and reclaims PCR primer, wherein, the nucleotide sequence such as SEQ ID of ShSWEET1 full length gene sequence amplification primers pair NO:2 and SEQ ID NO:Shown in 3；(4) PCR primer connection carrier, is converted, and sequencing, obtains ShSWEET1 gene fragments.

The fourth aspect of the invention is to provide the sugarcane saccharide transporter as described in one side of the invention ShSWEET1 genes position in the cell in application.

The fifth aspect of the invention is to provide the sugarcane saccharide transporter as described in one side of the invention Application of the ShSWEET1 genes in cold resistance of plant stress is improved.

The sixth aspect of the invention is to provide the sugarcane saccharide transporter as described in one side of the invention Application of the ShSWEET1 genes in the high anti-transgenic sugarcane of high sugared high yield is cultivated.

The seventh aspect of the invention is to provide a kind of expression vector, and it contains the sugarcane described in one side of the invention Saccharide transporter ShSWEET1 genes.

The eighth aspect of the invention is to provide a kind of primer pair, its nucleotide sequence such as SEQ ID NO:2 and SEQ ID NO:Shown in 3.The primer pair can be used for ShSWEET1 full length gene sequence amplifications.

The ninth aspect of the invention is to provide another primer pair, its nucleotide sequence such as SEQ ID NO:4 and SEQ ID NO:Shown in 5.

The tengh aspect of the invention is to provide another primer pair, its nucleotide sequence such as SEQ ID NO:6 and SEQ ID NO:Shown in 7.

Clone obtains ShSWEET1 genes to the present invention from sugarcane first, and the gene has the transhipment work(of saccharide transporter Can, it can be located in the cell membrane of cell, cold resistance of plant stress ability can be improved, it is raw under adverse condition to be advantageous to the plants such as sugarcane It is long, to disclose regulatory function of the sucrose transporting mechanism to growth and development of plants and quality-improving, to improve crop yield and improvement The genetic engineering research of quality provides science of heredity foundation.

Brief description of the drawings

Fig. 1 is the Total RNAs extraction electrophoretogram of sugarcane different parts.

Fig. 2 is the electrophoretogram of ShSWEET1 gene cDNAs total length amplification.

Fig. 3 is ShSWEET1 full length genes cDNA ORF analysis results.

Fig. 4 is ShSWEET1 transmembrane amino acids area prediction result.

Fig. 5 is SWEET1 amino acid Subcellular Localization prediction results.

Fig. 6 is sugarcane different tissues ShSWEET1 gene relative expression quantities.

Fig. 7 is ShSWEET1 gene relative expression quantities in different sugarcane disease leaves.

Fig. 8 is relative expression quantity under the cold stress of ShSWEET1 genes in sugarcane.

Fig. 9 is pShSWEET1-GFP vector construction procedure charts.

Figure 10 is pShSWEET1-GFP restriction enzyme digestion and electrophoresis figures.

Figure 11 is Subcellular Localization of the ShSWEET1 genes in onion endepidermis, wherein, A, C：(488nm is blue for GFP fluorescence Light excites)；D, F：White-light visualization：A/D：Turn there are pShSWEET1-GFP carrier epidermal cells；C/F：Turn have pCAMBIA1302 empty Carrier epidermal cell.

Figure 12 is plant expression vector pShSWEET1 restriction enzyme digestion and electrophoresis figures.

Figure 13 is the PCR testing results of transgene tobacco ShSWEET1 genes.

Figure 14 is the phenotype of the cold stress of tobacco, wherein, CK：Wild-type tobacco；1：ShSWEET1 transgene tobaccos.

Figure 15 is sugarcane genetically modified seedling cultivating process, wherein, A:The induction B of callus：Callus differentiates small again Seedling C：PPT screens D：Screening and culturing of taking root E：Turn ShSWEET1 resistant plant hardenings F:Turn the field planting of ShSWEET1 resistant plants.

Figure 16 is transformed plant Bar genetic test results, wherein, M：DL2 000marker；CK⁺:Vector plasmid；CK^-：It is non- Transfer-gen plant；1-5：Turn ShSWEET1 resistant plants.

Embodiment

With reference to the accompanying drawings, the present invention is further illustrated in conjunction with specific embodiments, to more fully understand this hair It is bright.The experimental method of unreceipted specific experiment condition in the following example, generally according to normal condition, molecular cloning (Molecular Cloning:A Laboratory Manual, 3rd ed.) or Yeast Genetics method experiment guide (Methods in Yeast Genetics:A Cold Spring Harbor Laboratory CourseManual, Adams A et al Compile, Cold Spring Harbor Laboratory, 1998 publish) described in condition, or according to the bar proposed by manufacturer Part.

1 experiment material

1.1 vegetable material

To plant in China tropic Agriculture Academy Sciences tropic Biotechnology Research Institute Lingao proving ground and Tropical China agriculture The new platform of industry academy of sciences Hai Kouyuan areas sugarcane sugared No. 22 (ROC22) is vegetable material, sugarcane ROC22 tissue-cultured seedling and Nicotiana tabacum for originally Laboratory provides.

1.2 bacterial strains and plasmid

Escherichia coli (Escherichia coli.DH5 α) competence is purchased from Fu Neng companies (Guangzhou)；Agrobacterium tumefaciems (Agrobacterium tumefaciens EHA105) preserves for this laboratory.PMD19-T, pMD19T simple carrier matter Grain is purchased from Takara companies；PCAMBIA3300-GUS, pCAMBIA1302 plant expression vector preserve for this laboratory.

1.3 enzymes and other reagents

Reagent RNA extracts kits are purchased from Invitrogen companies (U.S.)；PCR reagent, DNA glue QIAquick Gel Extraction Kit and small amount plasmid extraction kit are purchased from OMEGA companies (U.S.)；Reverse transcription reagent box, SYBR Green II Premix, rTaq enzyme, LA-Taq enzymes, restriction enzyme are purchased from TaKaRa companies (China, Dalian)；T4DNA ligases are purchased from Fermentas companies；Beta -mercaptoethanol, sucrose, ampicillin (Amp), streptomysin (Str), carboxylic become penicillin (Car), card That mycin (Kan), rifampin (Rif), careless fourth phosphine (Phosphinothricin, PPT), agarose are purchased from Sigma companies；It is conventional Chemical reagent for domestic analysis it is pure.

1.4 key instrument equipment

PCR amplification instrument T1Thermocycle (Biometra), grads PCR instrument (Eppendorf5331), Multipurpose high-speed Refrigerated centrifuge (Thermo), normal temperature centrifuge (HETTICHD-78532), automatic triple pure water distiller (Shanghai Asia Rong Shenghua Instrument plant SZ-97), thermostat water bath is constant-temperature table (N.JG-27EKISO), constant incubator (SANYO MRL-350), ultraviolet Spectrophotometer (Beijing Pu Xi general instrument Corp. T6), electrophoresis apparatus (LABNET EQ2705), gel imaging system (BIO- RAD), real-time fluorescence quantitative PCR instrument (StratageneMx3005P)

The preparation of 1.5 culture mediums

LB culture mediums (1L)：Peptone 10g, yeast extract 5g, NaCl10g, pH7.0, solid medium 1000mL add 15g agar powders, 121 DEG C, 25min autoclavings.

YEP culture mediums (1L)：Peptone 10g, yeast extract 10g, NaCl5g, pH7.0, solid medium 1000mL add 15g agar powders, 121 DEG C, 25min autoclavings.

Sugarcane callus Fiber differentiation M1 (1L)：MS+2.4-D 1mg/L+ sucrose 30g/L+ carragheens 8g/L, pH5.8.

Sugarcane differential medium M2 (1L)：MS+6-BA 1mg/L+KT 0.5mg/L+ sucrose 30g/L+ carragheen 8g/L, pH5.8。

Fluid nutrient medium MR (1L) is used in sugarcane conversion：1/5MS a great number of elements+MS other compositions+2.4D 1mg/L+ 10mmol/L fructose+10mmol/L glucose+30g/L multitudinous sugared (solid medium should add carragheen 8g/L), pH5.3.

Sugarcane rooting induction culture medium (1L)：MS (a great number of elements halves, other components unchangeds)+NAA 2mg/L+ sucrose 20g/L+ coconut palm water 100mL/L+ carragheen 8g/L+ activated carbons 0.2g/L, pH5.8.

2 experimental methods and experimental result

The clone of 2.1 sugarcane ShSWEET1 genes

(1) extraction of sugarcane root, stem, leaf total serum IgE

With reference to InvitrogenReagent extractions RNA method and step is carried out：Each 0.1g of root, stem, leaf is taken, Cut into pieces, be transferred in liquid nitrogen rapidly, with mortar grinder into powder.1mL Trizol are taken to add 1.5ml without enzyme centrifuge tube, mark Note.The good sample of liquid nitrogen grinding is quickly transferred in centrifuge tube, rapid oscillation 2min.After standing 5min at ambient temperature, add Enter 200 μ L chloroforms.After firmly overturning centrifuge tube mixing 30s repeatedly, after standing 10min at room temperature, 4 DEG C of condition 12000g centrifugations 15min.Draw supernatant and be transferred to new 1.5ml without (not rocking intermediate layer during absorption) in enzyme centrifuge tube.Add 500 μ l preservation Isopropanol in -20 DEG C of refrigerators, after thoroughly mixing, 10min is stood at -20 DEG C, 4 DEG C of condition 12000g centrifuge 15min.Abandon Clearly.1mL75% ethanol is added, gently vibrates centrifuge tube, suspend precipitation.4 DEG C of condition 7500g centrifuge 10min, abandon supernatant, ultra-clean Platform blows 5-10min, and liquid in pipe to be centrifuged is killed, and adds 40 μ LRnase-freeH₂O, 55-60 DEG C of dissolving 10min of water-bath. Put on ice, add Fermatas DNaseI buffer 5 μ L, DNaseI 5 μ L, 37 DEG C of warm bath 30min.Add 5 μ L 50mM EDTA, 65 DEG C of warm bath 10min, put -70 DEG C it is standby.

(2) RNA integralities and purity detecting

The 2 μ L agarose gel electrophoresis of Total RNA 1.2% is taken to detect the Total RNA with 1 μ L with through nucleic acid-protein Analysis-e/or determining RNA purity.Ratio through its purity of nucleic acid-protein analysis-e/or determining OD260/280 is 1.9~2.0, concentration In 200-600ng/ μ L.(Fig. 1) is detected by 1% agarose gel electrophoresis, the band of total serum IgE two respectively organized is clear, band Completely, 28SrRNA brightness is about 2 times of 18SrRNA, meets the requirement of follow-up test.

(3) reverse transcription synthesis cDNA chains

A. the Microtube pipes without enzyme are placed on and sequentially added in order on ice with following mixed liquor：

Template ribonucleic acid (below 5 μ g) 7.0 μ L (0.1ng-5 μ g)

Oligo(dT)18Primer(50μM) 1.0μL

RNase free ddH₂O up to 12.0μL

B.65 it is rapidly moved to more than 2min on ice after DEG C insulation 5min.

C.cDNA synthesis reaction solutions, add in order：

D. cDNA synthesis reaction solutions are added in RNA/ primer mixtures, gently mixed, simple centrifugation, 42 DEG C of incubation 1h.

E.70 DEG C, 10min terminating reactions.

F. reverse transcription cDNA be stored in -70 DEG C it is standby.

(4) ShSWEET1 full length genes sequence amplification

A. design of primers：Designed according to corn, sorghum saccharide transporter (Sugar efflux transporter) gene Primer is as follows：

B.PCR amplification systems are as follows：

C. micro- centrifugation after being well mixed, it is put into PCR instrument and enters performing PCR amplification, response procedures are：

(5) pcr amplification product gel electrophoresis

After pcr amplification product is added into 5 μ 6 × Loading of L buffer mixings, 1% agarose gel electrophoresis is carried out.Electricity 95V is pressed, electrophoresis 35min, is then photographed to record in gel imaging system, and cut the gel of doubtful purpose band.Expand piece Duan great little is about 800bp (such as Fig. 2).

(6) amplified fragments gel reclaims

The recovery of purpose band is carried out with reference to the gel reclaims kit specification of OMEGA companies.

(7) fragment and carrier T coupled reaction are reclaimed

Purpose band and pMD19-T carrier linked systems are as follows：

16 DEG C, connection is overnight.

(8) carrier conversion and sequencing are connected

Connection product (10 μ L) is added to the 1.5mL for containing 50 μ L competent cells (Escherichia coli.DH5 α) Centrifuge tube in, gently mixed with pipette tips, be placed on ice bath 30min on ice.42 DEG C of heat shock 90s, are transferred to place 2min on ice immediately.

900 μ L LB fluid nutrient mediums, 37 DEG C of constant incubator shaken cultivation 60min are added in liquid system mixed above. 10000rpm, 45s is centrifuged, discards the μ L of supernatant 500, then precipitation is gently mixed with pipette tips, be coated on preprepared and contain On 100 μ g/mL Amp LB culture mediums, 30min is stood in super-clean bench, after bacterium solution is absorbed by flat board, is trained in 37 DEG C of constant temperature Carton upside down flat board 12~16h of culture is supported, until forming single bacterium colony.About 10 single bacterium colonies of picking, are respectively put into 1mL and contain Amp and resist In the centrifuge tube of the LB liquid medium of property, 37 DEG C of constant incubator shaken cultivations are stayed overnight.Bacterium solution is dispensed, respectively about 200 μ L, one Manage for being sequenced, one manages the length scale that Insert Fragment in carrier is confirmed for PCR, remaining to save backup.1 μ L bacterium solutions are taken to make For pcr template, enter performing PCR amplification (method is with " ShSWEET1 full length genes sequence amplification "), serve marine growth engineering finite public affairs Department's sequencing, sequencing result show：It is 868bp that clone, which obtains clip size,.

2.2 sugarcane ShSWEET1 gene biological bioinformatics analysis

Blastp search comparisons are carried out to ShSWEET1 amino acid sequences on NCBI, find it and SWEET gene families It is homologous.According to http://www.ncbi.nlm.nih.gov/gorf/gorf.html is to ShSWEET1 gene cDNA full length sequences ORF analyses are carried out to find, the sequence possesses complete ORFs, length 732bp, from 98 initiation codon ATG to 829 TGA terminator codons are stopped, and encode 243 amino acid (Fig. 3) altogether.Compiled with online software NCBI CDS databases The conserved structure domain analysis of code albumen, it is found that ShSWEET1 contains the conservative of PQ-loop superfamily and MtN3/saliva Domain.ProtParam predicts that the relative molecular weight of the DNA encoding the protein is 26.93KDa, isoelectric point 8.87, overall average Hydropathy index is 0.916, and it is hydrophobic proteins to show the albumen.

ShSWEET1 amino acid sequences, which are carried out, with TMHMM Serverv.2.0 softwares carries out transmembrane structure prediction discovery (figure 4), ShSWEET1 albumen has 7 transmembrane regions, and transmembrane region is located at 15-37,58-80,85-107,116-138,148- respectively 170,177-199,204-226 totally 155 amino acid.It is similar to the eucaryote SWEET protein transmembrane structures reported before, because This infers that ShSWEET1 possesses the architectural feature of eucaryote SWEET albumen.Found with reference to Subcellular Localization software analysis ShSWEET1 is likely located on plasma membrane (Fig. 5), thus it is speculated that ShSWEET1 is probably a kind of plasmalemma protein.

The different plant SWEET protein amino acid sequences of 50 reported are chosen from ncbi database, with MEGA5.1 structures Build phylogenetic tree.It is amino acid sequence based on amino acid homology analysis shows ShSWEET1 gene codes and corn, millet, small The homology status of the monocotyledon ShSWEET family members such as wheat and two fringe false bromegrass approaches, respectively 91%, 89%, 85% and 83%.It is MtN3/saliva/ShSWEET family members to illustrate sugarcane ShSWEET1.

ShSWEET1 gene expression analysis in 2.3 sugarcane different tissues

ReferenceThe The Immature Leaves of Reagent extraction RNA methods extraction sugarcanes, climax leaves, prematurity stem, into Sick leaf and Stress treatment the Sugarcane Seedlings RNA of ripe stem, root and different Sugarcane Diseases, provided with reference to Fermentas companies anti- Transcript reagent box synthesizes cDNA.Uniform concentration and carry out RT-qPCR detections on real-time fluorescence quantitative PCR instrument Mx3005P afterwards Relative expression quantity of the ShSWEET1 genes under different tissues and Different stress.

A) RT-qPCR primers：

SW1F:5′-CCGCTCTCCGTGATGAAA-3′

SW1R:5′-GGAAGCCAGAGACAGGAA-3′

Sugarcane GAPDH internal control primers (quoted from Iskandar et al., 2004)：

P1：5′-CACGGCCACTGGAAG CA-3′

P2：5′-TCCTCAGGGTTCCTGATGCC-3′

B) PCR amplification system：Using Takara companiesThe kits of Premix Ex TaqTM II.

RT-qPCR amplification programs：95 DEG C, 3min；95 DEG C, 15s, 60 DEG C, 45s, 40 circulations；Each sample sets 3 Repeat, and do solubility curve.Reference gene amplification system and program are identical with target gene.Data use 2^-△△CTMethod point Phase separation is to quantitative values (Kenneth et al., 2001).

(1) ShSWEET1 gene expression analysis in sugarcane different tissues

Long-term acquisition is stretched in sugarcane to plant in China tropic Agriculture Academy Sciences tropic Biotechnology Research Institute's Lingao sugarcane examination The sugarcane plant different tissues sample in base is tested as test specimen.The healthy and strong sugarcane planting in the elongation phase is chosen during sample collection 9 plants of strain, every 3 plants are a repetition mixed sampling.The Immature Leaves, climax leaves, prematurity stem, ripe stem are gathered respectively.Prematurity Leaf is that sugarcane does not extract lobus cardiacus out；Climax leaves are fully deployed from top to bottom for sugarcane plant and+3 leaves of visible plump band.Prematurity Stem is the second section below the stem apical growing point of plant top；Ripe stem is sugarcane plant by lower and upper 3rd or 4 section.Root is to take Plant after ripening root.

RT-qPCR results are as shown in Figure 6：Root, prematurity stem, maturation of the ShSWEET1 genes in sugarcane elongation phase plant There is expression in stem, The Immature Leaves and climax leaves, illustrate that ShSWEET1 genes belong to constitutive expression in sugarcane.ShSWEET1 Gene expressed in climax leaves and ripe stem it is of a relatively high, it is each to organize relative expression quantity to be followed successively by：Climax leaves ＞ maturation stems ＞ is not Climax leaves ＞ root ＞ prematurity stems, it is respectively equivalent to 1~3.5 times of prematurity stem expression quantity.

(2) ShSWEET1 gene expression analysis under sugarcane different diseases

It is sweet in the crop field in China tropic Agriculture Academy Sciences tropic Biotechnology Research Institute Lingao sugarcane trial base planting Collection infects sugarcane brown streak, sugarcane red streak and smut of sugarcane and in the sugarcane plant leaf of morbidity mid-term respectively in sugarcane As sugarcane brown streak disease leaf, sugarcane red streak disease leaf, smut of sugarcane disease leaf；During sampling every kind of disease choose respectively typical case and The morbidity mid-term plant of single morbidity, each plant choose a piece of incidence of leaf, altogether selection 9, every 3 repetitions, 3 weights It is multiple.Using the sugarcane health plant leaf do not fallen ill as control.It is immediately placed in liquid nitrogen and preserves after the completion of leaf sample collection.

RT-qPCR results are as shown in Figure 7：In three kinds of sugarcane brown streak, smut of sugarcane, sugarcane red streak sick leaves ShSWEET1 genes relative expression quantity is all higher than the sugarcane top of health.Wherein ShSWEET1 gene expressions in sugarcane brown streak disease leaf Measure highest, quite healthy sugarcane top 2.6 times.Show when pathogen invades blade, may activation and induction ShSWEET1 Gene is expressed on mesophyll cell film, causes more sugar to be secreted into mesophyll cell gap by it, is the intrusion of bacterium and numerous Grow and a good environment is provided.

(3) Sugarcane Seedlings low-temperature treatment

Experiment is inoculated in MS culture mediums at 28 DEG C with sugar-cane tissue culture seedlings plant, and 16h illumination/8h is dark, and intensity of illumination is Preculture two weeks under the conditions of 3000lx, choose the sugar-cane tissue culture seedlings that leaf age is identical and growing way is consistent and be transferred to 28 in MS fluid nutrient mediums DEG C, 16h illumination/8h is dark, and intensity of illumination is 3000lx renewal cultivations two days, and then at 4 DEG C, 16h illumination/8h is dark, illumination Intensity carries out cold stress under the conditions of being 3000lx.It is sampled after cold stress 0h, 6h, 12h, 24h, 36h, 48h, 72h, 96h, often 9 plants of plant of sub-sampling, 3 plants are a repetition mixed sampling, 3 repetitions.It is immediately placed in liquid nitrogen and preserves after the completion of per sub-sampling.

QRT-PCR results are as shown in Figure 8：Cold stress can induce ShSWEET1 gene upregulations to express.4 DEG C of processing sugarcane groups Train seedling when, in 0-96h, ShSWEET1 with processing time the lasting increase of increase expression quantity.ShSWEET1 is in processing 48h When expressed by rapid induction, important, the possibility that shows that ShSWEET1 genes have the function that during sugarcane resists cold stress Transport soluble sugar is participated in sugarcane body to reduce osmotic pressure in sugarcane body, it will help maintain sugarcane under abiotic stress Normal growth.

Subcellular Localization of the 2.4 ShSWEET1 genes in onion endepidermis

(1) structure of GFP fusion vectors

A. to ShSWEET1 gene sequencings, find complete ORF and design the primer with restriction enzyme site at its both ends, under Trip primer removes terminator codon, as follows：

S2GF：5′-GGCCATGGATTGGGGTGATC-3′NcoⅠ

S2GR：5′-GGAGATCTTGTATGTGTGACTAGTAATGGCA-3′BglⅡ

B. using ShSWEET1-pMD19-T plasmids as template, primer (S2GF, S2GR) enter performing PCR amplification, amplification system and Program is as follows：

Response procedures are：95 DEG C of 5min, 95 DEG C of 1min, 62 DEG C of 1min, 72 DEG C of 1min, 35 circulations, 72 DEG C of 10min.PCR After reaction terminates, with 1% agarose gel electrophoresis.Purpose band is cut with scalpel in gel imaging system and reclaimed, then It is connected into pMD19T-simple carriers, is transformed into competence DH5 α, picking positive colony enters performing PCR identification, serves Hai Sheng Work is sequenced.

C. the bacterial strain of the monoclonal and the carrier containing pCAMBIA1302 to correct ShSWEET1-pMD19T is sequenced carries out plasmid Extraction.Plasmid extraction extracts examination on a small scale with reference to the E.Z.N.A Plasmid Miniprep Protocol plasmids of OMEGA companies Agent box specification is carried out.

D. double enzymes are carried out to plasmid ShSWEET1-pMD19T and pCAMBIA1302 with restriction enzyme NcoI, Bgl II Cut, digestion system is as follows：

E. then 1% agarose gel electrophoresis, carrier large fragment and target gene fragment are reclaimed, with T4DNA Ligase Connection, convert and enter competence DH5 α (pShSWEET1-GFP vector construction processes are as shown in Figure 9), picking positive colony, 37 DEG C The shaken cultivation grain of upgrading overnight, double digestion identification, electrophoresis excision purpose size strip (Figure 10), correct carrier are named as PShSWEET1-GFP fusion vectors.

Digestion system is as follows：

(2) preparation of Agrobacterium competence

A. Agrobacterium EHA105 bacterial strains are taken out from -80 ultra low temperature freezers, thaws on ice, agriculture bar is dipped in oese Bacterium, rule on YEP flat boards (Rif containing 10mg/mLStr+20mg/mL), 28 cultures 2 days.

B. choose and take single bacterium colony to be inoculated in containing 5ml YEP fluid nutrient mediums (Rif containing 10mg/mLStr+20mg/mL) In, 28 DEG C of 24~48h of shaken cultivation are untill bacterium solution muddiness.

C. the bacterium solution for taking 1mL to purify is inoculated into the triangular flask of the YEP fluid nutrient mediums containing 50mL antibiotic-frees, 28 DEG C 5~8h of shaken cultivation, untill surveying OD=0.5~0.6.

D. bacterium solution is poured into 80mL centrifuge tube from triangular flask, on ice cryostat 30min；

E.5000xg, 5min is centrifuged under the conditions of 4, collects thalline；

F. operated on super-clean bench, abandon supernatant, thalline is resuspended with 20mL precooling 50mmol/L CaCl2；

G. e is repeated.

H. operated on super-clean bench, abandon supernatant, suspended again with 1mL 50mM CaCl2, take 50 μ L bacterium solutions to be dispensed into In 1.5mL centrifuge tubes, liquid nitrogen flash freezer, -80 DEG C save backup

(3) conversion of expression vector

An Agrobacterium competent cell containing 50 μ L is taken from -80 DEG C of ultra low temperature freezers, is positioned over and thaws on ice；Take 4 μ L plasmids are added in the centrifuge tube of above-mentioned defrosting, the static 15min of ice bath, are subsequently placed into 5min in liquid nitrogen；In 28 DEG C of insulating boxs 5min is placed, adds 500 μ L YEP (not added with antibiotic) culture mediums, under the conditions of 28 DEG C, concussion and cultivate 3h；Draw 200 μ L bacterium Liquid is coated on tri- anti-flat boards of YEP (10mg/mL Str+20mg/mL Rif+50mg/mL Kan), is fallen in 28 DEG C of constant incubators Put culture 48h；Picking single bacterium colony is inoculated into containing 5ml YEP fluid nutrient mediums (10mg/mL Str+20mg/mL Rif+50mg/ ML Kan) in, under the conditions of 28 DEG C, 36~48h of shaken cultivation；Picking Agrobacterium positive colony, bacterium is shaken, with reference to escherichia coli plasmid Extract Agrobacterium plasmid, digestion PCR identifications.

(4) agriculture bacillus mediated transfection onion epidermis cell

Picking is distinguished on solid plate and has carried recombinant plasmid pShSWEET1-GFP's and empty plasmid pCAMBIA1302 Positive monoclonal, to 10mL YEP fluid nutrient mediums (10mg/mL Str+20mg/mL Rif+50mg/mL Kan), 28 DEG C, shake Swing overnight incubation.The 3-4mL that transfers stays overnight bacterium solution to 50mL YEP fluid nutrient mediums (10mg/mL Str+20mg/mL Rif+ 50mg/mL Kan), 28 DEG C, shaken cultivation to OD600=0.6-0.7.4000g/min centrifuges 2min, thalline is collected, with MS liquid Body culture medium (10mmol/LMgCl₂, 100 μm of ol/L AS) and suspension thalline, make OD600 values 1.0 or so.

Onion epidermis cell preculture：Fresh onion is chosen, removes outer scale about 3-4 layers, the immersion of 75% ethanol 10min, sterile pure water rinse three times, each 5-8min.Sterile scalpel is cut, and onion scale endepidermis is cut into 1cm²It is small Block, it is laid in close to mesophyll face on MS solid mediums (100 μm of ol/L AS).28 DEG C of photoperiod 14/10h, preculture 24h.Altogether Culture：Onion endepidermis after preculture immerses MS fluid nutrient mediums (10mmol/LMgCl₂, 100 μm of ol/L AS) suspend bacterium In liquid, 20-30min, under during which shake is several, one jiao is picked up, bacterium solution is filtered dry on filter paper, is laid in MS solid mediums (100 μ Mol/L AS) on.28 DEG C of photoperiod 16/8h, co-culture 24h.Onion endepidermis is taken out, washing is rocked with MS fluid nutrient mediums, The Agrobacterium of surface attachment is removed, tabletting film-making, in fluorescence microscopy Microscopic observation, is taken pictures.

Observing the Agrobacterium containing GFP fusion vectors under ultraviolet light using fluorescence microscope, to infect rear onion endepidermis thin GFP expression in born of the same parents, as a result as shown in figure 11, in each portions of Figure 11-C hollow carriers pCAMBIA1302 in cell Green fluorescence all can be observed in position, so in the cell without specific polarization, and Figure 11-A are the pShSWEET1- of conversion The onion cells of GFP carriers, there is green fluorescence on cell membrane, have obvious location feature, according to bioinformatic analysis ShSWEET1 is a kind of epicyte protein, therefore speculates that ShSWEET1 is positioned on cell membrane.

The research of 2.5 ShSWEET1 gene genetic transformation of tobacco

(1) plant expression vector pShSWEET1 is built

A. to ShSWEET1 gene sequencings, its complete ORF is found respectively and designs drawing with restriction enzyme site at both ends Thing, anti-sense primer removes terminator codon, as follows：

S23F：5′-GGGGATCC GCAAGTATCTTCCCTCGACG-3′BamHI

S23R：5′-GGGAGCTCTGGCATGCTCATGTATGTGTG-3′SacI

B. using ShSWEET1-pMD19-T plasmids as template, primer (S23F, S23R) enter performing PCR amplification, amplification system and Program is as follows：

C. response procedures are：95 DEG C of 5min, 95 DEG C of 1min, 60 DEG C of 1min, 72 DEG C of 1min, 35 circulations, 72 DEG C of 10min. After PCR reactions terminate, with 1% agarose gel electrophoresis.Purpose band is cut with scalpel in gel imaging system and reclaimed, so It is connected into afterwards in pMD19T-simple carriers, is transformed into competence DH5 α, picking positive colony enters performing PCR identification, serves sea Raw work sequencing.Correct carrier is named as pMD-ShSWEET1.

D. the bacterial strain of the monoclonal and the carrier containing pCAMBIA3300-GUS to correct pMD-ShSWEET1 is sequenced carries out plasmid Extraction, the same c of extraction step.

E. restriction enzyme BamH I, Sac I is used to carry out double enzymes to plasmid pMD-ShSWEET1 and pCAMBIA3300-GUS Cut, digestion system is as follows：

F. then with 1% agarose gel electrophoresis, recovery carrier large fragment and target gene fragment, use T4DNA Ligase connections, conversion enter competence DH5 α, picking positive colony, 37 DEG C of shaken cultivations grain of upgrading overnight, double digestion identification Purpose size strip (result such as Figure 12) is obtained, correct carrier is named as pShSWEET1 carriers.

Digestion system is as follows：

(2) culture of tobacco explant

Nicotiana tabacum (Nicotiam tabacum) seed with 95% alcohol soak 5s after, then with aseptic nipper by its It is transferred in 50% (V/V) liquor natrii hypochloritis's (adding a drop Tween-20), persistently stirs 20min.With rinsed with sterile water five It is secondary, seed is placed in aseptic filter paper and dried, is seeded on MS solid mediums, per 20 seeds of ware, 28 DEG C of illumination cultivations.One Seedling is changed in fresh MS culture mediums after week, 3 plants every bottle, wait grow up to have 3~4 Astilbo idestabularis (Hemsl.) Engler seedlings when, can be used for soak Dye.

(3) conversion of tobacco

EHA105 strains containing expression vector (are contained into 10mg/mL Str+20mg/mL Rif in YEP solid mediums + 50mg/mL Kan) in rule, 28 DEG C of incubated 2d.Picking monoclonal contains the YEP Liquid Cultures of same antibiotic in 5ml 200rpm in base, 28 DEG C incubated overnight.

2mL is taken to be incubated overnight liquid in 100mL YEP fluid nutrient mediums (containing same antibiotic), 200rpm, 28 DEG C of perseverances Temperature culture, until OD600 reaches 0.5~0.7.4 DEG C, 4000rpm are transferred in 50mL sterile centrifugation tubes, 5min is centrifuged, discards Supernatant, culture medium raffinate is suctioned out with sterile pipette tips, adds 100mL MR fluid nutrient mediums (containing 150 μm of ol/L AS) and thalline is resuspended, 220rpm, 28 DEG C of shaken cultivation 2h, you can obtain Agrobacterium-mediated Transformation and infect liquid.

Contaminate：Tobacco tender leaf is broken into diameter 5mm leaf dish with aseptic card punch, about 50 leaf dishes, conversion is put into and contaminates In bacterium solution, with shaking table jog 30rpm 3min, 10min is vacuumized.5-l0min is stored at room temperature, is pulled out leaf dish with sterile small spoon And blot surface moisture with aseptic filter paper.Leaf dish is finally seeded in 28 DEG C of dark co-cultivation 3d in MS solid mediums.

After co-cultivation, leaf dish is transferred on MS differential mediums (6-BA containing 1mg/L) and carries out differentiation culture, is changed weekly Once same culture medium is until differentiate seedling.

The seedling differentiated at leaf dish edge is cut to be subsequently placed in MS culture mediums and cultivated one week.

After one week, seedling is transferred in MS solid mediums (containing 2mg/LPPT) and carries out screening and culturing, is changed weekly once Identical culture medium, until filtering out resistance seedling.

Resistance seedling is put into culture of rootage and cultivated two weeks, hardening one week, is then planted in flowerpot.

(4) Molecular Detection of transgene tobacco

Extract the genomic DNA of tobacco in a small amount with CTAB methods.Extraction to transgene tobacco and wild-type leaves RNA and anti- Transcription.The PCR that target gene is carried out to obtained transgene tobacco is detected, and is detected with (S2GF, S2GR) as primer, with transgenosis The STb gene of tobacco leaf is as template, amplification program：95 DEG C of 5min, 95 DEG C of 40s, 60 DEG C of 40s, 72 DEG C of 50s, 34 circulations, 72℃10min.Using plant expression vector pShSWEET1 plasmids as positive control, wild-type tobacco and H₂O as negative control, Using primary dcreening operation obtain 20 plants turn ShSWEET1 resistance seedling DNA as template enter performing PCR amplification, the results showed that：In transgenic resistance seedling, 17 plants turn equal can expand of ShSWEET1 tobaccos and arrive the band consistent with positive control, and stripe size may each be about 750bp (Figure 13), false sun Property plant and non-transgenic tobacco be then proved to be objective gene sequence without respective strap, recovery sequencing, illustration purpose gene is whole Close in tobacco gene group.

(5) the cold Stress treatment of transgene tobacco

Select to develop transgene tobacco seedling and wild type control group seedling healthy and strong, that growing way is consistent under identical environment, put Enter in 4 DEG C of constant incubators, 16h illumination/8h is dark, and intensity of illumination is incubated 50d under the conditions of 3000lx, when one section Between observe the growing state of plant, take pictures and record growing state.It was found that compared with wild-type tobacco transgene tobacco growth it is fast, Blade is small and sharp, well developed root system, and nutrient growth is vigorous (Figure 14), shows under cold stress, and turning ShSWEET1 tobaccos may more hold Easily soluble sugar is transported to space between cells, maintains Cell Homeostasis to resist cold stimulation, makes its normal growth.

2.6 ShSWEET1 gene genetic Transformation of Sugarcane

(1) acquisition of transgenic sugarcane

Sugarcane apical meristem growing point spire is thinly sliced into the lucifuge on M1 culture mediums and induces 20-30d, gradual shape It is loose into quality, the extremely strong embryo callus of cell division differentiation capability (Figure 15 A), you can for converting.With containing After pShSWEET1 Agrobacteriums bacterium solution immersion callus, dark co-cultures 3-4d, and light culture screens 15d on M2 culture mediums, so After be transferred on subculture medium illumination and break up screening and culturing 20d, callus starts to grow seedling (Figure 15 B).Treat that seedling is grown To 1cm or so, be transferred to screening and culturing 30d, PPT screening concentrations on the root media containing PPT be 2.5mg/L (Figure 15 C and 10 plants of resistant plants for turning ShSWEET1 15D) are obtained altogether.Wait turning to clean seedling after ShSWEET1 resistant plants root growth is good Culture medium, cut off old leaf, move into hardening (Figure 15 E) in crystal mud and go forward side by side performing PCR detection, after 2 weeks by resistant plant extract kind in In flowerpot (Figure 15 F).

(2) ShSWEET1 is overexpressed resistant plant Bar detections

ShSWEET1 is extracted in a small amount with CTAB methods and is overexpressed resistant plant and the genomic DNA of non-transgenic sugarcane, to plant Anti-herbicide gene (Bar) the design primer that thing expression vector carries, expression vector plasmid is positive control, non-transgenic sugarcane For negative control, enter performing PCR augmentation detection, the results showed that：In transgenic resistance seedling, all transfer-gen plants can be expanded and arrived The band consistent with positive plasmid, respectively may be about 400bp specific band (Figure 16), and recovery sequencing is proved to be Bar gene sequences Row, show that Bar genes are had been integrated into sugarcane genome.

With reference to EnviroLogix QuickStixTMKit for(bar) the step of Cotton kits The detection of Bar gene coded proteins is carried out to transgenic sugarcane, as a result show turning ShSWEET1 sugarcanes plant and can examining for acquisition The expression of Bar genes is measured, it is consistent with PCR detection Bar genetic results.

(3) ShSWEET1 is overexpressed the detection of resistant plant target gene

Sense primer is designed with Ubi promoter sequences and gene regions design anti-sense primer separately designs ShSWEET1 and is overexpressed Resistant plant target gene detection primer, expanded by entering performing PCR to the garbled resistant plants of PPT, the results showed that obtain 10 plants are overexpressed in plant, and 8 plants turn ShSWEET1 plant detected magnitude about 750bp bands, recovered, connection conversion and sequencing ratio It is right, it is found that sequence unanimously, shows that ShSWEET1 genes are had been integrated into sugarcane genome with being expected.

The specific embodiment of the present invention is described in detail above, but it is intended only as example, it is of the invention and unlimited It is formed on particular embodiments described above.To those skilled in the art, it is any to the equivalent modifications that carry out of the present invention and Substitute also all among scope of the invention.Therefore, the impartial conversion made without departing from the spirit and scope of the invention and Modification, all should be contained within the scope of the invention.

SEQUENCE LISTING

<110>China tropic Agriculture Academy Sciences tropic Biotechnology Research Institute

<120>A kind of sugarcane saccharide transporter ShSWEET1 genes and its application

<130> 170119-1

<160> 8

<170> PatentIn version 3.3

<210> 1

<211> 770

<212> DNA

<213> Artificial

<220>

<223>ShSWEET1 genes

<400> 1

gcaagtatct tccctcgacg tccaggcacc atggattggg gtgatccggc attgacgagc 60

ttcgtcgccg actcgtcctt ccgccacctc tgctgctacg gcgccggaat cgcaggaaac 120

gtcttcgcct tcgtgctctt catctcccca ctccccacat tcaagcggat cgtccggaac 180

gggtccacgg agcagttctc ggccatgccg tacatctact cgctgctcaa ctgtctcatc 240

tgcatgtggt acggccttcc cttcgtctcc tacggcgtcg tcctcgtcgc caccgtcaac 300

tccatcggcg ccgtcttcca gctcgcatac accgccgtct tcatcgcctt cgccgacgcc 360

aagcagaggc tcaaggtctc tgctctcctg gccgccgtct ttgtggtgtt cggactgatt 420

gtgtttgtta gtttggcttt gttggatcac caaacccggc agatgttcgt cggatatctc 480

agcgtcgcat ccctcatatt catgttcgcg tcccccttgt caatcatcaa tctggtcatc 540

aggacgaaga gcgtggaata catgccattc tacttgtcat tatctatgtt tctgatgagt 600

gcatcattct tcggatacgg agtgctgctg cgtgatttct tcatatatat tccaaatggt 660

attggaacca tactgggtat cgtgcagttg atgctgtatg cctacttcag aaaaggatca 720

agcgaggaag ccaagctgcc attactagtc acacatacat gagcatgcca 770

<210> 2

<211> 23

<212> DNA

<213> Artificial

<220>

<223> S5S

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ctacttgctt gcatcagttg tct 23

<210> 3

<211> 21

<212> DNA

<213> Artificial

<220>

<223> S5AS

<400> 3

tggttccaaa tacagttcgt g 21

<210> 4

<211> 18

<212> DNA

<213> Artificial

<220>

<223> SW1F

<400> 4

ccgctctccg tgatgaaa 18

<210> 5

<211> 18

<212> DNA

<213> Artificial

<220>

<223> SW1R

<400> 5

ggaagccaga gacaggaa 18

<210> 6

<211> 28

<212> DNA

<213> Artificial

<220>

<223> S23F

<400> 6

ggggatccgc aagtatcttc cctcgacg 28

<210> 7

<211> 29

<212> DNA

<213> Artificial

<220>

<223> S23R

<400> 7

gggagctctg gcatgctcat gtatgtgtg 29

<210> 8

<211> 243

<212> PRT

<213> Artificial

<220>

<223>ShSWEET1 albumen

<400> 8

Met Asp Trp Gly Asp Pro Ala Leu Thr Ser Phe Val Ala Asp Ser Ser

1 5 10 15

Phe Arg His Leu Cys Cys Tyr Gly Ala Gly Ile Ala Gly Asn Val Phe

20 25 30

Ala Phe Val Leu Phe Ile Ser Pro Leu Pro Thr Phe Lys Arg Ile Val

35 40 45

Arg Asn Gly Ser Thr Glu Gln Phe Ser Ala Met Pro Tyr Ile Tyr Ser

50 55 60

Leu Leu Asn Cys Leu Ile Cys Met Trp Tyr Gly Leu Pro Phe Val Ser

65 70 75 80

Tyr Gly Val Val Leu Val Ala Thr Val Asn Ser Ile Gly Ala Val Phe

85 90 95

Gln Leu Ala Tyr Thr Ala Val Phe Ile Ala Phe Ala Asp Ala Lys Gln

100 105 110

Arg Leu Lys Val Ser Ala Leu Leu Ala Ala Val Phe Val Val Phe Gly

115 120 125

Leu Ile Val Phe Val Ser Leu Ala Leu Leu Asp His Gln Thr Arg Gln

130 135 140

Met Phe Val Gly Tyr Leu Ser Val Ala Ser Leu Ile Phe Met Phe Ala

145 150 155 160

Ser Pro Leu Ser Ile Ile Asn Leu Val Ile Arg Thr Lys Ser Val Glu

165 170 175

Tyr Met Pro Phe Tyr Leu Ser Leu Ser Met Phe Leu Met Ser Ala Ser

180 185 190

Phe Phe Gly Tyr Gly Val Leu Leu Arg Asp Phe Phe Ile Tyr Ile Pro

195 200 205

Asn Gly Ile Gly Thr Ile Leu Gly Ile Val Gln Leu Met Leu Tyr Ala

210 215 220

Tyr Phe Arg Lys Gly Ser Ser Glu Glu Ala Lys Leu Pro Leu Leu Val

225 230 235 240

Thr His Thr

Claims

A kind of 1. sugarcane saccharide transporter ShSWEET1 genes, it is characterised in that its nucleotide sequence such as SEQ ID NO:1 institute Show.
2. a kind of sugarcane saccharide transporter ShSWEET1, it is characterised in that it is the sugarcane saccharide transporter described in claim 1 The protein of ShSWEET1 gene codes, its nucleotide sequence such as SEQ ID NO:Shown in 8.
A kind of 3. cloning process of the sugarcane saccharide transporter ShSWEET1 genes described in claim 1, it is characterised in that including Following steps：

(1) total serum IgE is extracted from the root, stem and/or leaf of ripe sugarcane；

(2) reverse transcription is carried out as template using total serum IgE and obtains cDNA；

(3) ShSWEET1 full length gene sequence amplification primers pair are designed, enters performing PCR amplification, reclaims PCR primer, wherein, The nucleotide sequence such as SEQ ID NO of ShSWEET1 full length gene sequence amplification primers pair:2 and SEQ ID NO:Shown in 3；

(4) PCR primer connection carrier, is converted, and sequencing, obtains ShSWEET1 gene fragments.
4. sugarcane saccharide transporter ShSWEET1 genes as claimed in claim 1 position in the cell in application.
5. application of the sugarcane saccharide transporter ShSWEET1 genes as claimed in claim 1 in cold resistance of plant stress is improved.
6. sugarcane saccharide transporter ShSWEET1 genes as claimed in claim 1 are cultivating the high anti-transgenic sugarcane of high sugared high yield In application.
7. a kind of expression vector, it contains the sugarcane saccharide transporter ShSWEET1 genes described in claim 1.
A kind of 8. primer pair, it is characterised in that its nucleotide sequence such as SEQ ID NO:2 and SEQ ID NO:Shown in 3.
A kind of 9. primer pair, it is characterised in that its nucleotide sequence such as SEQ ID NO:4 and SEQ ID NO:Shown in 5.
A kind of 10. primer pair, it is characterised in that its nucleotide sequence such as SEQ ID NO:6 and SEQ ID NO:Shown in 7.