CN107723295B - Saccharum officinarum transport protein ShSWEET1 gene and application thereof - Google Patents

Abstract

The invention provides a saccharose transport protein ShSWEET1 gene, the nucleotide sequence of which is shown in SEQ ID NO. 1. The invention also provides a protein coded by the ShSWEET1 gene and application of the ShSWEET1 gene. The ShSWEET1 gene is cloned from sugarcane for the first time, has the transport function of sugar transport protein, can be positioned in cell membranes of cells, can improve the cold stress resistance of plants, is beneficial to the growth of the plants such as the sugarcane under the adverse condition, provides a genetic basis for disclosing the regulation function of a sucrose transport mechanism on the growth and development of the plants and the quality improvement and providing genetic engineering research on the improvement of the crop yield and the quality.

Description

Saccharum officinarum transport protein ShSWEET1 gene and application thereof

Technical Field

The invention belongs to the technical field of biology, and relates to a saccharose transporter ShSWEET1 gene and application thereof.

Background

Sugarcane (Saccharum officinarum L.) belongs to the gramineae, milo, sugarcane subfamily, Saccharum genus, annual or perennial root herbaceous plants, is homologous to corn and sorghum, originally produced in tropical and subtropical areas of new guinea or india, and is one of the most efficient plants for converting solar energy into chemical energy as a C4 plant. The sugarcane plants have high light saturation point, strong carbon dioxide fixing capacity, high photosynthetic rate, high photosynthetic intensity, high biomass, and abundant sugar content and can be used as biofuel. Over the past 30 years, sugarcane has received global attention as a superior potential alternative renewable energy source. Carbon partitioning is a key process of plant photosynthetic energy and material partitioning. In general, photosynthesis and carbon assimilation of C4 plants occur in the chloroplasts of mesophyll cells and in the bundle sheath cells. Photosynthetic source cells convert photosynthetically fixed carbon into sugars or sugar derivatives which are then transported long distances along the phloem to remote sink cells and consumed and stored in the sink cells (Poorterand Villar, 1997). Typically, 35% to 40% of the sugar is consumed by living cells for providing energy for cell growth (halandrao, 1999), plant development including cell expansion, division, differentiation, nutrient uptake and maintenance. Some are used as metabolic intermediates of cells, such as monosaccharides, amino acids, organic acids, etc., and the excess sugars are stored in vacuoles or reusable polymers (such as starch bodies), or constitute biological structural substances (such as cellulose, hemicellulose and lignin).

The high energy conversion capability of the sugarcane enables the sugarcane to become a high-yield feed crop with good prospect; meanwhile, molasses produced in the sugar production process of the sugarcane is more high-quality animal feed such as pigs and cattle, and bagasse can also be used as a paper-making raw material and an edible fungus cultivation raw material; sugarcane is also an important raw material for the light industry in China, and products taking cane sugar as a raw material and auxiliary materials are more than 2300 types of products in 56 types (Qin Wen et al, 2006); the sugarcane and sugarcane juice in the sugarcane are also superior health-care food, and the sugarcane is cool in nature and has the effects of clearing heat, dispelling the effects of alcohol, promoting urination, assisting spleen and stomach and nourishing. Sugarcane is a C4 plant with high photosynthetic efficiency, with high biological yield, which can produce about 7000L of alcohol per hectare per year on average in sugarcane high-yielding regions (Matsuoka et al, 2009), which is much 1-17 times higher than other crops, such as corn (4000L/hm2) and sweet sorghum (3000L/hm2) (Teetor et al, 2011; Hill et al, 2006). In brazil enjoying the reputation of "the country of green energy", the alcohol produced by sugarcane is nearly thousands of tons every year, and the new energy of the automobile mixed by alcohol and gasoline is enough for the third-generation automobiles in brazil to be completely used. In addition, sugarcane and sugarcane byproducts can be used for generating electricity by certain technical means so as to benefit human beings, and in brazil, the electricity generation by sugarcane raw materials exceeds the sum of water conservancy electricity generation, so that the sugarcane and sugarcane byproducts become important energy sources of brazil.

At present, a plurality of problems still exist in the planting and management of domestic sugarcane, and a great space still exists for improving the cultivation and management mode of sugarcane and increasing the biological yield of sugarcane. Meanwhile, the biggest bottleneck faced by sugarcane planting in China is the serious simplification trend of varieties, new wide-range, high-yield and high-sugar varieties are urgently required to be cultivated for production in production, the number of chromosomes of the sugarcane is large, the genome structure is complex, and the genetic basis of the production varieties is narrow, so that the time required for conventional seed selection is long, the total sugar content is difficult to improve, and the genetic engineering method only changes the distribution of carbon substances and does not integrally improve the content of cane sugar, so that the requirements of production and life cannot be met (Zhaotong, 2012). The research on sugar accumulation and distribution mechanisms in the sugarcane body finds out key control points in the sugar accumulation process, and then the improvement is an important way for realizing high yield and high sugar of the sugarcane. Provides scientific reference for improving the quality of the sugarcane and realizing the breeding target of high yield, high sugar content and high resistance of the sugarcane.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provides a saccharose transporter ShSWEET1, a coding gene thereof and application of the gene. Another purpose of the invention is to provide a cloning method of the sucrose transporter ShSWEET1 gene and a primer pair used by the cloning method.

The first aspect of the invention provides a gene of the sucrose transporter ShSWEET1, and the nucleotide sequence of the gene is shown in SEQ ID NO. 1.

The second aspect of the invention provides a sucrose transporter ShSWEET1, which is a protein encoded by the sucrose transporter ShSWEET1 gene of the first aspect of the invention, and the amino acid sequence of the protein is shown in SEQ ID NO. 8.

The third aspect of the invention provides a method for cloning the gene of the sucrose transporter ShSWEET1 in the first aspect of the invention, which comprises the following steps: (1) extracting total RNA from the roots, stems and/or leaves of mature sugarcane; (2) carrying out reverse transcription by taking the total RNA as a template to obtain cDNA; (3) designing a ShSWEET1 gene full-length sequence amplification primer pair, carrying out PCR amplification, and recovering a PCR product, wherein the nucleotide sequence of the ShSWEET1 gene full-length sequence amplification primer pair is shown as SEQ ID NO. 2 and SEQ ID NO. 3; (4) and connecting the PCR product with a vector, transforming and sequencing to obtain the gene segment of the ShSWEET1 gene.

In a fourth aspect, the invention provides the use of the gene of the sucrose transporter ShSWEET1 in locating cell membranes.

The fifth aspect of the invention provides the application of the gene of the sucrose transporter ShSWEET1 in improving the cold stress resistance of tobacco.

The sixth aspect of the invention provides the application of the gene ShSWEET1 of the cane sugar transport protein in the cultivation of high-sugar, high-yield and high-trans-resistance sugarcane.

In a seventh aspect, the invention provides an expression vector comprising the gene for the sucrose transporter ShSWEET1 according to the first aspect of the invention.

The eighth aspect of the present invention provides a primer pair, the nucleotide sequence of which is shown as SEQ ID NO. 2 and SEQ ID NO. 3. The primer pair can be used for amplification of the full-length sequence of the ShSWEET1 gene.

The ninth aspect of the present invention provides another primer pair, the nucleotide sequences of which are shown as SEQ ID NO. 4 and SEQ ID NO. 5.

The tenth aspect of the present invention provides another primer pair, whose nucleotide sequences are shown in SEQ ID NO. 6 and SEQ ID NO. 7.

The ShSWEET1 gene is cloned from sugarcane for the first time, has the transport function of sugar transport protein, can be positioned in cell membranes of cells, can improve the cold stress resistance of plants, is beneficial to the growth of the plants such as the sugarcane under the adverse condition, provides a genetic basis for disclosing the regulation function of a sucrose transport mechanism on the growth and development of the plants and the quality improvement and providing genetic engineering research on the improvement of the crop yield and the quality.

Drawings

FIG. 1 is an electrophoresis diagram of total RNA extraction from different parts of sugarcane.

FIG. 2 is an electrophoresis diagram of the full-length amplification of ShSWEET1 gene cDNA.

FIG. 3 shows the result of ORF analysis of the full-length cDNA of ShSWEET1 gene.

FIG. 4 shows the predicted results of the amino acid transmembrane region of ShSWEET 1.

FIG. 5 shows predicted results of SWEET1 amino acid subcellular localization.

FIG. 6 shows the relative expression of ShSWEET1 gene in different tissues of sugarcane.

FIG. 7 shows the relative expression of ShSWEET1 gene in different diseased leaves of sugarcane.

FIG. 8 shows the relative expression of ShSWEET1 gene under cold stress in sugarcane.

FIG. 9 is a diagram showing the construction process of pShSWEET1-GFP vector.

FIG. 10 shows the restriction electrophoresis of pShSWEET 1-GFP.

FIG. 11 is the subcellular localization of ShSWEET1 gene in onion inner epidermis, where A, C: GFP fluorescence (488nm blue excitation); d, F: and (3) white light observation: A/D: epidermal cells transfected with pShSWEET1-GFP vector; C/F: the cells were transfected with pCAMBIA1302 empty vector.

FIG. 12 shows the restriction enzyme electrophoresis of the plant expression vector pShSWEET 1.

FIG. 13 shows the PCR detection result of the transgenic tobacco ShSWEET1 gene.

FIG. 14 is a phenotype of cold stress of tobacco, wherein CK: wild-type tobacco; 1: ShSWEET1 transgenic tobacco.

FIG. 15 shows the cultivation process of sugarcane transgenic seedlings, wherein A is the induction of callus B: and (3) differentiating the callus to obtain a seedling C: PPT screening D: and (4) rooting, screening and culturing E: transformation of ShSWEET1 resistant plant and hardening-seedling F, and permanent planting of ShSWEET1 resistant plant.

FIG. 16 shows the Bar gene detection result of transformed plants, wherein M: DL 2000 marker; CK (CK)⁺A vector plasmid; CK (CK)^-: a non-transgenic plant; 1-5: transgenic plants resistant to ShSWEET 1.

Detailed Description

The invention will be better understood from the following description of specific embodiments with reference to the accompanying drawings. The experimental procedures for the specific experimental conditions not specified in the examples below are generally carried out according to conventional conditions, as described in Molecular Cloning (A Laboratory Manual,3rd ed.) or in the Yeast Genetics Laboratory Manual (methods in Yeast Genetics: A Cold Spring Harbor Laboratory Coursmasual, Adams A et al, Cold Spring Harbor Laboratory, 1998), or according to the conditions recommended by the manufacturer.

1 materials of the experiment

1.1 plant Material

New sugarcane No. 22 (ROC22) planted in a clinical high test base of the research institute of tropical biotechnology of the Chinese tropical academy of agricultural sciences and a seaport academy of the Chinese tropical academy of agricultural sciences is used as a plant material, and ROC22 tissue culture seedlings of sugarcane and common tobacco are provided for a laboratory.

1.2 strains and plasmids

Escherichia coli (Escherichia coli. DH5 α) was obtained from energy-rich company (Guangzhou), Agrobacterium tumefaciens (Agrobacterium tumefaciens EHA105) was stored in the laboratory, pMD19-T, pMD19T single vector plasmid was obtained from Takara, pCAMBIA3300-GUS, pCAMBIA1302 plant expression vectors were stored in the laboratory.

1.3 enzymes and other reagents

Reagent kit for the extraction of Reagent RNA was purchased from Invitrogen (USA), PCR Reagent kit, DNA gel recovery kit and plasmid miniprep kit were purchased from OMEGA (USA), reverse transcription kit, SYBR Green IIPremix, rTaq enzyme, LA-Taq enzyme, restriction enzyme were purchased from TaKaRa (China, Dalian), T4 DNA ligase was purchased from Fermentas, β -mercaptoethanol, sucrose, ampicillin (Amp), streptomycin (Str), carboxymutamycin (Car), kanamycin (Kan), rifampicin (Rif), Phosphinothricin (PPT), agarose were purchased from Sigma, and the usual chemical reagents were pure for domestic analysis.

1.4 Main instrumentation

PCR amplification instrument T1Thermocycle (biomera), gradient PCR instrument (Eppendorf5331), multipurpose high-speed refrigerated centrifuge (Thermo), normal temperature centrifuge (HETTICHD-78532), automatic triple pure water distiller (SZ-97 of Shanghai Yangrong Biochemical apparatus factory), constant temperature water bath pan, constant temperature shaking table (N.JG-27EKISO), constant temperature incubator (SANYO MRL-350), ultraviolet spectrophotometer (Beijing Pujingyou general apparatus company T6), electrophoresis apparatus (LAET BNEQ 2705), gel imaging system (BIO-RAD), real-time fluorescence quantitative PCR instrument (Stratagene Mx3005P)

1.5 preparation of the culture Medium

LB medium (1L): 10g of peptone, 5g of yeast extract, 10g of NaCl10g, pH7.0, 1000mL of solid medium with 15g of agar powder, and autoclaving at 121 ℃ for 25 min.

YEP medium (1L): 10g of peptone, 10g of yeast extract, 5g of NaCl5g, pH7.0, 1000mL of solid medium with 15g of agar powder, and autoclaving at 121 ℃ for 25 min.

Sugarcane callus induction culture M1 (1L): MS + 2.4-D1 mg/L + sucrose 30g/L + carrageenan 8g/L, pH5.8.

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

Liquid medium MR (1L) for sugarcane transformation: 1/5MS macroelements + MS other components +2.4D 1mg/L +10mmol/L fructose +10mmol/L glucose +30g/L sucrose (8 g/L carrageenan should be added into the solid culture medium), pH 5.3.

Sugarcane rooting induction medium (1L): MS (macroelement is halved, other components are unchanged) + NAA 2mg/L, sucrose 20g/L, coconut water 100mL/L, carrageenan 8g/L and active carbon 0.2g/L, and pH is 5.8.

2 method of experiment and results of experiment

2.1 cloning of the sugarcane ShSWEET1 Gene

(1) Extraction of total RNA of sugarcane root, stem and leaf

Reference Invitrogen

The steps of the method for extracting RNA by Reagent are as follows: 0.1g of each of the roots, stems and leaves was cut into small pieces, quickly transferred into liquid nitrogen, and ground into powder in a mortar. 1mL of Trizol was added to a 1.5mL enzyme free centrifuge tube and labeled. The liquid nitrogen-ground sample is quickly transferred to a centrifuge tube and quickly shaken for 2 min. After standing at room temperature for 5min, 200. mu.L of chloroform was added. The centrifuge tube was forcibly inverted and mixed for 30 seconds, and then left to stand at room temperature for 10min, and centrifuged at 12000g at 4 ℃ for 15 min. The supernatant was pipetted into a fresh 1.5ml enzyme-free centrifuge tube (without shaking the middle layer during pipetting). Adding 500 μ l isopropanol stored in refrigerator at-20 deg.C, mixing completely, standing at-20 deg.C for 10min, and centrifuging at 4 deg.C for 15min at 12000 g. The supernatant was discarded. 1mL of 75% ethanol was added, the tube was gently shaken, and the pellet was suspended. Centrifuging at 4 deg.C for 10min at 7500g, removing supernatant, blowing on a super clean bench for 5-10min, and adding 40 μ LRnase-freeH when liquid in the centrifugal tube is dried₂And O, dissolving in a water bath kettle at 55-60 ℃ for 10 min. Put on ice, add Fermatas DNaseI buffer 5. mu.L, DNaseI 5. mu.L, and incubate at 37 ℃ for 30 min. Adding 5 μ L50 mM EDTA, warm bathing at 65 deg.C for 10min, and standing at-70 deg.C for use.

(2) RNA integrity and purity testing

mu.L of Total RNA was subjected to 1.2% agarose gel electrophoresis and 1. mu.L of Total RNA was analyzed for RNA purity by a nucleic acid protein analyzer. The purity OD260/280 ratio measured by nucleic acid protein analyzer is 1.9-2.0, and the concentration is 600ng/μ L. Through 1% agarose gel electrophoresis detection (figure 1), two bands of total RNA of each tissue are clear and complete, and the brightness of 28SrRNA is about 2 times of 18SrRNA, thereby meeting the requirements of subsequent experiments.

(3) Reverse transcription to synthesize cDNA chain

a. The enzyme-free Microtube was placed on ice and the following mixed solutions were added sequentially:

template RNA (less than 5. mu.g) 7.0. mu.L (0.1 ng-5. mu.g)

Oligo(dT)18Primer(50μM) 1.0μL

RNase free ddH₂O up to 12.0μL

b.65 ℃ and 5min, and then quickly moving the mixture to ice for more than 2 min.

c, adding the reaction solution for cDNA synthesis:

d. the cDNA synthesis reaction was added to the RNA/primer mixture, gently mixed, centrifuged briefly, and incubated at 42 ℃ for 1 h.

e.70 ℃ and stop the reaction for 10 min.

f. The reverse transcribed cDNA was stored at-70 ℃ for future use.

(4) Amplification of full-length sequence of ShSWEET1 gene

a. Designing a primer: primers were designed based on the maize, sorghum Sugar transporter (Sugar effluxtransporter) genes as follows:

c. after being uniformly mixed, the mixture is subjected to microcentrifugation and is put into a PCR instrument for PCR amplification, and the reaction procedure is as follows:

(5) gel electrophoresis of PCR amplification products

The PCR amplification product was mixed with 5. mu.L of 6 Xloading buffer and subjected to 1% agarose gel electrophoresis. The voltage is 95V, electrophoresis is carried out for 35min, then photographing recording is carried out in a gel imaging system, and gel of a suspected target strip is cut off. The amplified fragment was approximately 800bp in size (FIG. 2).

(6) Gel recovery of amplified fragments

The target band was recovered by referring to the gel recovery kit (gel recovery kit) of OMEGA.

(7) Ligation of the recovered fragment with T vector

The connection system of the target strip and the pMD19-T vector is as follows:

ligation was carried out overnight at 16 ℃.

(8) Ligation vector transformation and sequencing

The ligation product (10. mu.L) was added to a 1.5mL centrifuge tube containing 50. mu.L of competent cells (Escherichia coli. DH5 α), gently mixed with a tip, placed on ice for 30min, heat-shocked at 42 ℃ for 90s, and immediately transferred to ice for 2 min.

Adding 900 μ L LB liquid culture medium into the above mixed solution system, and culturing at 37 deg.C for 60min with shaking. Centrifuging at 10000rpm for 45s, discarding 500 mu L of supernatant, gently mixing the supernatant with a gun head, precipitating, coating the precipitate on a LB culture medium which is prepared in advance and contains 100 mu g/mL Amp, standing in a super clean bench for 30min, and after bacterial liquid is absorbed by a flat plate, inverting the flat plate in a constant-temperature incubator at 37 ℃ for 12-16 h until a single colony is formed. About 10 single colonies were picked, and each colony was put into a 1mL centrifuge tube containing Amp-resistant liquid LB medium, and cultured overnight in a 37 ℃ incubator by shaking. The bacterial solution was aliquoted at about 200. mu.L each, one tube was used for sequencing, one tube was used for PCR confirmation of the length of the insert fragment in the vector, and the remainder was stored for future use. Taking 1 mu L of bacterial liquid as a PCR template, carrying out PCR amplification (the method is the same as that of the ShSWEET1 gene full-length sequence amplification), sending the PCR template to Shanghai bioengineering company Limited for sequencing, and the sequencing result shows that: the fragment obtained by cloning has the size of 868 bp.

2.2 bioinformatics analysis of the ShSWEET1 Gene from sugarcane

The amino acid sequence of ShSWEET1 was aligned by Blastp search at NCBI and found to be homologous to the SWEET gene family. Http// www.ncbi.nlm.nih.gov/gorf. html shows that the full-length cDNA sequence of ShSWEET1 gene has complete open reading frame, has the length of 732bp, and codes 243 amino acids from the 98 th start codon ATG to the 829 th TGA stop codon (FIG. 3). The conserved domain analysis of the encoded protein is carried out by using an online software NCBI CDS database, and the ShSWEET1 is found to contain the conserved domains of PQ-loop superfamily and MtN 3/saliva. ProtParam predicts that the relative molecular weight of the protein encoded by the gene is 26.93KDa, the isoelectric point is 8.87, and the total average hydrophobicity index is 0.916, which indicates that the protein is hydrophobic.

The TMHMServerv.2.0 software is used for carrying out transmembrane structure prediction on the ShSWEET1 amino acid sequence (FIG. 4), and the ShSWEET1 protein has 7 transmembrane regions which are respectively located at 15-37, 58-80, 85-107, 116-138, 148-170, 177-199 and 204-226-155 amino acids. Similar to the transmembrane structure of the eukaryotic SWEET protein reported previously, therefore, the ShSWEET1 is deduced to have the structural characteristics of the eukaryotic SWEET protein. Analysis by subcellular localization software revealed that ShSWEET1 could be located on the plasma membrane (FIG. 5), and ShSWEET1 was presumed to be a plasma membrane protein.

50 reported different plant SWEET protein amino acid sequences are selected from NCBI database, and MEGA5.1 is used for constructing a phylogenetic tree. Based on amino acid homology analysis, the amino acid sequence coded by the ShSWEET1 gene is close to the homology of monocotyledon ShSWEET family members such as corn, millet, wheat, brachypodium distachyon and the like, and is respectively 91%, 89%, 85% and 83%. The sugarcane ShSWEET1 is a member of the MtN3/saliva/ShSWEET family.

2.3 analysis of ShSWEET1 Gene expression in different tissues of sugarcane

Reference to

ReageThe nt RNA extraction method is used for extracting RNA of immature leaves, mature leaves, immature stems, mature stems and roots of sugarcane, diseased leaves of different sugarcane diseases and sugarcane seedling RNA subjected to stress treatment, and cDNA is synthesized by referring to a reverse transcription kit provided by Fermentas company. After the homogenization of the concentration, RT-qPCR is carried out on a real-time fluorescence quantitative PCR instrument Mx3005P to detect the relative expression quantity of the ShSWEET1 gene under different tissues and different stresses.

a) RT-qPCR primers:

SW1F:5′-CCGCTCTCCGTGATGAAA-3′

SW1R:5′-GGAAGCCAGAGACAGGAA-3′

sugarcane GAPDH internal reference primer (from Iskandar et al, 2004):

P1：5′-CACGGCCACTGGAAG CA-3′

P2：5′-TCCTCAGGGTTCCTGATGCC-3′

b) PCR amplification System: using Takara Inc

Premix Ex TaqTM II kit.

RT-qPCR amplification procedure: at 95 ℃ for 3 min; 95 ℃, 15s, 60 ℃, 45s, 40 cycles; 3 replicates were set for each sample and a dissolution curve was made. The internal reference gene amplification system and the procedure are the same as those of the target gene. Data all adopt 2^-△△CTThe method analyzes relative quantitative values (Kenneth et al, 2001).

(1) Analysis of ShSWEET1 gene expression in different tissues of sugarcane

Different tissue samples of sugarcane plants planted in a high sugarcane test base of the research institute of tropical biotechnology of the Chinese tropical agricultural academy of sciences are collected as test samples in the sugarcane elongation period. During sample collection, 9 robust sugarcane plants in the elongation stage are selected, and each 3 robust sugarcane plants are repeatedly mixed and sampled. Respectively collecting immature leaves, mature leaves, immature stems and mature stems. The immature leaves are heart leaves of the sugarcane which are not extracted; the mature leaves are +3 leaves of the sugarcane plants which are completely unfolded from top to bottom and can show a fat band. The immature stem is the second section below the growing point of the top of the upper stem of the plant; the mature stem is the 3rd or 4 th section from the lower part to the upper part of the sugarcane plant. The root is mature root after plant digging.

The RT-qPCR results are shown in FIG. 6: the ShSWEET1 gene is expressed in the root, immature stem, mature stem, immature leaf and mature leaf of sugarcane elongation-stage plants, which indicates that the ShSWEET1 gene is constitutively expressed in sugarcane. The expression of the ShSWEET1 gene is relatively high in mature leaves and mature stems, and the relative expression quantity of each tissue is as follows: mature leaves > mature stems > immature leaves > roots > immature stems, which are respectively 1-3.5 times of the expression amount of the immature stems.

(2) ShSWEET1 gene expression analysis under different diseases of sugarcane

Respectively collecting sugarcane plant leaves which are infected with sugarcane brown stripe disease, sugarcane red stripe disease and sugarcane smut and are in the middle disease stage as sugarcane brown stripe disease leaves, sugarcane red stripe disease leaves and sugarcane smut disease leaves in a field sugarcane planted in a sugarcane test base near a tropical biotechnological research institute of the Chinese tropical agricultural academy of sciences; during sampling, typical and single-morbidity plants in the middle of morbidity are selected for each disease, 9 diseased leaves are selected for each plant, and each plant is repeated for 3 times, wherein 9 diseased leaves are selected for each plant. Healthy sugarcane leaves which are not attacked are used as a control. And immediately putting the leaf samples into liquid nitrogen for preservation after the leaf samples are collected.

The RT-qPCR results are shown in FIG. 7: the relative expression quantity of the ShSWEET1 gene in three diseased leaves, namely sugarcane brown streak disease, sugarcane smut disease and sugarcane brown streak disease, is higher than that of healthy sugarcane leaves. The expression level of ShSWEET1 gene in the brown stripe disease leaves of the sugarcane is the highest and is 2.6 times of that of healthy sugarcane leaves. It is shown that when pathogenic bacteria invade the leaf, it is possible to activate and induce the expression of Shsweet1 gene on the mesophyll cell membrane, resulting in more sugar being secreted to the mesophyll cell space through the gene, and providing a good environment for the invasion and reproduction of bacteria.

(3) Low-temperature treatment of sugarcane seedlings

The sugarcane tissue culture seedling plant for the test is inoculated in an MS culture medium and is pre-cultured for two weeks under the conditions of 28 ℃, 16h illumination/8 h darkness and 3000lx illumination intensity, the sugarcane tissue culture seedlings with the same leaf age and consistent growth vigor are selected and are transferred into the MS liquid culture medium for 28 ℃, 16h illumination/8 h darkness and 3000lx illumination intensity, the culture is recovered for two days, and then cold stress is carried out under the conditions of 4 ℃, 16h illumination/8 h darkness and 3000lx illumination intensity. Sampling is carried out after cold stress is carried out for 0h, 6h, 12h, 24h, 36h, 48h, 72h and 96h, 9 plants are sampled each time, 3 plants are sampled in a repeated mixed mode, and 3 times are repeated. And immediately putting the sample into liquid nitrogen for preservation after each sampling is finished.

The qRT-PCR results are shown in FIG. 8: cold stress can induce the ShSWEET1 gene to up-regulate expression. When the sugarcane tissue culture seedlings are treated at 4 ℃, the expression level of ShSWEET1 is continuously increased along with the increase of the treatment time within 0-96 h. ShSWEET1 is rapidly induced to express when treated for 48h, which indicates that the ShSWEET1 gene plays an important role in the cold stress resistance of sugarcane, and is probably involved in transporting soluble sugar in the sugarcane body to reduce the osmotic pressure in the sugarcane body, so that the normal growth of the sugarcane under the abiotic stress can be maintained.

2.4 subcellular localization of the ShSWEET1 Gene in the onion inner epidermis

(1) Construction of GFP fusion vector

a. Analyzing the sequence of ShSWEET1 gene, finding out complete ORF and designing primers with enzyme cutting sites at both ends, and removing stop codon by downstream primer as follows:

S2GF：5′-GGCCATGGATTGGGGTGATC-3′NcoⅠ

S2GR：5′-GGAGATCTTGTATGTGTGACTAGTAATGGCA-3′BglⅡ

b. the ShSWEET1-pMD19-T plasmid is used as a template, primers (S2GF and S2GR) are used for PCR amplification, and an amplification system and a program are as follows:

the reaction program is that 95 ℃ 5min, 95 ℃ 1min, 62 ℃ 1min, 72 ℃ 1min, 35 cycles, 72 ℃ 10min, after the PCR reaction, 1% agarose gel electrophoresis is used, a scalpel is used in a gel imaging system to cut off a mesh belt and recover, then the mesh belt is connected into a pMD19T-simple vector and is transformed into competent DH5 α, a positive clone is selected for PCR identification, and the PCR identification is sent to the Shanghai biological engineering for sequencing.

c. Plasmid extraction was performed on a single clone of the correctly sequenced ShSWEET1-pMD19T and a strain containing the pCAMBIA1302 vector. Plasmid extraction was performed according to the instructions of the E.Z.N.A Plasmid Miniprep Protocol kit (OMEGA).

d. Plasmid ShSWEET1-pMD19T and pCAMBIA1302 were double digested with restriction enzymes NcoI, BglII as follows:

e. then carrying out electrophoresis on 1% agarose gel, recovering a large vector fragment and a target gene fragment, connecting the large vector fragment and the target gene fragment by using T4 DNAIlgase, transforming the large vector fragment and the target gene fragment into competent DH5 α (the construction process of the pShSWEET1-GFP vector is shown in figure 9), selecting a positive clone, carrying out shaking culture at 37 ℃ overnight to extract a plasmid, carrying out double enzyme digestion identification, carrying out electrophoretic excision on a target large band (figure 10), and naming the correct vector as the pShSWEET1-GFP fusion vector.

The enzyme digestion system is as follows:

(2) preparation of Agrobacterium competence

a. Agrobacterium EHA105 strain was removed from a-80 ultra-low temperature refrigerator, thawed on ice, dipped with an inoculating loop, streaked on YEP plates (containing 10mg/mLStr +20mg/mL Rif), and cultured for 28 days.

b. A single colony is picked and inoculated in a liquid culture medium containing 5mL YEP (containing 10mg/mLStr and 20mg/mL Rif), and is subjected to shaking culture at 28 ℃ for 24-48 h until the bacterial liquid is turbid.

c. Inoculating 1mL of the purified bacterial solution into a flask containing 50mL of YEP liquid medium without antibiotics, and performing shake culture at 28 ℃ for 5-8 h until OD is 0.5-0.6.

d. Pouring the bacterial liquid into a 80mL centrifuge tube from a triangular flask, and carrying out cold bath on ice for 30 min;

e.5000Xg, centrifuging for 5min under the condition of 4, and collecting thalli;

f. operating on a super clean bench, discarding the supernatant, and resuspending the thalli by using 20mL of precooled 50mmol/L CaCl 2;

g. and e is repeated.

h. Operating on a clean bench, discarding the supernatant, resuspending with 1mL of 50mM CaCl2, subpackaging 50 μ L of the bacterial solution into 1.5mL centrifuge tubes, quick freezing with liquid nitrogen, and storing at-80 deg.C for use

(3) Transformation of expression vectors

Taking one agrobacterium-infected competent cell containing 50 mu L from an ultra-low temperature refrigerator at minus 80 ℃, and unfreezing the agrobacterium-infected competent cell on ice; adding 4 μ L plasmid into the thawed centrifuge tube, standing in ice bath for 15min, and then placing into liquid nitrogen for 5 min; standing at 28 deg.C in a thermostat for 5min, adding 500 μ L YEP (without antibiotic) culture medium, and shake culturing at 28 deg.C for 3 hr; sucking 200 μ L of bacterial liquid, spreading on YEP three-antibody plate (10mg/mL Str +20mg/mL Rif +50mg/mL Kan), and performing inverted culture in 28 deg.C incubator for 48 h; selecting a single colony, inoculating the single colony into a liquid culture medium (10mg/mL Str +20mg/mL Rif +50mg/mL Kan) containing 5mL of YEP, and performing shaking culture at 28 ℃ for 36-48 h; selecting agrobacterium positive clone, shaking, extracting agrobacterium plasmid according to escherichia coli plasmid, and carrying out enzyme digestion PCR identification.

(4) Agrobacterium-mediated transfection of onion epidermal cells

Positive single clones carrying the recombinant plasmid pShSWEET1-GFP and the unloaded plasmid pCAMBIA1302 were picked up on solid plates, respectively, to 10mL YEP liquid medium (10mg/mL Str +20mg/mL Rif +50mg/mL Kan), and cultured overnight with shaking at 28 ℃. Transfer 3-4mL of overnight broth to 50mL of YEP broth (10mg/mL Str +20mg/mL Rif +50mg/mL Kan), and shake-culture at 28 ℃ until OD600 is 0.6-0.7. Centrifuging at 4000g/min for 2min, collecting thallus, and culturing in MS liquid culture medium (10 mmol/LMgCl)₂100. mu. mol/L AS) was suspended so that the OD600 value was about 1.0.

Pre-culturing onion epidermal cells: selecting fresh Bulbus Allii Cepae, removing outer layer scale 3-4 layers, soaking in 75% ethanol for 10min, rinsing with sterile pure water for three times each time5-8 min. Cutting with sterile scalpel to cut the inner skin of onion scale to 1cm²The small pieces, which were plated on MS solid medium (100. mu. mol/L AS) near the mesophyll surface. The light cycle was 14/10h at 28 ℃ and the preculture was carried out for 24 h. Co-culturing: immersing the inner epidermis of precultured onion in MS liquid medium (10 mmol/LMgCl)₂100. mu. mol/L AS) suspended in the culture medium, shaking several times during the period of 20-30min, holding a corner, draining the culture medium on filter paper, and plating on MS solid medium (100. mu. mol/L AS). The culture was incubated at 28 ℃ for 16/8h with a photoperiod of 24 h. Taking out the onion inner epidermis, shaking and washing the onion inner epidermis by using an MS liquid culture medium, removing agrobacterium attached to the surface, tabletting, observing under a fluorescence microscope, and taking a picture.

As a result of observing the expression of GFP in onion endothelial cells after infection with Agrobacterium containing a GFP fusion vector using a fluorescence microscope under ultraviolet light, as shown in FIG. 11, green fluorescence was observed at each site in the cells of the hollow vector pCAMBIA1302 in FIG. 11-C, and thus no specific localization was observed in the cells, whereas FIG. 11-A shows that the onion cells of the transformed pShSWEET1-GFP vector exhibited green fluorescence on the cell membrane, and had a distinct localization characteristic, and ShSWEET1 was a cell membrane protein according to bioinformatics analysis, and thus ShSWEET1 was presumed to be localized on the cell membrane.

2.5 study of tobacco genetically transformed with ShSWEET1 Gene

(1) Construction of plant expression vector pShSWEET1

a. Analyzing the sequence of ShSWEET1 gene, finding out complete ORF and designing primers with enzyme cutting sites at both ends, and removing stop codon with downstream primer as follows:

S23F：5′-GGGGATCC GCAAGTATCTTCCCTCGACG-3′BamHI

S23R：5′-GGGAGCTCTGGCATGCTCATGTATGTGTG-3′SacI

b. the ShSWEET1-pMD19-T plasmid is used as a template, primers (S23F and S23R) are used for PCR amplification, and an amplification system and a program are as follows:

c. the reaction program is that 95 ℃ 5min, 95 ℃ 1min, 60 ℃ 1min, 72 ℃ 1min, 35 cycles, 72 ℃ 10min, after the PCR reaction, 1% agarose gel electrophoresis is used, a scalpel is used in a gel imaging system to cut off and recover a target band, then the target band is connected into a pMD 19T-single vector, the target band is transformed into competent DH5 α, a positive clone is selected for PCR identification, the positive vector is sent to the Shanghai to be subjected to sequencing, and the correct vector is named as pMD-ShSWEET 1.

d. The single clone of pMD-ShSWEET1 with correct sequencing and the strain containing pCAMBIA3300-GUS vector were subjected to plasmid extraction, and the extraction procedure was the same as that of c.

e. The plasmids pMD-ShSWEET1 and pCAMBIA3300-GUS were double digested with the restriction enzymes BamHI, SacI as follows:

f. then, 1% agarose gel electrophoresis is carried out, a carrier large fragment and a target gene fragment are recovered, T4 DNAIgase is used for connection, transformation is carried out to enter a competence DH5 α, positive clone is selected, shaking culture is carried out at 37 ℃ overnight for plasmid extraction, a double enzyme digestion identification is carried out to obtain a target size band (the result is shown in figure 12), and the correct carrier is named as a pShSWEET1 carrier.

The enzyme digestion system is as follows:

(2) culture of tobacco explant

After soaking common tobacco (Nicotiam tabacum) seeds in 95% alcohol for 5s, the seeds were transferred to a 50% (V/V) sodium hypochlorite solution (plus one drop of Tween-20) with sterile forceps and stirred for 20 min. Rinsing with sterile water for five times, air drying the seeds in sterile filter paper, sowing the seeds on MS solid culture medium, culturing 20 seeds in each dish at 28 ℃ under illumination. And (3) after one week, the seedlings are changed into a fresh MS culture medium, 3 plants are planted in each bottle, and the seedlings can be used for dip-dyeing when 3-4 large-leaf seedlings are grown.

(3) Transformation of tobacco

EHA105 strain containing the expression vector was streaked in YEP solid medium (containing 10mg/mL Str +20mg/mL Rif +50mg/mL Kan) and incubated at 28 ℃ for 2 d. Single clones were picked up and incubated overnight at 28 ℃ in 5ml YEP broth containing the same antibiotic at 200 rpm.

2mL of overnight culture medium was incubated in 100mL of YEP broth (containing the same antibiotic) at 28 ℃ and 200rpm until OD600 reached 0.5-0.7. Transferring the strain into a 50mL sterile centrifuge tube, centrifuging for 5min at 4 ℃ and 4000rpm, discarding the supernatant, sucking out the residual liquid of the culture medium by using a sterile gun head, adding 100mL MR liquid culture medium (containing 150 mu mol/L AS) to resuspend the strain, and carrying out shaking culture at 220rpm and 28 ℃ for 2h to obtain the agrobacterium transformation infectious liquid.

Dip dyeing: beating the young tobacco leaves into leaf discs with the diameter of 5mm by using an aseptic puncher, placing about 50 leaf discs into the transformation dip-dyeing bacteria liquid, slightly shaking by using a shaking table at 30rpm for 3min, and vacuumizing for 10 min. Standing at room temperature for 5-l0min, taking out the leaf disc with a sterile spoon, and drying surface water with sterile filter paper. Finally, leaf disks were inoculated in MS solid medium and co-cultured in the dark at 28 ℃ for 3 d.

After co-culture, the leaf discs were transferred to MS differentiation medium (containing 1 mg/L6-BA) for differentiation culture, and the same medium was changed every week until seedlings were differentiated.

The differentiated plantlets at the edge of the leaf disc were cut and then placed in MS medium for one week.

After one week, the plantlets were transferred to MS solid medium (containing 2mg/LPPT) for selection culture, and the same medium was changed every week until resistant plantlets were selected.

The resistant seedlings are placed into rooting culture for two weeks, acclimatized for one week and then planted in flowerpots.

(4) Molecular detection of transgenic tobacco

The genome DNA of tobacco is extracted in a small amount by a CTAB method. And (3) extracting and reverse transcribing RNA of the transgenic tobacco and wild leaf. Performing PCR detection on the obtained transgenic tobacco for the target gene, wherein the detection takes (S2GF, S2GR) as a primer and takes the total DNA of the transgenic tobacco leaves as a template, and the amplification procedure is as follows: 5min at 95 ℃, 40s at 60 ℃, 50s at 72 ℃ for 34 cycles, and 10min at 72 ℃. By plant expression vectorsPlasmid pShSWEET1 as positive control, wild type tobacco and H₂And O is used as a negative control, 20 strains of ShSWEET 1-transferred resistant vaccine DNA obtained by primary screening are used as templates for PCR amplification, and the result shows that: in the transgenic resistant seedlings, 17 ShSWEET1 transgenic tobaccos can be amplified to strips consistent with positive control, the sizes of the strips are all about 750bp (figure 13), false positive plants and non-transgenic tobaccos do not have corresponding strips, and recovery sequencing proves that the target gene sequences are the target gene sequences, which indicates that the target genes are integrated into tobacco genomes.

(5) Transgenic tobacco cold stress treatment

Selecting transgenic tobacco seedlings which are strong in development and consistent in growth under the same environment and wild type control group seedlings, putting the transgenic tobacco seedlings and the wild type control group seedlings into a constant-temperature incubator at 4 ℃, culturing for 50 days under the conditions of 16h illumination/8 h darkness and 3000lx illumination intensity, observing the growth condition of plants at intervals, photographing and recording the growth condition. The transgenic tobacco was found to grow faster, have smaller and sharper leaves, developed roots and grow more nutritionally than the wild type tobacco (fig. 14), indicating that under cold stress, the ShSWEET1 transgenic tobacco may more readily transport soluble sugars to the intercellular spaces and maintain cellular homeostasis to resist cold stimulation and allow normal growth.

2.6 genetic transformation of sugarcane with ShSWEET1 Gene

(1) Obtaining transgenic sugarcane

The young leaves of the growing point of the apical meristem of the sugarcane are cut into slices and are induced for 20-30 days on an M1 culture medium in a dark place, and embryonic calluses with loose texture and extremely strong cell division and differentiation capability are gradually formed (figure 15A), so that the young leaves can be used for transformation. After the callus is soaked in the bacterial liquid containing pShSWEET1 Agrobacterium, the callus is cultured in the dark for 3-4 days, and is cultured and screened in the dark on an M2 culture medium for 15 days, and then is transferred to a subculture medium to be cultured for 20 days by illumination differentiation and screening, and the callus begins to grow seedlings (figure 15B). When the plantlets grow to about 1cm, the plantlets are transferred to a rooting medium containing PPT for screening and culturing for 30 days, and the PPT screening concentration is 2.5mg/L (shown in figures 15C and 15D), so that 10 resistant plants transformed into ShSWEET1 are obtained. After roots of the ShSWEET 1-resistant plants grow well, seedlings are cleaned of culture medium, old leaves are cut off, the seedlings are transplanted into crystal mud to be hardened (figure 15E) and subjected to PCR detection, and after 2 weeks, the resistant plants are pulled out and planted in flowerpots (figure 15F).

(2) Shsweet1 overexpression resistant plant Bar detection

The genomic DNA of ShSWEET1 overexpression resistant plants and non-transgenic sugarcane is extracted in a small quantity by using a CTAB method, a herbicide-resistant gene (Bar) carried by a plant expression vector is used for designing a primer, an expression vector plasmid is used as a positive control, the non-transgenic sugarcane is used as a negative control, PCR amplification detection is carried out, and the result shows that: in transgenic resistant seedlings, all transgenic plants amplified bands consistent with the positive plasmid, respectively, specific bands of about 400bp (FIG. 16), and recovery sequencing proved that the sequences were the Bar gene sequences, indicating that the Bar gene had been integrated into the sugarcane genome.

Reference to EnviroLogoix QuickStix^TMKit for Liberty

The Bar gene coding protein of transgenic sugarcane is detected by the (Bar) Cotton kit, and the result shows that the obtained transgenic ShSWEET1 sugarcane plant can detect the expression of the Bar gene and is consistent with the result of detecting the Bar gene by PCR.

(3) ShSWEET1 overexpression resistant plant target gene detection

An upstream primer designed by a Ubi promoter sequence and a downstream primer designed by a gene region are respectively used for designing a target gene detection primer of the ShSWEET1 overexpression resistant plant, PCR amplification is carried out on the PPT screened resistant plant, the result shows that 8 plants of 10 obtained overexpression plants are converted into ShSWEET1 plants to detect a band with the size of about 750bp, and the sequence is found to be consistent with the expectation through recovery, connection transformation and sequencing comparison, which shows that the ShSWEET1 gene is integrated into the sugarcane genome.

The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. Any equivalent modifications and substitutions to those skilled in the art are also within the scope of the present invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.

SEQUENCE LISTING

<110> research institute of tropical biotechnology of Chinese tropical academy of agricultural sciences

<120> cane sugar transport protein ShSWEET1 gene and application thereof

<130>170119-1

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<170>PatentIn version 3.3

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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

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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

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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 (10)

1. A gene of a sucrose transporter ShSWEET1 is characterized in that the nucleotide sequence is shown in SEQ ID NO. 1.

2. A sucrose transporter ShSWEET1, which is the protein coded by the sucrose transporter ShSWEET1 gene of claim 1, and the amino acid sequence of the protein is shown as SEQ ID NO. 8.

3. A method for cloning the ShSWEET1 gene as defined in claim 1, comprising the steps of:

(1) extracting total RNA from the roots, stems and/or leaves of mature sugarcane;

(2) carrying out reverse transcription by taking the total RNA as a template to obtain cDNA;

(3) designing a ShSWEET1 gene full-length sequence amplification primer pair, carrying out PCR amplification, and recovering a PCR product, wherein the nucleotide sequence of the ShSWEET1 gene full-length sequence amplification primer pair is shown as SEQ ID NO. 2 and SEQ ID NO. 3;

(4) and connecting the PCR product with a vector, transforming and sequencing to obtain the ShSWEET1 gene fragment.

4. The use of the gene of the sucrose transporter ShSWEET1 as defined in claim 1 for locating cell membrane.

5. The use of the sucralose transporter ShSWEET1 gene of claim 1 in improving the cold stress resistance of tobacco.

6. The use of the sucrose transporter ShSWEET1 gene as defined in claim 1 in cultivating high-sugar, high-yield, high-trans-resistance sugarcane.

7. An expression vector comprising the sucrose transporter ShSWEET1 gene of claim 1.

8. A primer pair is characterized in that the nucleotide sequence is shown as SEQ ID NO. 2 and SEQ ID NO. 3.

9. A primer pair is characterized in that the nucleotide sequence is shown as SEQ ID NO. 4 and SEQ ID NO. 5.

10. A primer pair is characterized in that the nucleotide sequence is shown as SEQ ID NO. 6 and SEQ ID NO. 7.

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