CN111154772B - Pear sugar transport gene PbSWEET4 and application thereof - Google Patents

Abstract

The invention discloses the application of the pear sugar transfer gene PbSWEET4 and its recombinant expression vector. A structural gene PbSWEET4 isolated from Dangshansu pear with sugar efflux function, the nucleic acid sequence of the gene is shown in SEQ ID No.1 of the sequence table, and the corresponding amino acid sequence is shown in the sequence table of SEQ ID No.2. The gene PbSWEET4 of the present invention was transformed into diploid forest strawberry and its function was verified. Taking wild-type strawberry as a control, the sucrose content in the leaves of the obtained transgenic strawberry plants was significantly reduced, and the leaves showed the phenomenon of premature senescence. It shows that the PbSWEET4 gene cloned in the present invention is a functional structural gene encoding a sugar transporter, has the function of effluxing soluble sugar, plays a negative regulatory role in sugar accumulation in leaves, and also participates in regulating the senescence process of leaves.

Description

Pear sugar transport gene PbSWEET4 and application thereof

Technical Field

The invention belongs to the field of plant genetic engineering, and relates to a pear sugar transport gene PbSWEET4, a recombinant expression vector and application thereof. In particular to a sweet potato family member PbSWEET4 gene which is related to the sugar transport of pears and is obtained by separating and cloning from Dangshan pear and application thereof.

Background

Pears (Pyrus) are perennial deciduous fruit trees of the genus Pyri (Rosaceae), are widely planted worldwide, and have important economic and social values. The edible quality of the pear fruit is an important factor for determining the value of the pear fruit, so that the improvement of the edible quality of the pear has important significance. The eating quality of the pear fruits is influenced by a plurality of factors, wherein sugar is one of important indexes constituting the fruit quality, and the sugar content of the pear fruits is increased, so that the sugar content is important for improving the pear quality. In recent years, the main cultivated varieties of Chinese pears have the problems of reduced sugar content, light flavor and the like due to variety degradation or poor management and the like, and the quality and the economic value of the fruits are seriously influenced. Therefore, quality improvement of sugar content of pear fruits has become one of the important targets of modern pear breeding. However, most of the research on sugar content of pear fruits focuses on the evaluation of sugar content of different pear varieties, and the functional research on sugar-related genes needs to be strengthened.

Sugars are first synthesized by leaf via photosynthesis, and then transported via phloem to the sink in the symplast or apoplast pathways (Oparka, 1990). Leaves are essential for the growth of most plants as the main site for sugar production by plants. Leaf senescence is the final stage of leaf development and is also an important component of the life cycle of deciduous fruit trees. The process involves a series of ordered changesIncluding degradation of macromolecules (e.g., proteins), transport of nutrients to actively growing organs (e.g., young leaves, developing seeds, and fruits), and the like. Leaf senescence determines the yield and quality of the fruit. If senescence occurs too early, the plants absorb CO ₂ Will eventually lead to a decrease in photosynthetic efficiency (Wingler et al, 2006). On the other hand, the nutrient cycle associated with senescence is inhibited (Himelblau and Amasino, 2001), which has a major influence on the development of the fruit. In arabidopsis, inhibition of the expression of AtTOR (rapamycin target protein) and SID2 (deletion of the salicylate synthase gene) leads to premature leaf senescence and reduced seed yield, while transgenic plants overexpressing NahG (expressing salicylate hydroxylase and capable of hydrolyzing salicylic acid) also show leaf senescence and seed reduction (depost et al, 2007 abreu and Munne-Bosch, 2009. In addition, transgenic tomato plants overexpressing SlNAP2 (NAC gene family senescence-promoting genes) exhibited premature leaf senescence, which in turn led to decreased fruit yield and soluble sugar content (Ma et al, 2018). RNA interference-INVINH 1 (invertase inhibitor) transgenic tomatoes have increased cell wall invertase activity, delaying leaf senescence, while increasing seed weight and sugar content (Jin et al, 2009). In conclusion, leaf development is crucial for the yield and quality of the fruit. However, the current research on the regulation mechanism of the influence of leaf senescence on fruit quality is still relatively deficient. Therefore, the method further discusses the relationship between leaf senescence and sugar metabolism, and has important theoretical and practical significance for revealing the mechanism of influence of leaf senescence on fruit quality and improving the fruit quality of pears.

It is well known that sugars may be involved in signal transduction, maintenance of osmotic pressure, constitute a carbon skeleton, or be stored in particular forms in fruits. In addition to this, sugars play an important role in stress. Sugar content is determined by a combination of processes such as synthesis, degradation, transport and storage, with transport being the more critical process (Katz et al, 2007). At present, three eukaryotic classes of sugar transporters have been found, respectively: glucose transporters (GLUT), sodium glucose transporters (SGLTs) and SWEET (Chen et al, 2015), where SWEET is a newly discovered class of sugar transporters. At present, no report related to the SWEET function in pears is found.

Disclosure of Invention

The invention aims to provide a SWEET gene with functions of sugar efflux and senescence promotion.

Another purpose of the invention is to provide application of the gene.

The purpose of the invention can be realized by the following technical scheme:

a structural gene PbSWEET4 which is separated from pear and has the function of sugar efflux belongs to the family of SWEET genes. The nucleic acid sequence of the gene is shown in a sequence table SEQ ID No.1 and comprises an open reading frame of 918 bp; 305 amino acids are coded, the coded amino acid sequence is shown in a sequence table SEQ ID No.2, the isoelectric point is 7.17, and the molecular weight is 34.2KDa.

The invention relates to a recombinant expression vector containing the PbSWEET4 gene.

The recombinant expression vector, preferably pMDC32, is obtained by inserting the gene PbSWEET4 as claimed in claim 1 into pMDC32 through Gateway reaction.

Host bacteria containing the PbSWEET4 gene of the invention.

The primer pair of the cDNA sequence of the PbSWEET4 gene is cloned, the sequence of an upstream primer PbSWEET4-F1 is shown as SEQ ID No.3, and the sequence of a downstream primer PbSWEET4-R1 is shown as SEQ ID No. 4.

The recombinant expression vector of PbSWEET4 disclosed by the invention is applied to promotion of sugar excretion and senescence of pear leaves.

The application comprises the steps of constructing a plant overexpression vector of the pear sugar transporter PbSWEET4 and converting diploid forest strawberries, taking wild strawberries as a control, and obviously reducing the sucrose content of leaves of obtained transgenic strawberry plants and showing the phenomenon of premature senility of the leaves.

Advantageous effects

Compared with the prior art, the invention has the following advantages and effects:

the discovery of the PbSWEET4 gene provides new genetic resources for promoting molecular breeding of pear sugar transport and realizing green agriculture, and the development and utilization of the genetic resources are beneficial to reducing agricultural cost and realizing agricultural friendliness.

2. The invention constructs a plant over-expression vector of the PbSWEET4 gene, transforms the pear PbSWEET4 gene into diploid strawberries by utilizing an agrobacterium-mediated genetic transformation method, and the obtained transgenic plants are analyzed by biological functions, which shows that the cloned PbSWEET4 gene promotes the efflux of sugar from strawberry leaves and simultaneously promotes the aging of leaves. The gene can be used for regulating the soluble sugar of the leaves of the transgenic plants and the senescence by the over-expression of the gene.

Description of the drawings:

FIG. 1 is the expression pattern analysis of the cloned pear PbSWEET4 gene in the leaf development process of different pear varieties. (A): 'Fengshui' (Pyrus pyrifolia N.cv.Hosui); (B): 'Korla bergamot pear' (Pyrus sinkiangensis Yu); (C): 'pear' (Pyrus bretschneideri Rehd. Cv. Yali); (D) 'Nanguo' (Pyrus ussuriensis Maxim). The expression patterns of the PbSWEET4 gene in leaves of different degrees of development (1-4 in the figure represent the degree from tender to mature) were analyzed by using 'plentiful water', 'Korla bergamot pear', 'Duck pear' and 'Nanguo' pears as test materials.

FIG. 2 shows the qualitative analysis result of GUS staining of transgenic Arabidopsis thaliana under the control of 2kb promoter of the cloned pear PbSWEET4 gene of the invention at different developmental stages. (A): seeding for 14 days; (B): 18 days after sowing; (C): 30 days after sowing; (D): 42 days after sowing.

FIG. 3 is a diagram showing the result of subcellular localization of the cloned pear PbSWEET4 gene in tobacco epidermal cells. (A): 35S imaging YFP (control) under fluorescence; (B): imaging of YFP (control) in the light field; (C): (A) imaging after superposition; (D): 35S, imaging YFP-PbSWEET4 under fluorescence; (E): 35S, imaging YFP-PbSWEET4 in a bright field; (F): and (D) and (E) imaging after superposition.

FIG. 4 is a graph showing the effect of over-expressing PbSWEET4 gene on strawberry leaf growth. (A): and (4) identifying the PbSWEET4 transgenic plant. (B): comparing the over-expressed PbSWEET4 gene plant with wild type; (C): the over-expression PbSWEET4 gene plant is compared with wild type leaves.

FIG. 5 shows the effect of over-expression of PbSWEET4 gene on soluble sugar and chlorophyll content in strawberry leaves. (A): the influence of the excessive expression of the PbSWEET4 gene on soluble sugar of leaves in strawberry plants; (B): the influence of the overexpression of the PbSWEET4 gene in strawberry plants on leaf chlorophyll. * Shows that the difference between the PbSWEET4 gene-transferred strain and the wild control reaches a significant level (P is less than or equal to 0.05).

Detailed Description

The present invention is described in detail below with reference to specific examples. From the following description and examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.

Example 1 analysis of the expression pattern of the pear PbSWEET4 gene during the development of pear leaves.

RNA was extracted from leaf of Dangshan pear, and genomic DNA contamination of the RNA was removed by DNase I (Invitrogen) digestion using CTAB method (Gasic et al, 2004), and first strand cDNA synthesis was performed using 1. Mu.g of RNA using a TOYOBO reverse transcription kit (purchased from Takara, inc., according to the kit instructions). The reverse transcribed first strand cDNA was used for real-time fluorescent quantitative PCR (qRT-PCR) of PbSWEET4. Using pear PbTublin (Pbr042345.1) as an internal reference, the nucleotide sequences of the primers were as follows:

forward primer TUB-F:5 'TGGGCTTTGCTCCTTAC-3' (SEQ ID No. 5)

Reverse primer TUB-R5

Designing a gene-specific qRT-PCR Primer pair in an open reading frame of the PbSWEET4 gene by using Primer 5.0, wherein the nucleotide sequence of the Primer is as follows:

forward primer PbSWEET4-F2: 5-

Reverse primer PbSWEET4-R2:5 'TCCTGCTTTCGGTTTCGGTA-3' (SEQ ID No. 8)

The SYBR Green kit (purchased from Roche, inc., according to the kit instructions) was used for qRT-PCR. The 20 μ L qRT-PCR reaction system included: 10 μ L of 2 × SYBR Premix ExTaq,0.25 μ L of forward primer, 0.25 μ L of reverse primer, 0.3 u LcDNA, 9.2L sterile double distilled water. PCR was carried out using a 96-well qRT-PCR plate (purchased from Roche) using a qRT-PCR instrument (model: lightCycler 480, roche). The qRT-PCR reaction program was: pre-denaturation at 95 ℃ for 10min, denaturation at 95 ℃ for 15 sec, annealing at 60 ℃ for 15 sec, extension at 72 ℃ for 20 sec, 40 thermal cycles. Repeating each sample for 3 times, calculating average Ct value of each cDNA sample, and passing through 2 ^-ΔΔCt The method (Livak and Schmittgen, 2001) calculates the relative expression level of PbSWEET4 gene.

Previous research results show that the expression level of PbSWEET4 is high in the late development stage of leaves (Li et al, 2017), and in order to verify whether the phenomenon is ubiquitous in pear leaves, the expression mode of PbSWEET4 in pear leaves of different varieties is detected. FIG. 1 is a diagram of the expression pattern of PbSWEET4 in different varieties of pear leaves at different developmental stages. As shown, pbSWEET4 showed the same expression pattern in four different pear varieties as leaf development progressed: the expression level was lower in young leaves and significantly increased in mature leaves (FIG. 1). Based on the above results, we speculate that PbSWEET4 may be associated with leaf development.

Example 2 cloning and vector construction of Pear PbSWEET4 Gene and its promoter

1. The method for extracting the total RNA of the pear leaves and synthesizing the cDNA is the same as the example 1. The forward primer sequence for amplifying PbSWEET4 is PbSWEET4-F1:5' -ATGGCTACAGTAGCAGAGACAGTCAC (SEQ ID No. 3), reverse primer sequence PbSWEET4-R1:5' -TCACACTGTCTGATGGTGTTTCAT (SEQ ID No. 4). High fidelity DNA polymerase for gene cloning (

Super-Fidelity DNA Polymerase (P505-d 1)) was purchased from Novowed Biotech. The amplification reaction system was 50. Mu.L, which included cDNA 200ng,2 XPhanta Max Buffer 25. Mu.L, 10mM dNTP 1. Mu.L, phanta Max Super-Fidelity DNA Polymerase 1. Mu.L, 10. Mu.M each of the above primers 2. Mu.L, plus ddH ₂ O to 50. Mu.L. The PCR reaction was performed on an Eppendorf amplification apparatus according to the following procedure: pre-denaturation at 95 deg.C for 3 min, denaturation at 95 deg.C for 15 s, annealing at 60 deg.C for 15 s, extension at 72 deg.C for 1 min, 35 thermal cycles, and 72 deg.CExtension for 5 min and storage at 4 ℃.

After the PCR product was detected by 1% agarose gel electrophoresis, the specific PCR amplified fragment was recovered by a rapid agarose gel DNA recovery kit (purchased from China, century Biotechnology Ltd.), and the procedure was referred to the instructions. The recovered and purified DNA was inserted into TOPO vector using TA cloning technique. Transformed into DH5 alpha Escherichia coli (Escherichia coli) competent cells (purchased from Pasteur Kay science Co., ltd., china) by heat shock method, cultured in LB solid medium containing 100. Mu.g/mL spectinomycin, screened for positive clones, propagated and sequenced (completed by Biotechnology engineering Co., ltd.). Sequencing-correct plasmid TOPO vector with PbSWEET4 full-length sequence was recombined into pMDC32 (for strawberry transformation) and pEarlyGate104 (for subcellular localization) overexpression vectors by LR enzyme, transformed into E.coli competent cells again by heat shock method, cultured in LB solid medium containing 50. Mu.g/mL kanamycin, positive clones were selected, amplified and sequenced. Sequencing results show that the full length of the PbSWEET4 gene is 918bp, the nucleic acid sequence of the PbSWEET4 gene is shown as a sequence table SEQ ID No.1, the PbSWEET4 gene codes 305 amino acids, the isoelectric point is 7.17, and the molecular weight is 34.2KDa. BLAST results analysis demonstrated that the newly obtained gene from pear is a member of SWEET gene family, and no relevant literature reports have been found about the specific functional studies of the gene, so we named the gene PbSWEET4. The recombinant vectors were named pMDC32-PbSWEET4 and pEarlyGate104-PbSWEET4, respectively.

2. DNA was extracted from the leaf of Dangshan pear by CTAB method (Chenglin poplar et al, 2014) and used for the amplification of the upstream promoter of PbSWEET4 gene (2 kb). The forward primer for amplifying the PbSWEET4 promoter was pPbSWEET4-F3:5' -TAGCTGAGGATGGTCAATGGGTTTA (SEQ ID No. 9), and the reverse primer is pPbSWEET4-R3:5' -ACCTTTCCAGAAAAATCAGCACACTGA (SEQ ID No. 10). High fidelity DNA polymerase for promoter cloning: (

Super-Fidelity DNA Polymerase (P505-d 1)) was purchased from Novowed Biotech. The reaction system for amplification is 5mu.L, including cDNA 200ng,2 × Phanta Max Buffer 25. Mu.L, 10mM dNTP 1. Mu.L, phanta Max Super-Fidelity DNA Polymerase 1. Mu.L, 10. Mu.M of the above primer 2. Mu.L, and ddH2O to 50. Mu.L. The PCR reaction was performed on an eppendorf amplification machine according to the following procedure: pre-denaturation at 95 ℃ for 3 min, denaturation at 95 ℃ for 15 sec, annealing at 60 ℃ for 15 sec, extension at 72 ℃ for 2 min, 35 thermal cycles, extension at 72 ℃ for 5 min, and storage at 4 ℃.

After the PCR product was detected by 1% agarose gel electrophoresis, the specific amplified fragment was recovered by a rapid agarose gel DNA recovery kit (purchased from China, century Biotechnology Ltd.), and the procedure was referred to the instructions. The recovered and purified DNA was inserted into TOPO vector using TA cloning technique. DH5 alpha E.coli (Escherichia coli) competent cells (purchased from Pasteur Kay science Co., ltd., china) were transformed by heat shock method, cultured in LB solid medium containing 100. Mu.g/mL spectinomycin, positive clones were selected, expanded and sequenced (by Biotechnology engineering Co., ltd.). The TOPO vector with PbSWEET4 promoter sequence is recombined into pMDC107 over-expression vector by LR enzyme, transformed into colibacillus by heat shock method, cultured in LB solid culture medium containing 50 ug/mL kanamycin, screened for vegetative clones, expanded and sequenced. The obtained nucleic acid sequence is shown in a sequence table SEQ ID No.11, and the recombinant vector is named as pMDC107-pPbSWEET4.

Example 3 qualitative analysis of GUS staining of transgenic Arabidopsis thaliana under the control of 2kb promoter of the pear PbSWEET4 Gene at different developmental stages

The PbSWEET4 promoter vector pMDC107-pPbSWEET4 was constructed in the same manner as in example 1. The final recombinant vector was transformed into Agrobacterium strain GV3101 by freeze-thaw method, then cultured in LB solid medium with 50. Mu.g/mL kanamycin, 100. Mu.g/mL rifampicin, and the correctly identified Agrobacterium strain was propagated using 10mL sterile centrifuge tubes until OD ₆₀₀ The value is about 1-1.2, and the mixture is centrifuged at 6000rpm for 10min to collect bacterial liquid. The vector was then transformed into wild type Arabidopsis plants by the floral dip method (Clough and Bent, 1998). GUS dye (purchased from Solibao, china) was used to treat southwest according to the instructionsMustard was stained at four stages from complete development of 4 rosette leaves (14 days after sowing) to complete maturity of arabidopsis thaliana (42 days after sowing). Finally, the plants were eluted with 25%,50%,70%,95% ethanol and observed.

GUS reveals GUS activity in Arabidopsis plants at each stage of development. The staining degree is continuously deepened along with the development of arabidopsis leaves, which indicates that the GUS activity in mature leaves is higher than that in young leaves (figure 2), and shows that the PbSWEET4 promoter has higher activity in old leaves, which is consistent with the expression pattern of PbSWEET4 in the leaf development process.

Example 4 subcellular localization of the PbSWEET4 Gene

The pEarlyGate104-PbSWEET4 vector was constructed in the same manner as in example 1. The final recombinant vector was transformed into Agrobacterium strain GV3101 by freeze-thaw method, then cultured in LB medium with 50. Mu.g/mL kanamycin, 100. Mu.g/mL rifampicin, and the correctly identified Agrobacterium strain was propagated using 10mL sterile centrifuge tubes until OD ₆₀₀ The value is about 1-1.2, and the mixture is centrifuged at 6000rpm for 10min to collect bacterial liquid. The procedure was carried out according to the method of Sperschneider (Sperschneider et al, 2017) as follows: the harvested Agrobacterium was resuspended in the infection solution (10 mM MgCl) ₂ 10mM EMS, pH 5.7, 200mM acetosyringone) to a final OD ₆₀₀ Is 0.8-1.2. Then, the resuspended suspension was placed on a shaker at room temperature (25 ℃) for 4 hours, after which the resuspension was injected with a 1mL syringe to the back of 3-4 week-old leaflet tobacco leaves. The injected tobacco leaves were cultured at 22 ℃ for 3 to 4 days, and then the epidermal cells of the injected tobacco leaves were observed using a confocal laser scanning microscope (Zeiss LSM 700, germany), photographed, and stored.

FIG. 3 is a subcellular localization map of PbSWEET4. YFP signal was observed on the cell membrane of the 35S-PbSWEET4-YFP fusion vector, whereas the empty control showed fluorescence in the cytoplasm and nucleus (FIG. 3). Our results indicate that PbSWEET4 encodes a membrane protein.

Example 5 genetic transformation of strawberry

The agrobacterium-mediated strawberry genetic transformation method refers to the method of Slovin et al (Slovin et al, 2009), and the specific operation steps are as follows:

1. and (3) disinfection and sterilization of stems and petioles: first sterilized in 70% ethanol for 30 seconds, then washed 3 times with sterile water, then sterilized with 1% sodium hypochlorite (20% bleach) for 10 minutes, and finally washed 4 times with sterile water.

2. Culturing agrobacterium tumefaciens: the pMDC32-PbSWEET4 vector was constructed as in example 1, the final recombinant vector was transformed into Agrobacterium strain GV3101 by freeze-thaw method, then cultured in LB medium with 50. Mu.g/mL kanamycin, 100. Mu.g/mL rifampicin, then the correctly identified Agrobacterium strain was cultured overnight at 28 ℃ and 220rpm in 50mL liquid medium using 10mL sterile centrifuge tubes until OD ₆₀₀ The value is around 0.5.

3. Infection transformation: the previously cultured Agrobacterium was centrifuged at 6000rpm for 10min to collect the bacterial liquid, which was then resuspended to OD in a coculture broth (1 XMS, pH 5.8,2% sucrose, 50. Mu.M acetosyringone) ₆₀₀ For 0.1, the resuspended agrobacteria were transferred to a sterile conical flask, the explants were immersed in the co-cultivation medium to which the inoculum was added and incubated for 20 minutes at room temperature. Then, the cells were blotted with a sterile filter paper and transferred to a solid medium (Table 1), and cultured in a dark environment at 25 ℃ for two days.

Hygromycin selection for resistant shoots: transgenic shoots were selected on selection medium containing 4mg/L hygromycin B. Explants were regenerated under a cold white fluorescent lamp under a 16 hour light, 8 hour dark photoperiod. Explants were checked daily for contamination and subcultured every 2 weeks.

Rooting induction and transplanting: when the strawberry explants formed different shoot buds, the whole mass was transferred to hormone-free rooting medium consisting of 0.5 × MS medium (pH 5.8), 1% glucose and 1% agar powder. Roots form within days to a month and individual plants can then be dissected from the sprouts. And (4) taking out the strawberry regeneration plant with a well-grown root system from the rooting culture medium, washing the root system with tap water, transplanting the strawberry regeneration plant into nutrient soil, and growing the strawberry regeneration plant under natural illumination at 25 ℃.

TABLE 1 culture medium for strawberry genetic transformation system

Example 6 identification of PbSWEET4 transgenic strawberry plants and determination of physiological indices

1. Screening of Positive plants

Strawberry regeneration plants were obtained according to the method described above in example 5, and total DNA of wild-type strawberries and transgenic strawberry leaves was extracted according to the method described in example 1.

The identification steps of the positive plants are as follows: the positive seedlings were identified by PCR amplification of the above DNAs with PbSWEET4 amplification primers (forward 1 and reverse 1, as shown in SEQ ID No.7 and SEQ ID No. 8), and the DNAs of leaves of strawberry which were not transformed by infection were used as controls. The PCR reaction procedure and system were carried out as described in example 1. As shown in FIG. 4-A, the strawberry leaves which are not infected and transformed do not amplify the target band, and the regenerated strawberry plants which can amplify the target band are preliminarily identified as positive transgenic strawberry lines.

2. Effect of PbSWEET4 Gene overexpression on strawberry plant growth

Compared with wild strawberry plants, pbSWEET4 transgenic strawberry plants at the same growth stage exhibited a premature leaf senescence phenotype, mainly manifested by yellowing of the leaf edges (fig. 4). The cloned pear PbSWEET4 gene is shown to be capable of making leaves senesce early.

Influence of PbSWEET4 gene overexpression on soluble sugar content of strawberry leaves

Soluble sugar content of leaves of PbSWEET4 transgenic strawberry plants was determined by using wild type strawberry leaves as a control.

The extraction steps of the soluble sugar are as follows: referring to Liu Lun et al (Liu et al, 2016), the specific procedures were as follows: accurately weighing 5.0g of leaves in a precooled mortar, grinding the leaves into powder by using liquid nitrogen, transferring the powder to a 10mL test tube, adding 8mL 80% ethanol, carrying out water bath at 37 ℃ for 25 minutes (shaking and mixing the powder every 5 minutes), fully extracting the powder by using ultrasonic waves for 10 minutes, centrifuging the powder at 12000rpm for 10 minutes, transferring the supernatant to a 25mL volumetric flask, repeating the steps for three times and fixing the volume. Taking 2mL of the extracting solution, evaporating to dryness by using a rotary evaporator (model: RE-3000, shanghai Yangrong biochemical instrument factory), dissolving by using 1mL of sterile double distilled water, and finally filtering by using a water filter with the diameter of 0.45 mu m, wherein the filtrate is used for determining the content of the soluble sugar. The content of soluble sugar is determined by high performance liquid chromatography (UPLC ACQUITY H-Class, waters), and the mobile phase is acetonitrile (1% ammonia water): water =85, flow rate 0.2mL/min, column temperature 45 ℃, sample introduction time 15 minutes, sample introduction volume 2 μ Ι _; the detector is ELSD, the carrier gas is nitrogen, the pressure is 25Psi, the drift tube is 55 ℃, and the atomizer is 25 ℃; the chromatographic column is UPLC ACQUITY BEH Amide 1.7um 2.1 x 100mm. The content was calculated from the peak area of the sample and the standard curve for each carbohydrate. Analysis results show that compared with wild strawberry leaves, the sucrose content of the transgenic strawberry is obviously reduced.

4. Influence of PbSWEET4 gene overexpression on strawberry leaf chlorophyll

Chlorophyll is degraded with leaf senescence (Hortenstein, 2006), so in order to further verify the effect of the pear PbSWEET4 gene on leaf senescence, we used wild strawberry leaves as a control, measured the chlorophyll content (i.e., SPAD value) of 30 transgenic strawberry leaves with a chlorophyll meter (purchased from Konikamenada, model SPAD-502), and plotted a box plot (FIG. 5). Analysis results show that the chlorophyll content of the PbSWEET4 transgenic strawberry leaves is obviously lower than that of the wild control.

Comprehensive analysis shows that the sucrose content and the chlorophyll content in the leaves of the PbSWEET4 over-expressed strawberry strain are obviously reduced, and the premature senility of the leaves of the plant appears, which shows that the PbSWEET4 gene of the pear has the functions of promoting sugar discharge and simultaneously promoting leaf senescence.

Primary references

1.Abreu,M.E.,and S.Munne-Bosch.(2009).Salicylic acid deficiency in NahG transgenic lines and sid2 mutants increases seed yield in the annual plant Arabidopsis thaliana.Journal of Experimental Botany 60(4):1261-1271.

2.Chandran,D.(2015).Co-option of developmentally regulated plant SWEET transporters for pathogen nutrition and abiotic stress tolerance.Iubmb Life 67,461-471.

3.Chen,L.Q.(2014).SWEET sugar transporters for phloem transport and pathogen nutrition.New Phytologist 201,1150-1155.

4.Chen,L.Q.,Cheung,L.S.,Feng,L.,Tanner,W.,and Frommer,W.B.(2015).Transport of sugars.Annual Review of Biochemistry 84,865-894.

5.Clough,S.J.,and Bent,A.F.(1998).Floral dip:a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana.Plant Journal 16,735-743.

6.Deprost,D.,L.Yao,R.Sormani,M.Moreau,G.Leterreux,M.Nicolai,M.Bedu,C.Robaglia,and C.Meyer.(2007).The Arabidopsis TOR kinase links plant growth,yield,stress resistance and mRNA translation.Embo Reports 8,864-870.

7.Gasic,K.,Hernandez,A.,and Korban,S.S.(2004).RNA extraction from different apple tissues rich in polyphenols and polysaccharides for cDNA library construction.Plant Molecular Biology Reporter 22,437-438.

8.Himelblau,E.,and Amasino,R.M.(2001).Nutrients mobilized from leaves of Arabidopsis thaliana during leaf senescence.Journal of Plant Physiology 158,1317-1323.

9.Hortensteiner,S.(2006).Chlorophyll degradation during senescence.Annual Review of Plant Biology 57,55-77.

10.Jin,Y.,Ni,D.A.,and Ruan,Y.L.(2009).Posttranslational elevation of cell wall invertase activity by silencing its inhibitor in tomato delays leaf senescence and increases seed weight and fruit hexose level.Plant Cell 21,2072-2089.

11.Li,J.M.,Qin,M.F.,Qiao,X.,Cheng,Y.S.,Li,X.L.,Zhang,H.P.,and Wu,J.(2017).A new Insight into the evolution and functional divergence of SWEET transporters in Chinese White Pear(Pyrus bretschneideri).Plant Cell Physiol 58,839-850.

12.Liu,L.,Chen,C.X.,Zhu,Y.F.,Xue,L.,Liu,Q.W.,Qi,K.J.,Zhang,S.L.,and Wu,J.(2016).Maternal inheritance has impact on organic acid content in progeny of pear(Pyrus spp.)fruit.Euphytica 209,305-321.

13.Livak,K.J.,and Schmittgen,T.D.(2001).Analysis of relative gene expression data using real-time quantitative PCR and the 2(T)(-Delta Delta C)method.Methods 25,402-408.

14.Ma,X.M.,Y.J.Zhang,V.Tureckova,G.P.Xue,A.R.Fernie,B.Mueller-Roeber,and S.Balazadeh.(2018).The NAC transcription factor SlNAP2 regulates leaf senescence and fruit yield in tomato.Plant Physiology 177,1286-1302.

15.Oparka,K.J.1990.What is phloem unloading.Plant Physiology 94,393-396.

16.Slovin,J.P.,Schmitt,K.,and Folta,K.M.(2009).An inbred line of the diploid strawberry Fragaria vesca f.semperflorens for genomic and molecular genetic studies in the Rosaceae.Plant Methods 5,15.

17.Sperschneider,J.,Catanzariti,A.M.,DeBoer,K.,Petre,B.,Gardiner,D.M.,Singh,K.B.,Dodds,P.N.,and Taylor,J.M.(2017).LOCALIZER:subcellular localization prediction of both plant and effector proteins in the plant cell.Sci Rep-Uk 7,44598.

18.Katz E,Fon M,Lee YJ,et al(2007).The citrus fruit proteome:insights into citrus fruit metabolism.Planta,226,989-1005.

19.Wingler,A.,Purdy,S.,MacLean,J.A.,and Pourtau,N.2006.The role of sugars in integrating environmental signals during the regulation of leaf senescence.Journal of Experimental Botany 57,391-399.

20. Chenling poplar, song Ming Shu, chaihong and Li Zhi Ming (2014). A general extraction method for improved plant genome DNA, plant classification and resource bulletin 36,375-380.

Sequence listing

<110> Nanjing university of agriculture

<120> pear sugar transporter PbSWEET4 and application thereof

<160> 11

<170> SIPOSequenceListing 1.0

<210> 1

<211> 918

<212> DNA

<213> Dangshan' pear (Pyrus)

<400> 1

atggctacag tagcagacag tcaccatcct ttggcattta catttggagt tctaggaaat 60

ctagtctcaa ccatggttta cttagcccca gtgccgacat tttatcgaat ttacaggaaa 120

aaatcgacag aaggattcca ctcggtgcca tatctggtag caatgttcag ttccatgctt 180

tggttctatt atgcgtcgct aaaaaagaat gctatgctgc tcatcaccat taactcattc 240

ggaagttttg cagagatgac ctacatcgtc atcttcgttg tgtatgcacc aagggatgct 300

aggaagctta cagtgaaatt atttggtatt atgaacgtgg gacttttcac cttgatcctt 360

gtcgtgtctc actttctagt gagtcgtgcg taccgggtcc cagttcttgg atggattaat 420

gttgccattt ctaccagtgt ttttgctgcg cccttaagca ttgtggcaca agttatccga 480

acaagaagtg tcgaattcat gccatttagg ttatcatttt tcctcactct gagtgccgtt 540

atgtggtttg catatggatt gttcctcaag gacatatgta ttgcaattcc aaacgttctg 600

ggttttgtgt tgggactgct tcagatgctg ctgtatgcga tgtaccgaaa ccgaaagcag 660

gagatactag aagatcatga gaaaaagcta ccggctgcta caccagatca cgtgaacaac 720

attgtgatca tagccacatt agcagcttcc gaggttcatc cggtggatgc tcaaccgaac 780

aatcgcaatg atgatggtga cgttaataat aacgcggtcg ttacagaggc aaaggagcat 840

gaacaaacgg atgatcatcg tcatgtggaa aatgcttccg tcgagcttca acctaatgaa 900

acaccatcag cagtgtga 918

<210> 2

<211> 305

<212> PRT

<213> Dangshan' pear (Pyrus)

<400> 2

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

1 5 10 15

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

20 25 30

Thr Phe Tyr Arg Ile Tyr Arg Lys Lys Ser Thr Glu Gly Phe His Ser

35 40 45

Val Pro Tyr Leu Val Ala Met Phe Ser Ser Met Leu Trp Phe Tyr Tyr

50 55 60

Ala Ser Leu Lys Lys Asn Ala Met Leu Leu Ile Thr Ile Asn Ser Phe

65 70 75 80

Gly Ser Phe Ala Glu Met Thr Tyr Ile Val Ile Phe Val Val Tyr Ala

85 90 95

Pro Arg Asp Ala Arg Lys Leu Thr Val Lys Leu Phe Gly Ile Met Asn

100 105 110

Val Gly Leu Phe Thr Leu Ile Leu Val Val Ser His Phe Leu Val Ser

115 120 125

Arg Ala Tyr Arg Val Pro Val Leu Gly Trp Ile Asn Val Ala Ile Ser

130 135 140

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

145 150 155 160

Thr Arg Ser Val Glu Phe Met Pro Phe Arg Leu Ser Phe Phe Leu Thr

165 170 175

Leu Ser Ala Val Met Trp Phe Ala Tyr Gly Leu Phe Leu Lys Asp Ile

180 185 190

Cys Ile Ala Ile Pro Asn Val Leu Gly Phe Val Leu Gly Leu Leu Gln

195 200 205

Met Leu Leu Tyr Ala Met Tyr Arg Asn Arg Lys Gln Glu Ile Leu Glu

210 215 220

Asp His Glu Lys Lys Leu Pro Ala Ala Thr Pro Asp His Val Asn Asn

225 230 235 240

Ile Val Ile Ile Ala Thr Leu Ala Ala Ser Glu Val His Pro Val Asp

245 250 255

Ala Gln Pro Asn Asn Arg Asn Asp Asp Gly Asp Val Asn Asn Asn Ala

260 265 270

Val Val Thr Glu Ala Lys Glu His Glu Gln Thr Asp Asp His Arg His

275 280 285

Val Glu Asn Ala Ser Val Glu Leu Gln Pro Asn Glu Thr Pro Ser Ala

290 295 300

Val

305

<210> 3

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atggctacag tagcagacag tcac 24

<210> 4

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

tcacactgct gatggtgttt cat 23

<210> 5

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

tgggctttgc tcctcttac 19

<210> 6

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

tcctgctttc ggtttcggta 20

<210> 7

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

gagtgccgtt atgtggtttg c 21

<210> 8

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

tcctgctttc ggtttcggta 20

<210> 9

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

tagctgagga tggtcaatgg gttta 25

<210> 10

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

accctttcca gaaaatcagc acactga 27

<210> 11

<211> 2000

<212> DNA

<213> Dangshan' pear (Pyrus)

<400> 11

ccctttccag aaaatcagca cactgaccta cagttctggc ttttggggtg aggaaaagaa 60

acttattttt cccaaatttg tcaaaatatc aacctaccta gcttagatta ctaatcaagc 120

acttggttga atatactgct caaattaaaa gtctgaaaaa cgcacgtatc aattagctct 180

taatttagct gtatttatct ttccaaatta gaaaatgtct caagttcaca tttcttgtta 240

tttcctcatt aatcaatgac gagttgttag tctagttatg aaacttgttt aaattttatc 300

atgcttacgt cgcgccttct cattgatcaa taacgagttg ttactctaat attattaaag 360

taacataact tataaaggat ctaaactcca gaaaaataaa agtatatcgc aaccaaatca 420

cacaaattaa tgaacgtcga tggaaatagc catgtacata tctagcaatc tgtccaaagg 480

ctcccagggt gtccacctag cattctcgaa tcccagccaa atgatagaga caagaacgag 540

taacaacatc atgattgtct tgtggctcat ctttaattat tcttttgtca taacttaaaa 600

cctccctccc tccgtcccca tctcataacc gcaaaaaata tgaaaaaagc tggccaggct 660

gcttggattg tggaatttga ttacttgaag aagaaaaaag tcagtcagat gaacccccga 720

tgcacacgaa accctctaaa tattgcatga acattgaagc actaccaaac aaacattcaa 780

cggcatagaa caaaagcttt gtgaacaata ttgtaaatct ttgagtgtgt gacttggaaa 840

gattgtttgt tgtaattgaa atattgtcag gttgtgttat tcaattcaat ttataataag 900

tatatttatt tgtgaggctc tacaagttga acatatcaaa tttgttgtct atattcttaa 960

gaaattatta ttgtcatttc aagaatttaa ttgtgcaatc caaactttct atatttaaaa 1020

aaatttatga gaagtgcata attaattttt ttagattgct aataataata atttattatt 1080

attataacac gtcgtggcaa gtgttccgag agtatatata tacaactata ttgtctgcgt 1140

gtgacttgtg agaatttaca agtgacaact agggctgaag cctgacgcgc caaggcgtgt 1200

ttcgtttagg gttttagaag atggagagaa acggtccaaa caatggccat acagtatgca 1260

ctagtgcttg gaattagaga tatagaagtc acgtgaatcg ggctctactc tggacagctt 1320

tgcggtctta gaagagatga gtaacgtaaa aaatcatatt cttattttag ttggaagaag 1380

ccacttgttt tttttttcaa agagcgtgga attcatgttt gattagaaaa aactcataaa 1440

aaattagtaa attagtgtcg attaaccaaa actataacta tataactctt cctaattcgc 1500

agttatggtg aaattaatta tttgaataat tatggtgatg atttggggga ctaccctaat 1560

tcctatccaa agtagtgtca agaagtgtgg tgaataatgc tctgcttttt ttttcttttt 1620

ttcttttttt ttgtggccgt tggatggagg ttacgcacac gtgatagagg ggcacgtgga 1680

acttggattt gtggttcatt gaatgagttc gttgagtagc ttttcattgt acgggaacat 1740

gacctggtac accaaatgtt ataatactag tgatttgata ttaaattttt ttttcccaat 1800

cacttgtatt atgacacttg atgtattaga cagtgttccc ggcacattga aaaaattctc 1860

gagagcatgg tacaccacct actaatcctc catctgtcat gcagccacaa tgagttcaat 1920

acgcacaccc tatttctttt tctttcactt tttgtgtgta tataaacaag ctgcgtaaac 1980

ccattgacca tcctcagcta 2000

Claims (4)

1. The nucleic acid sequence of the gene is shown as SEQ ID No.1PbSWEET4Application in regulating soluble sugar of strawberry leaf and aging.

2. Use according to claim 1, characterized in that the gene comprising the pear sugar transporter is constructedPbSWEET4The plant overexpression vector is converted into diploid forest strawberries, wild strawberries are used as a control, and the leaf sucrose content of the obtained transgenic strawberry plants is obviously reducedAnd the leaf exhibits the phenomenon of premature senescence.

3. Contains gene with nucleic acid sequence shown as SEQ ID No.1PbSWEET4The recombinant expression vector is applied to reducing the content of soluble sugar in strawberry leaves and promoting leaf senescence.

4. The use according to claim 3, wherein the recombinant expression vector is a pMDC32 vector, the gene of claim 1PbSWEET4Insertion of pMDC32 by Gateway reaction.

Images