CN111154772A

CN111154772A - Pear sugar transport gene PbSWEET4 and application thereof

Info

Publication number: CN111154772A
Application number: CN202010083362.7A
Authority: CN
Inventors: 吴俊�; 泥江萍; 李甲明; 张绍铃; 朱荣香; 刘海楠; 薛程; 张明月; 刘月园; 李晓龙
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2020-02-09
Filing date: 2020-02-09
Publication date: 2020-05-15
Anticipated expiration: 2040-02-09
Also published as: CN111154772B

Abstract

The invention discloses a pear sugar transport gene PbSWEET4 and application of a recombinant expression vector thereof. A structural gene PbSWEET4 separated from Dangshan pear and having sugar discharge function, the nucleic acid sequence of the gene is shown in a sequence table SEQ ID No.1, and the corresponding amino acid sequence is shown in a sequence table SEQ ID No. 2. The gene PbSWEET4 is transformed into diploid forest strawberries and subjected to functional verification, wild strawberries are used as a control, the sucrose content of leaves of obtained transgenic strawberry plants is obviously reduced, and the leaves show the phenomenon of premature senility. The cloned PbSWEET4 gene is shown to be a functional structural gene of coding sugar transporter, has the function of discharging soluble sugar, plays a role in negative regulation in the accumulation of leaf sugar, and simultaneously participates in the regulation of 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 transporter 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 enhanced.

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 locus 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. This process involves a series of ordered changes, including 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 CO₂Will reduce, eventually leading to a reduction 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) resulted in premature leaf senescence and reduced seed yield, while transgenic plants overexpressing NaHG (expressing salicylate hydroxylase and capable of hydrolyzing salicylic acid) also exhibited leaf senescence and seed reduction (Deprost et al, 2007; Abreu and Munne-Bosch, 2009). In addition, transgenic tomato plants overexpressing SlNAP2(NAC gene family senescence-promoting gene) exhibited premature leaf senescence, which in turn led to decreased fruit yield and soluble sugar content (Ma et al, 2018). RNA interference INVINH1 (invertase inhibitor) increased cell wall invertase activity in transgenic tomatoes delayed 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 sugar excretion and aging promotion functions.

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

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

a structural gene PbSWEET4 with sugar discharge function separated from pear belongs to the SWEET gene family. The nucleic acid sequence of the gene is shown as 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 34.2 KDa.

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 of claim 1 into pMDC32 through Gateway reaction.

A host bacterium containing the PbSWEET4 gene.

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 pear leaf sugar excretion and senescence.

The application comprises the steps of constructing a plant overexpression vector of the pear sugar transport gene 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 plant overexpression vector of the PbSWEET4 gene is constructed, the pear PbSWEET4 gene is transformed into diploid strawberries by utilizing an agrobacterium-mediated genetic transformation method, and the obtained transgenic plants are analyzed by biological functions, so that the cloned PbSWEET4 gene promotes the discharge of strawberry phyllospheres and simultaneously promotes leaf senescence. 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 development process of different pear varieties leaf blades. (A) The method comprises the following steps 'abundance' (Pyrus pyrifolia N.cv.Hosui); (B) the method comprises the following steps 'Korla bergamot pear' (Pyrussikangensis Yu); (C) the method comprises the following steps '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 young to mature) were analyzed using ` Fengshui `, ` Korla bergamot `, ` Duck `, and ` Nanguo ` pears as test material.

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 in different developmental stages. (A) The method comprises the following steps Seeding for 14 days; (B) the method comprises the following steps 18 days after sowing; (C) the method comprises the following steps 30 days after sowing; (D) the method comprises the following steps 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) The method comprises the following steps 35S imaging YFP (control) under fluorescence; (B) the method comprises the following steps 35S imaging YFP (control) in the light field; (C) the method comprises the following steps (A) Imaging after the superposition; (D) the method comprises the following steps 35S, imaging YFP-PbSWEET4 under fluorescence; (E) the method comprises the following steps 35S, imaging YFP-PbSWEET4 in a bright field; (F) the method comprises the following steps (D) And (E) imaging after superposition.

FIG. 4 shows the effect of over-expression of PbSWEET4 gene on strawberry leaf growth. (A) The method comprises the following steps Identification of PbSWEET4 transgenic plants. (B) The method comprises the following steps The over-expression PbSWEET4 gene plant is compared with the wild type; (C) the method comprises the following steps The over-expressed PbSWEET4 gene plant is compared with wild type leaf.

FIG. 5 shows the effect of over-expression of PbSWEET4 gene on soluble sugar and chlorophyll content of strawberry leaves. (A) The method comprises the following steps The influence of the overexpression of the PbSWEET4 gene in strawberry plants on soluble sugar of leaves; (B) the method comprises the following steps The influence of the overexpression of the PbSWEET4 gene in strawberry plants on leaf chlorophyll. Shows that the difference between the strain of the PbSWEET4 gene 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 leaves of Dangshan pear, genomic DNA contamination of the RNA was removed by DNase I (Invitrogen) digestion using the CTAB method (Gasic et al, 2004), and first strand cDNA was synthesized 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 PbSWEET 4. Using pear PbTublin (Pbr042345.1) as an internal reference, the nucleotide sequences of the primers were as follows:

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

Reverse primer TUB-R: 5'-CCTTCGTGCTCATCTTACC-3' (SEQ ID No.6)

A gene-specific qRT-PCR Primer pair was designed in the open reading frame of the PbSWEET4 gene using Primer 5.0, the nucleotide sequences of the primers were as follows:

forward primer PbSWEET4-F2: 5'-GAGTGCCGTTATGTGGTTTGC-3' (SEQ ID No.7)

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

The qRT-PCR was performed using SYBR Green kit (purchased from Roche, Inc., according to the kit instructions). The 20 mu LqRT-PCR reaction system comprises: 10 μ L of 2 XSSYBR Premix ExTaq, 0.25 μ L of forward primer, 0.25 μ L of reverse primer, 0.3 μ L of LcDNA, 9.2 μ L of sterile double distilled water. PCR was performed 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. Each sample was repeated 3 times, and the average Ct value of each cDNA sample was calculated and then passed 2^-ΔΔCtThe 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 pattern 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 related to leaf development.

Example 2 cloning and vector construction of the 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 PbSWEET 4-F1: 5' -ATGGCTACAGTAGCAGACAGTCAC (SEQ ID No.3), reverse primer sequence PbSWEET 4-R1: 5' -TCACACTGCTGATGGTGTTTCAT (SEQ ID No. 4). High fidelity DNA polymerase for gene cloning (

Super-Fidelity DNA Polymerase (P505-d1)) was purchased from Novowed Biotech. The reaction system for amplification was 50. mu.L, which included 200ng of cDNA, 25. mu.L of 2 XPPhanta Max Buffer, 1. mu.L of 10mM dNTP, 1. mu.L of Phanta Max Super-Fidelity DNA Polymerase, 2. mu.L of 10. mu.M of each of the above primers, plus ddH₂O to 50. mu.L. The PCR reaction was performed on an Eppendorf amplification apparatus 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 1 min, 35 thermal cycles, extension at 72 ℃ for 5 min, and storage at 4 ℃.

After the PCR products were detected by 1% agarose gel electrophoresis, specific PCR amplification fragments were recovered using a rapid agarose gel DNA recovery kit (purchased from china, japan biotechnology limited), referred to for use instructions, the recovered purified DNA was inserted into TOPO vectors using TA cloning techniques, transformed into DH5 α escherichia coli (escherchia) competent cells (purchased from basque science limited, china) using a heat shock method, and cultured in LB solid medium containing 100 μ g/mL spectinomycin, positive clones were screened, propagated and sequenced (completed by biologics engineering gmbh), correctly sequenced plasmids were recombined by LR enzyme with PbSWEET4 full length vector into pMDC32 (for strawberry transformation) and pEarlyGate104 (for subcellular localization) over-expression vectors, again named as escherichia coli competent cells using a heat shock method, and cultured in solid medium containing 50 μ g/mL isoelectric sequencing, the results of PbSWEET gate104 (for subcellular localization) overexpression sequence, the results of PbSWEET potato gene expression were found in PbSWEET potato competent cells, and the results of the PbSWEET potato gene amplification were found in PbSWEET potato gene expression vector, expressed as PbSWEET potato 16. the PbSWEET potato gene expression vector, the PbSWEET potato gene expression vector was found in PbSWEET potato gene expression, and the PbSWEET potato gene expression was found in PbSWEET potato gene expression, and the expression of PbSWEET potato gene expression, the expression of PbSWEET potato gene expression was found in PbSWEET potato gene expression, expressed as PbSWEET potato gene expression, expressed by PbSWEET potato gene expression, expressed in PbSWEET potato gene expression, expressed as PbSWEET potato 16. SWEET potato, expressed by PbSWEET potato, expressed as PbSWEET potato gene, expressed by PbSWEET potato gene, expressed in PbSWEET potato.

2. The leaf of Dangshan pear was extracted by CTAB method (Chenling poplar et al, 2014)Extracting DNA, and amplifying the promoter (2kb) at the upstream of PbSWEET4 gene. The forward primer for amplifying the PbSWEET4 promoter was pPbSWEET 4-F3: 5' -TAGCTGAGGATGGTCAATGGGTTTA (SEQ ID No.9), the reverse primer was pPbSWEET 4-R3: 5' -ACCCTTTCCAGAAAATCAGCACACTGA (SEQ ID No. 10). High fidelity DNA polymerase for promoter cloning: (

Super-Fidelity DNA Polymerase (P505-d1)) was purchased from Novowed Biotech. The reaction system for amplification was 50. mu.L, which included 200ng of cDNA, 25. mu.L of 2 × Phanta Max Buffer, 1. mu.L of 10mM dNTP, 1. mu.L of Phanta Max Super-Fidelity DNA Polymerase, 2. mu.L of 10. mu.M of the above primer, 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 ℃.

PCR products were detected by 1% agarose gel electrophoresis, and then specifically amplified fragments were recovered using a rapid agarose gel DNA recovery kit (purchased from Japan Biotechnology Ltd., China) using TA cloning technology to insert the recovered and purified DNA into TOPO vectors, transformed into competent cells of DH5 α E.coli (Escherichia coli) (purchased from Baische technologies Ltd., China) using a heat shock method, and cultured in LB solid medium containing 100. mu.g/mL spectinomycin, screening positive clones, expanded and sequenced (completed by Biotechnology Ltd.), plasmids with correct sequencing recombined PbSWEET4 promoter sequence into pMDC107 overexpression vector using LR enzyme, transformed into E.coli again using a heat shock method, and cultured in LB solid medium containing 50. mu.g/mL kanamycin, and screened as nutritional clones, and amplified and sequenced nucleic acid sequences as expressed in SEQ ID No. PbSWEET4, expressed in pMDC107, and pBlue-TOP vector containing 50. kanamycin.

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

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 OD₆₀₀The 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 dipping method (Clough and Bent, 1998). Four stages from complete development of 4 rosette leaves (14 days after sowing) to complete maturation of arabidopsis thaliana (42 days after sowing) were stained with GUS staining solution (purchased from solibao, china) according to the instructions. Finally, the plants were eluted with 25%, 50%, 70%, 95% ethanol and observed.

GUS reveals GUS activity at each stage of Arabidopsis plant development. The staining degree is increased continuously along with the development of Arabidopsis leaves, which indicates that GUS activity in mature leaves is higher than that in young leaves (FIG. 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 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 OD₆₀₀The value is about 1-1.2, and the mixture is centrifuged at 6000rpm for 10min to collect bacterial liquid. The specific procedure according to the method of Sperschneider (Sperschneider et al, 2017) was as follows: the harvested Agrobacterium was resuspended in the infection solution (10mM MgCl)₂10mM EMS, pH5.7, 200mM acetosyringone) to obtain the final OD₆₀₀Is 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 into the back of 3-4 weeks old leaflet tobacco leaves using a 1mL syringe. Culturing the injected tobacco at 22 deg.C for 3-4 days, and observing the injection with confocal laser scanning microscope (Zeiss LSM 700, Germany)The epidermal cells of the tobacco leaves are photographed and the tablets are preserved.

FIG. 3 is a subcellular localization map of PbSWEET 4. YFP signals were 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: 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 in 50mL liquid medium with 10mL sterile centrifuge tubes overnight at 28 ℃ at 220rpm until OD₆₀₀The value is about 0.5.

3. Infection transformation: the pre-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)₆₀₀At 0.1, the resuspended agrobacteria were transferred to a sterile conical flask, and the explants were immersed in a co-cultivation medium with added inoculum 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 taking out the strawberry regenerated plant with good root system growth from the rooting culture medium, washing the root system with tap water, transplanting the strawberry regenerated plant into nutrient soil, and growing the strawberry regenerated plant under natural illumination at 25 ℃.

TABLE 1 culture media 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 DNA with PbSWEET4 amplification primers (forward primer 1 and reverse primer 1, shown as SEQ ID No.7 and SEQ ID No.8), and the DNA of the strawberry leaves that were not infected and transformed was used as a control. 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.

Effect of PbSWEET4 Gene overexpression on strawberry plant growth

The PbSWEET4 transgenic strawberry plants at the same growth stage exhibited a premature leaf senescence phenotype, mainly manifested by yellowing of the leaf edges, compared to wild-type strawberry plants (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 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 of 80% ethanol, carrying out water bath at 37 ℃ for 25 minutes (shaking and mixing the powder every 5 minutes), carrying out ultrasonic wave full extraction for 10 minutes, centrifuging the mixture 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)), the mobile phase is acetonitrile (1% ammonia water): water 85:15, flow rate 0.2mL/min, column temperature 45 ℃, sample injection time 15 minutes, sample injection volume 2 uL; 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 1.7um2.1 x 100 mm. The amount of each carbohydrate 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.

Effect of PbSWEET4 gene overexpression on strawberry leaf chlorophyll

Chlorophyll is degraded with aging of leaves (Hortenstein, 2006), so in order to further verify the effect of the PbSWEET4 gene on leaf aging, wild strawberry leaves were used as a control, and chlorophyll content (i.e., SPAD value) of 30 transgenic strawberry leaves was measured by using a chlorophyll meter (purchased from Konika Mingta, model SPAD-502) and a box plot was plotted (FIG. 5). The analysis result shows that the chlorophyll content of the PbSWEET4 transgenic strawberry leaf 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 over-expressed strawberry strain of PbSWEET4 are obviously reduced, and the premature senility of the leaves of the plant appears, which shows that the PbSWEET4 gene of pear has the functions of promoting sugar discharge and simultaneously promoting the leaf senescence.

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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

1. A gene PbSWEET4 with sugar discharge function separated from pear is characterized in that the nucleic acid sequence is shown in a sequence table SEQ ID No.1, the cDNA full-length sequence is 918bp, and comprises an open reading frame of 918 bp.

2. The protein encoded by PbSWEET4 according to claim 1, wherein the amino acid sequence is represented by SEQ ID No.2 of the sequence Listing, which encodes 305 amino acids, has isoelectric point of 7.17 and molecular weight of 34.2 KDa.

3. A recombinant expression vector comprising the gene of claim 1.

4. The recombinant expression vector of claim 3, which is obtained by inserting the gene PbSWEET4 of claim 1 into pMDC32 through Gateway reaction, starting from pMDC 32.

5. A host bacterium containing the gene according to claim 1.

6. The cDNA sequence primer pair for cloning the gene PbSWEET4 as claimed in claim 1, characterized in that the sequence of the primer PbSWEET4-F1 is shown as SEQ ID No.3, and the sequence of the downstream primer PbSWEET4-R1 is shown as SEQ ID No. 4.

7. The use of the gene PbSWEET4 as defined in claim 1 for regulating soluble sugar in leaves and senescence.

8. The use according to claim 7, characterized in that a plant overexpression vector containing the pear sugar transporter gene PbSWEET4 is constructed and diploid forest strawberries are transformed, wild strawberries are used as a control, the leaf sucrose content of the obtained transgenic strawberry plants is obviously reduced, and the leaves show the phenomenon of premature senescence.

9. Use of the recombinant expression vector of claim 3 or 4 for reducing soluble sugar content in strawberry leaves and promoting leaf senescence.