Pear PbrCPK28 gene and its application

Abstract

This invention discloses the PbrCPK28 gene in pear and its applications. A PbrCPK28 gene, isolated from 'Dangshan Crisp Pear', regulates sugar accumulation in pear fruit. Its nucleotide sequence is shown in SEQ ID No. 1, and its encoded amino acid sequence is shown in SEQ ID No. 2. Transient overexpression in pear fruit confirmed that PbrCPK28 can alter the sugar content of the fruit. In-depth research on the PbrCPK28, PbrTST4, and PbrVHA-A1 genes, and the construction of the CPK-TST-VHA-A1 regulatory pathway, revealed the molecular basis of sugar accumulation in pear fruit. The analysis of the PbrCPK28 gene and its sugar regulation mechanism not only provides genetic resources for improving the sugar quality of pear fruit but also offers valuable reference for increasing the sugar content of fruits from other fruit trees.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering and relates to the pear PbrCPK28 gene and its applications. Specifically, it involves the isolation and cloning of a PbrCPK28 gene that regulates the sugar content of pear fruits from 'Dangshan Crisp Pear'. The CPK-TST-VHA-A1 regulatory pathway constructed in this invention reveals the molecular mechanism and genetic basis of sugar accumulation in pear fruits. Background Technology

[0002] Pear is a perennial woody plant belonging to the genus *Pyrus* of the subfamily Amygdaloideae in the family Rosaceae. It is the third largest fruit tree species in my country, with a long history of cultivation and a wide planting area. Currently, pears are divided into two main categories: Eastern pears, mainly including white pear (*P. bretschneideri*), sand pear (*P. pyrifolia*), autumn pear (*P. ussuriensis*), and Xinjiang pear (*P. siniangensis*); and Western pears, mainly European pears (*P. communis*). As a major pear exporter, my country has ranked first in the world in terms of pear cultivation area and yield in recent years. However, some traditional main pear varieties in my country have relatively poor fruit quality, exhibiting problems such as bland flavor and coarse flesh. Sugar content is a crucial factor affecting fruit quality. Comparisons of sucrose, glucose, fructose, and sorbitol revealed that fructose is the main reason for the significant difference in sugar content between cultivated and wild varieties. Therefore, increasing the fructose content of pears is essential for improving their quality. Therefore, clarifying the differences in fructose content and analyzing its regulatory mechanism is of great significance for improving the quality of pear fruits.

[0003] Genetic analysis was performed on the F1 generation of the cross between 'Xingao' and 'Hongxiangsu' pears. Interval mapping and QTL analysis were used to identify potential QTLs for soluble solids traits in pear fruits. Transcriptome data from different cultivated and wild varieties were analyzed, and combined with phenotypic data from sugar content measurements at fruit maturity, a gene, PbrCPK28, was identified. Transient overexpression in pear fruits confirmed that PbrCPK28 can alter fruit fructose content. Association analysis of transcriptomes from seven different pear varieties revealed that a sugar transporter with known gene function, PbrTST4, and the type V ATPase PbrVHA-A1 interact with PbrCPK28. PbrCPK28 phosphorylates both, and PbVHA-A1 provides the transport power for PbTST4, thereby promoting sugar efflux into vacuoles for storage, ultimately increasing the sugar content in the fruit. The discovery of this regulatory pathway supplements and improves the regulatory mechanism of fructose content in pear fruits, providing a theoretical basis for improving pear fruit quality. Summary of the Invention

[0004] The purpose of this invention is to provide a PbrCPK28 gene that increases fructose accumulation in pear fruit.

[0005] The purpose of this invention is to provide a regulatory pathway for controlling fructose accumulation in pear fruits.

[0006] Another objective of this invention is to provide the application of this gene and its regulatory pathway.

[0007] The objective of this invention can be achieved through the following technical solutions:

[0008] A PbrCPK28 gene isolated from 'Dangshan Crisp Pear' that affects the accumulation of fructose content in the fruit has a nucleotide sequence shown in SEQ ID No. 1, containing an open reading frame of 1659 bp and encoding 552 amino acids, the amino acid sequence of which is shown in SEQ ID No. 2 of the sequence listing.

[0009] A recombinant expression vector containing the PbrCPK28 gene described in this invention. The recombinant expression vector is constructed using Pearlygate104 as the final vector and the gateway system.

[0010] Host bacteria containing the PbrCPK28 gene described in this invention.

[0011] Primer pairs for cloning the PbrCPK28 gene cDNA sequence described in this invention, the upstream primer PbrCPK28-F1 sequence is shown in SEQ ID No. 3, and the downstream primer PbrCPK28-R1 sequence is shown in SEQ ID No. 4.

[0012] The application of the PbrCPK28 gene described in this invention in promoting the accumulation of fructose content in pear fruits.

[0013] The recombinant expression vector described in this invention is used to promote the accumulation of sugar content in pear fruits.

[0014] The regulatory pathway constructed by the PbrCPK28 gene and its interacting genes PbrTST4 and PbrVHA-A1 described in this invention is applied to promote the accumulation of sugar content in fruits; wherein the nucleotide sequence of the interacting gene PbrTST4 is shown in SEQ ID No. 5, and the amino acid sequence is shown in SEQ ID No. 6; the nucleotide sequence of the PbrVHA-A1 gene is shown in SEQ ID No. 7, and the amino acid sequence is shown in SEQ ID No. 8.

[0015] Beneficial effects

[0016] Compared with the prior art, the present invention has the following advantages and effects:

[0017] 1. The discovery of the PbrCPK28 gene provides a new genetic resource for molecular breeding of pear trees. The development and utilization of this genetic resource is beneficial to improving the quality of pear fruits and meeting consumers' demand for improved pear quality.

[0018] 2. The PbrCPK28 gene was functionally verified in pear fruit using Agrobacterium-mediated transformation. The results showed that the PbrCPK28 gene cloned in this invention has the function of regulating fruit sugar accumulation, providing a more efficient approach for molecular breeding.

[0019] 3. The CPK-TST-VHA-A1 regulatory pathway constructed in this invention reveals the molecular basis of sugar accumulation in pear fruit. This regulatory mechanism not only provides genetic resources for improving pear fruit quality but also offers a valuable reference for increasing the sugar content of fruits from other fruit trees. Attached Figure Description

[0020] Figure 1 QTL localization of PbrCPK28 of the present invention in the soluble solids of the F1 generation of the 'Xingao' and 'Hongxiangsu' hybrid.

[0021] Wherein: a: QTL mapping of soluble solids in pear fruit. b: Candidate genes included in linkage group 15 (LG15).

[0022] Figure 2 The RPKM values ​​of PbrCPK28 of the present invention are shown in (a) between different cultivated and wild species, and the fructose content of different cultivated and wild species is compared in (b).

[0023] Figure 3 Analysis of PbrCPK28 gene expression levels in different tissues and developmental stages of 'Dangshan Crisp Pear'. Note: DAFB: Days after full bloom; Stem: stem; Leaf: leaf; Pollen: pollen; Petal: petal; Ovary: ovary; Relative expression level: relative expression level of PbrCPK28 gene in different tissues, with 21 DAFB expression levels as 1.

[0024] Figure 4 This invention demonstrates the subcellular localization of the PbrCPK28 gene. Results showed that green fluorescence was observed on the root tip cell membrane of the PbrCPK28 Arabidopsis transgenic gene, indicating that the PbrCPK28 gene is localized on the cell membrane.

[0025] Figure 5Functional analysis of transient injection of the PbrCPK28 gene into pear fruit according to this invention. Specifically: a) Determination of sugar content at the injection site after transient transformation of 'Dangshan Crisp Pear' with 135DAFB. b) Gene expression analysis at the injection site after transient transformation of Dangshan Crisp Pear with 135DAFB.

[0026] Figure 6 This study presents an experimental analysis of the interaction between the gene PbrCPK28 and PbrTST4 in this invention.

[0027] The study included: a. In vitro validation of the interaction between PbrCPK28 and PbrTST4 using a yeast two-hybrid assay. b. Validation of the interaction between PbrCPK28 and PbrTST4 in *Nicotiana benthamiana* using an LCI assay. c. Validation of the interaction between PbrCPK28 and PbrTST4 in *Nicotiana benthamiana* using a BiFC assay. d. Demonstration of the PbrCPK28 phosphorylation site for PbrTST4.

[0028] Figure 7 This invention provides a functional analysis of the PbrTST4 gene transiently injected into pear fruit. Specifically: a) Determination of sugar content at the injection site after transient transformation of 'Dangshan Crisp Pear' with 135DAFB; b) Gene expression analysis at the injection site after transient transformation of Dangshan Crisp Pear with 135DAFB.

[0029] Figure 8 This study presents an experimental analysis of the interaction between the gene PbrCPK28 and PbrVHA-A1 in this invention.

[0030] The study included: a. In vitro validation of the interaction between PbrCPK28 and PbrVHA-A1 using a yeast two-hybrid assay. b. Validation of the interaction between PbrCPK28 and PbrVHA-A1 in *Nicotiana benthamiana* using an LCI assay. c. Validation of the interaction between PbCPK28 and PbVHA-A1 in *Nicotiana benthamiana* using a BiFC assay. d. Demonstration of the PbrCPK28 phosphorylation site for PbrVHA-A1.

[0031] Figure 9 Functional analysis of the PbrVHA-A1 gene transiently injected into pear fruit according to this invention. Specifically: a) Determination of sugar content at the injection site after transient transformation of 'Dangshan Crisp Pear' with 135DAFB. b) Gene expression analysis at the injection site after transient transformation of Dangshan Crisp Pear with 135DAFB.

[0032] Figure 10 A working model of how PbrCPK28, PbrTST4, and PbrVHA-A1 regulate sugar accumulation in pear fruit. Detailed Implementation

[0033] The present invention will now be described in detail with reference to specific embodiments. Based on the following description and these embodiments, those skilled in the art can determine the basic features of the present invention, and various changes and modifications can be made to the present invention to adapt it to various uses and conditions without departing from the spirit and scope of the invention.

[0034] Example 1: QTL mapping of the PbrCPK28 gene

[0035] QTL mapping was performed on physiological data using MapQTL 6.0 software. Since the soluble solids (SSC) trait of pear fruit conforms to the characteristics of quantitative trait inheritance, the phenotypic normality of the trait was tested using the SciPy.stats.shaprio(x) function. QTL mapping was performed using the interval mapping (IM) method; markers with LOD > 2.5 were identified as potential QTLs, and the "1-Lod Drop" method was used to determine candidate QTL intervals. For traits exhibiting non-normal distribution, the Kruskal-Wallis (KW) test was used for significance testing; markers with a Significance of < 0.005 were considered significant. The QTL (Im0214) for SSC was significantly mapped on LG15. Im0214 is located in the region of the highest log-to-digital ratio (LOD) peak, indicating that the main genetic determinants of the SSC trait are located in this region. Then, candidate genes upstream and downstream of Im0214 were extracted and manually screened. Among these candidate genes, the calcium-dependent protein kinase gene PbrCPK28 was classified as a candidate gene. Figure 1 ).

[0036] Example 2: Isolation, cloning, and construction of the overexpression vector for the PbrCPK28 gene

[0037] 3 μg of RNA from the early pulp of 'Dangshan Crisp Pear' was taken and reverse transcribed using a one-step gDNA removal and cDNA synthesis kit (Transgen, China), following the manufacturer's instructions. Primers SEQ ID NO.3 and SEQ ID NO.4 were designed using SnapGene software according to general primer design principles. A 50 μL reaction mixture included 200 ng cDNA, 1× buffer (TransStart FastPfu Buffer), 10 mM dNTPs, 1 U Taq polymerase (TransStart FastPfu DNA Polymerase) (the aforementioned buffer and Taq polymerase were purchased from TRANS), and 500 nM of the aforementioned primers. The PCR reaction was performed on an Eppendorf amplification instrument according to the following program: 95℃ pre-denaturation for 3 minutes; 95℃ denaturation for 15 seconds, 60℃ annealing for 15 seconds, 72℃ extension for 1 minute 30 seconds, 35 thermal cycles; 72℃ extension for 10 minutes; storage at 4℃. A single PCR band product was generated. After recovery and purification, the full-length sequence of PbrCPK28 was inserted into the TOPO vector using TA cloning technology. The vector was then transformed into *E. coli* DH5α using a heat shock method and in a solution containing 100 μg / mL... -1 sp + The PbrCPK28 gene was cultured in LB solid medium, positive clones were screened, amplified, and sequenced. Sequencing results showed that the full-length PbrCPK28 gene is 1659 bp, with its nucleotide sequence shown in SEQ ID NO.1, and it encodes a protein with 552 amino acid residues, with the sequence shown in SEQ ID NO.2. The correctly sequenced plasmid was recombined into the Pearlygate104 overexpression vector via an LR reaction using the TOPO vector containing the full-length PbrCPK28 sequence. This was then transformed again into *E. coli* DH5α and cultured in 50 μg / mL medium. -1 Kanamycin (K) + The culture was performed in LB solid medium, positive clones were screened, amplified, and sequenced. Plasmids with correct sequencing were transformed into Agrobacterium strain GV3101 using a freeze-thaw method, and then... (The sentence is incomplete and requires more context to translate accurately.) -1 K + 100 μg / mL -1 Rifampin + The culture was carried out in LB medium, and then the correctly identified Agrobacterium strain was expanded and propagated in 10 mL sterile centrifuge tubes and stored at -80℃ for later use.

[0038] Example 3: Spatiotemporal Expression Pattern Analysis of PbrCPK28 Gene

[0039] Different tissue samples of 'Dangshan Crisp Pear' were collected from orchards in Fengxian County, Jiangsu Province (2020). Total RNA was extracted using the CTAB method, and the quality of the extracted samples was detected by spectrophotometry and agarose gel electrophoresis. 3 μg of extracted total RNA was used for reverse transcription using a one-step DNA removal and cDNA synthesis kit (Transgen, China), following the manufacturer's instructions. The primers used for real-time PCR were gene-specific primer pairs: SEQ ID No. 5 and SEQ ID No. 6; GAPDH (Pbr036263.1) was used as an internal control gene. The real-time PCR kit was purchased from Roche. The instrument used for real-time PCR was a Roche 480 quantitative PCR instrument. The reaction system consisted of: 10 μL of 2×SYBR GreenI Master Mix, 0.4 μL of forward and reverse primers (10 μM), 1 μL of cDNA, and 8.2 μL of water. The reaction conditions were: denaturation at 95℃ for 3 min; pre-denaturation at 95℃ for 3 s, annealing at 60℃ for 10 s, and extension at 72℃ for 30 s, repeated for 45 cycles; melting curve analysis showed that the temperature increased by 1℃ every 5 s from 60℃ to 95℃.

[0040] Studies have shown that the fructose content of pear fruits gradually increases with the duration of fruit development. qRT-PCR results from different pear tissues indicated that the expression level of PbrCPK28 was significantly lower in the early stages of fruit development than in the later stages, and that PbrCPK28 expression gradually increased in fruits from 21 to 120 days after flowering. Figure 3 This indicates that the expression of PbrCPK28 is consistent with the time and space of fructose accumulation in fruit, and may be involved in the accumulation of fructose in pear fruit.

[0041] Example 4: Detection of transient transformation, sugar content, and gene expression levels in pear fruit

[0042] (1) Instantaneous transformation of pear fruit

[0043] Agrobacterium containing the PbrCPK28 overexpression vector was injected into 'Dangshan Crisp Pear' approximately 135 days after flowering using Agrobacterium-mediated transformation. The method is as follows:

[0044] 1. Activate Agrobacterium containing the correct plasmid on a solid culture medium and grow it in an incubator at 28°C for 48 hours;

[0045] 2. Add 30 mL of solution containing R to a 100 mL Erlenmeyer flask. + With K + Activated Agrobacterium was picked up with a pipette tip from the liquid LB medium and grown in a shaker at 28°C and 200 rpm for 12 hours.

[0046] 3. Pour the bacterial culture into a 50mL centrifuge tube, centrifuge at 6000rpm for 15 minutes, and collect all bacterial cells;

[0047] 4. Resuspend the precipitate with an appropriate amount of induction medium (10mM MgCl2, 10mM MES, 200mM acetylsalicylic acid, pH 5.6), OD600 = 0.8, 25℃, 80rpm for 4 hours;

[0048] 5. Inject the infection solution into pear fruits (135DAFB), injecting at least 10 fruits each time, and conduct three biological replicates of the experiment;

[0049] 6. After dark incubation for 24 hours, place the sample in an incubator with a photoperiod of 16 hours of light / 8 hours of darkness, and maintain the temperature at 22℃. After 6 days of incubation, collect pear fruit samples from the injection site.

[0050] (2) Detection of sugar content at the injection site

[0051] First, weigh 2g of pear fruit sample and grind it into powder using liquid nitrogen. Transfer the fruit sample powder to a 10mL centrifuge tube and add 80% ethanol. Then, incubate in a 37℃ water bath for 30min. After the water bath, extract the sample again in an ultrasonic bath for 10min, centrifuge (12000rpm, 4℃) for 10min, and repeat three times. Transfer the supernatant collected each time to a 25mL volumetric flask, and finally make up the volume of the extract to 25mL. Take 2mL of this extract and evaporate it to dryness in a rotary evaporator. Dissolve the extract in 1mL of ultrapure water and elute. Filter the extract through a 0.45μM aqueous filter membrane into a small vial for subsequent analysis. The determination was performed using high performance liquid chromatography (HPLC) on a WATERS 1525 system. The carbohydrate column was a Transgenomic COREGET-87C: 7.8mm x 300mm, 10μm, and the guard column was a Transgenomic CARB Sep Coregel 87C cartridge. The chromatographic conditions used were: column temperature 85℃, reference cell 35℃, flow rate 0.7 mL·min⁻¹; WATERS 2414 differential refractive index detector; mobile phase was degassed ultrapure water (18.2 mΩ·cm). Each injection volume was 2 μL. The content was calculated based on the peak area of ​​the sample and the standard curve of the fructose standard. The results are as follows: Figure 5 As shown in Figure a, the fructose content of the fruit increases after instantaneous injection of the PbrCPK28 gene.

[0052] (3) Detection of relative expression level of PbrCPK28 gene at injection site

[0053] RNA extraction, cDNA synthesis, and the system and procedures for quantitative real-time PCR were as described in Example 2. Results showed that overexpression of PbrCPK28 at the site of overexpression increased the expression level of the PbrCPK28 gene. Figure 5 b).

[0054] Example 5: Yeast two-hybrid experiments verified the interaction between PbrCPK28 and PbrTST4.

[0055] To elucidate how PbrCPK28 influences fruit sugar accumulation, correlation analysis was performed on transcriptome data from 35 developmental dynamics studies of five pear varieties. Combined with existing research, a vacuolar membrane monosaccharide transporter, PbrTST4, was identified. Validation was conducted using yeast two-hybrid (Y2H), BiFC, and LCI assays, revealing a strong interaction between PbrCPK28 and PbrTST4. Phosphorylation mass spectrometry confirmed that PbrCPK28 phosphorylates PbrTST4 at three sites (S277, T278, and S314), with S277 and S314 being highly conserved in other plants. Figure 6 To demonstrate the biological function of phosphorylated residues in PbrTST4, PbrTST4 and its phosphorylation site mutant were transiently expressed in pear fruits of 135 DAFB. The results showed that the PbrTST4 mutant had a lower sugar content and reduced transport function than PbrTST4. Figure 7 This observation suggests that phosphorylated residues are crucial for the function of PbrTST4.

[0056] Example 6: Yeast two-hybrid experiments verified the interaction between PbrCPK28 and PbrVHA-A1.

[0057] Previous results indicated that PbrTST4 is activated by a type V ATPase. We re-mined the transcriptome database of 35 fruit samples from five pear varieties at different developmental stages, and a type V ATPase gene (PbVHA-A1) was identified and used for further analysis. Subcellular localization results showed that PbrVHA-A1 is located on the vacuolar membrane, and qRT-PCR analysis of PbrVHA-A1 at different fruit developmental stages showed upregulated expression during fruit development. The interaction between PbrCPK28 and PbrVHA-A1 was demonstrated using Y2H, LCI, and BiFC experiments. Mass spectrometry confirmed that PbrCPK28 has the function of phosphorylating PbrVHA-A1. Figure 8 In 'Dangshan Crisp Pear' fruit with 135DAFB, injection of the PbrVHA-A1 overexpression vector resulted in a significant increase in fructose content at the injection site. Figure 9To demonstrate the biological function of phosphorylated residues in PbrVHA-A1, a phosphorylation site mutant of PbrVHA-A1 was expressed in pear fruit of 135DAFB. This mutant also showed reduced function, indicating that phosphorylated residues are also crucial for the function of PbrVHA-A1.

[0058] Example 7

[0059] Based on the research results of the above embodiments, a model of the PbrCPK28 regulatory pathway in pear fruit was constructed, see [link to study]. Figure 10 .

Claims

1. A gene isolated from 'Dangshan qu pear' for use in promoting sugar content accumulation in pear fruit. PbrCPK28 The CDS sequence of the gene is shown as SEQ ID No.

1. PbrCPK28 The CDS sequence of the gene is shown as SEQ ID No.

1.

2. A composition comprising the recombinant expression vector of claim 1 PbrCPK28 Use of a recombinant expression vector comprising a gene of claim 1 in promoting sugar content accumulation in pear fruit.

3. Use according to claim 2, characterized in that, The recombinant expression vector is Pearlygate 104 as the overexpression vector.

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