Function and application of kiwifruit pectin acetyl esterase 8 gene AcPAE8 in vitamin C metabolic regulation

Abstract

This invention belongs to the field of molecular breeding technology, and specifically discloses the kiwifruit pectin acetylesterase 8 gene. AcPAE8 Functions and applications of vitamin C in the regulation of metabolism. AcPAE8 This gene is a positive regulator of vitamin C metabolism in kiwifruit, controlling vitamin C accumulation. Overexpression of this gene significantly increases vitamin C content, while gene editing significantly decreases it. This invention identifies a gene related to vitamin C content through metabolic pathways. AcPAE8 All of the above functions are discovered for the first time. This gene exhibits high specificity and high regulatory efficiency, and can be used to target the improvement of vitamin C trait in kiwifruit, as well as to establish and improve molecular marker-assisted selection breeding systems.

Description

Technical Field

[0001] This invention belongs to the field of plant molecular breeding technology, specifically relating to a kiwifruit pectin acetylesterase 8 gene. AcPAE8 Application in vitamin C metabolism regulation and genetic improvement of kiwifruit. Background Technology

[0002] Kiwifruit is globally recognized as a fruit with high nutritional value, and its excellent health properties largely stem from its extremely high vitamin C content. Vitamin C is not only an essential water-soluble vitamin for the human body, possessing important physiological functions such as enhancing immunity, promoting collagen synthesis, and anti-oxidation; within plants, vitamin C, as a key antioxidant, directly participates in the scavenging of reactive oxygen species and the regulation of stress responses. Although New Zealand pioneered the commercial development of kiwifruit and dominated the early global industry landscape, China has rapidly emerged as the world's largest kiwifruit producer in recent years. However, the Chinese kiwifruit industry still faces the contradiction of strong consumer demand coupled with the need to improve fruit quality. Enhancing the flavor, nutrition, and post-harvest quality of kiwifruit is a key focus of kiwifruit research.

[0003] Current research on vitamin C metabolism in kiwifruit mainly relies on reverse genetics. Research using forward genetics strategies to systematically discover and verify key regulatory genes remains relatively scarce, hindering the establishment and improvement of marker-assisted selection (MAS) breeding systems. In hybridization breeding, the selection of superior lines is a core step. However, traditional breeding methods relying on phenotypic selection are difficult to implement and time-consuming, often requiring extensive time to develop new varieties. Furthermore, most phenotypic traits are quantitative traits heavily influenced by environmental factors, and their instability further reduces the accuracy of phenotypic selection-based breeding. In particular, the significant spatiotemporal specificity of vitamin C content in kiwifruit across species, different developmental stages, and tissue parts further increases the difficulty of breeding. Therefore, comprehensively utilizing forward genetics, multi-omics analysis, and molecular biology techniques to systematically elucidate the genetic basis and molecular regulatory mechanisms of vitamin C accumulation in kiwifruit is of significant theoretical importance for cultivating new varieties with high nutritional quality through molecular design breeding and has important practical value for promoting the quality and efficiency of my country's kiwifruit industry. Summary of the Invention

[0004] In view of the above background, the purpose of this invention is to provide a kiwifruit pectin acetylesterase 8 gene. AcPAE8 The study also explored the application of this gene in regulating vitamin C metabolism in kiwifruit, providing new molecular tools and technical solutions for the genetic improvement of kiwifruit.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides a kiwifruit pectin acetylesterase 8 gene. AcPAE8The AcPAE8 The gene is a positive regulator of vitamin C metabolism. Its complete open reading frame sequence is shown in SEQ ID NO.1, and the encoded amino acid sequence is shown in SEQ ID NO.2.

[0006] This invention provides AcPAE8 A gene recombinant vector, wherein the recombinant vector uses a plant expression vector as a backbone, and... AcPAE8 Multiple cloning site for gene insertion vectors. Preferably, the plant expression vector POE-3Flag-DN is used as the backbone, containing... AcPAE8 The gene removes the entire CDS sequence of the stop codon and the Kan resistance gene.

[0007] This invention provides a host cell containing the above-mentioned recombinant vector, wherein the host cell is an Agrobacterium cell (EHA105). This host cell can be used for... AcPAE8 Functional verification of genes and genetic transformation of kiwifruit.

[0008] This invention provides AcPAE8 A gene editing vector comprising a CRISPR-Cas9 editing system and two target sequences sgRNA1 and sgRNA2, the sequences of which are shown in SEQ ID NO. 9 and 10.

[0009] This invention also provides a method for regulating the vitamin C content of kiwifruit: [The method involves...] AcPAE8 The gene recombinant vector was introduced into kiwifruit leaves or callus tissue, enabling... AcPAE8 Gene overexpression increases the vitamin C content of kiwifruit; gene editing technology is used to edit the kiwifruit genome. AcPAE8 Genes, making the AcPAE8 The loss of gene function or reduced expression levels decreases the vitamin C content of kiwifruit.

[0010] This application utilizes forward genetics methods to screen and clone the kiwifruit pectin acetylesterase 8 gene for the first time. AcPAE8 This study clarified that it acts as a positive transcription factor regulating the vitamin C metabolism process in kiwifruit. Compared with existing technologies, this invention has the following beneficial effects: (1) High specificity and efficient regulation: AcPAE8 Genes are positive regulators of vitamin C metabolism in kiwifruit, and can be regulated through overexpression or gene editing techniques. AcPAE8 Gene expression can rapidly accumulate or deplete vitamin C, significantly shortening the breeding cycle compared to traditional hybridization breeding.

[0011] (2) Innovative methods and focus: This application integrates unique genetic materials with forward genetic mapping technology, which can help establish and improve the molecular marker-assisted selection breeding system; at the same time, this application breaks through the previous limitation of only focusing on synthetic pathway enzymes, focuses on vitamin C metabolism regulation, and supports the study of the nutritional quality mechanism of kiwifruit.

[0012] (3) Wide application and broad prospects: This gene and its regulation method can be applied to the genetic improvement of different kiwifruit varieties (such as Chinese kiwifruit, delicious kiwifruit, etc.), which has important scientific significance and practical value for promoting the quality and efficiency of my country's kiwifruit industry. Attached Figure Description

[0013] Figure 1 Vitamin content at four key fruit development stages in the F1 generation of the hybrid population. S1-S4 represent the rapid growth period, cell enlargement period, ripening period, and full ripening period, respectively.

[0014] Figure 2 Manhattan plot of QTL location of vitamin C content in hybrid population.

[0015] Figure 3 Based on the vitamin C content of the hybrid population, QTL analysis was conducted to locate candidate genes for the corresponding regions.

[0016] Figure 4 Prediction of AcPAE8 protein domains.

[0017] Figure 5 : Prediction of the 3D structure of AcPAE8 protein.

[0018] Figure 6 Map of the AcPAE8 protein subcellular localization recombinant vector GFP-AcPAE8.

[0019] Figure 7 Subcellular localization analysis of AcPAE8 protein.

[0020] Figure 8 Overexpression of recombinant vector OE- AcPAE8 The map.

[0021] Figure 9 Gene editing recombinant vector Cas9- AcPAE8 The map.

[0022] Figure 10 PCR testing AcPAE8 Screening results of plants with overexpressed genes.

[0023] Figure 11 qRT-PCR detection AcPAE8 Identification results of positive plants with gene overexpression.

[0024] Figure 12 CRISPR / Cas9 system editing AcPAE8 Gene sequencing results.

[0025] Figure 13 : Graph analysis results of vitamin C content in transgenic plants. Detailed Implementation

[0026] Example 1: Kiwifruit pectin acetylesterase 8 gene AcPAE8 Filtering 1.1 Experimental screening of progeny with high, medium, and low vitamin C content The experimental materials were sourced from the National Kiwifruit Germplasm Resource Nursery at Wuhan Botanical Garden, Chinese Academy of Sciences, using an F1 generation population constructed from a cross between *Actinidia serrata* and the male *Actinidia chinensis* 'Moshanxiong 7'. High-performance liquid chromatography (HPLC) was used to systematically determine the vitamin C content of 185 progeny plants at four key fruit development stages (rapid growth, cell enlargement, ripening, and full ripening). Key chromatographic parameters were set as follows: the mobile phase consisted of 0.1% metaphosphoric acid solution and acetonitrile at a ratio of 98:2 (v / v), and the flow rate was 0.5 mL / min. First, a vitamin C standard stock solution with a concentration of 1037 μg / mL was prepared. Then, under light-protected conditions, the solution was diluted with 0.1% metaphosphoric acid solution to prepare a series of standard solutions with concentrations ranging from 10.73 to 429.20 μg / mL, which were stored at -20℃ in the dark for establishing a standard curve. Accurately weigh 0.5 g of kiwifruit pulp and add 2 mL of 0.1% metaphosphate solution to homogenize thoroughly. Adjust the pH of the homogenate to 5.0-6.0 with 1 M NaOH, then incubate overnight at 4℃ in the dark for extraction. After extraction, centrifuge at 8000 r / min for 5 min, filter the supernatant through a 0.22 μm filter membrane, and collect it in a brown sample vial for instrument analysis. Each sample includes three independent biological replicates.

[0027] like Figure 1 The results showed that the vitamin C content of the offspring was continuously distributed, ranging from 7.0 to 141.0 mg / 100 g FW, with a range of more than 20 times. Finally, based on the vitamin C content at maturity, the offspring were divided into a high vitamin C content group (>81 mg / 100 g), a medium vitamin C content group (31-80 mg / 100 g), and a low vitamin C content group (<30 mg / 100 g).

[0028] 1.2 Constructing a high-density genetic linkage map and conducting QTL mapping Genomic DNA was extracted from the young leaves of hybrid parents and progeny using the CTAB method. DNA integrity, purity, and concentration were assessed by 1% agarose gel electrophoresis, NanoDrop2000, and Qubit3.0. Samples were then sent to BGI Genomics in Shenzhen for quality control, library construction, and sequencing. Population SNP variation detection and screening were performed using GATK software. Progeny genotyping was then performed based on progeny population and parental genotype data. Bin markers were developed using the sliding window method, and low-quality Bin markers shorter than 10 kb were removed. Linkage groups were identified using Lep-MAP3 software, and a high-precision genetic linkage map was constructed to determine the marker arrangement order and genetic distances between adjacent markers. Finally, using the R language QTL analysis package and the composite interval plotting method, we integrated phenotypic and genotypic data for whole-genome scanning to locate horticultural trait QTLs. After 1000 permutation tests, we determined the 99.5%, 95%, and 90% confidence level LOD thresholds and classified QTLs into three categories: highly significant, significant, and potential. We then combined the LOD peak value, confidence interval, and phenotypic variation explanation rate to complete the QTL evaluation and naming.

[0029] like Figure 2 The Manhattan plot results showed a significant association signal (LOD > 4.0) detected on chromosome 26, corresponding to an interval size of approximately 147.4 kb; Figure 3 The results showed that the gene annotation analysis within this QTL interval identified a total of 7 coding genes and 3 uncharacterized genes.

[0030] 1.3 Transcriptome sequencing to construct gene expression matrices and screen key genes Transcriptome sequencing services were provided by Benagene. The main steps included: extracting total RNA from samples and performing quality control; enriching and fragmenting mRNA; synthesizing double-stranded cDNA to construct a specific library; performing 150 bp paired-end sequencing on the Illumina platform; filtering raw data using FPKM software to remove low-quality sequences; ensuring at least 20 million effective reads per library; setting up three biological replicates; constructing a gene expression matrix based on the FPKM values ​​of the kiwifruit transcriptome; and finally identifying a key gene in the vitamin C metabolism regulation process—pectin acetylesterase 8. AcPAE8 Its complete open reading frame sequence is shown in SEQ ID NO.1, and the encoded amino acid sequence is shown in SEQ ID NO.2.

[0031] Example 2: Kiwifruit pectin acetylesterase 8 gene AcPAE8 Functional prediction 2.1 Prediction of the secondary and tertiary structures of the AcPAE8 protein sequence Protein secondary structure prediction was performed using the SMART v10 online platform in Normal analysis mode. The SMART, Pfam conserved domain library, SignalP 6.0 signal peptide, TMHMM 2.0 transmembrane region, and COILS coil-and-coil prediction modules were simultaneously enabled. The domain matching E-value threshold was set to 1×10⁻⁶. -5 Signal peptide determination: D-score ≥ 0.5; coiled-helix sliding window 28 aa, probability threshold 0.9. (Example) Figure 4 As shown, the AcPAE8 protein contains two important structural domains and possesses dual potential functions related to lipase signaling regulation and cytoskeleton and microtubule transport. The protein's three-dimensional structure and residue pairing error (PAE) analysis were performed using the ColabFold online platform, incorporating the AlphaFold2 monomer prediction model. Multiple sequence alignment was performed using the MMseqs2 database, employing a template-free free prediction mode. A heatmap of expected positional errors between residues was output, with an error scale range of 0–30 Å. A segmented, colored three-dimensional conformational model of the protein was simultaneously generated, directly outputting a visualization of the paired residue positional error matrix. Tertiary structure prediction revealed that the protein consists of a short, flexible N-terminal helical tail and a densely folded, structurally stable C-terminal globular domain (e.g., ...). Figure 5 ).

[0032] 2.2 Subcellular localization analysis of AcPAE8 protein 2.2.1 AcPAE8 Cloning of genes (1) Total RNA extraction: Total RNA was extracted from young leaves of 'Donghong' kiwifruit according to the instructions of the RNA extraction kit. The integrity and purity of the RNA were detected by 1% agarose gel electrophoresis and Eva 3200.

[0033] (2) cDNA synthesis: Using the extracted total RNA as a template, the genomic DNA was removed using a reverse transcription kit, and the first strand of cDNA was synthesized and stored at -20℃ for later use.

[0034] (3) Primer design: Specific primers (SEQ ID NO.3 and 4) were designed using Snapgene software and synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0035] (4) PCR amplification: PCR amplification was performed using cDNA as a template. PCR reaction system (20 μL): 10 μL of 2×Phanta FlashMaster Mix (Dye Plus), 1 μL each of forward and reverse primers (10 μM), 2 μL of cDNA template (100 ng), and ddH2O to 20 μL. PCR reaction conditions: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 1 min, 35 cycles; 72℃ final extension for 10 min, and storage at 4℃.

[0036] (5) Product recovery: The PCR product was detected by 1% agarose gel electrophoresis, the target band was cut out, and the target fragment was recovered using a gel recovery kit.

[0037] (6) Identification and sequencing of positive clones: The recovered target fragment was ligated into the pMD19-T vector and transformed into E. coli DH5α competent cells. The cells were plated on LB solid medium containing ampicillin (100 μg / mL) and incubated overnight at 37°C. Single colonies were picked for PCR identification. Positive clones were sent to Wuhan Aoke Dingsheng Biotechnology Co., Ltd. for sequencing. The sequencing results were analyzed using DNAMAN software to confirm the presence of the target fragment. AcPAE8 The complete open reading frame sequence of the gene (SEQ ID NO.1).

[0038] (7) Obtaining the target fragment: Select the single colony with correct sequencing for amplification, extract the plasmid according to the instructions of the plasmid extraction kit, and use the plasmid as a template to repeat steps (4) and (5) to finally obtain the target gene fragment.

[0039] 2.2.2 Construction of AcPAE8 protein subcellular localization recombinant vector Primers (SEQ ID NO. 5 and 6) were designed using Snapgene software and synthesized by Beijing Qingke Biotechnology Co., Ltd. The pCAMBIA1300-GFP vector was linearized using restriction endonucleases. The reaction mixture consisted of: 2 μL circular vector (1000 ng), 1 μL restriction endonuclease, 2 μL Cutsmart, and ddH2O to a final volume of 15 μL; reaction conditions: 37 ℃ for 4 hours. Homologous recombination of the target gene fragment with the linearized vector was then performed. The reaction mixture consisted of: 1 μL linear vector (200 ng), 1 μL target gene fragment (120 ng), 5 μL 2×Basic Assembly Mix, and ddH2O to a final volume of 10 μL; reaction conditions: 50 ℃ for 15 minutes. Finally, the... AcPAE8 The CDS fragment of the gene, with the stop codon removed, was inserted into the vector to construct GFP- AcPAE8 Recombinant plasmids (e.g.) Figure 6 ).

[0040] 2.2.3 Subcellular localization analysis of AcPAE8 protein The plasmid with the correct sequencing was transformed into Agrobacterium EHA105 strain, and single colonies were picked and activated to OD. 600 The fluorescence intensity was around 0.6 nm. After 3 hours of dark treatment at room temperature, the mixture was injected into the lower epidermal cells of tobacco leaves at a 1:1 (v / v) ratio. The cells were then placed in the dark for 12 hours, followed by 24 hours of light incubation. Fluorescence signals were then observed using a laser confocal 3D scanner (Leica TCSSP8, Germany). Figure 7 The results showed that the AcPAE8 protein plays a role in the endoplasmic reticulum.

[0041] Example 3: AcPAE8 Construction and genetic transformation of gene overexpression vectors and editing vectors 3.1 AcPAE8 Construction of gene overexpression vectors AcPAE8 The construction method of the gene overexpression vector is as described in 2.2.2. Primer sequences are shown in SEQ ID NO.7 and 8. The target fragment was ligated into the overexpression vector POE-3Flag-DN using the Clon Express Ultra One Step Cloning Kit (Novizan, China), thus constructing the OE-3Flag-DN overexpression vector. AcPAE8 The recombinant plasmid was then transformed into E. coli DH5α competent cells, positive clones were screened and sequenced to confirm the successful construction of the overexpression vector.

[0042] 3.2 AcPAE8 Gene editing vector construction Use the CRISPR RGEN tool (http: / / www.rgenome.net / ?tdsourcetag=s_pcqq_aiomsg) to filter. AcPAE8 Gene-specific sgRNAs (sequences shown in SEQ ID NO. 9 and 10) were cloned into a CRISPR / Cas9 vector using a ligation-by-cleaning restriction enzyme method (as shown in Table 1) (primer sequences shown in SEQ ID NO. 11 and 12), generating Cas9- AcePAE8 The vector was edited and then transformed into E. coli DH5α competent cells. Single clones were then selected for sequencing.

[0043] Table 1. Reaction system and conditions for constructing gene editing vectors

[0044] Reaction conditions: Step 1: Reaction at 37 ℃ for 30 min; Step 2: 15 cycles of reaction (37 ℃ for 2 min, 10 ℃ for 3 min, 20 ℃ for 5 min), followed by incubation at 37 ℃ for 2 h.

[0045] 3.3 Genetic transformation of kiwifruit 3.3.1 Agrobacterium-mediated transformation The recombinant plasmids with correct sequencing results as described in 3.1 and 3.2 were transformed into Agrobacterium tumefaciens EHA105. The specific procedure was as follows: Agrobacterium competent cells were removed from -80℃ and immediately placed on ice. After thawing, 10 μL of plasmid was added, gently mixed, and incubated on ice for 5 min, in liquid nitrogen for 5 min, in a 37℃ water bath for 5 min, and on ice for 5 min. Then, 700 μL of antibiotic-free LB broth was added to a clean bench, and the cells were incubated at 28℃ with shaking at 220 rpm for 3 h. After centrifugation at 6000 rpm for 1 min, 100 μL of the revived bacterial culture was taken from the clean bench and plated on LB agar plates (containing 50 mg / L kanamycin and 50 mg / L rifampin). The plates were then incubated upside down at 28℃ for 48 h. Single colonies were then picked for sequencing.

[0046] 3.3.2 Genetic transformation of kiwifruit The method for transgenic kiwifruit is as follows: Agrobacterium tumefaciens containing successfully transformed expression vectors and editing vectors were cultured on a large scale, the bacterial population was collected, and a suspension was prepared using MS medium (MS + acetylsalicylic acid 1.0 mg / L). -1 ) Adjusting OD 600 The pH value is 0.4~0.6, and it is stored in the dark for later use; take an appropriate amount of kiwi leaves, cut them into small pieces, mix them with the suspension, and shake for 12-15 minutes; use clean, sterile filter paper to dry the infected kiwi leaves, and inoculate them into co-medium (MS + 6-BA 2.0 mg·L⁻¹). -1 + NAA 0.5 mg·L -1 + TDZ 1.0 mg·L -1 + Acetyleugenone 1.0 mg / L -1 + 30g / L of sucrose -1 + 8g / L agar -1 The leaves were co-cultured on MS medium (pH 5.8) under dark conditions for 72 hours. After 72 hours, the leaves were rinsed with sterile water and air-dried, and then inoculated onto selection medium (MS + 6-BA 2.0 mg·L⁻¹). -1 + NAA 0.5 mg·L -1 + TDZ 1.0 mg·L -1 + 30g / L of sucrose -1 + 8g / L agar -1 + Termetidine 100 mg·L-1 In a medium containing G418 (pH 5.8), the leaf condition was observed. The medium was changed every two weeks for a total of four times until positive plants were selected for testing.

[0047] 3.3.3 Molecular identification of transgenic plants Total DNA and total RNA were extracted from overexpressing positive plants, and OE- was detected by conventional PCR (primer sequences as shown in SEQ ID NO. 13 and 14) and real-time quantitative PCR (qRT-PCR; primer sequences as shown in SEQ ID NO. 15 and 16). AcPAE8 The insertion status of the carrier and AcPAE8 Gene expression levels, results as follows Figure 10 As shown, three overexpression lines (OE-) were successfully constructed. AcPAE8 #2, OE- AcPAE8 #9、OE- AcPAE8 #12); Real-time quantitative PCR (qRT-PCR) results Figure 11 As shown, AcPAE8 Gene expression levels increased by approximately 5-fold, 15-fold, and 15.4-fold, respectively.

[0048] Total DNA was extracted from gene-edited positive plants and amplified by PCR. AcPAE8 DNA sequencing was performed on the target regions of the gene (primer sequences such as SEQ ID NO. 17 and 18), and the editing status was analyzed using the online tool Synthego. Results are as follows: Figure 12 As shown: One gene-edited line (pae8#10) was screened, which had varying degrees of fragment deletion in the sgRNA2 region.

[0049] 3.3.4 Phenotypic Identification of Transgenic Plants Following the vitamin C content determination method in section 1.1, the vitamin C content of all transgenic lines was measured. The results are as follows: Figure 13 As shown, the vitamin C content in the three overexpression lines increased significantly, by 119%, 166%, and 90.7%, respectively. Conversely, the vitamin C content in the gene-edited lines decreased significantly, by 40% compared to the wild-type control.

Claims

1. A gene for kiwifruit pectin acetylesterase 8 AcPAE8 Its characteristics are, The AcPAE8 The open reading frame nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The claim 1 AcPAE8 The protein encoded by the gene is characterized by, The amino acid sequence of the protein is shown in SEQ ID NO.

2.

3. A device comprising the contents of claim 1 AcPAE8 A gene recombination vector, characterized in that, The recombinant vector uses a plant expression vector as its backbone. AcPAE8 It is constructed from the multiple cloning site of the gene insertion vector.

4. The recombinant vector according to claim 3, characterized in that, The plant expression vector is the POE-3Flag-DN vector.

5. A host cell containing the recombinant vector of claim 3 or 4, characterized in that, The host cell is an Agrobacterium cell.

6. A method for increasing the vitamin C content of kiwifruit, characterized in that, The recombinant vector described in claim 3 is introduced into kiwifruit leaves or callus tissue, so that... AcPAE8 Gene overexpression.

7. A method for reducing the vitamin C content of kiwifruit, characterized in that, Using gene editing technology to edit the kiwifruit genome AcPAE8 Genes, making the AcPAE8 The gene is missing or its expression is reduced. AcPAE8 The open reading frame nucleotide sequence of the gene is shown in SEQ ID NO.

1.

8. The method according to claim 7, characterized in that, The sgRNA sequences are shown in SEQ ID NO. 9 and 10.

9. The claim 1 AcPAE8 The application of genes in kiwifruit breeding is characterized by, This is used to cultivate new kiwifruit varieties with increased vitamin C content.

10. The application according to claim 9, characterized in that, The kiwifruit variety mentioned applies to either Chinese kiwifruit or delicious kiwifruit.

Images