A phospholipase D (PLD) gene that can regulate the morphological development of bamboo fungus and its application

Abstract

This invention relates to a phospholipase D (PLD) gene that can regulate the morphological development of bamboo fungus and its application. The encoding nucleotide sequences of the phospholipase D gene are shown in SEQ ID NO:1 and SEQ ID NO:2, respectively. The protein encoded by SEQ ID NO:1 is the isoenzyme PLD-1, and its amino acid sequence is shown in SEQ ID NO:3; the protein encoded by SEQ ID NO:2 is the isoenzyme PLD-2, and its amino acid sequence is shown in SEQ ID NO:4. The application involves treating bamboo fungus bulbs with a phospholipase D inhibitor, which can inhibit the opening of the bulb cap. The full-length PLD gene for regulating the morphological development of bamboo fungus provided by this invention is mainly used to regulate the morphological development of bamboo fungus through the use of a PLD inhibitor (hexanal). The PLD inhibitor (hexanal) of this invention can effectively inhibit the morphological development of bamboo fungus bulbs after harvest, improve the preservation of bamboo fungus, and reduce transportation losses.

Description

Technical Field

[0001] This invention belongs to the field of regulating the morphological development of bamboo fungus, specifically involving a phospholipase D (PLD) gene that can regulate the morphological development of bamboo fungus and its application. Background Technology

[0002] Edible fungi are characterized by high water content and rapid respiration, resulting in short post-harvest shelf life and high loss rates. Among commercially available edible fungi, the morphological development of bamboo fungus during post-harvest preservation is particularly typical: harvested bamboo fungus spheres rapidly elongate their stipes and unfold into mature bamboo fungus under suitable temperature and humidity conditions, with the fruiting body elongating approximately 3-5 times within hours. Because mature bamboo fungus has a slender, hollow, network-like stipe, it is highly susceptible to breakage and spoilage, leading to losses during preservation and transportation. In contrast, bamboo fungus spheres are easy to transport and have a longer shelf life, making them a viable alternative for storage and transportation. Therefore, controlling the development of bamboo fungus spheres into mature bamboo fungus is crucial for post-harvest preservation management and preservation technology research.

[0003] Stipe elongation is driven by the swelling of stipe cells due to water absorption. Because the cell wall is a rigid structure, it needs to be dynamically hydrolyzed and synthesized during this process to continuously adapt to the increase in cell volume. If the cell wall remodeling process can be effectively blocked, cell volume will be locked, effectively inhibiting stipe elongation and thus delaying the maturation, senescence, and quality decline of the fruiting body. Currently, the phosphatidylinositol signaling pathway has been found to play a crucial role in the morphological development of bamboo fungus. Based on this, further elucidating the regulatory mechanism of bamboo fungus fruiting body morphological development is key to overcoming the bottleneck in postharvest preservation technology for bamboo fungus. Summary of the Invention

[0004] This invention proposes a phospholipase D (PLD) gene that can regulate the morphological development of bamboo fungus and its application.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A gene encoding phospholipase D that can regulate the morphological development of bamboo fungus is disclosed. The nucleotide sequences encoding phospholipase D are shown in SEQ ID NO:1 and SEQ ID NO:2, respectively.

[0007] Preferably, the protein encoded by SEQ ID NO:1 is isoenzyme PLD-1, and its amino acid sequence is shown in SEQ ID NO:3; the protein encoded by SEQ ID NO:2 is isoenzyme PLD-2, and its amino acid sequence is shown in SEQ ID NO:4.

[0008] Expression cassettes, recombinant vectors, transgenic cells, or recombinant bacteria containing the encoded gene.

[0009] Preferably, the application involves treating bamboo fungus balls with a phospholipase D inhibitor, which can inhibit the opening of the bamboo fungus balls.

[0010] Preferably, the inhibitor is hexanal.

[0011] More preferably, the treatment method is fumigation, and the acetaldehyde concentration is 20-30 μL / L.

[0012] The beneficial effects of this invention are:

[0013] 1. In this invention, the changes in gene expression in bamboo fungus were analyzed by transcriptomics. It was found that the gene expression of PLD in DM was 6.49 times and 6.81 times that of CK and DP, respectively. This indicates that the upregulation of PLD expression is accompanied by the morphological development of bamboo fungus and has a regulatory effect on the morphological development of bamboo fungus.

[0014] 2. In this invention, the morphological development of bamboo fungus was best inhibited by hexanal treatment after harvest, with a cap opening rate of 25%, compared to 83% in the control group. This result indicates that inhibiting PLD can suppress the morphological development of bamboo fungus.

[0015] 3. In this invention, transcriptomic analysis of CK, DHP, and DM showed that the gene expression level of PLD in DHP was only 2.45 times that in CK, and compared with DM, its expression was significantly downregulated by 0.40 times. This indicates that hexanal treatment inhibits PLD expression at the transcriptional level. Attached Figure Description

[0016] Figure 1 Morphological development of refrigerated bamboo fungus balls during induction treatment (A: Sample image; B: Mature opening rate; C: Two states of refrigerated bamboo fungus balls after induction treatment);

[0017] Figure 2 Changes in genes of the phosphatidylinositol signaling pathway in refrigerated bamboo fungus balls after induction treatment;

[0018] Figure 3 Morphological development of bamboo fungus after 8 hours of fumigation with different concentrations of hexanal (A: sample image; B: mature opening rate);

[0019] Figure 4 Morphological development of bamboo fungus after fumigation with 25 μL / L hexanal for different times (A: Sample image; B: Mature opening rate; C: Two morphologies of bamboo fungus after hexanal treatment and control induction treatment for 3 days).

[0020] Figure 5 Changes in PLD and phosphatidylinositol signaling pathway genes in bamboo fungus balls after hexanal treatment. Detailed Implementation

[0021] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0023] Example 1: Phospholipase D regulation of morphological development of cold-stored bamboo fungus pellets after induction treatment

[0024] I. Materials and Methods

[0025] 1. Materials and Reagents

[0026] Fresh bamboo fungus balls were harvested from Kunming, Yunnan Province. They were transported to the laboratory at low temperatures (4-10℃) immediately after being picked from the farmland. Bamboo fungus balls of uniform size and similar shape were selected for the experiment.

[0027] The RNA extraction kit (TRIzol® Reagent) was from Thermo Fisher Scientific, USA; the library preparation kit (Truseq™ RNA Sample Prep Kit) and sequencing kits (HiSeq X Reagent Kits, NovaSeq Reagent Kits) were from Illumina, USA; and the library recovery kit (Agencourt AMPure XP) was from Beckman Coulter, USA.

[0028] 2. Instruments and Equipment

[0029] Wonbio-96 multi-sample cryogenic grinder (Shanghai Wanbai Biotechnology Co., Ltd.); Agilent 2100 bioanalyzer (Agilent Technologies, USA); HiSeq Xten and NovaSeq 6000 sequencers (Illumina, USA); Nanodrop 2000 micro-volume UV spectrophotometer (Thermo Fisher Scientific, USA).

[0030] 3. Methods

[0031] 3.1 Sample Preparation

[0032] Eight samples of bamboo fungus fruiting bodies were used as one biological replicate, for a total of six biological replicates. The samples were stored for three days under suitable temperature and humidity conditions (26±2℃ and 95±3% relative humidity), and the morphological development of bamboo fungus fruiting bodies at different time points was photographed and compared. Bamboo fungus fruiting bodies were collected at 0 days, bamboo fungus fruiting bodies that had stopped developing were collected at 3 days, and mature bamboo fungus fruiting bodies were collected.

[0033] 3.2 Calculation of Mature Umbrella Opening Rate

[0034] We consider the normal maturation of bamboo fungus to be characterized by stipe elongation and cap opening. To more intuitively compare bamboo fungus balls that are developing normally with those that have stopped developing normally, we use the cap opening rate on day 3 to describe the development of the bamboo fungus balls. The formula for calculating the cap opening rate is:

[0035]

[0036] 3.3 Transcriptome Analysis

[0037] 3.3.1 RNA Extraction

[0038] Six to eight fruiting bodies at each stage of the following were collected: refrigerated bamboo fungus balls, bamboo fungus balls that had stopped developing after induction treatment, and mature bamboo fungus after induction treatment. After washing and wiping, the outer shell, polysaccharide layer, and spore layer of the fruiting bodies were peeled off. The stipe and cap portions were ground into powder in liquid nitrogen. Powders from the same stage were combined, with three replicates for each sample. Total RNA was extracted according to the TRI-zol assay kit instructions. 1 mL of MJzol Reagent was added to the sample, vortexed thoroughly, allowed to stand, and centrifuged to obtain the supernatant. 200 μL of chloroform was added to the supernatant, vortexed, allowed to stand, and centrifuged to obtain an aqueous phase containing RNA. 1.2 times the volume of anhydrous ethanol was added to the aqueous phase, mixed, and RNA was extracted using magnetic beads and a centrifuge column. Genomic DNA was removed using DNase I (TaKara). RNA degradation and contamination were monitored using a 1% agarose gel. The concentration and purity of the extracted RNA were then detected using Nanodrop 2000 and Agilent 2100.

[0039] 3.3.2 Sequencing and Assembly

[0040] mRNA was isolated from total RNA using Oligo(dT) magnetic beads and polyA for AT base pairing. Fragmentation buffer was added to randomly break the mRNA into fragments of approximately 300 bp. The mRNA was then reverse-engineered to synthesize cDNA. A cDNA library was constructed using the Illumina Truseq™ RNA sample prep Kit, followed by sequencing using an Illumina NovaSeq 6000. Before assembly, the raw data was filtered using fastp (https: / / github.com / OpenGene / fastp) to remove low-quality reads (reads containing indeterminate "N" bases or reads shorter than 30 bp), resulting in clean data. Since there was no reference genome for *Dictyophora indica*, de novo assembly was performed using Trinity (https: / / github.com / trinityrnaseq / trinityrnaseq / wiki), and the assembly results were optimized, filtered, and evaluated using TransRate (http: / / hibberdlab.com / transrate / ), CD-HIT (http: / / weizhongli-lab.org / cd-hit / ), and BUSCO (Benchmarking Universal Single-Copy Orthologs, http: / / busco.ezlab.org).

[0041] 3.3.3 Notes on Unigene

[0042] The longest assembled transcript was defined as Unigene, and then further functional annotations were obtained by comparing it with six major databases: DIAMOND and NCBI_NR (NCBI Non-Redundant Protein Library, ftp: / / ftp.ncbi.nlm.nih.gov / blast / db / ), Swiss-Prot (http: / / web.expasy.org / docs / swiss-prot_guideline.html), Pfam (http: / / pfam.xfam.org / ), COG (Clusters of Orthologous Groups of proteins, http: / / www.ncbi.nlm.nih.gov / COG / ), GO (Gene Ontology, http: / / www.geneontology.org), and KEGG (Kyoto Encyclopedia of Genes and Genomes, http: / / www.genome.jp / kegg / ).

[0043] 3.3.4 Quantitative comparison of Unigene

[0044] To identify differentially expressed genes among three states of bamboo fungus—refrigerated, immature, and mature—the mapping read counts of each assembled Unigene were calculated using RSEM (RNA-Seq by Expectation-Maximization) and normalized to TPM (Transcripts Per Million Reads) for gene expression analysis. Then, DEGseq2 was used to statistically analyze the raw counts obtained from RSEM, screening for differentially expressed genes with P < 0.05 and |log2FC| ≥ 2 between the two samples.

[0045] 3.3.5 Bioinformatics Analysis of Differentially Expressed Genes (DEGs)

[0046] Using the GO database, differentially expressed genes were classified according to their involvement in biological processes, cellular components, and molecular functions. GO enrichment analysis was performed on these differentially expressed genes using the software Goatools (https: / / github.com / tanghaibao / GOatools). Using the KEGG database, differentially expressed genes were classified according to their involvement in pathways or their functions. Genes from the gene set were displayed on a KEGG pathway map, showing the KEGG annotated pathways they participated in.

[0047] II. Results and Analysis

[0048] 1. Morphological development and maturity opening rate of refrigerated bamboo fungus balls

[0049] Peach-shaped bamboo fungus balls (CK) that had undergone 2 days of cold storage and transportation (4-10℃) were subjected to induction treatment (26±2℃, relative humidity 95±3%), and the opening rate with induction time was statistically analyzed. Figure 1 A). The results showed that the umbrella opening rate gradually increased with the extension of the induction treatment time. Figure 1 B). After 3 days of induction treatment, 81.25% of the bamboo fungus bulbs completed maturation and opened their caps to become mature bamboo fungus (DM), while 18.75% of the bamboo fungus bulbs failed to mature and opened their caps, remaining peach-shaped (DP). Figure 1 C). Due to the continued extension of the induction time, the quality of DP deteriorates and mold may occur. Therefore, it is considered that no further morphological development will occur.

[0050] 2. Phospholipase D may be a key enzyme regulating the morphological development of bamboo fungus.

[0051] Transcriptomics analysis results showed ( Figure 2 Six differentially expressed genes were annotated in the phosphatidylinositol signaling pathway, encoding PLD, CDP-diacylglycerol-inositol 3-phosphatidyltransferase (CDIPT, EC: 2.7.8.11), myotubule-associated protein 6 / 7 / 8 (MTMR6 / 7 / 8, EC: 3.1.3.64), phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase (PTEN), PI4K, and PLC. Figure 3 Compared with CK, PLD was significantly downregulated in DP and significantly upregulated in DM. Downregulation of PLD in DP led to inhibition of PE hydrolysis and reduced PA production; while downregulation of PI4K reduced the conversion rate of PI to PI(4)P. This suggests that the phosphatidylinositol signaling pathway in DP, due to PLD downregulation, resulted in insufficient PA accumulation, failing to activate downstream kinases and signaling molecules such as DG and IP(3,4,5)3. Upregulation of PLD in DM promoted PE hydrolysis, increased PA accumulation, and upregulation of CDIPT accelerated PI production. Simultaneously, upregulation of PI4K and PLC implied an accelerated production rate of the phosphatidylinositol pathway final products DG and I(1,4,5)P3. These results indicate that the phosphatidylinositol signaling pathway in DM is activated by PLD upregulation, triggering downstream cascade reactions and regulating the morphological development of *Dictyophora indica* at the gene level.

[0052] Example 2: Application of phospholipase D (PLD) (Effect of hexanal treatment on postharvest morphological development of bamboo fungus)

[0053] I. Materials and Methods

[0054] 1. Materials and Instruments

[0055] Hexanal analytical grade was purchased from the Exploration Platform.

[0056] 2. Instruments and Equipment

[0057] Same as Example 1

[0058] 3. Methods

[0059] 3.1 Sample Preparation

[0060] Experiment 2: Bamboo fungus balls were treated with three different concentrations of hexanal: 25 μL / L, 50 μL / L, or 100 μL / L. A blank control (CK) was used without any treatment. Each treatment group had three biological replicates. After cleaning the outer surface of the bamboo fungus balls, they were immediately sealed and placed in a 13L plastic box. The plastic box contained petri dishes and filter paper containing hexanal (25 μL / L, 50 μL / L, or 100 μL / L) to promote rapid evaporation; complete evaporation took approximately 2 hours. The bamboo fungus balls were exposed to hexanal vapor at room temperature for 8 hours. Untreated bamboo fungus balls were also stored in the plastic box for 8 hours. After 8 hours of exposure, the control and treated bamboo fungus balls were removed and stored under suitable temperature and humidity conditions (26±2℃ and 95±3% relative humidity), and photographs were taken for recording the results.

[0061] Experiment 3: In Experiment 3, a suitable hexanal concentration of 25 μL / L was determined. To screen for suitable hexanal fumigation times, 4 h, 6 h, and 8 h were selected as fumigation treatment times, with no treatment serving as a blank control (CK). After fumigation treatment, the procedures were the same as in Experiment 1.

[0062] The optimal concentration of hexanal and fumigation time were determined to be 25 μL / L and 6 h. Transcriptomic analysis will then be performed on the bamboo fungus balls fumigated with 25 μL / L hexanal for 6 h and the control group bamboo fungus balls. The main groupings are as follows: control bamboo fungus balls (CK group); hexanal-treated group (DHP group) which failed to open after induction treatment; and mature bamboo fungus control group (DM group) which opened after induction treatment.

[0063] 3.2 Calculation of Mature Umbrella Opening Rate

[0064] Same as Example 1

[0065] 3.3 Transcriptome Analysis

[0066] Same as Example 1

[0067] II. Results and Analysis

[0068] 1. The effect of treating bamboo fungus balls with a phospholipase D inhibitor (hexanal) on the opening rate of the bamboo fungus balls was investigated. The results showed that, compared with the untreated group, fumigation with different concentrations of hexanal (25 μL / L, 50 μL / L, and 100 μL / L, 8 h) significantly inhibited the opening of the bamboo fungus balls, exhibiting a dose-response effect. The opening rate decreased from 83% in the control group to 17-46% in the hexanal-treated group. Figure 3(A and B). After repeated trials, we found that 25 μL / L hexanal significantly inhibited the opening of bamboo fungus seeds. Therefore, we explored the effect of different fumigation times with hexanal at this concentration on the opening rate of bamboo fungus seeds. The results showed that fumigation times (4h, 6h, 8h) at a concentration of 25 μL / L hexanal had similar inhibitory effects on the opening rate of bamboo fungus seeds, with opening rates of 33.33%, 25%, and 29.17%, respectively. Among them, fumigation with 25 μL / L hexanal for 6h showed the best inhibitory effect, with an opening rate of 25% (…). Figure 4 (A and B).

[0069] 2. For example Figure 5 As shown, the gene expression levels of PLD in DM and DHP were significantly upregulated by 6.49-fold and 2.45-fold respectively compared to CK, while those in DHP were significantly reduced by 0.40-fold compared to DM. This indicates that hexanal treatment inhibits PLD expression at the transcriptional level. Furthermore, the gene expression level of phospholipase C (PLC), a key enzyme in the phosphatidylinositol signaling pathway, was significantly downregulated by 0.18-fold in DHP compared to DM, indicating that the phosphatidylinositol signaling pathway was not activated in DHP. In summary, hexanal treatment inhibited PLD expression at the transcriptional level and reduced the accumulation of phosphatidic acid, thus failing to activate the phosphatidylinositol signaling pathway and consequently failing to complete signal transduction, thereby inhibiting the morphological development of bamboo fungus. This suggests that PLD plays an important regulatory role in the morphological development of bamboo fungus.

[0070] Example 3: Full-length sequencing of the phospholipase D (PLD) gene in bamboo fungus

[0071] I. Bamboo Fungus Genome Sequencing Procedure

[0072] 1. DNA extraction and detection from bamboo fungus

[0073] To avoid contamination and reduced genome assembly, the outer shell, polysaccharide layer, and spore layer of the bamboo fungus were removed, and the stipe and veil were used as the primary DNA extraction samples. Genomic DNA was extracted from the samples using the GP1 extraction method, and the purity and integrity of the DNA were then detected by agarose gel electrophoresis, followed by quantification using Qubit.

[0074] 2. Library Construction

[0075] (1) SMRT Bell libraries were constructed using the SMRT Bell™ Template kit (version 2.0). DNA samples that passed electrophoresis were fragmented into target fragments of the required size for library construction using Covaris g-TUBE. After DNA damage repair and end repair, hairpin adapters were ligated to both ends of the DNA fragments using DNA adhesive enzyme. The DNA fragments were purified using AMPurePB magnetic beads. Fragments of specific sizes were screened using Blue Pippin. The concentration of the SMRT Bell library was screened using AMPurePB magnetic beads. After DNA damage repair, the SMRT Bell library was purified again using AMPurePB magnetic beads. The constructed library was quantified using Qubit concentration, and the insert size was detected using an Agilent 2100. Finally, sequencing was performed using the PacBio platform.

[0076] (2) DNA samples that passed electrophoresis were randomly fragmented into segments of approximately 350 bp using a Covaris ultrasonic disruptor. The processed DNA fragments were then... Ultra TM The DNA Library Prep Kit for Illumina (NEB, USA) completes the entire library preparation process, including end repair, A-tailing, sequencing adapter addition, purification, and PCR amplification. After library construction, preliminary quantification is performed using Qubit 2.0 to dilute the library to 2 ng / ul. Subsequently, the insert size is detected using an Agilent 2100. Once the insert size meets expectations, the effective concentration of the library is accurately quantified using Q-PCR to ensure library quality.

[0077] 4. Sequencing and Assembly

[0078] After the libraries passed the library inspection, different libraries were sequenced using PacBio Sequel II / PacBio Sequel IIe and Illumina NovaSeq PE150 according to their effective concentration and target data volume. Single-molecule real-time (SMRT) sequencing was performed. Low-quality reads were filtered using SMRT Link v8.0, and the filtered reads were assembled using Falcon software to generate sequences.

[0079] 5. Genomic composition analysis

[0080] After assembly, the composition of the sample genome was analyzed, including the prediction of coding genes, non-coding RNAs, and repetitive sequences. Since there was no reference genome for *Dictyophora indica*, de novo *Augustus* prediction was performed. GeneMarkS software (http: / / topaz.gatech.edu / ) was used for coding gene prediction, and Diamond software was used to compare the amino acid sequences of the target species with the NR database. The genes of the target species were combined with their corresponding functional annotation information to obtain the annotation results.

[0081] 6. Gene sequencing results showed that phospholipase D (PLD) in bamboo fungus has two isoenzymes (PLD-1 and PLD-2), with encoding gene sequence lengths of 2565 and 4641, respectively. The nucleotide sequence encoding PLD-1 obtained after sequencing is shown in SEQ ID NO. 1;

[0082] The nucleotide sequence encoding PLD-2 obtained after sequencing is shown in SEQ ID NO.2;

[0083] The PLD-1 gene encodes a protein with the following amino acid sequence (SEQ ID NO.3);

[0084] The PLD-2 gene encodes a protein with the following amino acid sequence (SEQ ID NO.4).

[0085] The above embodiments are merely preferred technical solutions of the present invention, but the scope of protection of the present invention is not limited thereto. The embodiments and features in the embodiments of this application can be arbitrarily combined with each other without conflict. The scope of protection of the present invention should be defined by the technical solutions described in the claims, including equivalent substitutions of the technical features described in the claims. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention are also covered within the scope of protection of the present invention.

Claims

1. The application of hexanal in regulating the morphological development of bamboo fungus, characterized in that, The application involves treating bamboo fungus balls with hexanal to inhibit their opening. The treatment method is fumigation, with an acetaldehyde concentration of 25 μL / L. The nucleotide sequences encoding the phospholipase D gene are shown in SEQ ID NO:1 and SEQ ID NO:2, respectively. The protein encoded by SEQ ID NO:1 is isoenzyme PLD-1, and its amino acid sequence is shown in SEQ ID NO:

3. The protein encoded by SEQ ID NO:2 is isoenzyme PLD-2, and its amino acid sequence is shown in SEQ ID NO:4.

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