Method for screening key genes of anti-monochamus alternatus clonal pinus massoniana based on genomics as well as obtained genes and application thereof

Abstract

The invention discloses a method for screening an anti-monochamus alternatus clonal pinus massoniana key gene based on genomics as well as an obtained gene and application thereof, and relates to the technical field of plant genetic engineering. The invention discloses a genomics-based method for screening an anti-monochamus alternatus clonal pinus massoniana key gene. The method comprises the following steps: screening a pinus massoniana anti-monochamus alternatus differential gene through a transcriptome; the masson pine anti-monochamus alternatus differential metabolites are screened out through the metabolome; according to a metabolome differential metabolite analysis result and in combination with a transcriptome differential gene analysis result, joint analysis is performed to construct an interaction network diagram, the pinus massoniana anti-monochamus alternatus key gene PmFLS is screened out, an overexpression strain of the pinus massoniana anti-monochamus alternatus key gene PmFLS is constructed, and the pinus massoniana anti-monochamus alternatus key gene PmFLS is obtained. Finally, a transgenic arabidopsis thaliana transgenic line with increased leaf area, advanced bolting time, increased total flavone content and increased FLS enzyme activity is obtained, and powerful support is provided for gene improvement of masson pine.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and more specifically, relates to a method for screening key genes of a Masson pine clone resistant to Monochamus alternatus based on genomics, and the obtained genes and applications thereof. Background Art

[0002] Masson pine (Pinus massoniana) is an important native tree species unique to China's subtropical regions. It has fast growth, strong adaptability and good forest restoration ability. At the same time, the comprehensive utilization rate of the whole tree of Masson pine is high, which gives it unique ecological functions and economic value. Therefore, it occupies a prominent position in my country's forestry construction and plays an indispensable role. However, during the growth and development of Masson pine, it faces a variety of biological stresses, especially the pine beetle (Monochamus alternatus).

[0003] As the main trunk-boring pest of Masson pine, the larvae of Monochamus alternatus bore into the trunk, destroying the host's conductive tissue, thereby hindering the normal transportation of water and nutrients, and eventually causing the host to die. At the same time, Monochamus alternatus is also a major vector of pine wood nematode disease, playing a key role in carrying, spreading and assisting pathogens to invade the host. When the adult Monochamus alternatus eats young branches to obtain nutrients, the pine wood nematodes (Bursaphelenchus xylophilus) carried in the body invade the host through the wounds, causing the host plant to gradually weaken and die. Monochamus alternatus has caused inestimable losses to my country and has been identified as a major quarantine forestry pest. At present, the methods for preventing and controlling Monochamus alternatus include silvicultural prevention and control, chemical prevention and control, physical prevention and control, and biological prevention and control, but these prevention and control methods have certain limitations due to the characteristics of strong concealment and long cycle time of the damage caused by Monochamus alternatus. Breeding insect-resistant varieties to improve the defense capabilities of host plants is an effective and sustainable method that can fundamentally solve the disease problem.

[0004] Since plants grow by fixing themselves to the ground through their roots, they cannot escape the continuous attack of insects. During the long process of evolution, plants have formed complex recognition and defense mechanisms, namely constitutive defense and induced defense, to avoid or reduce the degree of damage caused by herbivorous insects to themselves. The defense mechanism of plants is closely related to gene expression and metabolites. Therefore, understanding and effectively utilizing defense-related genes and metabolites is of great significance for Masson pine to resist Monochamus alternatus. The transcriptomics and metabolomics dual-omics analysis data can make up for the problems caused by factors such as data loss in single-omics data analysis, and achieve mutual verification at the transcriptional and metabolic levels, so as to be able to understand the plant defense response mechanism more deeply and comprehensively at the molecular and cellular levels. Previous studies have mainly focused on the induced defense mechanism, and there are few reports on the constitutive defense mechanism of host plants against insects.

[0005] Flavonol synthase (FLS) plays a crucial role in the flavonol synthesis pathway of plants. It is an enzyme that can catalyze the synthesis of flavonols, which are a type of flavonoid compound with various biological activities. The function of FLS in plants is mainly reflected in regulating the synthesis of flavonic acids and flavonols. It can catalyze the synthesis of flavonic acids, which are the precursors of flavonols. In the catalytic reaction, FLS uses phenylalanine and rutinose as substrates to generate flavonic acids. This reaction is an oxidation reaction that requires the use of reduced glutathione as a cofactor. FLS is a single oxidase with a molecular weight of about 42 kDa and has two conserved subunits, one of which is the catalytic subunit and the other is the oxidase subunit. Flavonols in host plants can stimulate or repel the feeding and oviposition behaviors of longhorn beetles. Therefore, studying the FLS gene in Masson pine can also provide new ideas for cultivating highly resistant Masson pine. Summary of the Invention

[0006] Aiming at the above problems existing in the prior art, the technical problems to be solved by the present invention are as follows: to provide a method for screening key genes of Masson pine clones resistant to Monochamus alternatus based on multi-omics; to provide the key gene PmFLS of Masson pine resistant to Monochamus alternatus; to provide the expression protein of the key gene PmFLS of Masson pine resistant to Monochamus alternatus; and to provide the application of the key gene PmFLS of Masson pine resistant to Monochamus alternatus for regulating plant growth, regulating the total flavonoid content in plants, and regulating the activity of flavonol synthase in plants.

[0007] To solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0008] Method for screening key genes of resistant clones of Masson pine against Monochamus alternatus based on genomics, which includes screening differential genes of Masson pine resistant to Monochamus alternatus through transcriptome; screening differential metabolites of Masson pine resistant to Monochamus alternatus through metabolome; according to the analysis results of metabolome differential metabolites, combining with the analysis results of transcriptome differential genes, conducting joint analysis to construct an interaction network diagram, and screening out the key gene PmFLS of Masson pine resistant to Monochamus alternatus. The steps include:

[0009] 1) Respectively extract total RNA from needles of highly resistant clones and susceptible clones of Masson pine, and synthesize cDNA;

[0010] 2) Construct a cDNA library for cDNA library normalization, second-generation sequencing and assembly to obtain reference sequences;

[0011] 3) Align the clean reads obtained from the second-generation sequencing to the reference sequences, and after obtaining the alignment results, conduct differential expression analysis to screen out differential genes in the transcriptome of Masson pine resistant to Monochamus alternatus;

[0012] 4) Extract metabolic samples from needle extracts of highly resistant clones and susceptible clones of Masson pine, and then conduct liquid chromatography-tandem mass spectrometry analysis;

[0013] 5) Based on the self-built database of Guangzhou GeneDenovo Biotechnology Co., Ltd. and the public database of metabolite information, adopt the multiple reaction monitoring mode, conduct quantitative analysis on metabolic samples according to the daughter ion pairs, and conduct qualitative analysis on metabolic samples according to the parent ion, daughter ion, retention time, declustering voltage and collision energy;

[0014] 6) Combine the VIP value of the OPLS-DA in the multivariate statistical analysis and the P value of the T test in the univariate statistical analysis to screen out differential metabolites in the metabolome; the threshold for significant differences is: VIP≥1 in the OPLS-DA model and T-testP<0.05;

[0015] 7) Conduct joint analysis on the metabolome differential metabolite data in step 6) and the transcriptome differential gene data in step 3), jointly conduct KEGG pathway database mapping, and screen out the key gene PmFLS of Masson pine resistant to Monochamus alternatus.

[0016] Step 3) is to use the Bowtie2 software to align the clean reads obtained from the Illumina NovaSeq 6000 high-throughput sequencing platform to the reference sequences; normalize to TPM as an index for measuring the transcriptional expression level, and screen differential expression genes with the criteria of |log2FC|>log22 and FDR<0.05.

[0017] The key gene PmFLS of Masson pine resistant to Monochamus alternatus has a nucleotide sequence as shown in SEQ ID NO. 1.

[0018] The expressed protein of the key gene PmFLS of Pinus massoniana against Monochamus alternatus has an amino acid sequence as shown in SEQ ID NO. 2.

[0019] Application of the PmFLS gene of Pinus massoniana in regulating plant growth.

[0020] Application of the PmFLS gene of Pinus massoniana in regulating plant leaf area.

[0021] Application of the PmFLS gene of Pinus massoniana in regulating plant bolting time.

[0022] Application of the PmFLS gene of Pinus massoniana in regulating the total flavonoid content in plants.

[0023] Application of the PmFLS gene of Pinus massoniana in regulating the activity of flavonol synthase in plants.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0025] 1) The present invention screens out the differential genes of Pinus massoniana against Monochamus alternatus through transcriptome; screens out the differential metabolites of Pinus massoniana against Monochamus alternatus through metabolome; according to the analysis results of metabolome differential metabolites, combined with the analysis results of transcriptome differential genes, a co-analysis is carried out to construct an interaction network diagram, and finally the key gene PmFLS of Pinus massoniana against Monochamus alternatus is screened out, and its nucleotide sequence is as shown in SEQ ID NO. 1.

[0026] 2) The present invention constructs an overexpression line of the key gene PmFLS of Pinus massoniana against Monochamus alternatus. The results show that the leaves of transgenic Arabidopsis are much larger than those of the wild type, and the transgenic lines have bolted when Arabidopsis grows to 25 days, while the wild type has not bolted.

[0027] 3) The present invention constructs an overexpression line of the key gene PmFLS of Pinus massoniana against Monochamus alternatus. The results show that the total flavonoid content in the transgenic Arabidopsis lines is significantly higher than that of the wild type.

[0028] 4) The present invention constructs an overexpression line of the key gene PmFLS of Pinus massoniana against Monochamus alternatus. The results show that the FLS enzyme activity in the transgenic Arabidopsis lines is significantly higher than that of the wild type. Description of the Drawings

[0029] Figure 1 It is an electrophoresis diagram for detecting total RNA of Pinus massoniana by 1% agarose gel electrophoresis (lanes 1-5: MK27; lanes 6-10: MK30; lanes 11-15: MK94);

[0030] Figure 2 It is a principal component analysis diagram of the transcriptomes of different resistant MK27, MK30, and MK94 of Pinus massoniana;

[0031] Figure 3 is the heat map of differentially expressed genes;

[0032] Figure 4 is the KEGG enrichment analysis map of up-regulated (A) and down-regulated (B) differentially expressed genes;

[0033] Figure 5 is the principal component analysis map of the metabolomes of different resistant MK27, MK30, and MK94 of Pinus massoniana;

[0034] Figure 6 is the heat map of differential metabolites;

[0035] Figure 7 is the species analysis map of differential metabolites;

[0036] Figure 8 is the KEGG enrichment analysis map of differential metabolites;

[0037] Figure 9 is the omics joint analysis map based on the KEGG pathway;

[0038] Figure 10 is the agarose gel electrophoresis map of cloning the PmFLS gene;

[0039] Figure 11 is the relative expression level map of the PmFLS gene in different tissues of Pinus massoniana;

[0040] Figure 12 is the gDNA agarose gel electrophoresis detection map of the WT and OE-PmFLS lines;

[0041] Figure 13 is the relative expression level map of the PmFLS gene in the WT and OE-PmFLS lines;

[0042] Figure 14 is the phenotypic observation map of the transgenic and wild-type lines after being transferred to soil for 21 days;

[0043] Figure 15 is the bolting state observation map of the transgenic and wild-type lines after being transferred to soil for 25 days;

[0044] Figure 16 is the total flavonoid content map in the WT and L2, L3, L8 lines;

[0045] Figure 17 is the statistical chart of enzyme activity in the WT and L2, L3, L8 lines. Detailed implementation methods

[0046] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described below in conjunction with specific embodiments. In the following embodiments, unless otherwise specified in detail, the technical means used are all conventional means well-known to those skilled in the art. For the molecular biology experimental methods not specifically described, they can be referred to the conventional methods in the art or carried out according to the kits and product manuals.

[0047] The 10-year-old highly resistant and susceptible clonal plants of Pinus massoniana used in the present invention were all planted in the Washan State-owned Forest Farm of the Quanjiao County Forestry Bureau, Chuzhou City, Anhui Province, China. For plant materials, the tender parts of plants with normal growth and development were preferentially selected. Plants with consistent growth vigor were selected from them as the source of a biological replicate sample to avoid differences caused by excessive individual differences. Sample tissues were collected from the corresponding same positions (such as the same direction of light) of different plants (from one source of biological replicate) and mixed. During collection, the samples were wiped or rinsed clean with RNase-free water, and then the surface of the samples was blotted dry with absorbent paper. They were quickly placed into 50 mL centrifuge tubes, frozen in liquid nitrogen for 5 - 10 minutes, transported briefly with dry ice, and stored in a -80 °C refrigerator for subsequent processing.

[0048] Example 1 Obtaining Differentially Expressed Genes by De Novo Transcriptome Sequencing

[0049] 1. Total RNA Extraction and cDNA Obtaining

[0050] Using the FastPure Universal Plant Total RNA Isolation Kit (RC411 - 01, Vazyme Biotech, Nanjing, China), the total RNA of highly resistant and susceptible needles of Pinus massoniana was extracted respectively by the silica column purification technology method. The purity of the RNA (OD 260 / 280 ratio) was detected using a NanoDrop 2000 spectrophotometer (Thermo, USA), and the detected concentration was between 260 ng / μL - 360 ng / μL, and OD 260 / 280 , OD 260 / 230 was between 1.8 - 2.1. The integrity of the total RNA and the presence of DNA contamination were preliminarily evaluated by 1.5% agarose gel electrophoresis. The results were as Figure 1 shown, and the bands were clear. Finally, a Qubit 2.0 Fluorometer (Thermo, USA) and an Agilent 2100 bioanalyzer (Agilent, USA) were used to accurately detect the RNA concentration and integrity respectively for the construction of high-throughput sequencing libraries.

[0051] The specific operation steps for RNA extraction are as follows:

[0052] 1) Take an appropriate amount of plant tissue ground in liquid nitrogen and immediately add 600 μL of Buffer PSL. Vortex vigorously for 30 seconds to fully mix the sample with the lysis buffer. Centrifuge at 12,000 rpm for 5 minutes and immediately proceed with the following steps.

[0053] 2) Transfer approximately 500 μL of the supernatant to FastPure gDNA-Filter Columns III and centrifuge at 12,000 rpm for 30 seconds to collect the filtrate.

[0054] 3) Add anhydrous ethanol with a volume 0.5 times that of the filtrate (about 250 μL, adjusted according to the actual situation of the supernatant) to the collection tube and mix by oscillation for 15 seconds.

[0055] 4) Transfer the above mixture to FastPure RNA Columns V and centrifuge at 12,000 rpm for 30 seconds. Discard the filtrate.

[0056] 5) Add 700 μL of Buffer RWA to FastPure RNA Columns V and centrifuge at 12,000 rpm for 30 seconds. Discard the filtrate.

[0057] 6) Add 500 μL of Buffer RWB (previously mixed with 48 mL of anhydrous ethanol) to FastPure RNA Columns V and centrifuge at 12,000 rpm for 30 seconds. Discard the filtrate.

[0058] 7) Repeat step 6.

[0059] 8) Place FastPure RNA Columns V back into the collection tube and centrifuge at 12,000 rpm for 2 minutes.

[0060] 9) Transfer FastPure RNA Columns V to a new 1.5 mL RNase-free Collection Tube. Pipette 30 - 100 μL of RNase-free ddH2O (preheated at 65 °C) onto the center of the adsorption column membrane and centrifuge at 12,000 rpm for 1 minute to obtain the RNA solution.

[0061] The cDNA used in subsequent gene cloning and quantification is obtained by reverse transcribing the extracted Pinus massoniana needle RNA using the Hifair ® Ⅲ 1st Strand cDNA Synthesis SuperMix for qPCR reverse transcription kit from Shanghai Yisheng Biotechnology Co., Ltd. The specific operation is carried out according to the instructions.

[0062] 2. Library construction and sequencing

[0063] After extracting total RNA, eukaryotic mRNAs with polyA tails were enriched with Oligo(dT) beads, and prokaryotic mRNAs were enriched by removing rRNA using the Ribo-ZeroTM Magnetic Kit (Epicentre, Madison, WI, USA). Then, the enriched mRNA was fragmented with fragmentation buffer as a template, and the first-strand cDNA was synthesized in the M-MuLV reverse transcriptase system with random oligonucleotide primers. The second-strand cDNA was mainly synthesized using dNTPs as raw materials in the DNA polymerase I system by degrading the RNA strand with RNaseH. The cDNA fragments were purified, the ends were repaired, A bases were added, and they were ligated to the Illumina sequencing adapter using the QiaQuick PCR Extraction Kit (Qiagen, Venlo, the Netherlands). cDNA of about 200 bp was selected by agarose gel electrophoresis, amplified by PCR, and sequenced using the Illumina NovaSeq 6000 of Gene Denovo Biotechnology Co. (Guangzhou, China). Reads were further filtered using fastp (version 0.18.0), mainly including removing reads containing adapters, removing reads containing more than 10% unknown nucleotides (N), and removing low-quality reads containing more than 50% low-quality bases (q value ≤ 20) to obtain clean reads. Subsequently, the Trinity software was used to assemble the clean reads to obtain the reference sequence for subsequent analysis.

[0064] 3. Screening and analysis of differentially expressed genes

[0065] The clean reads obtained from the Illumina NovaSeq 6000 high-throughput sequencing platform were mapped to the reference sequence using Bowtie 2 software, and then normalized to TPM as an index for measuring the transcriptional expression level. Differentially expressed genes were screened with the criteria of |log2FC| > log22 and FDR < 0.05. Based on the reference sequence to which the clean reads were mapped, DESeq2 software was used for differential expression analysis, and the screening criteria for differentially expressed genes were set as the false discovery rate (FDR) parameter less than 0.05 and the absolute fold change ≥ 2. Principal component analysis (PCA) was carried out using R (http: / / www.r-project.org / ). The Gene Ontology (GO) database (http: / / www.geneontology.org / ) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http: / / www.genome.jp / kegg / ) were used to perform enrichment analysis on the differential genes (with FDR ≤ 0.05 as the threshold).

[0066] The results are as Figure 2 shown. PCA analysis was performed on the gene expression profiles of all samples. The percentage of the interpretation values of the first principal component PC1 and the second principal component PC2 in the transcriptome were 71.2% and 24% respectively, indicating high repeatability within groups and large differences between groups of samples.

[0067] The results are as Figure 3 shown. The 3114 differentially expressed genes identified were presented in the form of a heatmap. There were significant changes in transcripts among different resistant clones, indicating obvious differences in gene expression profiles, which were related to the differences in resistance.

[0068] The results are as Figure 4 shown. The differentially expressed genes upregulated in the comparison between susceptible and highly resistant clones were mainly enriched in the MAPK signaling pathway - plant, plant - pathogen interaction, plant hormone signal transduction, and thiamine metabolism pathway. The downregulated differentially expressed genes were mainly related to plant - pathogen interaction and the MAPK signaling pathway - plant. This indicates that plant - pathogen interaction and the MAPK signaling pathway - plant are related to the resistance of Pinus massoniana to Monochamus alternatus. In addition, there were common enrichment pathways in the downregulated genes, such as flavonoid biosynthesis, flavone and flavonol biosynthesis, and phenylpropanoid biosynthesis, which were related to the resistance of Pinus massoniana to Monochamus alternatus.

[0069] Example 2 Obtaining differential metabolites by broad-target metabolomics

[0070] 1. Metabolite extraction

[0071] After freeze-drying the samples, they were ground for 1.5 minutes at 30 Hz using a mixer mill (MM 400, Retsch GmbH, Düsseldorf, Germany) with zirconia beads. 100 mg of the sample was taken, resuspended in pre-cooled 80% methanol and 0.1% formic acid, incubated on ice for 5 minutes, centrifuged at 4 °C at 15,000 g for 20 minutes. A portion of the supernatant was diluted with LC-MS grade water to a final concentration containing 53% methanol. The sample was transferred to a fresh Eppendorf tube, centrifuged at 4 °C at 15,000 g for 20 minutes, and finally the supernatant was injected into the LC-MS / MS system for analysis.

[0072] 2. LC-MS Analysis

[0073] Liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis was performed at GeneDenovo Biotechnology Co., Ltd. (Guangzhou, China) using an ExionLC™ AD system (SCIEX) and a QTRAP® 6500+ mass spectrometer (SCIEX). The sample was passed through an Xselect HSS T3 (2.1×150 mm, 2.5 μL) column at a flow rate of 0.4 mL / min, and a 20-minute linear gradient was used for injection in positive / negative polarity modes. The mobile phases were solvent A (0.1% formic acid-water) and solvent B (0.1% formic acid-acetonitrile). The solvent gradient was set as follows: 2% B for 2 minutes; 2 - 100% B for 15 minutes; 100% B for 17 minutes; 100 - 2% B for 17.1 minutes; 2% B for 20 minutes. The QTRAP® 6500+ mass spectrometer was operated in positive polarity mode with a curtain gas of 35 psi, the collision gas set to medium, the ion spray voltage of 5500 V, the temperature of 550 °C, the ion source gas 1 of 60, and the ion source gas 2 of 60. The QTRAP® 6500+ mass spectrometer was operated in negative polarity mode with a curtain gas of 35 psi, the collision gas set to medium, the ion spray voltage of -4500 V, the temperature of 550 °C, the ion source gas 1 of 60, and the ion source gas 2 of 60.

[0074] The results are as Figure 5 shown. PCA analysis generally reflects the overall metabolic differences between groups of samples and the degree of variability within samples of the same group. The two-dimensional PCA plot shows that the three biological replicates of each clone cluster together, indicating a high repeatability among the same clones. The metabolic profiles between the two clones show a separation trend, indicating significant differences in metabolites between groups.

[0075] 3. Qualitative and Quantitative Analysis of Metabolites

[0076] The detection of experimental samples was carried out in multiple reaction monitoring mode based on the self-built database of Guangzhou Gene Denovo Biotechnology Co., Ltd. Daughter ions (Q3) were used for metabolite quantification, and parent ions (Q1), daughter ions (Q3), retention time (RT), declustering potential (DP), and collision energy (CE) were used for metabolite qualitative analysis. The data files generated by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) were processed using SCIEX OS version 1.4 to integrate and correct peaks. The main parameter settings were as follows: the minimum peak height was 500, the signal-to-noise ratio was 5, and the Gaussian smoothing width was 1. The peak area of each chromatographic peak represented the relative content of the corresponding substance, and finally, the integrated data of all chromatographic peak areas were exported to obtain the qualitative and quantitative results of metabolites.

[0077] 4. Screening of differential metabolites

[0078] Combined with the VIP value of multivariate statistical analysis OPLS-DA and the P value of univariate statistical analysis T-test to screen differential metabolites between different groups. The threshold for significant differences was: VIP≥1 in the OPLS-DA model and T-test P<0.05. Heat map visualization and classification were performed on all the above differential metabolites, and through KEGG enrichment analysis, the key metabolic pathways where the metabolites were located were found.

[0079] The results showed that the clustering heat map visualization of 39 differential metabolites demonstrated significant differences in metabolites between susceptible and highly resistant clones ( Figure 6 ); the 39 differential metabolites could be divided into 13 categories, mainly including flavonoids, amino acids and their derivatives, lipids, etc. Among them, the flavonoid compounds accounted for about 53.8%, with the largest proportion ( Figure 7 ); the top 20 KEGG terms showed that the differential metabolites were mainly enriched in the biosynthesis of flavonoids and flavonols, the biosynthesis of phenylpropanoids, and the biosynthetic pathways of secondary metabolites. Among them, the participation in the biosynthesis of flavonoids and flavonols had the most significant difference, indicating that this pathway was the key metabolic pathway for the defense of Pinus massoniana against Monochamus alternatus ( Figure 8 ).

[0080] Example 3 Transcriptome and metabolome combined analysis

[0081] Based on the results of differential metabolite analysis and combined with the results of differential gene analysis in the transcriptome, a pathway functional model was used to jointly analyze the two sets of data on gene expression levels and metabolite abundances, identify the KEGG pathways that were significantly enriched in common between the transcriptome and metabolome, search for associated genes and metabolites with significant changes in expression in the common pathways, and screen out key candidate genes and metabolites. The Pearson correlation coefficient between gene expression levels and metabolite abundances was calculated, and according to the magnitude of the correlation coefficient, a correlation network diagram was used to present the correlation between related differential genes and flavonoid metabolites.

[0082] The results are as Figure 9 shown. There were a total of 16 commonly enriched pathways in the KEGG enrichment analysis. Among them, 7 differentially expressed genes and 1 differential metabolite were involved in flavonoid biosynthesis, 2 differentially expressed genes and 4 differential metabolites were related to the biosynthesis of flavones and flavonols, 2 differentially expressed genes and 1 differential metabolite were enriched in the phenylpropanoid metabolic pathway, and 2 differentially expressed genes and 1 differential metabolite were involved in the biosynthesis of phenylalanine, tyrosine, and tryptophan.

[0083] In summary, a total of 14 key genes related to the flavonoid synthesis pathway were screened by combining the transcriptome and metabolome. One of the genes encoding flavonol synthase, Unigene0050303, was named PmFLS.

[0084] Example 4

[0085] 1. Total RNA extraction and reverse transcription

[0086] The FastPure Universal Plant Total RNA Isolation Kit (RC411 - 01, Vazyme Biotech, Nanjing, China) was used to extract the total RNA of highly resistant and susceptible Masson pine needles respectively by the silica column purification technology method. 1.5% agarose gel electrophoresis was used to preliminarily evaluate the integrity of the total RNA, and the Qubit 2.0 Fluorometer (Thermo, USA) and Agilent 2100 bioanalyzer (Agilent, USA) were used to accurately detect the RNA concentration and integrity respectively. The extracted Masson pine needle RNA was reverse transcribed into cDNA using the Hifair ® Ⅲ 1st Strand cDNA Synthesis SuperMix for qPCR reverse transcription kit, and reverse transcription was carried out according to the recommended system in the instruction manual.

[0087] 2. Cloning of the PmFLS gene

[0088] Specific primers were designed according to the identified PmFLS gene sequence. The cloning primer sequences of the PmFLS gene are as follows:

[0089] PmFLS-F: 5'-ATGAATGATCGCATAGCAGG-3',

[0090] PmFLS-R: 5'-CTACTGGGGCAGCTTGTT-3'.

[0091] The PCR reaction system was: 2.5 μL of cDNA; 2.5 μL each of primers F / R; 25 μL of 2× high-fidelity enzyme; 17.5 μL of ddH2O.

[0092] The PCR reaction conditions were: reaction at 98 °C for 1 min; denaturation at 95 °C for 15 s, annealing at 58 °C for 5 s, extension at 72 °C for 15 s, for 35 cycles; and then extension at 72 °C for 3.5 min.

[0093] The PCR product was mixed with 6× loading buffer and then loaded into the gel, and electrophoresed together with the Marker at 180 V for 15 min. After electrophoresis, the gel was placed in a gel imaging system for observation ( Figure 10 ). The product purification kit (DC301) from Novizan was used for product purification to obtain the target gene.

[0094] Finally, the ORF of the PmFLS gene obtained by sequencing was 1125 bp, and its nucleotide sequence was as shown in SEQ ID NO.1, and the sequence of its expressed protein was as shown in SEQ ID NO.2.

[0095] 3. Tissue quantitative analysis of the PmFLS gene in Pinus massoniana

[0096] Specific primers for real-time fluorescence quantitative PCR were designed, and the internal reference gene was TUA of Pinus massoniana. The primer sequences are as follows:

[0097] PmFLS-q-F: 5´-GTGCCAGGACTTCAAGTGTTCAATG-3´,

[0098] PmFLS-q-R: 5´-TTGCTCACCACGCTCCTATGC-3´;

[0099] PmTUA-q-F: 5´-CAAACTTGGTCCCGTATCCTC-3´,

[0100] PmTUA-q-R: 5´-CACAGAAAGCTGCTCATGGTAA-3´.

[0101] Using the 2× Real-Time PCR amplification pre-solution from Shanghai Yisheng Biotechnology Co., Ltd., the experiment was carried out on the StepOnePlus Real-Time PCR System. The reaction solution was prepared on ice, adding 10 μL of Hieff UNICON Universal Blue qPCR SYBR Green Master Mix, 2 μL of template cDNA from each tissue, 0.4 μL of each primer (concentration 10 μM), and 7.2 μL of sterile and enzyme-free ultrapure water. The samples were pipetted onto a 96-well plate one by one in an ice box, and after centrifugation for 1 min, the program was started. The program settings were: pre-denaturation at 95 °C for 2 min; 40 cycles of denaturation at 95 °C for 10 s and annealing at 60 °C for 30 s; finally, the melting curve was collected using the default program of the instrument.

[0102] The results are as Figure 11 shown. PmFLS was expressed in all organs of Pinus massoniana, with the most prominent expression in needles, while the expression in stems and roots was relatively low. In summary, PmFLS plays a key role in the needles of Pinus massoniana.

[0103] Example 5

[0104] 1. Construction of pCAMBIA1302-PmFLS vector

[0105] Primers were designed using the nucleotide sequence of the purified PmFLS gene from Pinus massoniana and the sequences of the used vector and restriction sites. The primer sequences are as follows:

[0106] 1302-PmFLS-F:

[0107] 5´-ACTCTTGACCATGGTAGATCTATGAATGATCGCATAGCAGGC-3´,

[0108] 1302-PmFLS-R:

[0109] 5´-AAGTTCTTCTCCTTTACTAGTCTGGGGCAGCTTGTTGATCT-3´;

[0110] The pCAMBIA1302 vector plasmid was linearized using the restriction endonucleases NcoI and SpeⅠ. The specific steps were:

[0111] The restriction enzyme reaction system is as follows: 1 μg of pCAMBIA1302 vector plasmid, 5 μL of 10×Quick Cut Buffer, 1 μL of NcoI; 1 μL of SpeⅠ, supplemented with ddH2O to 50 μL. Then, it is incubated in a PCR instrument at 37°C for 30 min, 85°C for 20 min, and 4°C ∞. The PCR product is transformed into competent Escherichia coli DH5α; it is cultured overnight on an LB solid plate containing kanamycin (50 mg / L). Single colonies are picked and cultured in an LB liquid medium containing the corresponding resistance for 5 - 6 h. The colonies are identified by PCR and sequencing. The plasmid with correct sequencing is transferred into Agrobacterium for Arabidopsis transformation.

[0112] 2. Cultivation of Arabidopsis thaliana

[0113] 1) Disinfection of Arabidopsis thaliana seeds: Take an appropriate amount of wild-type Arabidopsis thaliana seeds and put them into a sterilized EP tube, add

[0114] an appropriate amount of 75% ethanol, shake and disinfect for 30 s and then aspirate; add the same amount of 15% sodium hypochlorite, disinfect for 30 s and then aspirate; add the same amount of deionized water, shake and wash and then aspirate and place the seeds in a new EP tube; repeat the water washing four times.

[0115] 2) Cultivation of Arabidopsis thaliana: Place the disinfected seeds in a suspension, and evenly sow them into 1 / 2 MS medium using a pipette; seal the medium well and place it in a 4°C refrigerator for vernalization for 3 days; place the vernalized medium in a 23°C constant temperature incubator for 7 days; when the Arabidopsis thaliana seedlings grow 2 true leaves, transplant the Arabidopsis thaliana into the previously prepared substrate culture soil and place it in a light incubator for cultivation. The parameters set for the light incubator are: 23°C constant temperature, light intensity 12000 Lux, humidity 75%, and light time 16 h / d; use it for transformation before Arabidopsis thaliana enters the full flowering stage.

[0116] 3. Transformation of Agrobacterium

[0117] Transform the recombinant vector into competent Agrobacterium GV3101, pick a single colony and inoculate it into an LB liquid medium, shake and culture at 28°C for 2 d, centrifuge at 5000 rpm for 5 min, collect the bacteria, and suspend them with a 5% sucrose solution; use full-length primers for colony PCR to screen for positive clones, and store the positive clones at 4°C for later use. Healthy Arabidopsis thaliana to be planted grows to flowering. When the OD of the positive clone detected by PCR 600 reaches 0.8, Arabidopsis thaliana floral organ immersion transformation is carried out. The specific steps are as follows:

[0118] 1) Before immersion, add Silwet L-77 with a concentration of 0.05% (500 μL / L) to the Agrobacterium suspension solution, and shake out the foam;

[0119] 2) Immerse the aerial parts of Arabidopsis thaliana in the Agrobacterium suspension solution for 30 s, gently shaking during this period;

[0120] 3) Place the immersed Arabidopsis thaliana flat in a tray, cover it with plastic wrap for moisture retention, and seal it with tin foil to avoid light for 24 h;

[0121] 4) Uncover the tin foil and culture under normal conditions. Stop watering when the seeds are mature;

[0122] 5) Collect the seeds of Arabidopsis thaliana as T1 generation seeds, and dry them at 37 °C for one week and then store.

[0123] 4. Screening of T1 generation positive plants of Arabidopsis thaliana transformed with PmFLS gene and obtaining of T3 generation transgenic seedlings

[0124] 1) PCR detection of T1 generation transgenic Arabidopsis thaliana

[0125] The seeds of Arabidopsis thaliana transformed by Agrobacterium infection were disinfected and sown in 1 / 2 MS medium containing hygromycin (50 mg / L). After vernalization for 3 d and germination, they were transferred to a light culture room to observe the growth changes of the plants. Due to the influence of hygromycin, the seedlings of non-transgenic plants and control group plants gradually turned yellow and withered, while the transgenic seedlings grew normally. About 10 d later, all the transgenic plants and control group plants turned yellow and died. After screening with hygromycin, 15 plants were obtained and named L1 to L8 respectively. After extracting the genomic DNA of these 8 transgenic Arabidopsis thaliana lines with a kit (using the gDNA extraction kit DC104 of Novoprotein), PCR detection was carried out. The PCR system (50 μL) was: 19 μL ddH2O, 25 μL 2×Taq PCR MASTER Mix, 2 μL PmFLS-R (10 μM), 2 μL 1302-PmFLS-R (10 μM), 2 μL DNA. The PCR program was: 95 °C for 5 min; 95 °C for 15 s, 58 °C for 15 s, 72 °C for 1 min, 35 cycles; 72 °C for 5 min; store at 4 °C.

[0126] The results were as Figure 12 shown. Bands were obtained for L1 to L8 respectively, indicating that the gene PmFLS was successfully transferred.

[0127] 2) Obtaining of T3 generation transgenic Arabidopsis thaliana

[0128] After disinfecting the above-mentioned T1 generation seeds, they were sown into 1 / 2 MS medium containing hygromycin (50 mg / L). After vernalization for 3 days, they were placed in a suitable environment for cultivation (the cultivation conditions were the same as above). Then they were transplanted into substrate soil, and the harvested seeds were T2 generation transgenic Arabidopsis seeds. The obtained T2 generation transgenic Arabidopsis seeds were cultivated according to the above steps to screen T3 generation homozygous transgenic Arabidopsis seeds and plants. After transplanting into substrate soil, they were placed in a light incubator for cultivation (the set parameters were the same as above).

[0129] 3) Determination of the expression level of PmFLS gene in T3 generation transgenic Arabidopsis

[0130] Total RNA of transgenic and wild-type Arabidopsis leaves was extracted and reverse transcribed into cDNA. Using cDNA as a template, RT-qPCR detection was performed with PmFLS-q-F / R.

[0131] The results were as Figure 13 shown. The relative expression level of PmFLS gene in transgenic Arabidopsis was significantly higher than that in the wild type. It indicated that the PmFLS gene had been successfully transferred into Arabidopsis.

[0132] Example 6

[0133] 1. Phenotype observation of transgenic Arabidopsis

[0134] Three overexpression lines L2, L3, L8 with the highest relative expression levels and wild type WT were selected for phenotype observation. The leaf sizes of wild-type and transgenic Arabidopsis transplanted into soil and grown for 21 days were observed and counted.

[0135] The results were as Figure 14 and Figure 15 shown. The leaves of transgenic Arabidopsis were much larger than those of the wild type ( Figure 14 ). Continuing the observation, it was found that when Arabidopsis grew to 25 days old, the transgenic lines had bolted, while the wild type had not bolted ( Figure 15 ).

[0136] 2. Total flavonoid content of transgenic and wild-type Arabidopsis

[0137] Take about 0.1 g of fresh sample (if the moisture is sufficient, the sample sampling mass can be increased). Using a total flavonoid kit (purchased from Shanghai Huding Biological Company), the total flavonoid content was determined by the NaNO2-Al(NO3)3-NaOH colorimetric method, that is, in an alkaline nitrite solution, flavonoid compounds form a red complex with aluminum ions that has a characteristic absorption peak at 510 nm. The absorbance value of the reaction product at 510 nm was measured to calculate the total flavonoid content in the sample. The specific operation was carried out according to the instructions.

[0138] Result calculation:

[0139] Standard curve equation: y = 1.6277x - 0.0049, where x is the standard concentration (mg / mL) and y is ΔA.

[0140] Total flavonoid content (mg / g dry weight) = [((ΔA + 0.0049) ÷ 1.6277 × V1)] ÷ (V1 ÷ V × W) × D = 0.6 × (ΔA + 0.0049) ÷ W × V × D.

[0141] Where: V is the volume of the extraction solution, 1.5 mL; V1 is the volume of the sample in the reaction, 50 μL = 0.05 mL; W is the sample mass, g; D is the dilution factor, which is 1 if not diluted.

[0142] The results are as Figure 16 shown, and the total flavonoid content in the transgenic Arabidopsis thaliana lines is significantly higher than that in the wild type.

[0143] 3. FLS enzyme activity of transgenic and wild-type Arabidopsis thaliana

[0144] The enzyme activity was measured using a plant flavonol synthase (FLS) ELISA kit (DB191X-Pt) (purchased from Shanghai Huding Biological Company). The specific operation was carried out according to the instructions. The calculation method is as follows: with the concentration of the standard as the abscissa and the OD value as the ordinate, draw a standard curve on the coordinate paper. According to the OD value of the sample, find the corresponding concentration from the standard curve; then multiply by the dilution factor; or calculate the curve regression equation of the standard curve using the concentration and OD value of the standard, substitute the OD value of the sample into the equation, calculate the sample concentration, and then multiply by the dilution factor to obtain the actual concentration of the sample.

[0145] The results are as Figure 17 shown, and the FLS enzyme activity in the transgenic Arabidopsis thaliana lines is significantly higher than that in the wild type.

[0146] The above description is illustrative rather than restrictive for the present invention. Those of ordinary skill in the art understand that many modifications, variations or equivalents can be made without departing from the spirit and scope defined by the appended claims, and all of them will fall within the protection scope of the present invention.

Claims

1. A method for screening key genes of clonal masson pines resistant to Monochamus alternatus based on genomics, characterized in that, Differentially expressed genes related to the resistance of Pinus massoniana to Monochamus alternatus were screened by transcriptome analysis; differentially accumulated metabolites related to the resistance of Pinus massoniana to Monochamus alternatus were screened by metabolome analysis; based on the results of metabolome differentially accumulated metabolite analysis and combined with the results of transcriptome differentially expressed gene analysis, a co-analysis was performed to construct an interaction network diagram, and the key gene PmFLS related to the resistance of Pinus massoniana to Monochamus alternatus was screened out.

2. The method according to claim 1, wherein Differentially expressed genes were obtained by de novo transcriptome sequencing. The steps include: 1) Total RNA was extracted from the needles of highly resistant and susceptible clones of Pinus massoniana, respectively, and cDNA was synthesized. 2) A cDNA library was constructed for cDNA library normalization, second-generation sequencing and assembly to obtain reference sequences. 3) The clean reads obtained from the second-generation sequencing were aligned to the reference sequences. After obtaining the alignment results, differential expression analysis was performed to screen out the differentially expressed genes in the transcriptome of Pinus massoniana resistant to Monochamus alternatus. 4) Needle extracts from highly resistant and susceptible clones of Pinus massoniana were used for metabolite sample extraction, followed by liquid chromatography-tandem mass spectrometry analysis. 5) Based on the in-house database established by Guangzhou GeneDenovo Biotechnology Co., Ltd. and the public database of metabolite information, the multiple reaction monitoring mode was adopted. Quantitative analysis of metabolite samples was performed according to the product ions, and qualitative analysis of metabolite samples was performed according to the precursor ions, product ions, retention time, declustering voltage and collision energy. 6) Combining the VIP value of the multivariate statistical analysis OPLS-DA and the P value of the univariate statistical analysis T-test, differentially accumulated metabolites in the metabolome were screened out. The threshold for significant differences was: VIP≥1 in the OPLS-DA model and T-test P<0.

05. 7) The metabolome differentially accumulated metabolite data in step 6) and the transcriptome differentially expressed gene data in step 3) were jointly analyzed, and a KEGG pathway database mapping was performed together to screen out the key gene PmFLS related to the resistance of Pinus massoniana to Monochamus alternatus.

3. The method according to claim 2, characterized in that, In step 3), the Bowtie2 software was used to align the clean reads obtained from the Illumina NovaSeq 6000 high-throughput sequencing platform to the reference sequences; the transcripts were normalized to TPM as an index for measuring transcriptional expression levels, and differentially expressed genes were screened using the criteria of |log2FC|>log22 and FDR<0.

05.

4. The key gene PmFLS related to the resistance of Pinus massoniana to Monochamus alternatus screened by the method according to any one of claims 1-3, the nucleotide sequence of which is shown in SEQ ID NO.

1.

5. The expressed protein of the key gene PmFLS related to the resistance of Pinus massoniana to Monochamus alternatus according to claim 4, the amino acid sequence of which is shown in SEQ ID NO.

2.

6. Use of the PmFLS gene of Pinus massoniana according to claim 4 in regulating plant growth.

7. Use of the PmFLS gene of Pinus massoniana according to claim 4 in regulating the leaf area of plants.

8. Use of the PmFLS gene of Pinus massoniana according to claim 4 in regulating the bolting time of plants.

9. Use of the PmFLS gene of Pinus massoniana according to claim 4 in regulating the total flavonoid content in plants.

10. Use of the PmFLS gene of Pinus massoniana according to claim 4 in regulating the flavonol synthase activity in plants.

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