A zhennan macaw rot-resistant marker gene PzPLR1 and its amplification primers and applications

Abstract

The invention provides a macaw rot-resistant marker gene PzPLR1 The invention relates to amplification primers and applications thereof, and belongs to the field of agricultural biotechnology. PzPLR1 The nucleotide sequence is shown in SEQ ID NO: 1. The 100% rot-resistant marker gene of Phoebe zhennan PzPLR1 It is differentially expressed in different tissues and organs, with relatively high expression in roots and stems, and is positively correlated with the level of corrosion resistance. PzPLR1 The expression level of the gene can accurately reflect the decay resistance of Phoebe zhennan tissues and organs. Compared with traditional methods that rely on plant morphology or physiological indicators, the present invention has higher accuracy, stronger scientificity and good repeatability in judging the decay resistance of tree tissues and organs.

Description

Technical Field

[0001] The invention belongs to the field of agricultural biotechnology, and specifically relates to a rot-resistant marker gene of Phoebe zhennan PzPLR1 Amplification primers and their applications. Background Art

[0002] Phoebe nanmu (scientific name: Phoebe zhennan ) is also known as Nanshu and Zhennan, and is a species of the genus Nan in the Lauraceae family. It is a large tree that can reach 30 meters when mature. Its wood is hard and expensive, and is mostly used in shipbuilding and palaces. Zhennan wood is not easy to deform or crack, is easy to process, and is resistant to decay. It has a gorgeous and varied texture and is currently recognized by the domestic wood industry as the best tree species of "golden nanmu".

[0003] In recent years, forestry experts and scholars believe that the excellent decay resistance of Phoebe zhennan is mainly related to the production of secondary metabolites of Phoebe zhennan, which mainly include flavonoids, terpenes and isoquinoline alkaloids. The expression of some key genes of secondary metabolites makes Phoebe zhennan wood have extremely high decay resistance, which can protect the roots, stems and other tissues and organs of Phoebe zhennan from being eaten by pests all year round.

[0004] In production practice, if the selected materials are corroded by harmful bacteria, resulting in wood rot, it will cause huge economic losses. Therefore, in the evaluation of the quality of Phoebe zhennan wood, the selection of wood materials with strong individual corrosion resistance is crucial for the selection of subsequent processing methods, and it is necessary to select materials with strong corrosion resistance more quickly and conveniently. To determine whether the wood of a tree has strong corrosion resistance, or to detect whether the wood is corroded, the traditional method is usually to cut the wood and observe whether there are traces of bacterial corrosion or holes bitten by pests inside the wood. However, the results are often affected by the cutting site, environment and human factors and are not accurate, and it is not possible to detect surviving Phoebe zhennan plants, so the scope of application is poor. In recent years, with the deepening of molecular biology research, many important regulatory factors related to plant corrosion resistance have been discovered, but marker genes that can determine the corrosion resistance of plant tissues and organs have not yet been found in Phoebe zhennan. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a marker gene for the decay resistance of Phoebe zhennan PzPLR1 , which can efficiently and accurately determine the decay resistance of tree tissues and organs, and provide scientific and effective detection targets for determining the decay resistance of trees.

[0006] The invention provides a macaw rot-resistant marker gene PzPLR1 , the nucleotide sequence is shown in SEQ ID NO: 1.

[0007] The present invention provides a method for detecting the rot-resistant marker gene of Phoebe zhennan by real-time fluorescence quantitative PCR. PzPLR1Primers were used, including a forward primer having a nucleotide sequence as shown in SEQ ID NO:2 and a reverse primer having a nucleotide sequence as shown in SEQ ID NO:3.

[0008] The invention provides a kit for detecting the corrosion resistance of Phoebe zhennan, comprising a qPCR reaction reagent and the primers.

[0009] Preferably, it also includes an internal reference gene primer;

[0010] The internal reference gene primers include a forward primer whose nucleotide sequence is shown in SEQ ID NO: 5 and a reverse primer whose nucleotide sequence is shown in SEQ ID NO: 6.

[0011] Preferably, it also includes a reverse transcription reagent and / or an RNA extraction reagent.

[0012] The invention provides application of the primer or the kit in judging the corrosion resistance of Phoebe zhennan.

[0013] The present invention provides a method for PzPLR1 The method for judging the corrosion resistance of Phoebe zhennan tissues and organs comprises the following steps:

[0014] Detecting genes in the sample to be tested using the primers PzPLR1 The expression level of the gene in the sample to be tested PzPLR1 The expression level and the reference sample level are used to determine the corrosion resistance of the sample to be tested:

[0015] If the gene in the sample to be tested PzPLR1 If the expression level of the test sample is not significantly different from that in the reference sample, the corrosion resistance of the tissue and organ of the test sample is the same or similar to that of the reference sample;

[0016] If the gene in the sample to be tested PzPLR1 If the expression level of is significantly lower than that of the reference sample, the corrosion resistance of the sample to be tested is lower than that of the reference sample;

[0017] If the gene in the sample to be tested PzPLR1 If the expression level of the test sample is significantly higher than that of the reference sample, the corrosion resistance of the test sample is higher than that of the reference sample.

[0018] Preferably, the reference sample includes one or more of the root, stem, leaf, flower and fruit tissue organs of Phoebe zhennan.

[0019] Preferably, the detection of genes in the sample to be tested PzPLR1 The reaction system for the expression level of 10×SuperRealPreMix Plus was 10µL, 100 μM forward primer 0.8µL, 100 μM reverse primer 0.8µL, cDNA 1µL and ddH 2O7.4µL.

[0020] Preferably, the detection of genes in the sample to be tested PzPLR1 The reaction program for expression level was pre-denaturation at 95 °C for 2 min; 94 °C for 5 s, 60 °C for 30 s, and 39 cycles; the reaction program for melting curve analysis was 65-95 °C with an increase of 0.5 °C every 5 s.

[0021] The invention provides a macaw rot-resistant marker gene PzPLR1 The nucleotide sequence is shown in SEQ ID NO: 1. The present invention uses different tissues and organs of Phoebe zhennan as sample materials, and obtains the Phoebe zhennan decay resistance marker gene through high-throughput sequencing and analysis screening. PzPLR1 The expression level of 1777 gene in different tissues and organs of Phoebe zhennan was different, with the highest expression level in roots and stems, the highest expression level in flowers, and the lowest expression level in leaves and fruits. PzPLR1 The expression level of was positively correlated with the decay resistance of Phoebe zhennan. Therefore, by detecting the decay resistance marker gene of Phoebe zhennan PzPLR1 The expression level of PzPLR can accurately reflect the corrosion resistance of Phoebe zhennan tissues and organs. Because PzPLR is a pine resin reductase, it is involved in the synthesis of lignin, a corrosion-resistant substance of Phoebe zhennan. PzPLR1 The detection can effectively reflect the corrosion resistance of wood. Compared with the traditional judgment method that relies on cutting wood, the corrosion resistance marker gene of Phoebe zhennan provided by the present invention can effectively reflect the corrosion resistance of wood. PzPLR1 The judgment of the tree's resistance to decay is more accurate, more scientific and more repeatable. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Real-time fluorescence quantitative PCR was used to detect genes in roots, stems, leaves, flowers and fruits of Phoebe zhennan. PzPLR1 The expression results of

[0023] Figure 2 Real-time fluorescence quantitative PCR was used to detect genes in roots, stems, leaves, flowers and fruits of Phoebe zhennan. PzPLR1 The expression results; the horizontal axis: qPCR fluorescence intensity; the vertical axis: different tissues of Phoebe zhennan;

[0024] Figure 3 The real-time fluorescence quantitative PCR test results of the two samples to be tested. DETAILED DESCRIPTION

[0025] The invention provides a macaw rot-resistant marker gene PzPLR1 , the nucleotide sequence is as shown in SEQ ID NO: 1 (ATGGGAAAGAGCAAAGTTCTTGTGGTGGGGGGAACTGGTTACATAGGC

[0026] AAGAGAATTGTTAAGGCAAGCTTAGCTCAAGGTCATGAGACATATGTCCTCCACCGGCCGGAGATCGGCGTCGACATCGAAAAGGTCCAGATGCTATTGTCATTCAAGGAGCAAGGGGCCCGCCTCATCGAGGGATCATTTTCCGATCACCGGAGCCTAGTCGATGCCGTGAAACAGGTGGATGTGGTCATTTGTACTATATCGGGTGTTCACATAAGGAGCCACCACATCCTCCCGCAGCTTAAGCTCGTCGACGCCATTAAAGAAGCTGGCAATATCAAGCGATTCTTGCCATCCGAGTTTGGCACAGATCCGGCTAGGATGGCTGATGCATTGGAGCCTGGAAGAGTGACATTCGACGATAAAATGGTAGTGAGAAAGGCGATTCAAGACGCAGGGATTCCCTTCACTTACGTCTCTGCCAACTGCTTTGCGGGCTATTTTGTTGGGGGCCTCTGTCAACCTGGTAGAATTATACCTTCAAGGGAGAGTGTCTGCTTGTTTGGAGATGGTAATGTCAAAGCTATTTTTGTAGATGAAGATGACATTTCTACCTACACCGTCAAAATTATAGACGACCCAAGAACGCTGAACAAGACACTTTACCTCCGGCCCCCTGAAAACATCCTCTCCCAGAGAGAGGTAGTCAAGATGTGGGAGAAACTCATTGGAAAAGAACTCGAGAAAACTAGTATATCCGCAGAGGAATTCCTTGCTCAAATGAAAGGTCTGGATTACGCACAGCAGGTGGGTTTGGGACATTACTACCATGTTTGCTTTGATGGTTGTCTTGCAAATTTCGAAATAGGAGAAGAGGGAGAAGAGGCTTCACAGTTGTATCCAGAGGTTAACTATACTCGAGTGAAGGACTATCTCAAACGCTATCTATAG) as shown.

[0027] In the present invention, the Phoebe zhennan decay-resistant marker gene PzPLR1The amino acid sequence of the encoded protein is shown in SEQ ID NO: 4 (MGKSKVLVVGGTGYIGKRIVKASLAQGHETYVLHRPEIGVDI

[0028] 。EKVQMLLSFKEQGARLIEGSFSDHRSLVDAVKQVDVVICTISGVHIRSHHILPQLKLVDAIKEAGNIKRFLPSEFGTDPARMADALEPGRVTFDDKMVVRKAIQDAGIPFTYVSANCFAGYFVGGLCQPGRIIPSRESVCLFGDGNVKAIFVDEDDISTYTVKIIDDPRTLNKTLYLRPPENILSQREVVKMWEKLIGKELEKTSISAEEFLAQMKGLDYAQQVGLGHYYHVCFDGCLANFEIGEEGEEASQLYPEVNYTRVKDYLKRYL).

[0029] The present invention provides a method for screening a macaw rot-resistant marker gene, comprising the following steps:

[0030] Different tissues and organs of Phoebe zhennan were used as sample materials. RNA extraction, library construction, and transcriptome sequencing were performed to obtain transcriptome data of different tissues and organs of Phoebe zhennan.

[0031] The transcriptome data of different tissues and organs of the Phoebe zhennan were compared with the reference genome, and after obtaining the matching data, gene expression analysis and weighted gene co-expression network analysis were performed to obtain the Phoebe zhennan decay resistance marker gene. 。

[0032] The present invention uses different tissues and organs of Phoebe zhennan as sample materials, and obtains transcriptome data of different tissues and organs of Phoebe zhennan through RNA extraction, library construction, and transcriptome sequencing.

[0033] In the present invention, the different tissues and organs of Phoebe zhennan preferably include roots, stems, leaves, flowers and fruits of Phoebe zhennan. The present invention can quickly and efficiently screen out the decay-resistant marker genes of different tissues and organs by selecting reasonable sampling sites. The present invention does not specifically limit the RNA extraction, library construction, and transcriptome sequencing, and can be carried out using conventional RNA extraction methods, library construction methods, and transcriptome sequencing platforms in the art. In an embodiment of the present invention, the acquisition of transcriptome data of different tissues and organs of Phoebe zhennan was entrusted to Annoroad Gene Technology Co., Ltd.

[0034] After obtaining the transcriptome data of different tissues and organs of Phoebe zhennan, the present invention compares the transcriptome data of different tissues and organs of Phoebe zhennan with the reference genome, and after obtaining the matching data, performs gene expression analysis and weighted gene co-expression network analysis to obtain the Phoebe zhennan decay resistance marker gene.

[0035] In the present invention, before the comparison, it is preferred to further include quality control of the transcriptome data of different tissues and organs of the P. zhennan to obtain high-quality quality control data. The quality control method is preferably to filter out reads with a base N ratio greater than 10%, filter out reads with a quality value Q ≤ 20 that accounts for more than 50% of the entire read, truncate the adapter and the subsequent part, and filter out reads shorter than 50 bp after truncation of the adapter.

[0036] In the present invention, the reference genome is preferably the Lindera megaphyllagenome (https: / / www.ncbi.nlm.nih.gov / bioproject / PRJNA850554). The transcriptome data after quality control is compared with the reference genome to obtain matching data. The present invention has no particular limitation on the comparison method, and a comparison method known in the art can be used.

[0037] In the present invention, the gene expression analysis preferably includes the following steps: mapping the clean reads of each sample to the reference genome to obtain the expression level of mRNA, calculating the expression abundance of each uigene using the RNA-Seq data quantification software Kallisto, and selecting the normalization method of TPM to characterize the expression of the gene. The present invention has no special restrictions on the method of weighted gene co-expression network analysis, and the weighted gene co-expression network analysis method known in the art can be used.

[0038] In the present invention, the selected zhennan rot-resistant marker gene PzPLR1 To verify, RT-qPCR was used to detect genes PzPLR1 The expression levels of the gene in roots, stems, leaves, flowers and fruits of Phoebe zhennan were analyzed. PzPLR1 The expression level was positively correlated with the corrosion resistance concentration of each tissue and organ. Specifically, the expression level was higher in roots and stems, relatively higher in flowers, and relatively lower in leaves and fruits.

[0039] The invention provides a kit for detecting the corrosion resistance of Phoebe zhennan, comprising a real-time fluorescence quantitative PCR method for detecting the corrosion resistance marker gene of Phoebe zhennan PzPLR1Primers and qPCR reaction reagents are used; the primers include a forward primer with a nucleotide sequence as shown in SEQ ID NO: 2 (CGCCTCATCGAGGGATCATT) and a reverse primer with a nucleotide sequence as shown in SEQ ID NO: 3 (ACTCTTCCAGGCTCCAATGC).

[0040] In the present invention, the detection kit preferably further comprises an internal reference gene primer. The present invention has no particular limitation on the type of the internal reference gene, and any internal reference gene known in the art may be used, for example PzActin Gene. PzActin The gene amplification primers preferably include a forward primer having a nucleotide sequence as shown in SEQ ID NO: 5 (GGCATCCGGCAGCTCTTC) and a reverse primer having a nucleotide sequence as shown in SEQ ID NO: 6 (AAGTTGTTGGCGGCGTCTT).

[0041] In the present invention, the detection kit preferably further comprises a reverse transcription reagent and / or an RNA extraction reagent. The present invention has no particular limitation on the source of the reverse transcription reagent and / or the RNA extraction reagent, and the source of the reverse transcription reagent and / or the RNA extraction reagent known in the art can be used.

[0042] Based on the decay resistance marker gene of Phoebe zhennan PzPLR1 The expression level is positively correlated with the corrosion resistance of Phoebe zhennan. The present invention provides the use of the primer or the Phoebe zhennan corrosion resistance detection kit in judging the corrosion resistance of Phoebe zhennan.

[0043] In the present invention, the method of judging the decay resistance of Phoebe zhennan preferably includes judging the decay resistance in different tissues and organs of Phoebe zhennan, preferably roots, stems, leaves, flowers, and fruits, more preferably roots, stems, and flowers, and most preferably roots. The method of judging the decay resistance of Phoebe zhennan is preferably RT-qPCR detection technology.

[0044] In the present invention, the level of the decay resistance of Phoebe zhennan is preferably based on the content of lignans. PzPLR1 There are large differences in the expression levels of genes in the samples of roots, stems, leaves, flowers and fruits of Phoebe zhennan. The expression levels are higher in roots and stems, relatively higher in flowers, and relatively lower in leaves and fruits. In addition, there are literatures that show that (Phoebe zhennan PLR Gene family identification and expression analysis of response hormones), the content of lignans in plants is closely related to plant decay resistance, which is due to PLR The function of the gene, therefore PzPLR1 The expression level of the gene is positively correlated with the corrosion resistance of tissues and organs.

[0045] The present invention provides a method for PzPLR1The method for judging the corrosion resistance of Phoebe zhennan tissues and organs comprises the following steps:

[0046] Detecting genes in the sample to be tested using the primers PzPLR1 The expression level of the gene in the sample to be tested PzPLR1 The expression level and the reference sample level are used to determine the corrosion resistance of the sample to be tested:

[0047] If the gene in the sample to be tested PzPLR1 If the expression level of the test sample is not significantly different from that in the reference sample, the corrosion resistance of the tissue and organ of the test sample is the same or similar to that of the reference sample;

[0048] If the gene in the sample to be tested PzPLR1 If the expression level of is significantly lower than that of the reference sample, the corrosion resistance of the sample to be tested is lower than that of the reference sample;

[0049] If the gene in the sample to be tested PzPLR1 If the expression level of the test sample is significantly higher than that of the reference sample, the corrosion resistance of the test sample is higher than that of the reference sample.

[0050] In the present invention, the reference sample preferably includes one or more of the root, stem, leaf, flower and fruit tissues and organs of Phoebe zhennan. The same tissues and organs in the reference sample are selected according to the tissue and organ type of the sample to be tested.

[0051] In the present invention, the amplification of the zhennan rot resistance marker gene PzPLR1 The primers preferably include a forward primer with a nucleotide sequence as shown in SEQ ID NO: 2 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO: 3. PzPLR1 The optimal reaction system for gene expression is 2×SuperReal PreMix Plus 10µL, 100 μM forward primer 0.8µL, 100 μM reverse primer 0.8µL, cDNA 1µL and ddH 2 O 7.4µL. Detection of the sample PzPLR1 The reaction program for gene expression is preferably pre-denaturation at 95°C for 2 min; 94°C for 5 s, 60°C for 30 s, and 39 cycles; the melting curve analysis reaction program is set to 65~95°C with an increase of 0.5°C every 5 s.

[0052] In the embodiment of the present invention, it is verified by experiments that by amplifying the rot-resistant marker gene of the sample to be tested, PzPLR1 By measuring the expression level of the test sample and comparing it with the reference sample, the ability of the test sample to resist corrosion can be accurately and quickly identified, which greatly simplifies the detection of antiseptic wood.

[0053] The following is a description of the decay resistance marker gene of Phoebe zhennan provided by the present invention in combination with the embodiments.PzPLR1 , amplification primers and kits and their applications are described in detail, but they should not be understood as limiting the scope of protection of the present invention.

[0054] Example 1

[0055] Screening method for corrosion resistance marker genes

[0056] 1) Sampling of different tissues and organs of Phoebe zhennan

[0057] The samples were collected at the Forest Seed Station in Yongchuan District, Chongqing (106°E, 29°N). The roots, stems, leaves, flowers and fruits of Phoebe zhennan were sampled. Each group of samples had three biological replicates. All the materials were quickly frozen with liquid nitrogen immediately after collection and stored in a -80 ℃ ultra-low temperature refrigerator.

[0058] 2) RNA extraction, library construction, and transcriptome sequencing

[0059] The total RNA of the reference samples was extracted by the Trizol method. After quality inspection, Annoroad Gene Technology Co., Ltd. was commissioned to construct the transcriptome library and perform high-throughput sequencing on the Illumina nova-6000 platform.

[0060] 3) Data quality control and reference genome comparison

[0061] Perform data quality control on raw reads, filter reads with base N ratio greater than 10%, filter reads with quality value Q ≤ 20 that account for more than 50% of the entire read, truncate the adapter and the part after it, and filter reads shorter than 50 bp after truncation of the adapter to obtain high quality clean reads.

[0062] The high-quality genome of Lindera megaphylla genome (https: / / www.ncbi.nlm.nih.gov / bioproject / PRJNA850554), a closely related species of Phoebe zhennan, was used as the reference genome, and the quality control data were compared with it to obtain the matching data (mapped reads) for subsequent transcript assembly and expression analysis.

[0063] 4) Gene expression analysis

[0064] The clean reads of each sample were mapped to the reference genome of P. nigromaculata to obtain the expression level of mRNA. The RNA-Seq data quantification software Kallisto was used to calculate the expression abundance of each uigene, and the TPM (Transcript per million) normalization method was selected to characterize the expression of the gene.

[0065] 5) Weighted gene co-expression network analysis

[0066] Genes with expression levels of TPM<1 in all samples were filtered out, and the filtered gene set was soft-thresholded with "power = 15" to construct a gene network. The parameters for network construction and module merging were "deepSplit = 2; minModuleSize = 100; mergeCutHeight = 0.20". The obtained modules were analyzed for correlation between modules and between samples and modules. The former was clustered by module gene expression, and the latter was calculated by Pearson correlation analysis to calculate the correlation between module eigenvalues ​​and sample expression matrices. Finally, PCA analysis was performed on gene expression in the module, and PC1 was used to represent the module index, namely the module eigenvector (MEs), and the module was screened based on the change trend of MEs values. The top 1% genes of the two modules were retained to construct the hub gene network. The network was visualized and the hub genes were screened using Cytoscape software. The subnetwork was extracted using the MCODE plug-in, and then the CytoHubba plug-in was combined with the intersection of 12 algorithms such as MMC (including Degree, Edge Percolated component, Maximum neighborhood component, Density of MaximumNeighborhood Component, Maximum Clique Centrality, Botteleneck, EcCentricity, Closeness, Radiality, Betweenness and Stress) to obtain the corrosion-resistant marker genes. PzPLR1 , PzPLR1 The sequence of the gene is shown in SEQ ID NO:1.

[0067] Example 2

[0068] Verification of Corrosion Resistance Marker Genes

[0069] 1) Primer design

[0070] Corrosion resistance marker gene PzPLR1 The CDS sequence of the primers was designed using the NCBI online primer design website Primer-BLAST.PzPLR1 The primer sequences for the genes are:

[0071] Upstream primer: CGCCTCATCGAGGGATCATT (SEQ ID NO: 2);

[0072] Downstream primer: ACTCTTCCAGGCTCCAATGC (SEQ ID NO: 3).

[0073] The internal reference gene is PzActin Gene, primer sequences are as follows:

[0074] Internal reference upstream primer: GGCATCCGGCAGCTCTTC (SEQ ID NO: 5);

[0075] Internal reference downstream primer: AAGTTGTTGGCGGCGTCTT (SEQ ID NO: 6).

[0076] 2) Sampling of different tissues and organs of Phoebe zhennan

[0077] The sampling parts were roots, stems, leaves, flowers and fruits of Phoebe zhennan. The above materials were immediately frozen with liquid nitrogen after collection and stored in a -80 ℃ ultra-low temperature refrigerator.

[0078] 3) Extraction of total RNA from samples

[0079] The total RNA of the samples collected in step 2) was extracted using RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (DP441) from Tiangen Biochemical Technology (Beijing) Co., Ltd. The RNA concentration and integrity were determined using a Nano micro-spectrophotometer and 1% agarose gel electrophoresis, respectively.

[0080] 4) Reverse transcription of total RNA from samples

[0081] In an RNase free PCR tube, complete the reverse transcription reaction of the total RNA obtained in step 3) and remove the genomic DNA. Prepare the reverse transcription system according to Table 1 below:

[0082] Table 1 Reverse transcription system

[0083]

[0084] The above system was gently mixed, incubated in a PCR instrument at 42 °C for 15 min, and then heated at 85 °C for 5 s to completely inactivate RT / RI Enzyme and gDNA Remover. The product was stored at -20 °C.

[0085] 5) PzPLR1 Real-time fluorescence quantitative PCR verification of genes

[0086] The cDNA generated by reverse transcription in step 4) was diluted 30 times and used as a template, and qRT-PCR was performed according to the qRT-PCR reaction system and reaction conditions in Table 2 below:

[0087] Table 2 qRT-PCR reaction system

[0088]

[0089] The SYBR Green chimeric fluorescence method was used for the reaction. The pre-denaturation reaction program was set at 95°C for 2 min, the PCR reaction program was set at 94°C for 5 s, 60°C for 30 s, and 39 cycles; the melting curve analysis reaction program was set at 65-95°C with an increase of 0.5°C every 5 s. Each sample was repeated 3 times in technical replicates and 3 times in biological replicates, and an independent internal reference was added to each plate sample. PzPLR1 .

[0090] PzPLR1 The results of real-time fluorescence quantitative PCR of genes in roots, stems, leaves, flowers and fruits of Phoebe zhennan were as follows: Figure 2 and Table 3. Compared with the transcriptome sequencing results ( Figure 1 )compared to, Figure 2 Root, stem, leaf, flower and fruit samples of Phoebe zhennan PzPLR1 The gene expression trends were very similar, except for PzPLR1 The gene expression level is relatively high. Overall, the qPCR test results and the transcriptome data have a general trend that is consistent.

[0091] Table 3 qPCR test results

[0092]

[0093] The existing technology (Study on the improvement of decay resistance and color of poplar wood by heat-treated wood extracts and its mechanism of action, Bi Zhenju, Northwest A&F University, 2023; Antibacterial activity of Phoebe heartwood extracts, Li Jiqing, Journal of Forest and Environment, 2017) tested the antibacterial properties of different medium extracts of Phoebe zhennan and poplar wood, and the media such as methanol, ethyl acetate, and petroleum ether used in the technology have been proven to be able to extract lignans in plants (Study on the extraction process of total lignans from camphor leaves and their antibacterial activity; Study on lignan components in milk mulberry). The research results show that, except for the acetone and methanol medium extraction of poplar wood, which has a high inhibitory effect on Glehnia littoralis (inhibition rate = 89.24%), the inhibition rates of the other five medium extracts on Glehnia littoralis are all lower than 50%, and some even have an inhibition rate lower than 0% (petroleum ether and hot water). The study of extracts from different media of Phoebe zhennan wood found that acetone and methanol were also the extraction media with the highest antibacterial rate, with inhibition rates of 67.78% and 72.59% against Glechoma spp. respectively; at the same time, compared with poplar wood, the extracts from the other three media (ethyl acetate, petroleum ether and distilled water) of Phoebe zhennan wood had relatively high inhibition rates against Glechoma spp., with inhibition rates of 53.96%, 53.81% and 39.2% against Glechoma spp. respectively (poplar was 23.33%, -46.7% and -24.21%). Therefore, the above results show that the antibacterial ability of Phoebe zhennan wood is much higher than that of poplar wood.

[0094] Example 3

[0095] Machilus zhennan Rot Resistance Marker Gene PzPLR1 Application in judging the decay resistance of roots, stems, leaves, flowers and fruits of Phoebe zhennan

[0096] 1. Primers

[0097] PpPLR1 The primer sequences for the genes are:

[0098] Upstream primer: CGCCTCATCGAGGGATCATT (SEQ ID NO: 2);

[0099] Downstream primer: ACTCTTCCAGGCTCCAATGC (SEQ ID NO: 3);

[0100] The internal reference gene is PzActin Gene, primer sequences are as follows:

[0101] Internal reference upstream primer: GGCATCCGGCAGCTCTTC (SEQ ID NO: 5);

[0102] Internal reference downstream primer: AAGTTGTTGGCGGCGTCTT (SEQ ID NO: 6).

[0103] 2. Samples to be tested

[0104] The first sample material to be tested is a young root of Phoebe zhennan, which is about 30-40 years old; the second sample material to be tested is a young root of Populus, which is about 30-40 years old.

[0105] 3. Setting of reference samples

[0106] The young roots of Phoebe zhennan, which are about 40 years old, are used as reference sample material 1; the young roots of Populus, which are about 30 years old, are used as reference sample material 2.

[0107] 4. Detection Methods

[0108] 4.1 Extraction of RNA from Phoebe zhennan

[0109] Use Adela EASYspin Plus Plant RNA Kit for extraction, and follow the instructions for specific steps.

[0110] The RNA concentration and integrity were determined by Nano micro-spectrophotometer and 1% agarose gel electrophoresis, respectively.

[0111] 4.2 Synthesis of first-strand cDNA

[0112] Prepare the reverse transcription reaction system according to the reagent addition amounts in Table 4.

[0113] Table 4 Reverse transcription reaction system

[0114]

[0115] After gently mixing according to the above system, incubate at 42°C for 15 min and heat at 85°C for 5 s.

[0116] 4.3 Real-time fluorescence quantitative PCR

[0117] The real-time fluorescence quantitative PCR reaction system was prepared according to the reagent addition amounts in Table 5.

[0118] Table 5 Reaction system of real-time fluorescence quantitative PCR

[0119]

[0120] The SYBR Green chimeric fluorescence method was used for the reaction. The pre-denaturation reaction program was set at 95 °C for 2 min, the PCR reaction program was set at 94 °C for 5 s, 60 °C for 30 s, and 39 cycles. The melting curve analysis reaction program was set at 65-95 °C with an increase of 0.5 °C every 5 s. Each sample was repeated 3 times in technical replicates and 3 times in biological replicates, and an independent internal reference gene was added to each plate sample.

[0121] Test results see Figure 3 . Since the expression of anti-corrosion substances between the wood of Phoebe zhennan and the wood of poplar is quite different, the anti-corrosion results of the two samples to be tested are judged according to the results of real-time fluorescence quantitative PCR of the test sample material 1, the test sample material 2 and the reference sample. The reference sample material 1 is the root of a 40-year-old Phoebe zhennan. PzPLR1 There is no significant difference between the expression level of and reference sample 1. Therefore, the test sample material 1 is a zhennan material with strong corrosion resistance. PpPLR1 There is no significant difference between the gene and the reference sample material 2, indicating that the sample material 2 to be tested is judged to be a poplar material. PzPLR1 It is scientifically feasible to judge the decay resistance of Phoebe zhennan based on the expression level of .

[0122] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A marker gene for rot resistance in Phoebe zhennan PZPLR1 , characterized in that, The nucleotide sequence is shown in SEQ ID NO:

1.

2. A real-time fluorescence quantitative PCR method for detecting the rot resistance marker gene of Phoebe zhennan described in claim 1 PZPLR1 Using a primer, characterized in that It comprises a forward primer having a nucleotide sequence as shown in SEQ ID NO: 2 and a reverse primer having a nucleotide sequence as shown in SEQ ID NO:

3.

3. A kit for detecting the corrosion resistance of Phoebe zhennan, characterized in that: It comprises qPCR reaction reagents and the primers according to claim 2.

4. The zhennan macaw corrosion resistance detection kit according to claim 3, characterized in that: Also included are primers for internal reference genes; The internal reference gene primers include a forward primer whose nucleotide sequence is shown in SEQ ID NO: 5 and a reverse primer whose nucleotide sequence is shown in SEQ ID NO:

6.

5. The zhennan macaw corrosion resistance detection kit according to claim 3 or 4, characterized in that: Reverse transcription reagents and / or RNA extraction reagents are also included.

6. Use of the primer according to claim 2 or the kit according to any one of claims 3 to 5 in determining the corrosion resistance of Phoebe zhennan.

7. A method for producing a rot-resistant marker gene based on the Phoebe zhennan described in claim 1 PZPLR1 The method for judging the decay resistance of Phoebe zhennan tissues and organs is characterized in that: The following steps are involved: Detecting genes in the sample to be tested using the primers described in claim 2 PZPLR1 The expression level of the gene in the sample to be tested PZPLR1 The expression level and the reference sample level are used to determine the corrosion resistance of the sample to be tested: If the gene in the sample to be tested PZPLR1 If the expression level of the test sample is not significantly different from that in the reference sample, the corrosion resistance of the tissue and organ of the test sample is the same or similar to that of the reference sample; If the gene in the sample to be tested PZPLR1 If the expression level of is significantly lower than that of the reference sample, the corrosion resistance of the sample to be tested is lower than that of the reference sample; If the gene in the sample to be tested PZPLR1 If the expression level of the test sample is significantly higher than that of the reference sample, the corrosion resistance of the test sample is higher than that of the reference sample.

8. The method according to claim 7, characterized in that: The reference samples include one or more of the root, stem, leaf, flower and fruit tissue organs of Phoebe zhennan.

9. The method according to claim 7, characterized in that: The method for detecting genes in the sample to be tested PZPLR1 The reaction system for the expression level was 2×SuperReal PreMix Plus 10µL, 100 μM forward primer 0.8µL, 100 μM reverse primer 0.8µL, cDNA 1µL and ddH2O 7.4µL.

10. The method according to any one of claims 7 to 9, characterized in that: The method for detecting genes in the sample to be tested PZPLR1 The reaction program for expression level was pre-denaturation at 95 °C for 2 min; 94 °C for 5 s, 60 °C for 30 s, and 39 cycles; the reaction program for melting curve analysis was 65-95 °C with an increase of 0.5 °C every 5 s.

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