Application of PtoERD3 gene structure variation in evaluation of lignin content of poplar

Abstract

The invention discloses an application of PtoERD3 gene structure variation in evaluation of poplar lignin content, the structure variation is located in an upstream promoter region of a poplar PtoERD3 gene, is a 54bp chromosome structure variation SV fragment, and has a nucleotide sequence as shown in SEQ ID NO.1. The invention also discloses an application of the PtoERD3 gene structure variation in evaluation of poplar lignin content. By measuring the structural variation, the lignin content of the poplar can be accurately judged, superior plants with high-quality wood quality characters can be accurately and efficiently screened in the early growth stage of the poplar, the breeding period is effectively shortened, and a theoretical support is provided for molecular design breeding of the poplar wood quality.

Description

Technical Field

[0001] The present invention relates to the field of molecular biology technology, in particular to molecular markers related to poplar lignin content, and especially to an application of a PtoERD3 gene structural variation in evaluating poplar lignin content. Background Art

[0002] Populus tomentosa is an important fast-growing timber species in northern my country. The lignin content and composition of its wood directly influences pulping efficiency for papermaking and its potential for biomass energy conversion. In recent years, structural variation (SV) has become an important entry point for understanding the mechanisms of wood quality in Populus tomentosa due to its significant impact on gene expression regulation and phenotypic diversity.

[0003] Structural variation involves deletions, duplications, inversions, and translocations of large chromosomal segments (≥50 bp). These variations can simultaneously affect multiple genes or regulatory elements, leading to altered gene expression, changes in protein structure, or chromosomal rearrangements. The effects on complex traits are often more pronounced and diverse. Lignin is a key component of plant secondary cell walls, and its biosynthesis is coordinated by genes encoding enzymes such as cinnamyl alcohol dehydrogenase (CAD) and caffeoyl-CoA methyltransferase (CCoAOMT) in the phenylpropanoid metabolic pathway. The significant variability in lignin content and composition among natural populations of Populus tomentosa suggests the existence of complex molecular mechanisms within its genetic regulatory network.

[0004] Currently, relevant research lacks functional analysis of lignin-related SVs. Therefore, with the continuous development of modern sequencing and molecular breeding technologies, it is necessary to develop key SV molecular markers that can affect lignin content and determine the genetic effects of key SV molecular markers on tree wood traits. This will enable rapid and accurate screening of new forest germplasm with high-quality wood species and provide theoretical support for molecular design breeding of Populus tomentosa wood quality. Summary of the Invention

[0005] In order to overcome the above problems, the inventors, based on the strategy of genome-wide association analysis, relied on the white poplar germplasm resource population and used GEMMA (v0.98.5) software to detect SV sites significantly associated with lignin content at the genome-wide level. A 54bp insertion variant SV fragment was detected, located in the promoter region of the PtoERD3 gene, having a nucleotide sequence as shown in SEQ ID NO.1, or a nucleotide sequence with more than 90% homology to the nucleotide sequence. By using this insertion variant SV fragment as a molecular marker for detecting the lignin content of poplars, it is possible to accurately judge the lignin content of poplars, accurately and efficiently screen superior plants with high-quality wood quality traits in the early growth stage of poplars, significantly shorten the breeding cycle, and provide theoretical support for molecular design breeding of poplar wood quality, thereby completing the present invention.

[0006] Specifically, the purpose of the present invention is to provide the following aspects:

[0007] In the first aspect, a molecular marker related to the lignin content of poplar is provided, wherein the molecular marker is a 54 bp chromosome structural variation SV fragment.

[0008] The chromosome structural variation SV fragment has a nucleotide sequence as shown in SEQ ID NO. 1, or a nucleotide sequence having more than 90% homology with the nucleotide sequence.

[0009] In a second aspect, a primer pair for detecting the molecular marker described in the first aspect is provided, wherein the primer pair comprises primer P1 and primer P2, primer P1 has a sequence as shown in SEQ ID NO.3, and primer P2 has a sequence as shown in SEQ ID NO.4.

[0010] In a third aspect, there is provided an application of the molecular marker described in the first aspect in detecting the lignin content of poplars, the application comprising the following steps:

[0011] First, PCR amplification was performed using the genomic DNA of the tested poplar as a template;

[0012] Then, based on the amplified product, it is determined whether it contains molecular markers, and then the lignin content of the poplar is determined.

[0013] In a fourth aspect, the present invention provides an application of the molecular marker described in the first aspect in regulating the lignin content of poplars, wherein the regulation of the lignin content of poplars by the molecular marker is achieved by overexpressing the PtoERD3 gene or silencing the PtoERD3 gene.

[0014] Lignin content was reduced by overexpressing the PtoERD3 gene in poplar plants, while lignin content was increased by silencing the PtoERD3 gene in poplar plants.

[0015] In a fifth aspect, a method for genetic improvement of poplars is provided, wherein the genetic improvement is to increase the lignin content of poplars. The method comprises the steps of successive breeding of poplar individuals that do not have the molecular marker described in the first aspect, and eliminating poplar individuals that have the molecular marker.

[0016] The beneficial effects of the present invention include:

[0017] (1) The molecular marker related to poplar lignin content provided by the present invention is located in the upstream promoter region of the poplar PtoERD3 gene and negatively regulates poplar lignin content. By measuring this structural variation, it is possible to accurately determine the lignin content of poplars, accurately and efficiently screen for superior plants with high-quality wood quality traits in the early stages of poplar growth, and significantly shorten the breeding cycle;

[0018] (2) The primer pairs for detecting molecular markers provided by the present invention can be effectively used in molecular marker-assisted breeding, thereby enabling the screening of high-quality tree species with high lignin content in a short time, at low cost, and with high accuracy;

[0019] (3) The application of the molecular markers provided by the present invention in regulating the lignin content of poplars is achieved by overexpressing the PtoERD3 gene or silencing the PtoERD3 gene. The PtoERD3 gene silencing mutant can significantly increase the lignin content of poplars, which is of great significance for breeding poplar plants with excellent wood quality traits and innovating poplar germplasm resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The figure shows the position of the most significant SV in the promoter region of the PtoERD3 gene in Example 1; Figure 2 The verification electropherogram of the candidate individual SV in Example 2 is shown; Figure 3 Graph showing the fluorescence signal results of transiently transcribed SV in Example 3; Figure 4 The figure shows the quantitative analysis results of the relative fluorescence activity of transiently transcribed SVs in Example 3; Figure 5 The transcription levels of PtoERD3 in overexpressing plants (OE-2, OE-3, and OE-5) detected by RT-qPCR in Example 6 are shown, wherein the error bars represent standard deviations; Figure 6 The transcription level of PtoERD3 in the interference silenced plants (RNAi1, RNAi6, and RNAi8) detected by RT-qPCR in Example 6 is shown, wherein the error bars represent standard deviations; Figure 7 The phenotypes of the interference silenced plants (denoted as RNAi-PtoERD3), the phenotypes of the wild-type Populus tomentosa (denoted as WT), and the phenotypes of the PtoERD3 overexpressing plants (denoted as OE-PtoERD3) are shown; Figure 8 The plant heights of RNAi-PtoERD3, WT, and OE-PtoERD3 are shown; Figure 9 The diameters of RNAi-PtoERD3, WT, and OE-PtoERD3 are shown; Figure 10 The lignin contents of RNAi-PtoERD3, WT, and OE-PtoERD3 are shown. DETAILED DESCRIPTION

[0021] The present invention will be further described in detail below through preferred embodiments and examples. Through these descriptions, the characteristics and advantages of the present invention will become more clear and distinct.

[0022] The word “exemplary” is used exclusively herein to mean “serving as an example, example, or illustration.” Any embodiment described herein as “exemplary” is not necessarily to be construed as preferred or advantageous over other embodiments.

[0023] The inventors have discovered through research that the ERD3 gene (Early Responsive to Dehydration 3 gene), as an important member of the plant stress response gene family, exhibits unique functions in regulating poplar lignin metabolism. This gene can negatively regulate lignin content and improve the quality traits of poplar wood.

[0024] Therefore, in order to quickly and accurately screen new forest germplasm with excellent wood species, the first aspect of the present invention provides a molecular marker related to the lignin content of poplar, which is a 54bp chromosome structural variation SV fragment.

[0025] The chromosome structural variation SV fragment has a nucleotide sequence as shown in SEQ ID NO. 1, or a nucleotide sequence having more than 90% homology with the nucleotide sequence.

[0026] Preferably, the nucleotide sequence of the chromosome structural variation SV fragment is shown as SEQ ID NO.1.

[0027] According to a preferred embodiment of the present invention, the molecular marker is located in the upstream promoter region of the poplar PtoERD3 gene, and the poplar is preferably Populus tomentosa. The sequence of the upstream promoter of the PtoERD3 gene is shown in SEQ ID NO.2.

[0028] Among them, poplars are divided into the Populus alba, Populus nigra, Populus euphratica and Populus macrophylla schools. Among them, Populus tomentosa in the Populus tomentosa school is a native tree species unique to China and is widely distributed, with the middle and lower reaches of the Yellow River as the central distribution area. It occupies an important position in forestry production and ecological environment construction in northern my country and is a pioneer tree species for forest cultivation in the northern region. Therefore, Populus tomentosa is preferably selected as the research object in the present invention.

[0029] In a further preferred embodiment, the molecular marker is located at 1725 bp of the nucleotide sequence shown in SEQ ID NO.2.

[0030] Based on the strategy of genome-wide association analysis, the present inventors identified a structural variation (SV) associated with the lignin content of poplar, which negatively regulates the lignin content of poplar.

[0031] According to a preferred embodiment of the present invention, when the structural variation exists in the upstream promoter region of the poplar PtoERD3 gene, the lignin content is low; when the structural variation does not exist in the upstream promoter region of the poplar PtoERD3 gene, the lignin content is high.

[0032] Therefore, by measuring this structural variation, it is possible to accurately determine the lignin content of poplar trees, accurately and efficiently screen superior plants with high-quality wood quality traits in the early stages of poplar growth, and significantly shorten the breeding cycle.

[0033] According to a preferred embodiment of the present invention, the molecular marker is obtained by a method comprising the following steps:

[0034] First, the association analysis between SV loci and poplar lignin content was performed to obtain significantly associated SV loci;

[0035] Then, the obtained significantly associated SV sites were annotated and their positions were obtained.

[0036] In a further preferred embodiment, GEMMA software was used to perform SVGWAS analysis on 49,380 high-confidence SVs and the lignin content of 302 Populus tomentosa materials, and the PCA matrix was added to correct the population structure. 38 significant SV sites were screened, and the most significantly associated site was annotated to the promoter region of PtoERD3 (p = 1.02E-8), specifically an insertion-type structural variation of 54 bp located at the 1725 bp of the nucleotide sequence shown in SEQ ID NO.2.

[0037] According to a preferred embodiment of the present invention, the method further comprises the step of verifying the obtained SV.

[0038] Preferably, the verification is as follows: randomly selecting several individuals from the Populus tomentosa germplasm resource bank, extracting genomic DNA to perform SV verification, and then evaluating the lignin content of the selected individuals.

[0039] In the present invention, the Populus tomentosa germplasm resource bank is preferably a Populus tomentosa hybrid population germplasm resource bank planted in Guan County, Shandong Province, and the number of selected individuals is 24.

[0040] Verification revealed that individuals without the mutation (i.e., individuals lacking the SV segment) had a lignin content of 22.3±0.6%. When the candidate individuals were homozygous for the mutation, the candidate poplars had the lowest lignin content, with the lignin content (18.2±1.2%) being 18.39% lower than that of individuals without the mutation (22.3±0.6%). When the candidate individuals were heterozygous for the mutation, the lignin content (18.89±0.6%) was 15.29% lower than that of individuals without the mutation. "No mutation" refers to individuals with a homozygous genotype lacking the SV, meaning both alleles lack the SV (wild-type homozygous); "homozygous mutation" refers to individuals with a homozygous genotype lacking the structural variant (SV), meaning both alleles contain the SV; and "heterozygous mutation" refers to individuals with both alleles containing and not containing the SV, forming a heterozygous genotype.

[0041] That is, the lignin content of homozygous mutant plants with SV mutation was reduced by 18.39% compared with homozygous plants without SV mutation; the lignin content of heterozygous mutant plants was reduced by 15.29% compared with homozygous plants without SV mutation.

[0042] Furthermore, considering that the molecular marker described in the present invention is located in the promoter region of the PtoERD3 gene, in order to clarify whether the promoter insertion mutation of the gene affects its promoter activity, the present invention preferably uses a dual luciferase experiment to carry out verification work.

[0043] Verification showed that SV can significantly enhance the transcription of PtoERD3 and positively regulate the expression of PtoERD3.

[0044] The second aspect of the present invention provides a primer pair for detecting the molecular marker described in the first aspect, wherein the primer pair comprises primer P1 and primer P2, primer P1 has a sequence as shown in SEQ ID NO.3, and primer P2 has a sequence as shown in SEQ ID NO.4.

[0045] In the present invention, the above primer pairs can be used to effectively perform PCR amplification on molecular markers related to the lignin content of poplars, and then detect the molecular markers by electrophoresis.

[0046] In the present invention, in order to eliminate the quality problem of the DNA template of the poplar to be detected and improve the accuracy of detection, primers P1 and P2 are preferably designed according to the upstream and downstream sequences of the SV position to obtain an amplification product of a specific length.

[0047] Preferably, primer pairs that add 1 to 20 bases to the 5' and 3' ends of the nucleotide sequences shown in SEQ ID NO. 3 and SEQ ID NO. 4, respectively, and can produce substantially identical DNA fragments (the DNA sequences between the upstream primer and the downstream primer are identical) are all included in the primer pairs of the present invention.

[0048] According to a preferred embodiment of the present invention, the genomic DNA of the poplar to be tested is used as a template, primer pairs P1 and P2 are used to perform PCR amplification, and then the amplified product is identified.

[0049] Preferably, after identification, when the amplified product contains the molecular marker described in the first aspect, the poplar lignin content is low; when the amplified product does not contain the molecular marker described in the first aspect, the poplar lignin content is high.

[0050] More preferably, the amplified products are subjected to electrophoresis identification. When a single band of 308 bp is amplified, it indicates that the plant has the structural variation and the poplar lignin content is the lowest; when a single band of 254 bp is amplified, it indicates that the plant does not have the structural variation and the poplar lignin content is the highest; when double bands of 308 bp and 254 bp are amplified, it indicates that the plant has a heterozygous mutation and the poplar lignin content is between the above two.

[0051] Therefore, the primer pair for detecting molecular markers can be effectively used in molecular marker-assisted breeding, thereby enabling the screening of high-quality tree species with high lignin content in a short time, at low cost and with high accuracy.

[0052] The third aspect of the present invention provides a use of the molecular marker described in the first aspect in detecting the lignin content of poplar.

[0053] Preferably, the application comprises the following steps:

[0054] First, PCR amplification was performed using the genomic DNA of the poplar to be tested as a template.

[0055] Preferably, the primer pair used in the PCR amplification is primer P1 and primer P2.

[0056] Then, based on the amplified product, it is determined whether it contains molecular markers, and then the lignin content of the poplar is determined.

[0057] Preferably, the amplified product is detected to determine whether it has the SV structural variation described in the first aspect. If the SV structural variation does not exist, the poplar lignin content is high; if the SV structural variation exists, the poplar lignin content is low.

[0058] According to a preferred embodiment of the present invention, the amplified product was subjected to electrophoresis analysis to obtain three electrophoresis results: a single band of 308 bp, a single band of 254 bp, and a double band of 308 bp / 254 bp;

[0059] When a single band of 308 bp was amplified, the poplar lignin content was determined to be the lowest; when a single band of 254 bp was amplified, the poplar lignin content was determined to be the highest; when double bands of 308 bp and 254 bp were amplified, the poplar lignin content was determined to be between the above two.

[0060] The fourth aspect of the present invention provides a use of the molecular marker described in the first aspect in regulating the lignin content of poplar.

[0061] As mentioned above, the molecular marker of the present invention can significantly enhance the transcription of PtoERD3 and positively regulate the expression of PtoERD3. Therefore, the regulation of the lignin content of poplar by the molecular marker is achieved by overexpressing the PtoERD3 gene or silencing the PtoERD3 gene, that is: reducing the lignin content by overexpressing the PtoERD3 gene in poplar plants, and increasing the lignin content by silencing the PtoERD3 gene in poplar plants.

[0062] According to a preferred embodiment of the present invention, the PtoERD3 gene includes the nucleotide sequence shown as SEQ ID NO.5, and preferably the nucleotide sequence of the PtoERD3 gene is shown as SEQ ID NO.5.

[0063] According to a preferred embodiment of the present invention, the method for reducing the lignin content of poplars by molecular markers comprises the following steps:

[0064] Step 1: construct an overexpression vector of the PtoERD3 gene and transform it into Agrobacterium to obtain a positive overexpression engineered bacterium.

[0065] According to a preferred embodiment of the present invention, the overexpression vector is obtained by constructing the CDS sequence of the PtoERD3 gene into the pBI121 vector, and the CDS sequence of the PtoERD3 gene is shown as SEQ ID NO.6.

[0066] In a further preferred embodiment, the CDS sequence of the PtoERD3 gene is obtained by overexpression primers P3 and P4, the sequence of the overexpression primer P3 is shown as SEQ ID NO.7, and the sequence of the overexpression primer P4 is shown as SEQ ID NO.8.

[0067] Step 2: using positive overexpression engineered bacteria to infect poplar materials, and obtaining overexpression positive seedlings after cultivation and identification.

[0068] Preferably, the poplar material is infected by the callus method.

[0069] According to a preferred embodiment of the present invention, the identification of overexpression-positive seedlings includes identification at the DNA level and identification at the transcription level.

[0070] In a further preferred embodiment, the identification at the DNA level is PCR identification, and the reaction procedure is: 95°C for 2 min; (94°C for 30 s; 58°C for 30 s; 72°C for 60 s) 35 cycles; 72°C for 5 min; 4°C∞.

[0071] The plants that were positive by PCR identification were PtoERD3 gene overexpression plants.

[0072] According to a preferred embodiment of the present invention, the identification of the transcription level is carried out by RT-qPCR experiment, and the primer pair used is PtoERD3-qPCR-F and PtoERD3-qPCR-R, wherein the sequence of primer PtoERD3-qPCR-F is shown in SEQ ID NO.9, and the sequence of primer PtoERD3-qPCR-R is shown in SEQ ID NO.10.

[0073] In a further preferred embodiment, the reaction procedure for the transcription level identification is: 95°C for 3 min; 40 cycles of (95°C for 5 sec; 60°C for 30 sec; 72°C for 15 sec); 72°C for 5 min; 4°C∞.

[0074] In the present invention, the transcription level was detected and it was found that the overexpression plants numbered OE-2, OE-3 and OE-5 had higher expression levels, which were 99.5 times, 151.3 times and 130 times that of the wild-type plant (WT), respectively.

[0075] Step 3: Transplant the overexpression positive seedlings to obtain poplar plants with reduced lignin content.

[0076] According to a preferred embodiment of the present invention, the method for increasing the lignin content of poplar by molecular markers comprises the following steps:

[0077] Step i, constructing an interference silencing vector for the PtoERD3 gene and transforming it into Agrobacterium to obtain a positive gene silencing engineered bacterium;

[0078] Step ii, infecting poplar materials with positive gene silencing engineered bacteria, culturing transgenic seedlings, and identifying gene silencing positive seedlings;

[0079] Step iii: transplanting the gene-silencing positive seedlings to obtain poplar plants with increased lignin content.

[0080] Wherein, in step i, the interference silencing vector of the PtoERD3 gene is obtained by constructing the interference fragment of the PtoERD3 gene into the pBI121 vector. Preferably, the nucleotide sequence of the interference fragment of the PtoERD3 gene is shown in SEQ ID NO.11.

[0081] Preferably, the interference fragment of the PtoERD3 gene is obtained by interfering with silencing primers P5 and P6, the sequence of the interfering with silencing primer P5 is shown as SEQ ID NO.12, and the sequence of the interfering with silencing primer P6 is shown as SEQ ID NO.13.

[0082] In step ii, the identification of the gene silencing positive seedlings includes identification at the DNA level and identification at the transcription level.

[0083] Preferably, the identification of transcription levels is performed by RT-qPCR experiments, and the primer pairs used are PtoERD3-qPCR-F and PtoERD3-qPCR-R, wherein the nucleotide sequence of primer PtoERD3-qPCR-F is shown in SEQ ID NO.9, and the nucleotide sequence of primer PtoERD3-qPCR-R is shown in SEQ ID NO.10.

[0084] In a further preferred embodiment, the reaction procedure for the transcription level identification is: 95°C for 3 min; 40 cycles of (95°C for 5 sec; 60°C for 30 sec; 72°C for 15 sec); 72°C for 5 min; 4°C∞.

[0085] In the present invention, the detection of transcriptional levels revealed that the silenced plants numbered RNAi-1, RNAi-6, and RNAi-8 had higher expression levels, which were 0.06 times, 0.12 times, and 0.11 times that of the wild-type plant (WT), respectively.

[0086] In the present invention, the PtoERD3 gene silencing mutant can significantly increase the lignin content of poplar, which is of great significance for breeding poplar plants with excellent wood quality traits and innovating poplar germplasm resources.

[0087] The fifth aspect of the present invention provides a method for genetic improvement of poplars, wherein the genetic improvement is to increase the lignin content of poplars. The method includes the steps of successive breeding of poplar individuals that do not have the molecular marker described in the first aspect, and eliminating poplar individuals that have the molecular marker.

[0088] The present invention utilizes a 54 bp inserted variant SV fragment in the upstream promoter region of the PtoERD3 gene as a molecular marker for detecting the lignin content of poplars, which can quickly and accurately distinguish the lignin content of poplar materials and provide a powerful tool for poplar breeding.

[0089] Example

[0090] The present invention is further described below through specific examples. However, these examples are merely exemplary and do not constitute any limitation to the scope of protection of the present invention.

[0091] Unless otherwise specified, the reagents involved in the following examples are all conventional reagents available on the market, the methods used are all commonly used methods in this technical field, and the parameters are all conventional settings.

[0092] Example 1 Obtaining structural variation SV related to lignin content of Populus tomentosa

[0093] The molecular markers related to the lignin content of Populus tomentosa were obtained by the following steps:

[0094] Using GEMMA (v0.98.5) software, SVGWAS analysis was performed on 49,380 high-confidence SVs and the lignin content of 302 Populus tomentosa materials at the whole genome level. The PCA matrix was added to correct the population structure, and 38 significant SV sites were screened. One of the significantly associated sites was annotated to the promoter region of PtoERD3 (p = 1.02E-8). The nucleotide sequence of the PtoERD3 gene is shown in SEQ ID NO.5, and the nucleotide sequence of its promoter region is shown in SEQ ID NO.2. The significantly associated SV site is located at 1725bp of the nucleotide sequence shown in SEQ ID NO.2. Figure 1 Its nucleotide sequence is shown in SEQ ID NO.1.

[0095] Among them, 49,380 high-confidence SVs were obtained by using the Manta software to identify population structural variations.

[0096] 302 Populus tomentosa accessions were collected from 83 locations across the Yellow River distribution area and planted in Guanxian County, Shandong Province, China.

[0097] The lignin content was determined based on the Klason standard method developed by the National Renewable Energy Laboratory (NREL) of the United States. The specific operation is as follows: the ground sample is first placed in a 72% sulfuric acid solution for hydrolysis, and then diluted and hydrolyzed under dilute acid conditions. At this time, most of the lignin remains in solid form, and the lignin content is calculated by weighing the solid mass.

[0098] GWAS results were obtained using the R software package CMplot (https: / / github.com / YinLiLin / CMplot).

[0099] Example 2 SV-based molecular markers of Populus tomentosa lignin content and identification methods thereof

[0100] To eliminate quality issues with the DNA template of the poplar trees to be tested, upstream and downstream primers for amplifying the SV sequence were designed according to the upstream and downstream positions of the SV. Taking all the principles of primer design into consideration, primer blast tools (NCBI, https: / / blast.ncbi.nlm.nih.gov) were used for primer design. The resulting primers SV-F (primer P1) and SV-R (primer P2) are shown in SEQ ID NO. 3 and SEQ ID NO. 4, respectively.

[0101] Then, 24 individuals were randomly selected from the germplasm resource bank of Populus tomentosa hybrid population planted in Guanxian County, Shandong Province, and genomic DNA was extracted. They were numbered 1 to 24 and SV was verified to evaluate the lignin content of the samples.

[0102] The specific steps are as follows:

[0103] (1) Crude extraction of Populus tomentosa DNA to be tested:

[0104] (i) Sample processing: Place one leaf to be extracted in a 2 mL centrifuge tube, add one grinding bead, and freeze in liquid nitrogen. Then, grind in a tissue crusher for 5 min until the leaf is powdered.

[0105] (ii) Buffer treatment: Add 500 μL of TBS buffer to the tube, mix thoroughly, and heat in a 65°C water bath for 10 min.

[0106] (iii) Initial centrifugation and precipitation: Centrifuge at 12,000 rpm for 15 min. Transfer the supernatant to a fresh tube. Add an equal volume of isopropanol and let it stand at room temperature for 30 min until a white flocculent DNA precipitate appears.

[0107] (iv) Nucleic acid collection: Centrifuge at 12000 rpm for 15 min, carefully discard the supernatant, and add 1 mL of 75% ethanol to wash the precipitate.

[0108] (v) Ethanol removal: Centrifuge at 7500 rpm for 10 min and completely remove any residual liquid (residues on the tube wall can be removed using a pipette).

[0109] (vi) Drying and dissolving: Dry in a 40°C oven for 2 h, add 50 μL ddH2O to dissolve the DNA precipitate, and store in a -20°C refrigerator until use.

[0110] (2) Identification of SV

[0111] Using the Populus tomentosa DNA obtained in step (1) above as a template, PCR amplification was performed. The reaction system is shown in Table 1:

[0112] Table 1

[0113]

[0114]

[0115] The reaction conditions were: 95°C for 3 min; 35 cycles of (94°C for 30 s; 58°C for 30 s; 72°C for 60 s); 72°C for 5 min; 4°C∞.

[0116] Amplification results such as Figure 2 As shown, the ability to amplify a 308 bp band indicates the presence of SV, the amplification of only a 254 bp band indicates the absence of SV, and the amplification of two bands indicates a heterozygous mutation.

[0117] (3) Comparison of identification results based on SV molecular markers and lignin content determination

[0118] The lignin content of 24 randomly selected Populus tomentosa individuals was compared with the SV-based molecular markers. The results are shown in Table 2:

[0119] Table 2

[0120] Plant number Mutation Type Lignin content 1 Homozygous variant 17.5％ 2 Homozygous variant 18.0％ 3 Homozygous variant 19.0％ 4 Homozygous variant 16.8％ 5 Homozygous variant 18.5％ 6 Homozygous variant 17.2％ 7 Homozygous variant 20.4％ 8 Homozygous variant 18.2％ 9 No variation 21.5％ 10 No variation 22.0％ 11 No variation 23.0％ 12 No variation 21.8％ 13 No variation 22.5％ 14 No variation 22.2％ 15 No variation 21.9％ 16 No variation 22.8％ 17 heterozygous variant 18.6％ 18 heterozygous variant 19.2％ 19 heterozygous variant 18.8％ 20 heterozygous variant 19.5％ 21 No variation 23.0％ 22 heterozygous variant 17.8％ 23 heterozygous variant 18.9％ 24 heterozygous variant 19.4％

[0121] Among them, homozygous variation refers to the homozygous genotype of an individual carrying a structural variation (SV), that is, both alleles have the SV; no variation refers to the homozygous genotype of an individual carrying the absence of the SV, that is, both alleles do not contain the SV (wild type homozygous); heterozygous variation refers to the individual carrying alleles with and without the SV at the same time, forming a heterozygous genotype.

[0122] As can be seen from Table 2, the lignin content of individuals without mutations is 22.3±0.6%; when the candidate individual is a homozygous mutation, the lignin content of the candidate poplar individual is the lowest, which is reflected in the lignin content of the candidate individual (18.2±1.2%) being 18.39% lower than that of individuals without mutations (22.3±0.6%); when the candidate individual is a heterozygous mutation, the lignin content (18.89%±0.6%) is 15.29% lower than that of individuals without mutations.

[0123] Example 3 Analysis of the regulatory effect of structural variant SV on PtoERD3 gene promoter activity

[0124] Given that the SV in the present invention is located in the promoter region of the PtoERD3 gene, in order to clarify whether the promoter insertion mutation of the PtoERD3 gene affects its promoter activity, a dual luciferase assay was used to conduct verification work. The specific operation is as follows:

[0125] (1) Cloning of two promoters of the PtoERD3 gene (with and without SV fragment insertion)

[0126] (i) DNA was extracted from poplar samples with and without SV fragment insertion according to the method shown in Example 2.

[0127] (ii) Referring to the nucleotide sequence of the PtoERD3 gene promoter (shown in SEQ ID NO. 2), the primer design method was the same as in Example 2 to obtain primers P7 and P8 (the sequence of P7 is shown in SEQ ID NO. 14, and the sequence of P8 is shown in SEQ ID NO. 15).

[0128] The extracted DNA was used as a template and PCR amplification was performed using Phanta Max Super-Fidelity DNA Polymerase from Nanjing Vazyme Company. The PCR reaction system (50 μL) is shown in Table 3:

[0129] Table 3

[0130]

[0131] PCR amplification program: 95°C for 3 min; 36 cycles of (95°C for 25 s; 57°C for 30 s; 72°C for 60 s (amplification efficiency 45 s / kb)); 72°C for 5 min; 4°C∞.

[0132] (iii) The PCR product was subjected to agarose gel electrophoresis and, if confirmed, purified using a DNA Clean-up Kit from Jiangsu Kangwei Century Biotechnology Co., Ltd. After purification, the purity and concentration of the purified DNA product were determined using an instrument.

[0133] (2) Construction of pGreenII 0800-Pro-LUC and pGreenII 0800-Pro+SV-LUC vectors

[0134] The pGreenII 0800-LUC plasmid was digested with SalI endonuclease. The digestion reaction system is shown in Table 4:

[0135] Table 4

[0136]

[0137] The enzyme digestion reaction conditions are: 37°C water bath for 7-9 hours. The plasmid after enzyme digestion is recovered by gel recovery as a vector backbone for later use and stored at -20°C.

[0138] (3) Connection conversion

[0139] (3.1) Ligation of expression vector

[0140] The two promoter fragments were separately cloned into the 0800 vector using the Uniclone One Step Seamless Cloning Kit from Beijing Jinsha Biotechnology Co., Ltd. to generate recombinant vectors. Ligation was performed at 50°C for 10 minutes, and the ligation products were used to transform competent E. coli DH5α cells.

[0141] (3.2) Transformation of E. coli DH5α competent cells

[0142] Take 50μl DH5α competent cells, add 5μl ligation product, flick to mix, and let it stand on ice for 30 minutes. Subsequently, place the mixture in a 42°C water bath for 45 seconds, quickly transfer to ice and cool for 2 minutes, and avoid shaking during the process to ensure transformation efficiency. Next, add 700μl of sterile LB liquid culture medium without antibiotics, blow to mix evenly, and place it on a shaker at 37°C and 220rpm for 15 minutes to revive the bacteria. After recovery, collect the bacteria by centrifugation at 6000rpm for 1 minute, retain 100μl of supernatant to resuspend the bacteria, spread it on LB solid plate culture medium containing kanamycin (100mg / ml), and invert and culture at 37°C for 14-16 hours.

[0143] (3.3) Identification of positive clones by PCR

[0144] Using a sterile pipette tip, single colonies from the pGreenII 0800-Pro-LUC and pGreenII 0800-Pro+SV-LUC plates were picked and placed in 250 μl of LB liquid medium containing kanamycin. Cultured at 37°C with shaking at 200 rpm / min for approximately 3 hours to serve as amplification templates. Separately, positive and negative controls were set up using the purified PCR product and ddH2O (double-distilled water) as templates. PCR amplification was performed using Taq Plus Master Mix from Nanjing Vazyme.

[0145] The PCR reaction system is shown in Table 5:

[0146] Table 5

[0147]

[0148]

[0149] The sequence of primer 0800-R is shown in SEQ ID NO.16.

[0150] The reaction conditions were: 95°C for 3 min; 35 cycles of (94°C for 30 s; 58°C for 30 s; 72°C for 60 s); 72°C for 5 min; 4°C∞.

[0151] The PCR products were detected by 1% agarose gel electrophoresis, and the colonies that could amplify the band with the same size as the positive control were considered positive clones.

[0152] (3.4) Extraction of positive clone plasmids

[0153] PCR-positive clones were transferred to 6 ml of LB liquid medium containing kanamycin and cultured overnight at 37°C with shaking at 200 rpm. Plasmids were extracted using a plasmid extraction kit from Jiangsu Kangwei Reagent Co., Ltd. and sequenced by Beijing Ruibo Xingke Biotechnology Co., Ltd. Vector construction was completed after sequence alignment was confirmed.

[0154] (4) Transformation of recombinant plasmid into Agrobacterium

[0155] (4.1) Add about 1 μg of pGreenII 0800-Pro-LUC and pGreenII 0800-Pro+SV-LUC recombinant plasmids to 100 μL of Agrobacterium GV3101 competent cells and mix gently.

[0156] (4.2) Place on ice for 5 minutes, quick-freeze in liquid nitrogen for 1 minute, immediately place in a 37°C water bath for 5 minutes, and then place in an ice bath for 5 minutes.

[0157] (4.3) Add 700 μL of antibiotic-free YEP liquid medium to the bacterial suspension, mix thoroughly, and incubate with shaking at 28°C and 200 rpm for 2-3 hours. After the recovery incubation, centrifuge the suspension at 6000 rpm for 1 minute, discard a portion of the supernatant, and retain 100 μL of the supernatant to mix with the suspension. Using a disposable sterile applicator, evenly spread the suspension onto the surface of YEP solid medium containing 50 mg / L rifampicin and 50 mg / L kanamycin. Incubate the suspension in an inverted position at 28°C in an incubator for 72-90 hours.

[0158] (4.4) After single colonies have grown, several monoclonal plaques were picked with a sterile pipette tip and placed in a 2 mL centrifuge tube containing 250 μL of YEP liquid medium (supplemented with Rif and Kana at a 1:1000 ratio). The culture was shaken at 30°C and 200 rpm for 2 h. After the culture was complete, the bacterial suspension was aspirated and identified by PCR using the same method as described in Example 2. Correctly identified positive bacterial suspensions were added to 50% glycerol, quickly frozen in liquid nitrogen, and stored at -80°C for subsequent experiments.

[0159] (5) Tobacco transient transformation experiment

[0160] (5.1) Remove Agrobacterium tumefaciens containing pGreenII 0800-Pro-LUC and pGreenII 0800-Pro+SV-LUC plasmids from the -80°C freezer, streak onto YEP solid medium (1:1000 supplemented with Rif and Kana), seal the plate, and incubate inverted in a dark incubator at 28°C for 2-3 days.

[0161] (5.2) Use a sterile pipette to pick a single colony from the plate and inoculate it into 3 mL of YEP liquid medium (1:1000 supplemented with Rif and Kana). Incubate overnight on a shaker at 30°C, 200 rpm. Transfer 1 mL of the bacterial suspension to a 250 mL sterile Erlenmeyer flask containing 100 mL of YEP liquid medium (1:1000 supplemented with Rif and Kana). Incubate on a shaker at 30°C, 200 rpm, for 4-5 hours, until the OD600 reaches 0.3-0.5.

[0162] (5.3) In a clean bench, transfer 100 mL of the bacterial suspension to two 50 mL sterile centrifuge tubes and centrifuge at 2560 g at 4°C for 20 min to collect the cells. In a clean bench, discard the supernatant and resuspend the cells in 100 mL of resuspension solution in a sterile wide-mouth bottle. This suspension will be used for subsequent tobacco transient transformation experiments. The resuspension solution formula is shown in Table 6:

[0163] Table 6

[0164]

[0165] (6.4) The resuspended pGreenII 0800-Pro-LUC and pGreenII 0800-Pro+SV-LUC bacterial solutions were allowed to stand at room temperature for 3-4 hours. Select tobacco leaves with good growth and inject the mixed bacterial solution into the lower epidermis of the leaves using a needleless syringe. The infected tobacco plants were placed in a greenhouse and cultured under 16h light / 8h dark conditions for 48-72 hours. The images were taken using a molecular imaging system (IVIS Lumina XRMS Series III, PerkinElmer, Waltham, USA). The results are shown in Figure 5. Figure 3 As shown. Figure 3 It can be seen that the fluorescence signal of pGreenII 0800-Pro+SV-LUC is significantly higher than that of pGreenII 0800-Pro-LUC, proving that the SV can significantly enhance the transcriptional activation activity.

[0166] Approximately 100 mg of infected tobacco leaf tissue was collected, ground with liquid nitrogen, and luciferase activity was detected using a Dual-Luciferase Reporter Assay System (Promega, Madison, USA) according to the kit instructions. The luminescence intensity (LUC) of firefly luciferase was measured using a multifunctional microplate reader (Varioskan LUX, Thermo Fisher, Waltham, USA). 100 μL Stop & Reagent, measure the luminescence intensity (REN) of Renilla luciferase. Using Renilla luciferase as an internal reference, calculate the ratio of the luminescence intensity of firefly luciferase to that of Renilla luciferase (LUC / REN) to measure the activation activity of SV on PtoERD3. The experiment was repeated at least 3 times, with 3 biological replicates each time. A control experiment was performed using an empty plasmid according to the above method, and the results were as follows: Figure 4 As shown. Figure 4 It can be seen that SV can significantly enhance the transcription of PtoERD3 and positively regulate the expression of PtoERD3.

[0167] Example 4 Construction of PtoERD3 gene overexpression vector and interference silencing vector

[0168] (1) Enzyme digestion of vector

[0169] The selected overexpression vector is pBI121 vector, which is 13629 bp in total and contains a strong 35S promoter (CaMV35S), is kanamycin-resistant (Kana), and can be digested with XbaI. The enzyme digestion reaction system is shown in Table 7:

[0170] Table 7

[0171]

[0172] The enzyme digestion reaction conditions are: 37°C water bath for 7-9 hours. The plasmid after enzyme digestion is recovered by gel recovery as a vector backbone for later use and stored at -20°C.

[0173] The enzyme-digested product was subjected to gel recovery and purification using the Kangwei Century Gel Extraction Kit and purification using the Kangwei Century DNA Clean-up Kit.

[0174] (2) RNA from wild-type white poplar was extracted using a plant RNA extraction kit from Beijing Quanshijin Biotechnology Co., Ltd.; the RNA was reverse transcribed into cDNA using a HiScript III 1st Strand cDNA Synthesis Kit (Nanjing Novozyme Biotechnology Co., Ltd.), and the cDNA obtained by reverse transcription was used as a template. Based on the CDS sequence of the PtoERD3 gene (as shown in SEQ ID NO.6), the overexpression and interference silencing primers were designed according to the same principles as in Example 2, and the overexpression primers P3 and P4 (sequences shown in SEQ ID NO.7 and SEQ ID NO.8) were obtained. The PCR experimental reaction system (50 μL) is shown in Table 8. The interference silencing primers were P5 and P6 (sequences shown in SEQ ID NO.12 and SEQ ID NO.13), and the PCR experimental reaction system (50 μL) is shown in Table 9.

[0175] Table 8

[0176]

[0177] Table 9

[0178]

[0179]

[0180] The reaction conditions were as follows: 95°C for 3 min; 35 cycles of (94°C for 30 s; 58°C for 30 s; 72°C for 60 s); 72°C for 5 min; and 4°C∞.

[0181] The PCR products were subjected to agarose gel electrophoresis and, if confirmed, purified using a DNA Clean-up Kit from Jiangsu Kangwei Century Biotechnology Co., Ltd. After purification, the purity and concentration of the purified DNA products were determined using an instrument.

[0182] Finally, the CDS sequence of the PtoERD3 gene was obtained, as shown in SEQ ID NO.6, and the nucleotide sequence of the interference fragment was shown in SEQ ID NO.11.

[0183] (3) The expression vector was connected, the competent E. coli DH5α cells were transformed, and the positive clones were identified by PCR of the bacterial solution. The PCR reaction system for overexpression of the PtoERD3 gene is shown in Table 10:

[0184] Table 10

[0185]

[0186] Among them, the sequence of SKP2a-F is shown as SEQ ID NO.17.

[0187] The PtoERD3 gene interference silencing PCR reaction system is shown in Table 11:

[0188] Table 11

[0189]

[0190] The conditions for PtoERD3 gene overexpression PCR reaction and interference silencing PCR reaction were: 95℃2min; (94℃30s; 58℃30s; 72℃60s) 35 cycles; 72℃5min; 4℃∞.

[0191] The PCR product was detected by 1% agarose gel electrophoresis. The colony that could amplify the same size band as the positive control was considered a positive clone. The vector construction was completed after the sequence was determined and aligned.

[0192] Example 5 Genetic transformation of PtoERD3 gene

[0193] (1) Transformation of Agrobacterium with recombinant plasmid

[0194] The method for transforming Agrobacterium with the recombinant plasmid is as described in Example 3.

[0195] (2) Activation of Agrobacterium

[0196] (2.1) Remove the Agrobacterium solution containing the overexpression vector plasmid and the interference silencing vector plasmid from the -80°C refrigerator, streak inoculate each onto YEP solid medium (1:1000 supplemented with Rif and Kana), seal the plate, and culture inverted in a dark incubator at 28°C for 2-3 days.

[0197] (2.2) Use a sterile pipette to pick a single colony from the plate and inoculate it into 3 mL of YEP liquid medium (1:1000 supplemented with Rif and Kana). Incubate overnight on a shaker at 30°C, 200 rpm. Transfer 1 mL of the bacterial suspension to a 250 mL sterile Erlenmeyer flask containing 100 mL of YEP liquid medium (1:1000 supplemented with Rif and Kana). Incubate on a shaker at 30°C, 200 rpm, for 4-5 hours, until the OD600 reaches 0.3-0.5.

[0198] (2.3) In a laminar flow hood, transfer 100 mL of bacterial suspension to two 50 mL sterile centrifuge tubes and centrifuge at 2560 g at 4°C for 20 min to collect the cells. In a laminar flow hood, discard the supernatant and resuspend the cells in 100 mL of resuspension buffer in a sterile wide-mouth bottle. This suspension will be used for subsequent infection experiments.

[0199] The resuspension was prepared according to the following ingredients and amounts: WPM (2.37 g) + VB 1 (0.9 mg) + MES (0.5 g) + sucrose (20 g) + 2,4-D (0.1 mg) + AS (200 mM) + ddH2O (to a volume of 1 L).

[0200] (3) Infection by wound healing method

[0201] Select leaves from healthy and sterile seedlings (dark green leaves with a thick texture), remove the petioles with a sterile scalpel, and leave the growing callus on the petioles. Make 2-3 wounds horizontally on the main vein of the leaves and spread them face down on the callus culture medium (CIM). Culture them in the dark at 25°C. After the leaves have grown for 20 to 30 days, white and loose callus will grow on the wounds. Peel the callus off the leaves, cut them into pieces the size of soybeans, and place them in new callus culture medium. Transfer all the prepared callus to a sterile wide-mouth bottle containing the bacterial solution and place it in a shaker at 28°C and 160rpm for infection for 15-20 minutes.

[0202] Among them, callus culture medium (CIM) was prepared according to the following ingredients and amounts: WPM (2.37 g) + 2,4-D (0.1 mg) + KT (0.1 mg) + MES (0.5 g) + agar (7.5 g) + ddH2O (concentrated to 1 L).

[0203] (4) Co-culture

[0204] In a clean bench, calli were removed from the bacterial solution with tweezers, excess bacterial solution was absorbed with filter paper, and the infected calli were spread onto co-culture medium (WPMC) and cultured upside down at 25°C in the dark for 2 days.

[0205] The co-culture medium was prepared according to the following ingredients and amounts: WPM (2.37 g) + VB 1 (0.9 mg) + sucrose (20 g) + MES (0.5 g) + AS (200 mM) + agar (7.5 g) + ddH2O (concentrated to 1 L).

[0206] (5) Differentiation culture

[0207] After dark culture, appropriate plant resistance is selected according to the carrier and differentiation medium containing corresponding antibiotics is prepared.

[0208] The calli are transferred to differentiation medium (WPMD) and the medium is changed every 20 days for the first time, and every 10 days thereafter. During this period, the calli will turn green, harden, and then partially turn red. This stage lasts for about two months and is maintained in a 25°C light incubator.

[0209] Among them, the co-culture medium was prepared according to the following components and amounts: WPM (2.37 g) + VB 1 (0.9 mg) + sucrose (20 g) + MES (0.5 g) + AS (200 mM) + 6-BA (0.5 mg) + NAA (0.1 mg) + TDZ (0.002 mg) + TMT (250 mg) + Cef (250 mg) + kana (20 mg) + agar (7.5 g) + ddH2O (fixed to 1 L).

[0210] (6) Root induction of clustered shoots

[0211] After the buds grow to about half a centimeter, use sterile forceps or a scalpel to cut them off (pay attention to the growth point of the buds to avoid selecting buds that have differentiated from the same cell) and place them in bud elongation medium (WPML) for growth. After a period of elongation culture, when the buds reach 1-2 centimeters, use sterile forceps or a scalpel to cut each bud individually and place them on rooting medium for rooting. After about 10 days, the buds will grow roots and grow into complete plants through rooting culture.

[0212] Among them, the elongation medium was prepared according to the following ingredients and amounts: WPM (2.37 g) + IBA (0.2 mg) + sucrose (20 g) + 6-BA (0.5 mg) + TMT (250 mg) + Cef (250 mg) agar (7.5 g) + ddH2O (concentrated to 1 L).

[0213] Example 6 Identification of PtoERD3 Gene Overexpression and Interference Silencing Plants

[0214] (1) Crude DNA extraction of wild-type Populus tomentosa, PtoERD3 gene overexpressing plants and PtoERD3 gene interference silenced plants was performed using the method described in Example 2.

[0215] (2) Identification of PtoERD3 gene overexpression plants and PtoERD3 gene silencing plants:

[0216] (2.1) DNA level identification

[0217] PCR identification was performed using Taq Plus Master Mix using crudely extracted gDNA from overexpressing plants and gDNA from interference-silenced plants as templates, respectively. The PCR reaction system was referred to Tables 10 and 11 in Example 4. The PCR products were directly subjected to electrophoresis to check whether the bands were correct. If correct, they were PtoERD3 gene-overexpressing plants or PtoERD3 gene-silenced plants.

[0218] (2.2) Identification of transcriptional levels

[0219] RT-qPCR was used to detect the transcription level of PtoERD3 in overexpression and interference silenced plants. The specific steps are as follows:

[0220] (i) Extract RNA from leaf tissue to be tested and reverse transcribe to obtain template cDNA. The RNA extraction and reverse transcription methods are the same as those in Example 4. The reaction system (20 μL) is shown in Table 12:

[0221] Table 12

[0222]

[0223] The reaction procedure was: 50°C for 5 min; 85°C for 5 sec.

[0224] (ii) RT-qPCR experiments were performed using 2×ChamQ SYBR Color qPCR Master Mix. In this example, the following fluorescent quantitative PCR (polymerase chain reaction) primers were designed based on the PtoERD3 gene sequence of Populus tomentosa. The primers are named PtoERD3-qPCR-F and PtoERD3-qPCR-R, and their nucleotide sequences are shown in SEQ ID NO. 9 and SEQ ID NO. 10, respectively. The nucleotide sequences of the internal reference primers Actin-F and Actin-R are shown in SEQ ID NO. 18 and SEQ ID NO. 19, respectively.

[0225] The reaction system (20 μL) is shown in Table 13:

[0226] Table 13

[0227]

[0228] The PCR reaction program was as follows: 95°C for 3 min; 40 cycles of (95°C for 5 sec; 60°C for 30 sec; 72°C for 15 sec); 72°C for 5 min; and 4°C∞.

[0229] The overexpression detection results of RT-qPCR are as follows Figure 5 As shown by Figure 5 It can be seen that the overexpression plants numbered OE-2, OE-3 and OE-5 have higher expression levels, which are 99.5 times, 151.3 times and 130 times that of the wild-type plant (WT), respectively.

[0230] The results of RT-qPCR interference silencing detection are as follows Figure 6 As shown by Figure 6 It can be seen that the overexpression plants numbered RNAi-1, RNAi-6 and RNAi-8 have higher expression levels, which are 0.06 times, 0.12 times and 0.11 times that of the wild-type plant (WT), respectively.

[0231] Example 7 Phenotypic Analysis of PtoERD3 Gene Overexpression Plants and Interference Silencing Plants

[0232] PtoERD3 gene overexpressing plants, interference silenced plants and wild-type Populus tomentosa were planted in a greenhouse at the same time, and the plants were phenotypically measured and photographed.

[0233] like Figure 7 As shown, from left to right are the phenotypes of the PtoERD3 overexpressing plant (denoted as OE-PtoERD3) of the present invention, the phenotype of the wild type white poplar (denoted as WT), and the phenotype of the interference silenced plant (denoted as RNAi-PtoERD3). Figure 7It can be seen that compared with wild-type Populus tomentosa, the PtoERD3-silenced plants were taller and had a thicker ground diameter, while the growth of PtoERD3-overexpressing plants was significantly inhibited, with shorter plant height and thinner ground diameter. Figure 8 and Figure 9 The results of plant height and ground diameter analysis of overexpression plants, silenced plants and wild-type Populus tomentosa are shown respectively.

[0234] Furthermore, the Klason method was used to determine the lignin content of the plants. Figure 10 As shown in the figure, the lignin content of PtoERD3-silenced plants increased by 36% compared with the wild type, while the lignin content of PtoERD3-overexpressed plants decreased by 17.1% compared with the wild type. These results indicate that the PtoERD3 gene negatively regulates the lignin content of poplar.

[0235] The present invention has been described in detail above with reference to specific embodiments and exemplary examples. However, these descriptions are not to be construed as limiting the present invention. Those skilled in the art will appreciate that, without departing from the spirit and scope of the present invention, various equivalent substitutions, modifications, or improvements may be made to the technical solutions and implementations of the present invention, all of which fall within the scope of the present invention.

Claims

1. A molecular marker related to the lignin content of poplar, characterized in that: The molecular marker is a 54bp chromosome structural variation SV fragment, The chromosome structural variation SV fragment has a nucleotide sequence as shown in SEQ ID NO. 1, or has a nucleotide sequence having more than 90% homology with the nucleotide sequence.

2. The molecular marker according to claim 1, characterized in that The molecular marker is located in the upstream promoter region of the poplar PtoERD3 gene, and the nucleotide sequence of the upstream promoter of the PtoERD3 gene is shown in SEQ ID NO.

2.

3. A primer pair for detecting the molecular marker according to claim 1, characterized in that: The primer pair includes primer P1 and primer P2, primer P1 has a sequence as shown in SEQ ID NO.3, and primer P2 has a sequence as shown in SEQ ID NO.

4.

4. The primer pair according to claim 3, characterized in that Using the genomic DNA of the poplar to be tested as a template, primer pairs P1 and P2 were used to amplify the molecular markers related to the lignin content of the poplar, and then the identification was carried out. If the amplified product contains the molecular marker described in claim 1, the poplar lignin content is low; if the amplified product does not contain the molecular marker described in claim 1, the poplar lignin content is high.

5. Use of the molecular marker according to claim 1 in detecting the lignin content of poplar, characterized in that: The application comprises the following steps: First, PCR amplification was performed using the genomic DNA of the tested poplar as a template; Then, based on the amplified product, it is determined whether it contains molecular markers, and then the lignin content of the poplar is determined.

6. Use of the molecular marker according to claim 1 in regulating the content of lignin in poplar, characterized in that: The regulation of the lignin content of poplar by the molecular marker is achieved by overexpressing the PtoERD3 gene or silencing the PtoERD3 gene. Lignin content was reduced by overexpressing the PtoERD3 gene in poplar plants, while lignin content was increased by silencing the PtoERD3 gene in poplar plants.

7. The use according to claim 6, characterized in that The method for reducing the lignin content of poplars by molecular markers includes the following steps: Step 1: construct an overexpression vector of the PtoERD3 gene and transform it into Agrobacterium to obtain a positive overexpression engineered bacterium; Step 2: Infecting poplar materials with positive overexpression engineered bacteria, and obtaining overexpression positive seedlings through cultivation and identification; Step 3: Transplant the overexpression positive seedlings to obtain poplar plants with reduced lignin content.

8. The use according to claim 6, characterized in that The method for increasing the lignin content of poplars by molecular markers comprises the following steps: Step i, constructing an interference silencing vector for the PtoERD3 gene and transforming it into Agrobacterium to obtain a positive gene silencing engineered bacterium; Step ii, infecting poplar materials with positive gene silencing engineered bacteria, culturing transgenic seedlings, and obtaining gene silencing positive seedlings after identification; Step iii: transplanting the gene-silencing positive seedlings to obtain poplar plants with increased lignin content.

9. The use according to claim 8, characterized in that In step i, the interference silencing vector of the PtoERD3 gene is obtained by constructing the interference fragment of the PtoERD3 gene into the pBI121 vector. The nucleotide sequence of the interference fragment of the PtoERD3 gene is shown in SEQ ID NO.

11.

10. A method for genetic improvement of poplars, characterized in that: The genetic improvement is to increase the wood content of poplar. The method comprises the steps of successive breeding of poplar individuals that do not have the molecular marker according to claim 1, and eliminating poplar individuals that have the molecular marker.

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