Application of Populus tomentosa PtrSCZ1 and PtrSCZ3 genes in regulating development of tree vascular cambium

Abstract

The application belongs to the technical field of genetic engineering, and particularly relates to application of Populus davidiana PtrSCZ1 and PtrSCZ3 genes in regulation of tree vascular cambium development. The problem of tree vascular cambium development is solved. The application includes application of a recombinant vector or an engineering bacterium of the Populus davidiana PtrSCZ1 and PtrSCZ3 genes in regulation of tree vascular cambium development. The application obtains Populus davidiana transgenic plants with overexpression of PtrSCZ1 and PtrSCZ3 through a Populus davidiana genetic transformation system mediated by Agrobacterium. It is found through a series of technical means that overexpression of the PtrSCZ1 and PtrSCZ3 transcription factors makes the Populus davidiana grow slowly, and the number of vascular cambium cell layers is increased. The research content of the application has important theoretical guiding significance for cultivation of high-quality wood tree new varieties.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically involving the application of the PtrSCZ1 and PtrSCZ3 genes of Populus tomentosa in regulating the development of the vascular cambium in trees. Background Technology

[0002] Timber is a natural, renewable resource and has long been widely used as an important raw material in industries such as construction, papermaking, and furniture manufacturing. Simultaneously, timber is also an important source of biomass energy. Compared to other raw materials, timber is environmentally friendly, safe, economical, and efficient. Using timber as a primary raw material to meet various needs of human production and daily life aligns with the requirements of sustainable development. In addition to economic benefits, timber, as an important byproduct of tree secondary growth, also possesses significant ecological benefits, such as carbon sequestration.

[0003] Wood, also known as secondary xylem, originates from the dynamic and continuous biological processes of vascular cambium activity. Through division and differentiation, the vascular cambium continuously produces xylem and phloem cells to both sides. Xylem cells undergo cell expansion, secondary cell wall biosynthesis, and programmed cell death to become secondary xylem (i.e., wood). This process is precisely regulated by both internal and external factors of the tree, such as plant hormones, local peptide signaling, and various interacting regulatory factors. Transcription factors play a central regulatory role in the development of the vascular cambium, with some even acting as marker genes. Previous studies have found that WOX4, as a marker gene for the vascular cambium, is a core transcription factor regulating vascular cambium division activity. Ethylene and TDIF signaling jointly regulate WOX4 expression, thus controlling cambium activity. The regulatory role of WOX4 is also conserved in the woody plant poplar. Poplar lines with suppressed expression of PttWOX4a / b show reduced vascular cambium division activity and decreased secondary xylem production. The number of cambium proliferation zone cell layers was significantly reduced in the poplar mutant ptrwox4a / ptrwox4b, which co-knockout PtrWOX4a and its homolog PtrWOX4b.

[0004] Nevertheless, current research still reveals very little about the key transcription factors in the development of the vascular cambium in trees. Identifying the key regulatory factors of the tree vascular cambium and revealing the molecular regulatory mechanisms of vascular cambium development during wood formation will provide important candidate genes and theoretical references for using molecular breeding techniques to cultivate new tree varieties that meet human needs.

[0005] Therefore, it is urgent to propose the application of the PtrSCZ1 and PtrSCZ3 genes in regulating the development of the vascular cambium in Populus tomentosa to solve the above-mentioned technical problems. Summary of the Invention

[0006] This invention is of great significance for the research and development of regulation of the vascular cambium in trees and the cultivation of new timber varieties. A brief overview of the invention is provided below to offer a basic understanding of certain aspects of it. It should be understood that this overview is not an exhaustive summary of the invention. It is not intended to identify key or essential parts of the invention, nor is it intended to limit the scope of the invention.

[0007] The technical solution of this invention:

[0008] Application of the PtrSCZ1 and PtrSCZ3 genes of Populus tomentosa in regulating the development of the vascular cambium in trees, and application of the recombinant vector or engineered bacteria of the PtrSCZ1 and PtrSCZ3 genes of Populus tomentosa in regulating the development of the vascular cambium in trees.

[0009] Preferred: Overexpression of the PtrSCZ1 and PtrSCZ3 genes slows down the growth of Populus tomentosa, increases the number of vascular cambium layers, and advances the secondary development of xylem and phloem.

[0010] Preferably, the nucleotide sequences of the Populus tomentosa PtrSCZ1 and PtrSCZ3 genes are shown in SEQ ID No. 1 and SEQ ID No. 2.

[0011] Preferably, the amino acid sequences of the proteins encoded by the Populus tomentosa PtrSCZ1 and PtrSCZ3 genes are shown in SEQ ID No. 3 and SEQ ID No. 4.

[0012] Preferably, the recombinant vector is a plant expression vector pBI121 in which the PtrSCZ1 and PtrSCZ3 genes of Populus tomentosa are recombined.

[0013] Preferred: The engineered bacteria is one of Escherichia coli or Agrobacterium.

[0014] A method for increasing the number of vascular layers in Populus tomentosa involves constructing a recombinant vector containing the Populus tomentosa PtrSCZ1 and PtrSCZ3 genes as described in claim 1, and transforming Populus tomentosa to obtain transgenic plants.

[0015] Preferred: The plant expression vector used for constructing the recombinant vector is pBI121.

[0016] Preferred method: Agrobacterium-mediated transformation.

[0017] The present invention has the following beneficial effects:

[0018] This invention obtained transgenic poplar plants overexpressing PtrSCZ1 and PtrSCZ3 through an Agrobacterium-mediated Populus tomentosa genetic transformation system.

[0019] This invention utilizes a series of technical methods to analyze and discover that overexpression of PtrSCZ1 and PtrSCZ3 transcription factors slows down the growth of Populus tomentosa and increases the number of vascular cambium cell layers.

[0020] The research content of this invention has important theoretical guiding significance for cultivating new varieties of high-quality timber trees. Attached Figure Description

[0021] Figure 1 This is a graph showing the results of transcriptional level identification in Populus tomentosa plants overexpressing PtrSCZ1 and PtrSCZ3.

[0022] Figure 2 This is a graph showing the growth status of wild-type Populus tomentosa plants after 4 months of greenhouse growth following overexpression of PtrSCZ1 and PtrSCZ3.

[0023] Figure 3 This is a graph showing the results of the growth phenotype analysis of PtrSCZ1 and PtrSCZ3 overexpression and wild-type Populus tomentosa plants after 4 months of greenhouse growth.

[0024] Figure 4 The image shows the results of cross-sectional cell morphology observation of the 5th, 6th, and 8th stem nodes of Populus tomentosa plants overexpressing PtrSCZ1 and PtrSCZ3.

[0025] Figure 5 This is a diagram showing the morphological observation of the cambium at the 8th node of a *Populus pubescens* plant overexpressing PtrSCZ1 and PtrSCZ3.

[0026] Figure 6 This is a statistical result of the number of cambium cell layers in the 5th, 6th, and 8th stem nodes of Populus tomentosa plants overexpressing PtrSCZ1 and PtrSCZ3 according to the present invention.

[0027] Figure 1 In the table: OE-PtrSCZ1-L1, OE-PtrSCZ1-L4 and OE-PtrSCZ3-L3, OE-PtrSCZ1-L12 represent two different lines of plants overexpressing PtrSCZ1 and PtrSCZ3, respectively; WT represents wild-type Populus tomentosa; error bars represent the standard error calculated from at least three biological replicates; asterisks represent t-test results; **P<0.01;

[0028] Figure 2 In the text: OE-PtrSCZ1-L1, OE-PtrSCZ1-L4 and OE-PtrSCZ3-L3, OE-PtrSCZ1-L12 represent two different strains of plants overexpressing PtrSCZ1 and PtrSCZ3, respectively. WT represents wild-type Populus tomentosa. The scale bar is 10cm.

[0029] Figure 3In the table, OE-PtrSCZ1-L1, OE-PtrSCZ1-L4, OE-PtrSCZ3-L3, and OE-PtrSCZ1-L12 represent two different lines of plants overexpressing PtrSCZ1 and PtrSCZ3, respectively. WT represents wild-type Populus tomentosa. Error bars represent the standard error calculated from at least 5 biological replicates. Asterisks represent t-test results. **P<0.01.

[0030] Figure 4 Medium scale = 500 μm;

[0031] Figure 5 In the middle, WT represents wild-type poplar, P represents phloem cells, C represents cambium cells, X represents xylem cells, and the scale bar is 25 μm.

[0032] Figure 6 In the text, WT represents wild-type Populus tomentosa, the error bar represents the standard error calculated by at least three biological replicates, the asterisk represents the t-test result, *P<0.05, **P<0.01. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of this invention clearer, the invention is described below with reference to specific embodiments shown in the accompanying drawings. However, it should be understood that these descriptions are merely exemplary and not intended to limit the scope of the invention. Furthermore, descriptions of well-known structures and technologies are omitted in the following description to avoid unnecessarily obscuring the concept of the invention.

[0034] Specific implementation method one: Combining Figures 1-6 This embodiment describes the application of the PtrSCZ1 and PtrSCZ3 genes of Populus tomentosa in regulating the development of the vascular cambium in trees, including the application of recombinant vectors or engineered bacteria of the PtrSCZ1 and PtrSCZ3 genes in regulating the development of the vascular cambium in trees.

[0035] Specific Implementation Method Two: Combining Figures 1-6 This embodiment describes the application of the PtrSCZ1 and PtrSCZ3 genes in regulating the development of the vascular cambium in Populus tomentosa. Overexpression of the PtrSCZ1 and PtrSCZ3 genes slows down the growth of Populus tomentosa, increases the number of vascular cambium layers, and advances the secondary development of xylem and phloem.

[0036] Specific implementation method three: Combining Figures 1-6 This embodiment describes the application of the PtrSCZ1 and PtrSCZ3 genes of Populus tomentosa in regulating the development of the vascular cambium in trees. The nucleotide sequences of the PtrSCZ1 and PtrSCZ3 genes of Populus tomentosa are shown in SEQ ID No. 1 and SEQ ID No. 2.

[0037] Specific implementation method four: Combination Figures 1-6 This embodiment describes the application of the Populus tomentosa PtrSCZ1 and PtrSCZ3 genes in regulating the development of the vascular cambium in trees. The amino acid sequences of the proteins encoded by the Populus tomentosa PtrSCZ1 and PtrSCZ3 genes are shown in SEQ ID No. 3 and SEQ ID No. 4.

[0038] Specific Implementation Method Five: Combining Figures 1-6 This embodiment describes the application of the Populus tomentosa PtrSCZ1 and PtrSCZ3 genes in regulating the development of the vascular cambium in trees. The recombinant vector is the Populus tomentosa PtrSCZ1 and PtrSCZ3 genes recombined into the plant expression vector pBI121.

[0039] Specific Implementation Method Six: Combination Figures 1-6 This embodiment describes the application of the PtrSCZ1 and PtrSCZ3 genes of Populus tomentosa in regulating the development of the vascular cambium in trees. The engineered bacteria is one of Escherichia coli and Agrobacterium.

[0040] Specific implementation method seven: Combination Figures 1-6 This embodiment describes a method for increasing the number of vascular layers in Populus tomentosa, which involves constructing a recombinant vector containing the Populus tomentosa PtrSCZ1 and PtrSCZ3 genes as described in claim 1, and transforming Populus tomentosa to obtain transgenic plants.

[0041] Specific implementation method eight: Combination Figures 1-6 This embodiment describes a method for increasing the number of vascular layers in Populus tomentosa, wherein the plant expression vector used in the construction of the recombinant vector is pBI121.

[0042] Specific Implementation Method Nine: Combining Figures 1-6 This embodiment describes a method for increasing the number of vascular layers in Populus tomentosa, wherein the transformation method is an Agrobacterium-mediated transformation method.

[0043] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials, reagents, enzymes, competent cells, plasmids, etc. used are all commercially available.

[0044] Example 1: Obtaining the PtrSCZ1 and PtrSCZ3 transcription factor genes from Populus tomentosa and constructing plant expression vectors.

[0045] 1. Cloning of PtrSCZ1 and PtrSCZ3 gene sequences

[0046] Total RNA was extracted from wild-type Populus trichocarpa Nisqually-1 genotype plants using the Qiagen Plant RNA Extraction Kit, and cDNA was obtained by reverse transcription using the Takara Reverse Transcription Kit (RR047A).

[0047] Referring to the genome sequence information of Populus tomentosa provided on the phytozome website, gene-specific primers were designed at both ends of the target gene sequence and the primers were synthesized by a biotechnology company. The upstream and downstream primer sequences for cloning the PtrSCZ1 and PtrSCZ3 genes are shown in Table 1.

[0048] Table 1. Upstream and downstream primer sequences used for cloning the PtrSCZ1 and PtrSCZ3 genes.

[0049]

[0050]

[0051] Note: The underlined part represents the enzyme cleavage site, and the bases preceding the enzyme cleavage site are protective bases.

[0052] The target sequence was obtained by polymerase chain reaction (PCR) using the primers described above. The PCR product was then separated into target bands by agarose gel electrophoresis. The target fragment was recovered from the gel using a Qiagen gel extraction kit.

[0053] 2. Construction of plant expression vectors

[0054] The recovered target gene DNA fragment and the pBI121 plant expression vector were subjected to double enzyme digestion. The specific reaction system and reaction conditions are as follows:

[0055] Target DNA fragment digestion system:

[0056]

[0057] pBI121 vector enzyme digestion system:

[0058]

[0059] Enzyme digestion conditions:

[0060] 37℃ for 1 hour

[0061] After enzyme digestion, the target band was separated by electrophoresis and recovered using a Qiagen gel extraction kit. The target gene was then inserted into the plant expression vector pBI121 using T4 ligase. The specific reaction system and conditions are as follows:

[0062] T4 connection system:

[0063]

[0064] Connection conditions:

[0065] 4℃ overnight

[0066] Thaw the TOP10 competent cells on ice, add all the ligation products to the competent cells, and mix thoroughly. Heat shock at 42°C for 1 minute, place on ice for 3 minutes, add antibiotic-free LB medium, and activate at 37°C and 200 rpm for 1 hour. Collect the cells at low speed and spread them on solid LB medium (containing 50 mg / L kanamycin), and incubate overnight at 37°C. The next day, perform PCR identification of the obtained single clones using the vector primers, as shown in Table 2.

[0067] Table 2 Primer sequences used for monoclonal detection in plant expression vector construction.

[0068]

[0069] PCR products were analyzed by electrophoresis to identify positive clones. Positive single clones were picked and cultured overnight in 5 mL LB medium (kanamycin, 50 mg / L) at 37°C, 220 rpm. The next day, the bacterial strain was preserved and collected by centrifugation. Plasmids were extracted, and the extracted plasmids were sequenced using primers 35SP1 and NosT2, following the instructions of the Qiagen plasmid extraction kit.

[0070] Based on the Populus hairy-fruited genomic sequence information provided by the phytozome website, the target genes were obtained using PCR and ligated into the pBI121 vector. Sequencing analysis revealed that the obtained PtrSCZ1 and PtrSCZ3 genes are 810 bp (SEQ ID NO.1) and 816 bp (SEQ ID NO.2), respectively, encoding 270 amino acids (SEQ ID NO.3) and 272 amino acids (SEQ ID NO.4), which are consistent with the Populus hairy-fruited genomic sequence information provided by the phytozome website, indicating that we successfully obtained the Populus hairy-fruited genomic sequence PtrSCZ1 and PtrSCZ3 genes.

[0071] SEQ ID NO.1:

[0072] ATGGCTTTTACAGTGGATAGATGTGAAGAGATGGTGTTTACTGTGGAGTCTCAAAAGGCAGTTCCTGCACCATTCTTAACAAAAACATATCAGCTGGTTGATGATCCTCTTACTGACCACATTGTGTCTTGGGGTGATGATGAAACCAGCTTTGTTGTGTGGAGACCTCCTGAGTTTTCAAGGGATCTTCTTCCCAACTATTTCAAGCACAACAACTTCTCAAGCTTCGTCAGGCAGCTCAATACCTATGGATTCAAGAAGGTAGTAGCTGACAGATGGGAGTTTGCAAATGAGTACTTCAGAAAAGGAGCAAAGCACTTGTTATCTGAGATCCACAGGAGAAAAACATCCCAACACCATCACCAGCACTACCCTGAGCAACCACCCCAATTTTTCCAACCAGAAGATGGTTTTAGTTGGATCGACCCTCCATTTCAATCTCCAAAATCAAGTACTGACATCCTAACTGCACTCTCAGAAGATAATCAGCGACTGAGAAGGAAAAACTGCATGCTTTTATCAGAACTCTCCCACATGAAGAACCTCTATAATGACATTATCTACTTCATCCAAAACCATGTAAAACCAATGCCCTATGAGCAAAAGGCTTATAATGCAGCACCTAAGCTAATAGAACTGGGCTCTTCATGTCAGGATCAAACCATTTGTTCTAGCATTCAAAGAGCTAAGAATGGTACTGTTTTGGGTCAGCATTCATTGACATTAAGTACTGAGGAATCAACTAGTCCTGTGAAGCTCTTTGGAGTCCCTCTCAGTGGCAATAAAAGGCTGCATCCAGAAGTGATCGAT

[0073] SEQ ID NO.2:

[0074] ATGGCATTTACAGTGGATAGGTGTGAAGAGATGGTGTTTACTGTGGAGTCTCAAAAGGCAGTTCCTGCACCCTTCTTAACAAAAACTTACCAGCTAGTTGATGACCCTCTTACAGACCACGTTGTCTCTTGGGGTGATGATGAAACCACCTTTGTTGTGTGGAGACCTCCTGAGTTTGCTAGGGAACTCCTCCCCAATTACTTCAAGCACAACAACTTCTCAAGCTTTGTCAGGCAGCTCAACACCTACGGATTCAAGAAGGTAGTAACTGACAGGTGGGAGTTTGCAAATGAGTACTTCAGAAAAGGAGCAAAGCAGTTGCTATCTGAGATTCACAGGAGAAAAACAATATCCCAGCACCATCACCAACACTACCCTGACCAAGCAACCCAATTTCTCCAATCAGAAGATCATGGTTTTGGTTGGATTGATCCTCCATTTCCATCTCCAAAACCAAATGTTGATCATATCCTAACTGCCCTCTCAGAAGACAACCAGAAACTGAGAAGGAAAAACTGTATGCTTTTATCAGAACTCTCCCACATGAAGAATCTCTATAATGACATTATCTACTTCATCCAAAACCATGTAAAACCAGTGCCCAATGAGCAAAAAGCTTACAATACAGTACCCAAGCTTATAGAGCCGGGTTCTTCATGTCAAGATCAAACCATTTGTTTTGGCGTTCAAAGGGCTAAGAATGGTGTTTTGGGTAAGCATTCATTAACATTTAGTACTGAGGAATCATCTAGTCCAGTGAAGCTTTTTGGAGTCCCTCTCATTGATAACAAAAGGCTGCATCCAGAAGCGATTGAA

[0075] SEQ ID NO.3:

[0076] MAFTVDRCEEMVFTVESQKAVPAPFLTKTYQLVDDPLTDHIVSWGDDETSFVVWRPPEFSRDLLPNYFKHNNFSSFVRQLNTYGFKKVVADRWEFANEYFRKGAKHLLSEIHRRKTSQHHHQHYPEQPPQFFQPE DGFSWIDPPFQSPKSSSTDILTALSEDNQRLRRKNCMLLSELSHMKNLYNDIIYFIQNHVKPMPYEQKAYNAAPKLIELGSSCQDQTICSSIQRAKNGTVLGQHSLTLSTEESTSPVKLFGVPLSGNKRLHPEVID

[0077] SEQ ID NO.4:

[0078] MAFTVDRCEEMVFTVESQKAVPAPFLTKTYQLVDDPLTDHVVSWGDDETTFVVWRPPEFARELLPNYFKHNNFSSFVRQLNTYGFKKVVTDRWEFANEYFRKGAKQLLSEIHRRKTISQHHHQHYPDQATQFLQSE DHGFGWIDPPFPSPKPNVDHILTALSEDNQKLRRKNCMLLSELSHMKNLYNDIIYFIQNHVKPVPNEQKAYNTVPKLIEPGSSCQDQTICFGVQRAKNGVLGKHSLTFSTEESSSPVKLFGVPLIDNKRLHPEAIE

[0079] Example 2: Obtaining Populus tomentosa plants overexpressing PtrSCZ1 and PtrSCZ3

[0080] 1. Freeze-thaw transformation of Agrobacterium

[0081] Step 1: Take out Agrobacterium competent cells and place them on ice. Add about 30 ng of the plasmid constructed in Example 1 to the Agrobacterium competent cells, mix well, and then incubate on ice for 30 min. Immediately place them in liquid nitrogen and freeze for 1 min, then immediately place them in a water bath at 37°C for 3 min.

[0082] Step 2: Add 1 mL of liquid LB and incubate at 28℃ and 180-200 rpm for 2-3 hours;

[0083] Step 3: Incubate briefly for 15 seconds, remove the supernatant, retain 300-400 μL of supernatant, resuspend, spread on LB (kanamycin + gentamicin) solid medium, and incubate at 28°C for about 48 hours;

[0084] Step 4: Select large clones and inoculate them into 5 mL of LB (kanamycin + gentamicin) liquid medium, and incubate overnight at 220 rpm and 28°C.

[0085] Step 5: Perform PCR using bacterial culture as a template to confirm positive clones. Refer to Example 1 for specific methods.

[0086] Step 6: Preserve positive clones by mixing 500 mL of bacterial culture with 500 mL of 50% glycerol, flash freezing in liquid nitrogen, and storing at -80°C.

[0087] 2. Agrobacterium-mediated genetic transformation of Populus tomentosa

[0088] Step 1: Take 10 μL of Agrobacterium tumefaciens containing the plant expression vector and add it to 5 mL of resistant LB medium (50 mg / L kanamycin + 50 mg / L gentamicin). Incubate overnight at 28°C and 220 rpm.

[0089] Step 2: The next day, take 100 μL of the cultured bacterial solution and add it to 50 mL of resistant LB liquid medium (50 mg / L kanamycin + 50 mg / L gentamicin), incubate at 28℃ and 220 rpm until OD600 = 0.4, and set aside for later use;

[0090] Step 3: Take tender stem segments from 3-6 month old poplar seedlings in greenhouses and disinfect them in 10% sodium hypochlorite for 20 minutes;

[0091] Step 4: Thoroughly wash and disinfect the explant stem segments with sterile water;

[0092] Step 5: Cut the stem segments into small pieces of about 5mm and place them into the cultured bacterial solution for 5 minutes;

[0093] Step 6: Remove the stem segments and place them on CIM1 medium, then incubate in the dark for 2 days;

[0094] Step 7: Two days later, thoroughly wash the explant stem segments with sterile water and place them on CIM2 medium for incubation in the dark, changing the medium every 2 weeks.

[0095] Step 8: After culturing in the dark on CIM2 medium for 2 months, transfer to SIM1 medium and culture under light. After one month, transfer to SIM2 medium and culture under light.

[0096] Step 9: After the resistant buds have grown, insert them into the rooting medium to induce rooting.

[0097] 3. Identification of transgenic plants

[0098] Step 1: Plant the resistant plants together with wild-type plants of the same condition in the soil and cultivate them until they are 4 months old. Collect the cambium tissue of the resistant plants and the wild-type control.

[0099] Step 2: Perform RNA extraction. Refer to the instructions for the Plant RNA Extraction Kit (Qiagen) for specific steps.

[0100] Step 3: The obtained RNA was used to determine its concentration and quality using NanoDrop.

[0101] Step 4: Refer to the instructions and use OligdT primers to reverse transcribe the obtained RNA to obtain cDNA;

[0102] Step 5: Dilute the obtained cDNA 10-fold and perform RT-qPCR. The internal control is PtrActin7. Primer sequences are shown in Table 3.

[0103] Quantitative PCR reaction system 15μL:

[0104]

[0105] Step Six: Use 2 △△Ct The method is used to calculate quantitative results.

[0106] Table 3 Primer sequences used for identifying transgenic plants

[0107]

[0108] Two independent resistant lines were obtained through Agrobacterium-mediated genetic transformation, named OE-PtrSCZ1-L1, OE-PtrSCZ1-L4, and OE-PtrSCZ3-L3, respectively. Transcriptional analysis revealed that, compared to wild-type Populus tomentosa, the transcriptional levels of the PtrSCZ1 and PtrSCZ3 genes were significantly increased in the resistant lines. Figure 1 This indicates that transgenic Populus tomentosa plants with overexpression of the PtrSCZ1 and PtrSCZ3 genes were successfully obtained. The growth status of PtrSCZ1 and PtrSCZ3 overexpressed plants and wild-type plants after 4 months of greenhouse growth is shown below. Figure 2 As shown.

[0109] Example 3: Application of the PtrSCZ1 and PtrSCZ3 genes in regulating the development of xylem vascular cambium in Populus tomentosa

[0110] 1. Phenotypic analysis of the growth of transgenic and wild-type Populus tomentosa obtained in Example 2.

[0111] Growth phenotypic analysis includes the following indicators:

[0112] Tree height measurement: The height from the soil surface to the terminal bud was measured on 4-month-old wild-type and transgenic greenhouse plants that overexpressed PtrSCZ1 and PtrSCZ3, with at least 5 biological replicates for both wild-type and transgenic plants.

[0113] Number of stem segments: The number of stem segments was counted for 4-month-old wild-type and transgenic greenhouse plants that overexpressed PtrSCZ1 and PtrSCZ3, with at least 5 biological replicates for both wild-type and transgenic plants.

[0114] Basal stem measurement: The diameter at the soil surface was measured using vernier calipers on 4-month-old wild-type and transgenic greenhouse plants that overexpressed PtrSCZ1 and PtrSCZ3, with at least 5 biological replicates for both wild-type and transgenic plants.

[0115] 2. Statistical analysis of the number of cambium cell layers was performed on the transgenic and wild-type Populus tomentosa plants obtained in Example 2.

[0116] Paraffin sections and histochemical staining were performed on the 5th, 6th, and 8th stem segments of wild-type and PtrSCZ1 and PtrSCZ3 overexpressing plants grown in a greenhouse for 4 months. Toluidine blue was used as the staining agent. Statistical analysis of cells was performed using an M8 scanner (Precipoint) and ViewPoint software. The number of cambium cell layers in each stem segment was counted, with 30 cell files collected from each segment. Statistical data processing and analysis were performed using SPSS software. The results showed that the number of cambium cell layers in the overexpressing plants was significantly higher than that in the wild type (see...). Figure 4 , Figure 5 For example, in the 8th stem node, the wild type has an average of about 6 layers of cambium cells, while transgenic plants that overexpress PtrSCZ1 and PtrSCZ3 have about 1 to 2 more layers (see...). Figure 6 This shows that the PtrSCZ1 and PtrSCZ3 genes affect the secondary growth of trees by regulating cambium development.

[0117] It should be noted that in the above embodiments, as long as the technical solutions are not contradictory, they can be permuted and combined. Those skilled in the art can exhaust all possibilities based on the mathematical knowledge of permutation and combination. Therefore, the present invention will not describe the technical solutions after permutation and combination one by one, but it should be understood that the technical solutions after permutation and combination have been disclosed by the present invention.

[0118] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The application of the PtrSCZ1 and PtrSCZ3 genes in regulating the development of the vascular cambium in Populus tomentosa, characterized by: The nucleotide sequences of the PtrSCZ1 and PtrSCZ3 genes of Populus tomentosa are shown in SEQ ID No. 1 and SEQ ID No.

2. Overexpression of the PtrSCZ1 and PtrSCZ3 genes slows the growth of Populus tomentosa, increases the number of vascular cambium layers, and advances the secondary development of xylem and phloem. The development of the vascular cambium of Populus tomentosa can be regulated by recombinant vectors or engineered bacteria containing the PtrSCZ1 and PtrSCZ3 genes of Populus tomentosa.

2. The application of the PtrSCZ1 and PtrSCZ3 genes of Populus tomentosa according to claim 1 in regulating the development of the vascular cambium in trees, characterized in that: The recombinant vector is formed by recombining the PtrSCZ1 and PtrSCZ3 genes of Populus tomentosa into the plant expression vector pBI121.

3. The application of the PtrSCZ1 and PtrSCZ3 genes of Populus tomentosa according to claim 1 in regulating the development of the vascular cambium of trees, wherein the engineered bacteria is one of Escherichia coli and Agrobacterium.

4. A method for increasing the number of vascular layers in Populus hairgrass, characterized in that: A recombinant vector containing the PtrSCZ1 and PtrSCZ3 genes of Populus tomentosa as described in claim 1 was constructed, and Populus tomentosa was transformed to obtain transgenic plants.

5. The method for increasing the number of vascular layers in Populus tomentosa according to claim 4, characterized in that: The plant expression vector used to construct the recombinant vector was pBI121.

6. The method for increasing the number of vascular layers in Populus tomentosa according to claim 4, characterized in that: The transformation method is the Agrobacterium-mediated transformation method.

Images