Application of populus trichocarpa PtrSCZ1 and PtrSCZ3 genes in regulation and control of development of vascular cambium of trees

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to application of populus trichocarpa PtrSCZ1 and PtrSCZ3 genes in regulation and control of development of a tree vascular cambium. The problem of tree vascular cambium development is solved. Comprising the application of the recombinant vector or the engineering bacterium of the populus trichocarpa PtrSCZ1 and PtrSCZ3 genes in regulating and controlling the development of the vascular cambium of the tree. According to the invention, PtrSCZ1 and PtrSCZ3 overexpressed populus trichocarpa transgenic plants are obtained through an agrobacterium-mediated populus trichocarpa genetic transformation system, and a series of technical means are utilized for analysis to find that the overexpression of PtrSCZ1 and PtrSCZ3 transcription factors enables the growth of the populus trichocarpa to be slow, the number of vascular cambium cell layers is increased, and the growth of the populus trichocarpa is accelerated. The research content provided by the invention has important theoretical guiding significance for cultivating new varieties of high-quality woods and forests.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and specifically relates to the application of PtrSCZ1 and PtrSCZ3 genes of Populus trichocarpa in regulating the development of vascular cambium of trees. Background Art

[0002] Wood is a natural renewable resource and has been widely used as an important raw material in industries such as construction, papermaking and furniture making. At the same time, wood is also an important biomass energy source. Compared with other raw materials, wood is environmentally friendly, safe, economical and efficient. Using wood as the main raw material to meet the various needs of human production and life is in line with the requirements of sustainable development. In addition to economic benefits, wood, as an important product of secondary growth of trees, also has huge ecological benefits such as carbon sinks.

[0003] Wood, also known as secondary xylem, originates from the dynamic and continuous biological process of vascular cambium activity. The vascular cambium continuously produces xylem cells and phloem cells on both sides through division and differentiation. Xylem cells become secondary xylem (i.e. wood) through cell expansion, biosynthesis of secondary cell walls and programmed cell death. The above processes are precisely regulated by factors both internal and external to the tree, such as plant hormones, local peptide signals and their multiple interacting regulatory factors. Among them, transcription factors play a core regulatory role in regulating the development of vascular cambium, and some transcription factors play the role of marker genes. Previous studies have found that WOX4, as a marker gene of vascular cambium, is a core transcription factor that regulates the division activity of vascular cambium. Ethylene and TDIF signal transduction jointly regulate the expression of WOX4 to regulate the activity of cambium. The regulatory role of WOX4 is also conserved in the woody plant poplar. In the suppressed expression lines of poplar PttWOX4a / b, the division activity of vascular cambium is reduced and the production of secondary xylem is reduced. The number of cell layers in the proliferative zone of the cambium was significantly reduced in the Populus trichocarpa mutant ptrwox4a / ptrwox4b, in which PtrWOX4a and its homologous gene PtrWOX4b were co-knocked out.

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

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

[0006] The present invention is of great significance for the research and development of regulating the vascular cambium of trees and cultivating new wood varieties. A brief summary of the present invention is given below to provide a basic understanding of certain aspects of the present invention. It should be understood that this summary is not an exhaustive summary of the present invention. It is not intended to determine the key or important parts of the present invention, nor is it intended to limit the scope of the present invention.

[0007] The technical solution of the present invention:

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

[0009] Preferably: overexpression of PtrSCZ1 and PtrSCZ3 genes slows the growth of Populus trichocarpa, increases the number of vascular cambium layers, and advances the secondary development of xylem and phloem.

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

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

[0012] Preferably, the recombinant vector is obtained by recombining the PtrSCZ1 and PtrSCZ3 genes of Populus trichocarpa into the plant expression vector pBI121.

[0013] Preferably, the engineered bacteria is one of Escherichia coli and Agrobacterium tumefaciens.

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

[0015] Preferably, the plant expression vector used for constructing the recombinant vector is pBI121.

[0016] Preferably, the transformation method is Agrobacterium transformation method.

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

[0018] The present invention obtains PtrSCZ1 and PtrSCZ3 overexpressing Populus trichocarpa transgenic plants through Agrobacterium-mediated Populus trichocarpa genetic transformation system;

[0019] The present invention uses a series of technical means to analyze and find that the overexpression of PtrSCZ1 and PtrSCZ3 transcription factors slows the growth of Populus trichocarpa and increases the number of vascular cambium cell layers;

[0020] The research content of the present invention has important theoretical guiding significance for cultivating new varieties of high-quality timber trees. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a diagram showing the results of the identification of transcriptional levels in PtrSCZ1 and PtrSCZ3 overexpressing Populus trichocarpa plants;

[0022] Figure 2 This is a graph showing the growth status of PtrSCZ1 and PtrSCZ3 overexpressing and wild-type Populus trichocarpa plants after 4 months of greenhouse growth;

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

[0024] Figure 4 This is the result of observation of cell morphology in the cross section of the 5th, 6th and 8th stem nodes of PtrSCZ1 and PtrSCZ3 overexpressing Populus trichocarpa plants;

[0025] Figure 5 This is the result of observation of the morphology of the cambium at the 8th stem node of PtrSCZ1 and PtrSCZ3 overexpressing Populus trichocarpa plants;

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

[0027] Figure 1 Middle: OE-PtrSCZ1-L1, OE-PtrSCZ1-L4 and OE-PtrSCZ3-L3, OE-PtrSCZ1-L12 represent two different lines of PtrSCZ1 and PtrSCZ3 overexpressing plants, respectively. WT represents wild-type Populus trichocarpa. Error bars represent standard errors calculated from at least three biological replicates. Asterisks represent t-test results. **P < 0.01.

[0028] Figure 2 Middle: OE-PtrSCZ1-L1, OE-PtrSCZ1-L4, OE-PtrSCZ3-L3, OE-PtrSCZ1-L12 represent two different lines of PtrSCZ1 and PtrSCZ3 overexpressing plants, respectively. WT represents wild-type Populus trichocarpa. Scale bar is 10 cm.

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

[0030] Figure 4 Middle scale bar = 500 μm;

[0031] Figure 5 WT in the middle represents wild-type Populus trichocarpa, P represents phloem cells, C represents cambium cells, X represents xylem cells, scale bar = 25 μm;

[0032] Figure 6 WT represents wild-type Populus trichocarpa, error bars represent standard errors calculated from at least three biological replicates, asterisks represent t-test results, *P < 0.05, **P < 0.01. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is described below by the specific embodiments shown in the accompanying drawings. However, it should be understood that these descriptions are only exemplary and are not intended to limit the scope of the present invention. In addition, in the following description, the description of well-known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present invention.

[0034] Specific implementation method 1: Combination Figure 1-Figure 6 The present embodiment is described, and the application of the PtrSCZ1 and PtrSCZ3 genes of Populus trichocarpa in the present embodiment in regulating the development of the vascular cambium of trees, including the application of the recombinant vectors or engineered bacteria of the PtrSCZ1 and PtrSCZ3 genes of Populus trichocarpa in regulating the development of the vascular cambium of trees.

[0035] Specific implementation method 2: Combination Figure 1-Figure 6 The present embodiment is described, and the application of the PtrSCZ1 and PtrSCZ3 genes of Populus trichocarpa in the present embodiment in regulating the development of the vascular cambium of trees, through the overexpression of the PtrSCZ1 and PtrSCZ3 genes, the growth of Populus trichocarpa is retarded, the number of vascular cambium layers is increased, and the secondary development of xylem and phloem is advanced.

[0036] Specific implementation method three: Combination Figure 1-Figure 6 The present embodiment is described, and the application of the PtrSCZ1 and PtrSCZ3 genes of Populus trichocarpa in the present embodiment in regulating the development of the vascular cambium of trees, and the nucleotide sequences of the PtrSCZ1 and PtrSCZ3 genes of Populus trichocarpa are shown in SEQ ID No.1 and SEQ ID No.2.

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

[0038] Specific implementation method five: Combination Figure 1-Figure 6 The present embodiment is described. The application of the PtrSCZ1 and PtrSCZ3 genes of Populus trichocarpa in the present embodiment in regulating the development of the vascular cambium of trees is described. The recombinant vector is the recombinant PtrSCZ1 and PtrSCZ3 genes of Populus trichocarpa into the plant expression vector pBI121.

[0039] Specific implementation method six: Combination Figure 1-Figure 6 The present embodiment is described. The PtrSCZ1 and PtrSCZ3 genes of Populus trichocarpa are used in regulating the development of vascular cambium of trees. The engineered bacteria is one of Escherichia coli and Agrobacterium tumefaciens.

[0040] Specific implementation method seven: Combination Figure 1-Figure 6 The present embodiment is described as a method for increasing the number of vascular layers of Populus trichocarpa. A recombinant vector containing the PtrSCZ1 and PtrSCZ3 genes of Populus trichocarpa as claimed in claim 1 is constructed and transformed into Populus trichocarpa to obtain transgenic plants.

[0041] Specific implementation method eight: Combination Figure 1-Figure 6 The present embodiment is described as a method for increasing the number of vascular layers of Populus trichocarpa. The plant expression vector used for constructing the recombinant vector is pBI121.

[0042] Specific implementation method nine: Combination Figure 1-Figure 6 The present embodiment is described. The present embodiment provides a method for increasing the number of vascular layers of Populus trichocarpa, wherein the transformation method is an Agrobacterium transformation method.

[0043] The experimental methods involved in the following examples are all conventional methods unless otherwise specified, and the materials, reagents, enzymes, competent cells, plasmids, etc. used can be obtained from commercial channels unless otherwise specified.

[0044] Example 1 Obtaining the transcription factor genes of Populus trichocarpa PtrSCZ1 and PtrSCZ3 and constructing the plant expression vector

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

[0046] The total RNA of wild-type plants of Populus trichocarpa Nisqually-1 genotype was extracted using the plant RNA extraction kit of Qiagen Company, and the cDNA was obtained by reverse transcription using the Takara reverse transcription kit (RR047A).

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

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

[0049]

[0050]

[0051] Note: The underlined part represents the restriction site, and the protection base is in front of the restriction site.

[0052] The primers were used to obtain the target sequence through polymerase chain reaction (PCR), and the obtained PCR product was subjected to agarose gel electrophoresis to separate the target band. The target fragment was recovered by gel recovery kit of Qiagen Company.

[0053] 2. Construction of plant expression vector

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

[0055] Target DNA fragment digestion system:

[0056]

[0057] pBI121 vector restriction enzyme system:

[0058]

[0059] Enzyme digestion conditions:

[0060] 37℃ 1 hour

[0061] After enzyme digestion, the target band was separated by electrophoresis and recovered by Qiagen gel recovery kit. The target gene was loaded 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] Take out the TOP10 competent cells and thaw them on ice, add all the above ligation products to the competent cells and mix them evenly. Heat shock at 42°C for 1 minute, place on ice for 3 minutes, add LB medium without resistance, and activate at 37°C and 200rpm for one hour. Collect the bacteria at a low speed, spread the bacteria on solid LB medium (containing kanamycin 50mg / L), and culture at 37°C overnight. The next day, use the vector primers to perform PCR identification on the obtained monoclonal clones. The primers are shown in Table 2;

[0067] Table 2 Primer sequences for monoclonal detection of plant expression vectors

[0068]

[0069] The PCR products were analyzed by electrophoresis to determine the positive clones. The positive single clones were picked and cultured in 5 mL LB medium (kanamycin, 50 mg / L) at 220 rpm and 37 ° C overnight. The next day, the strains were preserved and centrifuged to collect the bacteria, and the plasmids were extracted. The extracted plasmids were sequenced with primers 35SP1 and NosT2. The specific process was referred to the Qiagen plasmid extraction kit instructions.

[0070] According to the genome sequence information of Populus trichocarpa provided by phytozome website, the target gene was obtained by PCR and connected to pBI121 vector. Sequencing analysis showed that the PtrSCZ1 and PtrSCZ3 genes we obtained were 810bp (SEQ ID NO.1) and 816bp (SEQ ID NO.2), encoding 270 amino acids (SEQ ID NO.3) and 272 amino acids (SEQID NO.4), respectively, which were consistent with the sequence information of Populus trichocarpa PtrSCZ1 and PtrSCZ3 provided by phytozome website, indicating that we successfully obtained the PtrSCZ1 and PtrSCZ3 genes of Populus trichocarpa.

[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 trichocarpa PtrSCZ1 and PtrSCZ3 overexpression plants

[0080] 1. Freeze-thaw transformation of Agrobacterium

[0081] Step 1: Take out the Agrobacterium competent cells and put them on ice, add about 30 ng of the plasmid constructed in Example 1 to the Agrobacterium competent cells, mix well, and place them in an ice bath for 30 minutes, immediately put them in liquid nitrogen, freeze them for 1 minute, and then immediately put them in a 37°C water bath for 3 minutes;

[0082] Step 2: Add 1 mL of liquid LB and culture at 28°C, 180-200 rpm for 2-3 hours;

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

[0084] Step 4: Pick a large clone, inoculate it into 5 mL LB (kanamycin + gentamicin) liquid medium, and culture it overnight at 220 rpm and 28°C;

[0085] Step 5: Perform PCR using the bacterial solution as a template to confirm the positive clones. The specific method is as described in Example 1.

[0086] Step 6: Preserve positive clones: 500 mL bacterial solution + 500 mL 50% glycerol, mix well, freeze quickly in liquid nitrogen, and store at -80°C.

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

[0088] Step 1: Take 10 μL of Agrobacterium containing the plant expression vector and add it to 5 mL of resistant LB (kanamycin 50 mg / L + gentamicin 50 mg / L) medium, and culture 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 (kanamycin 50 mg / L + gentamicin 50 mg / L), culture at 28°C, 220 rpm until OD600 = 0.4, and set aside;

[0090] Step 3: Take the tender stem segments of 3-6 month old greenhouse seedlings of Populus trichocarpa and disinfect them in 10% sodium hypochlorite for 20 minutes;

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

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

[0093] Step 6: Take out the stem segments, place them on CIM1 medium, and culture them in the dark for 2 days;

[0094] Step 7: Two days later, wash the explant stem segments thoroughly with sterile water and place them on CIM2 medium. Culture them in the dark and change 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 grow, 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 status in the soil, cultivate them until they are 4 months old, and collect the cambium tissues of the resistant plants and wild-type controls;

[0099] Step 2: Extract RNA. For specific steps, refer to the instructions of the Plant RNA Extraction Kit (Qiagen);

[0100] Step 3: The obtained RNA is measured for concentration and quality using NanoDrop;

[0101] Step 4: Refer to the instructions to reverse transcribe the obtained RNA using the OligdT primer to obtain cDNA;

[0102] Step 5: The obtained cDNA was diluted 10 times and RT-qPCR was performed, the internal reference was PtrActin7, and the primer sequences were shown in Table 3;

[0103] Quantitative PCR reaction system 15 μL:

[0104]

[0105] Step 6: Use 2 △△Ct The quantitative results were calculated.

[0106] Table 3 Primer sequences used for identification of transgenic plants

[0107]

[0108] Two independent resistant strains were obtained by Agrobacterium-mediated genetic transformation, named OE-PtrSCZ1-L1, OE-PtrSCZ1-L4, OE-PtrSCZ3-L3, and OE-PtrSCZ1-L12. After transcriptional level identification, the transcription levels of PtrSCZ1 and PtrSCZ3 genes in resistant plants were significantly increased compared with those in wild-type plants of Populus trichocarpa ( Figure 1 ), indicating that transgenic P. trichocarpa plants overexpressing PtrSCZ1 and PtrSCZ3 genes were successfully obtained. The growth status of PtrSCZ1 and PtrSCZ3 overexpressed and wild-type plants after 4 months of greenhouse growth is shown in Figure 2. Figure 2 shown.

[0109] Example 3 Application of PtrSCZ1 and PtrSCZ3 genes in regulating the development of wood vascular cambium

[0110] 1. Growth phenotype analysis of the transgenic and wild-type Populus trichocarpa plants obtained in Example 2

[0111] Growth phenotyping includes the following indicators:

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

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

[0114] Basal stem measurement: The diameter of 4-month-old wild-type and transgenic greenhouse plants overexpressing PtrSCZ1 and PtrSCZ3 was measured at the soil surface using a vernier caliper. There were at least 5 biological replicates for wild-type plants and transgenic plants.

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

[0116] The 5th, 6th and 8th nodes of the stems of the wild type and PtrSCZ1 and PtrSCZ3 overexpressing plants grown in the greenhouse for 4 months were respectively taken for paraffin sectioning and histochemical staining analysis, and the dye used was toluidine blue. The statistical analysis of cells in the present invention was performed using an M8 scanner (Precipoint) and ViewPoint software. The number of cambium cell layers in each stem node was counted, and 30 cell files were counted for each stem node. SPSS software was used for statistical data processing and analysis. 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 had an average of about 6 layers of cambium cells, while the transgenic plants overexpressing PtrSCZ1 and PtrSCZ3 had an increase of about 1 to 2 layers (see Figure 6 ), it can be seen that PtrSCZ1 and PtrSCZ3 genes affect the secondary growth of trees by regulating the development of cambium.

[0117] It should be noted that in the above embodiments, as long as the technical solutions are not contradictory, they can be arranged and combined, and those skilled in the art can exhaust all possibilities based on the mathematical knowledge of arrangement and combination. Therefore, the present invention will no longer describe the technical solutions after arrangement and combination one by one, but it should be understood that the technical solutions after arrangement and combination have been disclosed by the present invention.

[0118] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Application of the PtrSCZ1 and PtrSCZ3 genes of Populus trichocarpa in regulating the development of vascular cambium of trees, characterized in that: The application of the recombinant vector or engineered bacteria of the PtrSCZ1 and PtrSCZ3 genes of Populus trichocarpa in regulating the development of the vascular cambium of trees.

2. The use of the PtrSCZ1 and PtrSCZ3 genes of Populus trichocarpa in regulating the development of vascular cambium of trees according to claim 1, characterized in that: Overexpression of PtrSCZ1 and PtrSCZ3 genes slowed the growth of Populus trichocarpa, increased the number of vascular cambium layers, and advanced the secondary development of xylem and phloem.

3. The use of the PtrSCZ1 and PtrSCZ3 genes of Populus trichocarpa in regulating the development of vascular cambium of trees according to claim 1, characterized in that: The nucleotide sequences of the PtrSCZ1 and PtrSCZ3 genes of Populus trichocarpa are shown in SEQ ID No.1 and SEQ ID No.

2.

4. The use of the PtrSCZ1 and PtrSCZ3 genes of Populus trichocarpa in regulating the development of vascular cambium of trees according to claim 1, characterized in that: The amino acid sequences of the proteins encoded by the PtrSCZ1 and PtrSCZ3 genes of Populus trichocarpa are shown in SEQ ID No.3 and SEQ ID No.

4.

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

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

7. A method for increasing the number of vascular layers of Populus trichocarpa, characterized in that: A recombinant vector containing the PtrSCZ1 and PtrSCZ3 genes of Populus trichocarpa as claimed in claim 1 is constructed and transformed into Populus trichocarpa to obtain transgenic plants.

8. The method for increasing the number of vascular layers of Populus trichocarpa according to claim 7, characterized in that: The plant expression vector used for constructing the recombinant vector is pBI121.

9. The method for increasing the number of vascular layers of Populus trichocarpa according to claim 7, characterized in that: The transformation method is an Agrobacterium transformation method.

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