Application of PtrMYB167 and PtrHox52 in tree high yield and drought tolerance polybreeding

Abstract

The application belongs to the technical field of genetic engineering, and particularly relates to application of Populus davidiana PtrMYB167 and PtrHox52 genes in tree high-yield drought-tolerant polybreeding. The application solves the problem that common tree improvement technologies cannot simultaneously consider enhancing drought tolerance and improving growth. The application includes nucleotide sequences such as PtrMYB167 of Populus davidiana, homologous genes PtrMYB90 and PtrHox52 genes of Populus davidiana, a recombinant vector containing the genes or fragments thereof, or an engineering bacteria containing the genes or fragments thereof, and application of the genes in tree high-yield drought-tolerant polybreeding. The application obtains polybreeding transgenic Populus davidiana plants with simultaneous improvement of growth and drought tolerance, and provides a new thought and strategy for cultivating new forest tree germplasm with high quality, high yield and high resistance.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to Populus tomentosa. PtrMYB167 and PtrHox52 Application of genes in high-yield and drought-resistant hybrid breeding of trees. Background Technology

[0002] Forests are a critical resource for human survival, providing a sustainable biological resource for our lives and social development. Drought is the most frequent and damaging environmental stressor in plant life activities. Under drought stress, slowing down growth and development rates and reducing biomass are the main strategies for plants to adapt to stress. Therefore, there is often a negative correlation between plant growth and development and drought resistance. Polymer breeding can integrate multiple superior genes into a single variety, thereby obtaining new germplasm resources with multiple resistances or superior traits, and is an effective breeding strategy for creating superior germplasm. Currently, polymer breeding technology mainly includes three approaches: traditional polymer breeding, polymer breeding using molecular marker-assisted selection, and polymer breeding through genetic transformation. Among them, genetic transformation polymer breeding utilizes gene editing technology to modify the genes controlling corresponding traits, which is an effective way to change plant characteristics and obtain new forest germplasm with target traits.

[0003] However, traditional gene editing technology can only improve a single trait of trees. Generally, improvements that enhance drought resistance are often accompanied by the negative effect of reduced growth.

[0004] Therefore, it is urgent to propose the theory of Populus tomentosa. PtrMYB167 and PtrHox52 The application of genes in high-yield and drought-resistant hybrid breeding of trees can solve the above-mentioned technical problems. Summary of the Invention

[0005] The purpose of this invention is to address the problem that commonly used forest improvement techniques cannot simultaneously enhance drought resistance and increase biomass. 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.

[0006] The technical solution of the present invention: hairy poplar PtrMYB167 and PtrHox52 Application of genes in high-yield and drought-resistant hybrid breeding of trees, with nucleotide sequences such as SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3 of Populus hairy-fruited tamarisk. PtrMYB167 and its homologous genes PtrMYB90 and PtrHox52Application of genes, recombinant vectors containing the gene or its fragments, or engineered bacteria containing the gene or its fragments in high-yield and drought-resistant aggregate breeding of trees.

[0007] Preferred: Hairy Poplar PtrMYB167 , PtrMYB90 and PtrHox52 The amino acid sequence of the gene-encoded protein is shown in SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 6.

[0008] Preferred: Simultaneous knockout of hairy poplar PtrMYB167 and PtrMYB90 Application of gene recombinant vectors in increasing the biomass of Populus tomentosa.

[0009] Preferred: PtrHox52 Application of gene recombinant vectors in enhancing the drought resistance of Populus tomentosa.

[0010] Preferred: Knockout PtrMYB167 , PtrMYB90 and overexpression PtrHox52 Genes significantly increase the biomass of poplar trees and enhance their drought resistance.

[0011] Preferred: The recombinant vector is a vector that simultaneously knocks out poplar fruit. PtrMYB167 and PtrMYB90 The gRNA of the gene was recombined into the plant expression vector pEgP237.

[0012] Preferred: The recombinant vector is poplar hairy fruit poplar. PtrHox52 Genes were recombined into the plant expression vector pCAMBIA1300.

[0013] Preferred: Simultaneous knockout of hairy poplar PtrMYB167 and PtrMYB90 Application of genetically engineered bacteria in increasing tree biomass.

[0014] Preferred: Hairy Poplar PtrHox52 Application of genetically engineered bacteria in enhancing the drought resistance of trees.

[0015] A method to increase tree biomass and enhance drought resistance involves constructing the aforementioned scheme while simultaneously knocking out Populus tomentosa. PtrMYB167 and PtrMYB90 Gene recombination vectors were used to transform hairy poplar trees to obtain... ptrmyb167 / 090 Genetically modified plants; Constructing a poplar tree containing the hairy fruit poplar as described in claim 1 PtrHox52 Gene recombination vectors, transformation ptrmyb167 / 090 Transgenic materials, to obtain transgenic plants with polymeric traits The present invention has the following beneficial effects: This invention yielded transgenic poplar plants with improved growth and drought resistance, providing new ideas and strategies for cultivating high-quality, high-yield, and highly resistant new forest tree germplasm. Attached Figure Description

[0016] Figure 1 for ptrmyb167 / 090 Image showing DNA level identification results of mutant plants; M represents DNA Marker DL 2000bp, 1 represents pEgP237- PtrMYB167 / 090 Positive control using recombinant plasmid as template; samples 2-5 are identification samples using resistant plant genomic DNA as template; sample 6 is a negative control using WT genomic DNA as template; and sample 7 is a negative control using deionized water as template.

[0017] Figure 2 for ptrmyb167 / 090 / 35S:PtrHox52 Figure showing the results of DNA level identification in plants expressing polymerase; where M is DNA Marker DL 2000; 1 is the negative control using WT genomic DNA as a template; 2 is pCAMBIA1300- PtrHox52 Positive control using recombinant plasmid as template; 3-5 are identification samples using genomic DNA of resistant plants as template; 6 is a negative control using deionized water as template; Figure 3 for ptrmyb167 / 090 / 35S:PtrHox52 Polymer expression in the xylem of Populus tomentosa PtrHox52 Relative gene expression levels; among which, ptrmyb167 / 090 Representatives simultaneously knocked out PtrMYB167 and PtrMYB090 The mutant poplar plants, L2, L38, and L39 represent three different lines of the aggregate expression plants. The error bars represent the standard error calculated from at least three biological replicates, and the asterisks indicate... t Test results,** P <0.01; Figure 4 for ptrmyb167 / 090 / 35S:PtrHox52 Polymer expression and growth of Populus tomentosa plants in the control group; the control group consisted of: WT wild-type, OE-PtrHox52 overexpression and ptrmyb167 / 090 Mutant Populus hairy-fruited, scale bar 10 cm; Figure 5 for ptrmyb167 / 090 / 35S:PtrHox52 Phenotypic analysis results of *Populus hairy fruit* plants grown in greenhouse for 60, 90, and 120 days, showing the expression of polymerases and the growth phenotype of the control group. The control group consisted of WT wild-type plants. OE-PtrHox52 Overexpressing plants and ptrmyb167 / 090Mutant plants; where A represents the statistical result of plant height; B represents the statistical result of ground diameter; C represents the statistical result of stem node number; D represents the statistical result of the statistical result of the 10th stem node length; error bars represent the standard error calculated by at least three biological replicates, and asterisks indicate... t Test results,** P <0.01, ***P <0.001; Figure 6 for ptrmyb167 / 090 / 35S:PtrHox52 Biomass results of *Populus hairy fruit* plants expressing polymers and in the control group after 120 days of greenhouse growth; the control group consisted of: WT wild-type, OE-PtrHox52 overexpression and ptrmyb167 / 090 Mutant Populus tomentosa plants; error bars represent standard errors calculated from at least three biological replicates, asterisks indicate... t Test results,** P <0.01, ***P <0.001; Figure 7 for ptrmyb167 / 090 / 35S:PtrHox52 Figure showing the results of drought stress treatment on *Populus hairy fruit* with polymer expression and control groups; the control group consisted of: WT wild-type, OE-PtrHox52 overexpression and ptrmyb167 / 090 Mutant Populus hairy-fruited, scale bar 10 cm; Figure 8 for ptrmyb167 / 090 / 35S:PtrHox52 The statistical results of the survival rate of *Populus hairy fruit* plants after drought stress were obtained from the aggregation expression and control groups. The control group consisted of: WT wild-type, OE-PtrHox52 overexpression and ptrmyb167 / 090 The mutant *Populus pubescens*, with error bars representing the standard error calculated from at least three biological replicates, and asterisks indicating... t Test results,** P <0.01, ***P <0.001. Detailed Implementation

[0018] 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.

[0019] Specific implementation method one: Combining Figures 1-8 This embodiment describes the hairy poplar of this embodiment. PtrMYB167 and PtrHox52 Application of genes in high-yield and drought-resistant hybrid breeding of trees, with nucleotide sequences such as SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3 of Populus tomentosa. PtrMYB167 and its homologous genes PtrMYB90 and PtrHox52 Application of genes, recombinant vectors containing the gene or its fragments, or engineered bacteria containing the gene or its fragments in high-yield and drought-resistant aggregate breeding of trees.

[0020] Specific Implementation Method Two: Combining Figures 1-8 This embodiment describes the hairy poplar of this embodiment. PtrMYB167 and PtrHox52 Application of genes in high-yield and drought-resistant hybrid breeding of trees, Populus tomentosa PtrMYB167 , PtrMYB90 and PtrHox52 The amino acid sequence of the gene-encoded protein is shown in SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 6.

[0021] Specific implementation method three: Combining Figures 1-8 This embodiment describes the hairy poplar of this embodiment. PtrMYB167 and PtrHox52 Application of genes in high-yield and drought-resistant hybrid breeding of trees, while knocking out hairy-fruited poplar PtrMYB167 and PtrMYB90 Application of gene recombinant vectors in increasing the biomass of Populus tomentosa.

[0022] Specific implementation method four: Combination Figures 1-8 This embodiment describes the hairy poplar of this embodiment. PtrMYB167 and PtrHox52 Application of genes in high-yield and drought-resistant hybrid breeding of trees PtrHox52 Application of gene recombinant vectors in enhancing the drought resistance of Populus tomentosa.

[0023] Specific Implementation Method Five: Combining Figures 1-8 This embodiment describes the hairy poplar of this embodiment. PtrMYB167 and PtrHox52 Application of gene knockout in high-yield and drought-resistant hybrid breeding of trees PtrMYB167 , PtrMYB90 and through expression PtrHox52 Genes significantly increase the biomass of poplar trees and enhance their drought resistance.

[0024] Specific Implementation Method Six: Combination Figures 1-8 This embodiment describes the hairy poplar of this embodiment. PtrMYB167 and PtrHox52 The application of genes in high-yield and drought-resistant hybrid breeding of trees, wherein the recombinant vector is a vector that simultaneously knocks out hairy-fruited poplar. PtrMYB167 and PtrMYB90 The gRNA of the gene was recombined into the plant expression vector pEgP237.

[0025] The pEgP237 vector is a plant expression vector based on the CRISPR / Cas9 system. Its backbone sequence can be found in published literature (e.g., Zhang et al., Nature Protocols, 2019), and it contains a 35S promoter, a gRNA insertion site (BsaI restriction site), and a kanamycin resistance gene. Those skilled in the art can obtain or construct this vector from existing resources using conventional molecular cloning methods.

[0026] Specific implementation method seven: Combining Figures 1-8 This embodiment describes the hairy poplar of this embodiment. PtrMYB167 and PtrHox52 Application of genes in high-yield and drought-resistant hybridization breeding of trees, the recombinant vector is Populus tomentosa PtrHox52 Gene recombination into the plant expression vector pCAMBIA1300 Specific implementation method eight: Combination Figures 1-8 This embodiment describes the hairy poplar of this embodiment. PtrMYB167 and PtrHox52 Application of genes in high-yield and drought-resistant hybrid breeding of trees, while knocking out hairy-fruited poplar PtrMYB167 and PtrMYB90 Application of genetically engineered bacteria in increasing tree biomass.

[0027] Specific Implementation Method Nine: Combining Figures 1-8 This embodiment describes the hairy poplar of this embodiment. PtrMYB167 and PtrHox52 Application of genes in high-yield and drought-resistant hybrid breeding of trees, Populus tomentosa PtrHox52 Application of genetically engineered bacteria in enhancing the drought resistance of trees.

[0028] The engineered bacteria include Escherichia coli and Agrobacterium.

[0029] Specific Implementation Method Ten: Combining Figures 1-8 This embodiment describes a method for increasing tree biomass while enhancing drought resistance, comprising constructing a method for simultaneously knocking out hairy-fruited poplar trees as described in claim 1. PtrMYB167 and PtrMYB90 Gene recombination vectors were used to transform hairy poplar trees to obtain... ptrmyb167 / 090 Genetically modified plants; Constructing a poplar tree containing the hairy fruit poplar as described in claim 1 PtrHox52 Gene recombination vectors, transformation ptrmyb167 / 090 Transgenic materials are used to obtain transgenic plants with polymeric traits.

[0030] The plant expression vectors used to construct the recombinant vector were pEgP237 and pCAMBIA1300.

[0031] The transformation method was Agrobacterium-mediated genetic transformation of Populus tomentosa.

[0032] 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.

[0033] Example 1: Populus tomentosa ptrmyb167 / 090 Creation of mutant plants (1) Construction of pEgP237 plant expression vector On the website (http: / / crispr.hzau.edu.cn / CRISPR2 / ), based on the gRNA selection principle, a 20 bp long sequence with high homology and specificity is selected from the exon region of the gene as a co-knockout sequence. PtrMYB167 and homologous genes PtrMYB090 The selected gRNA has the sequence: GAATCATTGGCATGTCATCA. The selected gRNA was annealed using primers; the sequences are shown in Table 1. Table 1 Primer sequences used for gRNA renaturation

[0034] Note: The underlined part represents Bsa I restriction site.

[0035] gRNA renaturation reaction system: Ex-taq Buffer 2 μL pEgP237- PtrMYB167 / 090 -F9 μL pEgP237- PtrMYB167 / 090 -F9 μL gRNA renaturation reaction conditions: 94℃ for 30 seconds 72℃ for 2 min 37℃ for 2 min 28℃ for 2 min The product was diluted 100 times and stored at -20℃ for later use.

[0036] The renatured gRNA was constructed into the pEgP237 vector. First, the pEgP237 vector was digested with a single enzyme; the reaction system and conditions are as follows: Enzyme digestion system: Adjust the volume to 50 μL using ddH2O. Cutsmart 5 μL Bsa I5 μL pEgP237 vector 2 mg Enzyme digestion conditions: 37℃ for 2 hours After enzyme digestion, the target band was separated by electrophoresis and recovered using a gel extraction kit. The renatured product was then loaded into the pEgP237 vector using T4 ligase. The specific reaction system and conditions are as follows: T4 connection system: T4 enzyme 1 μL T4 buffer 1 μL 4 μL of refolded product 4 μL of pEgP237 vector Connection requirements: 4℃ overnight Thaw TOP10 competent cells on ice. Add at least 5 μL of ligation product 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 220 rpm for 1 hour. Collect 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 on the obtained single clones. Primers are shown in Table 2. Table 2 Primer sequences used for single-clone detection of pEgP237 plant expression vector

[0037] Positive single clones were picked and cultured overnight in 5 mL LB medium (kanamycin, 50 mg / L) at 220 rpm and 37°C. The next day, the bacterial strain was preserved and sequenced. Sequencing analysis confirmed that the gRNA had been successfully inserted into the vector for the creation of [a specific product / organism / etc.]. ptrmyb167 / 090 The pEgP237 plant expression vector for mutant plants has been successfully constructed.

[0038] (2) Freeze-thaw transformation of Agrobacterium ① Take out Agrobacterium competent cells and place them on ice. Add about 100 ng of plasmid 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.

[0039] ② Add 500 μL of liquid YEP and incubate at 28℃ and 180-200 rpm for 2-3 h.

[0040] ③ Incubate briefly for 15 seconds, remove the supernatant, retain 300-400 μL of the supernatant, resuspend, spread on YEP (kanamycin + gentamicin + rifampin) solid medium, and incubate at 28℃ for about 48 h.

[0041] ④ Perform PCR using a single clone as a template to confirm the positive clone. Refer to Example 1 for the specific method.

[0042] ⑤ Select positive clones and inoculate them into 5 mL of YEP (kanamycin + gentamicin + rifampin) liquid medium, and incubate overnight at 28°C and 180 rpm.

[0043] ⑥ To preserve positive clones, mix 500 μL of bacterial culture with 500 μL of 50% glycerol, flash freeze in liquid nitrogen, and store at -80℃.

[0044] (3) Agrobacterium-mediated genetic transformation of Populus tomentosa ① Streak the constructed Agrobacterium culture onto a YEP (kanamycin + gentamicin + rifampin) plate, and incubate the plate at 28°C for two days.

[0045] ② Pick single colonies and inoculate them into 20 mL of YEP (kanamycin + gentamicin + rifampin) liquid medium. Incubate overnight at 28°C and 180 rpm until OD reaches 100%. 600 =0.9. Take 1 mL of the cultured bacterial suspension and add it to 50 mL of YEP (kanamycin + gentamicin + rifampin) liquid medium. Incubate at 28℃ and 200 rpm until OD reaches 0.9. 600 =0.6, centrifuge the bacterial cells, and resuspend them in suspension to OD. 600 =0.4, reserved.

[0046] ③ Cut the stem segments of wild-type Populus tomentosa tissue culture seedlings into small segments of about 1 cm, put them into the bacterial solution, and infect for 20 minutes.

[0047] ④ Remove the stem segments and place them on a co-culture medium. Incubate in the dark for 2 days.

[0048] ⑤ Two days later, rinse the co-cultured stem segments with cephalosporin solution (250 mg / L) for 5 minutes, and then rinse twice with sterile water for 3 minutes each time.

[0049] ⑥ Place the cleaned stem segments on a selective culture medium (cephalosporin 250 mg / L + kanamycin 25 mg / L) and incubate under light.

[0050] ⑦ After the resistant buds emerge, insert the resistant buds into a rooting medium containing kanamycin resistance (20 mg / L) to screen for positive plants.

[0051] (4) Identification of transgenic plants ① Extract DNA from leaves of resistant plants. Refer to the instructions for the Plant DNA Extraction Kit (TIANGEN) for specific steps.

[0052] ② Using the above DNA as a template, with M13F and pEgP237- PtrMYB167 / 090-R was used as the primer for PCR amplification. The primer sequences are shown in Table 2. The recombinant plasmid was used as a template for positive control, and wild-type DNA and water were used as templates for negative control. After DNA-level identification, the band position was correct. Figure 1 This indicates that the recombinant plasmid has been successfully integrated into the Populus tomentosa genome.

[0053] ③ In order to further determine ptrmyb167 / 090 The specific editing details of the mutant plant were investigated. DNA was extracted from the third leaf of the mutant plant and used as a template for amplification. PtrMYB167 and PtrMYB090 Genes, including gRNA, were amplified. The amplification primers are shown in Table 3. PtrMYB167 The theoretical gene amplification sequence is 1012 bp (SEQ ID NO.7). PtrMYB90 The theoretical gene amplification sequence was 974 bp (SEQ ID NO. 8). This sequence was constructed into the pMD18-T vector and transformed into *E. coli*. Thirty single clones were selected from each plant and sent to the company for sequencing, and the results were compared. The results are shown in Table 4. Compared to the wild-type sequence, ptrmyb167 / 090-L4 mutant plants PtrMYB090 There are two types of gene editing: inserting one base into one strand of DNA and deleting nine bases into the other strand. PtrMYB167 There is a type of gene editing where a strand of a gene cannot express the PtrMYB167 protein normally due to a frameshift caused by the deletion of a base gene. ptrmyb167 / 090-L5 mutant plants PtrMYB090 There is only one type of gene editing: inserting one base into one strand of DNA. PtrMYB167 Two gene editing scenarios were observed: a 13-base deletion on one strand and a 5-base deletion on the other. Both editing scenarios demonstrated successful acquisition of the desired results. ptrmyb167 / 090 Mutant plant.

[0054] Table 3 Identification PtrMYB90 / 167 Primer sequences for gene editing

[0055] Table 4 ptrmyb167 / 090 gRNA editing status of gene mutants

[0056] Note: The underlined part represents the insertion or replacement of a base, and "-" represents the absence of a base.

[0057] SEQ ID NO.7: SEQ ID NO.8: GTGCTATAGCTATAGCTAGGGTTCATATATTCAAGTACTATAGAAATATCGTGTTTTTGGTTCTTGGGATTGGATTCTTTGATTGTTGTATTTAGAAGATTAAAAAAAATGTGTACTAGAGGCCATTGGAGGCCTGCAGAAGATGAGAAGCTTAAGGAGTTGGTTGAGAAGTATGGCCCTCATAATTGGAACGCGATCGCCGAAAAGCTTCATGGAAGATCAGGTGAAAATTTAAACCTAAAGAGAAACTAATTTGTATCCTCACAAATATTTTCATTTCAATATTTTTTTTATGTTGTTATTTAGAAACAACTAGGGTTTGGTACTGTATGCAAAAGTTGAGATATACTTATAACTAATGGAAATTAAAAGGGAAGCTAACTATGACTGTGACTAATAACTAACAAAACGAAGTTTGAACAAATTGAATCAGGGAAGAGTTGTAGGTTGAGATGGTTTAATCAGTTGGATCCAAGAATCAATAGAAGTCCTTTTACCGAAGAGGAAGAAGAAAGACTTCTTGCTTCTCACCGGATCCATGGGAATAGATGGGCAGTTATTGCAAGGCTTTTCCCAGGTCGCACCGATAATGCAGTGAAGAATCATTGGCATGTCATCATGGCAAGAAGATATAGAGAAAGGTCTAGGCTTCATGCAAAAAGAACTGCTCAAGCTTTGGTAAACGAGCAAAAATTCTCTTCAAAACAAGACATGCAGATAAATTGTGAGACGAGGAGCTTTTCTTCATTTGTCAAGAAATATTGTGAGAAATTCGGCCAATATCCTTTGATCACTCACAGCTACTTACCGGCCTTTTGGAAAGAGTTCTACAATGACGATCTAAGTAATTGTAAAGGTAACTAGATCGAGCTAGCATCATGGTGATGTGCTATATATATACAGTTCGGGTTCTAGTTTTACAATATTGATAATAATTTCTTAGCTCTTATTGCTCATGTTCCTTACATACATGG Example 2: Populus trichocarpaptrmyb167 / 090 / 35S:PtrHox52 Creation of plants expressing aggregation Will PtrHox52 The full-length CDS sequence of the gene was constructed into a plant expression vector. Primers were designed and synthesized by a biotechnology company. The primer sequences for constructing the pCAMBIA1300 plant expression vector are shown in Table 5.

[0058] Table 5 Primer sequences used to construct the pCAMBIA1300 plant expression vector

[0059] Note: The underlined part represents the enzyme cleavage site, and the bases preceding the enzyme cleavage site are protective bases.

[0060] 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 gel recovery kit.

[0061] The recovered target gene DNA fragment and the pCAMBIA1300 plant expression vector were subjected to double enzyme digestion. The specific reaction system and reaction conditions are as follows: Target DNA fragment digestion system: Adjust the volume to 20 μL using ddH2O. Cutsmart 2 μL KpnI 1 μL SalI 1 μL 1 mg of target gene DNA fragment pCAMBIA1300 vector enzyme digestion system: Adjust the volume to 50 μL using ddH2O. Cutsmart 5 μL KpnI 2 μL SalI 2 μL pCAMBIA1300 vector 2 mg Enzyme digestion conditions: 37℃ for 2 hours After enzyme digestion, the target band was separated by electrophoresis and recovered using a gel extraction kit. The target gene was then inserted into the plant expression vector pCAMBIA1300 using T4 ligase. The specific reaction system and conditions are as follows: T4 connection system: T4 enzyme 1 μL T4 buffer 1 μL Gene fragment 4 μL (concentration 10-100 ng / μL) 4 μL of pCAMBIA1300 vector (concentration 10-100 ng / μL) Connection conditions: 4℃ overnight Thaw TOP10 competent cells on ice. Add at least 5 μL of ligation product 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 220 rpm for 1 hour. Collect 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 on the obtained single clones. Primers are shown in Table 6. Table 6 Primer sequences for monoclonal detection used in the construction of pC1300 plant expression vector

[0062] PCR products were analyzed by electrophoresis to identify positive clones. Positive single clones were picked and incubated overnight at 37°C (220 rpm) in 5 mL LB medium (kanamycin, 50 mg / L). The next day, the bacterial culture was preserved, and sequencing was performed using primer 35SP1.

[0063] Sequencing analysis revealed that the inserted fragment, totaling 717 bp (SEQ ID NO.3), was identical to that of *Populus hairy fruit* provided by the phytozome website. PtrHox52 The sequence information is consistent, indicating that we have successfully obtained the fusion of *Populus pilosa*. PtrHox52 pCAMBIA1300 plant expression vector for the gene.

[0064] SEQ ID NO.3: ATGAGGAAGCGACAAGTGGTGGTAAGAAGATCAGAAGAACCTTCAAGAAGCTCAACGACTTCTTCTTTCACTATCAGGAATGTTAAATACGGAGAGTGCCAAAAGAATCATGCTGCTGGAGTTGGAGGTTACGCTGTTGATGGG TGCAGAGAGTTCATGGCAAGTGGTGAAGAAGGCACAGCTGCTGCACTCACTTGTGCGGCTTGTGGTTGCCATAGGAACTTCCACAGAAGGGAAGTGGAAACAGAGGTGGCATGTGATTGTTCTTCACCTTCTTCAAATGGTAAT (2) Freeze-thaw transformation of Agrobacterium The specific method is as described in Example 1 (2): Agrobacterium-mediated genetic transformation of Populus tomentosa. The specific method is the same as in Example 1, except that the material being transformed is... ptrmyb167 / 090 Tissue culture seedlings of mutant plants were screened for resistance to hygromycin using a selection medium (cephalosporin 250 mg / L + hygromycin 5 mg / L) and a rooting medium for resistance (hygromycin 5 mg / L).

[0065] (3) Identification of transgenic plants ① Extract DNA from leaves of resistant plants. Refer to the instructions for the Plant DNA Extraction Kit (TIANGEN) for specific steps.

[0066] ② Using the above-mentioned DNA as a template, PCR amplification was performed using F-terminal and R-terminal primers of hygromycin. The primer sequences are shown in Table 7. The recombinant plasmid was used as a positive control, and wild-type DNA and water were used as negative controls. After DNA-level identification, the band positions were correct, indicating that the recombinant plasmid had been successfully integrated into the Populus hairgrass genome.

[0067] Table 7 Primer sequences used for DNA level identification in transgenic plants

[0068] ③ Plant the resistant plants in a greenhouse to simultaneously knock out the resistant plants. PtrMYB167 and PtrMYB090 The transgenic plants of the gene served as controls. After culturing for 4 months, xylem tissues from both resistant and control plants were collected for RNA extraction. The specific steps were performed according to the instructions of the Plant RNA Extraction Kit (Omega). The concentration and quality of the obtained RNA were determined using NanoDrop.

[0069] ④ Refer to the instruction manual and use OlidT primers to reverse transcribe the obtained RNA to obtain cDNA.

[0070] ⑤ Dilute the obtained cDNA 10-fold and perform RT-qPCR, with the internal control being... PtrActin 7 The primer sequences are shown in Table 8.

[0071] Quantitative PCR reaction system 15 μL: 0.6 μL of 5 μM 5' end primer 0.6 μL of 5 μM 3' end primer FastStart Universal SYBR Green Master7.5 μL 6.3 μL of cDNA ⑥ Use 2 △△Ct The method is used to calculate the quantitative results.

[0072] Table 8 Primer sequences used for identifying transcriptional levels in transgenic plants

[0073] After transcriptional level identification, compared with control plants, PtrHox52 The transcriptional level of the gene was significantly increased in resistant plants. Based on these results, Agrobacterium-mediated genetic transformation successfully yielded [the desired gene]. ptrmyb167 / 090 / 35S: PtrHox52 The three transgenic independent lines of the aggregated expression plant were named L2, L38, and L39, respectively. Among them, L39 had the highest expression fold, so it was selected for subsequent growth phenotype and drought resistance analysis.

[0074] Example 3: Populus tomentosa ptrmyb167 / 090 / 35S:PtrHox52 Analysis of plant growth and drought tolerance of polymer expression (1) The L39 polymer expression plant obtained in Example 2 and the control group were simultaneously planted in a greenhouse for growth phenotypic analysis. The control group consisted of plants that were simultaneously knocked out. PtrMYB167 and PtrMYB090 genetically modified plants, PtrHox52 Overexpressing plants and wild-type plants. The condition of polymerase-expressing plants and control groups after 4 months of growth in a greenhouse.

[0075] Growth phenotypic analysis includes the following indicators: Tree height measurement: The height from the soil surface to the terminal bud was measured in aggregate expression plants and control plants at 60, 90 and 120 days of growth, with at least 10 biological replicates in aggregate expression plants and control plants.

[0076] Stem diameter measurement: The diameter of the aggregate expression plants and control plants at the soil surface was measured using vernier calipers at 60, 90 and 120 days of growth, with at least 10 biological replicates for each aggregate expression plant and control group.

[0077] Number of stem segments: The number of stem segments was counted for the polymer expression plants and control plants after 60, 90 and 120 days of growth, respectively. There were at least 5 biological replicates for the polymer expression plants and control plants.

[0078] Node length: The length of the 10th node was recorded for the polymer expression plants and control plants after 60, 90 and 120 days of growth, respectively. There were at least 10 biological replicates for the polymer expression plants and control plants.

[0079] Through observation, it was found that ptrmyb167 / 090 / 35S:PtrHox52 The plants expressing the aggregate expression showed a significant growth advantage. Statistical analysis data showed that... OE-PtrHox52 The growth of overexpressing plants was not significantly different from that of wild-type plants, while ptrmyb167 / 090 The mutant plants were significantly taller, had a greater diameter at ground level, more stem nodes, and a longer tenth stem node than the wild type. ptrmyb167 / 090 / 35S:PtrHox52 The above indicators in the aggregate expression plants were higher than those in the wild type, but did not exceed them.ptrmyb167 / 090 Mutant plants. Based on the above results, ptrmyb167 / 090 / 35S:PtrHox52 The plants expressing the polymer were still able to maintain a good growth advantage.

[0080] (2) Biomass statistics were performed on the polymorphic expression plants obtained in Example 2. Harvest plants grown in greenhouses for 120 days ptrmyb167 / 090 / 35S:PtrHox52 Polymer expression plants and control group (simultaneously knocked out) PtrMYB167 and PtrMYB090 genetically modified plants, PtrHox52 Whole plants (overexpressing plants and wild-type plants) were used as materials for biomass statistics. Biomass was divided into three parts: leaves and stems (aboveground parts), intact roots (belowground parts), and peeled stems (woody parts). The total weight of each part in its fresh state was weighed and recorded as fresh weight. The materials were then air-dried or oven-dried at 40-60℃ until all moisture was evaporated, and weighed again as dry weight. Biomass statistics showed that both the overexpressing plants and the control group met the requirement of three biological replicates. The results indicated that, compared to the wild type, ptrmyb167 / 090 The mutant plants had significantly higher fresh and dry weights in both above-ground and underground parts, as well as in their stems, compared to the wild type. ptrmyb167 / 090 / 35S:PtrHox52 The above-ground fresh and dry weights of the aggregate-expressing plants were slightly higher than those of the wild-type plants. OE-PtrHox52 There was no significant difference in biomass between the overexpression plants and the wild-type plants. In conclusion... ptrmyb167 / 090 / 35S:PtrHox52 Plants expressing aggregates can still maintain the dominant trait of high biomass.

[0081] (3) Drought resistance analysis of the polymer expression plants obtained in Example 2. The experimental subjects were plants that had been grown in a greenhouse for 120 days. ptrmyb167 / 090 / 35S:PtrHox52 Polymer expression plants and control group (simultaneously knocked out) PtrMYB167 and PtrMYB090 genetically modified plants, PtrHox52 Nine plants from each group (overexpressing plants and wild-type plants) were selected, and after thorough watering, watering was stopped. These plants were subjected to drought stress treatment. Soil moisture content was measured daily for each plant during the drought stress period. When the soil moisture content reached approximately 15% after 12 days of drought, the plants were rehydrated. The survival rate of the plants three days after rehydration was statistically analyzed. The results showed that: OE-PtrHox52 The survival rate of overexpressing plants after rehydration was significantly higher than that of wild-type plants. ptrmyb167 / 090 / 35S:PtrHox52 The survival rate of plants expressing polymerases was between OE- PtrHox52 and ptrmyb167 / 090 Among transgenic plants. Based on the above results, in ptrmyb167 / 090 Overexpression on transgenic plants PtrHox52 Subsequently, the plants expressing the aggregation showed a significant increase in their adaptability to drought stress.

[0082] Sequence Listing SEQ ID No.1 ATGTGCACTAGAGGACATTGGAGGCCTGCTGAGGATGAGAAACTTAAGGAATTGGTTGAAAAGTATGGTCCTCATAATTGGAACGCCATTGCCGAAAAGCTTCAAGGAAGATCAGGGAAGAGTTGTAGGTTGAGATGGTTTAATCAGCTGGATCCAAGAATCAATAGAAGCCCGTTTACAGAAGAGGAAGAAGAAAGACTACTTGCTTCCCACAGGATTCATGGGAATAGATGGGCAATTATTGCAAGATTTTTCCCTGGTCGCACCGATAATGCAGTGAAGAATCATTGGCATGTCATCATGGCAAGAAGATATAGAGAGAGGTCTAGACTTCATGCAAAAAGGGCTGCTCAAACTTTGGTAAATGATAACAAATTATCCTCAAAACAAGATCACATGCACATGGATTGTGAGACGAGGAATTTTTCTTCATTTTCCAAGAAATATTGTGAAAAATATGGCCAATATCCTATGGTTACTCACAGCTACTTACCGGCCTTTTGCAAAGAGTTCTACAATGAAGATCCAAGTCATTGTGAAGATCAAAGTCGGCCGATTGAGTTTTATGATTTTCTCCAAGTAAACACTGACTCCAATAAAAGTGAAGTGATAGACAATGCAAGAAGAGATGATGAGGAGGTAGATCAGCAGGAAGCCTTGGAAAATAATCAGAGCAAGGCTGATGTTCCATTCATTGATTTTTTCTCTGTTAATGGCAAATCCTCATCATAA SEQ ID No.2 ATGTGTACTAGAGGCCATTGGAGGCCTGCAGAAGATGAGAAGCTTAAGGAGTTGGTTGAGAAGTATGGTCCTCATAATTGGAACGCGATCGCCGAAAAGCTTCATGGAAGATCAGGGAAGAGTTGTAGGTTGAGATGGTTTAATCAGTTGGATCCAAGAATCAATAGAAGTCCTTTTACCGAAGAGGAAGAAGAAAGACTTCTTGCTTCTCACCGGATCCATGGGAATAGATGGGCAGTTATTGCAAGGCTTTTCCCAGGTCGCACCGATAATGCAGTGAAGAATCATTGGCATGTCATCATGGCAAGAAGATATAGAGAAAGGTCTAGGCTTCATGCAAAAAAGAACTGCTCAAG CTTTGGTAAACGAGCAAAAATTCTCTTCAAAACAAGACATGCAGATAAATTGTGAGACGAGGAGCTTTTCTTCATTTGTCAAGAAATATTGTGAGAAATTCGGCCAATATCCTTTGATCACTCACAGCTACTTACCGGCCTTTTGGAAAGAGTTCTACAATGACGATCTAAGTAATTGTGAAGATCAAAATCGGCCCATTGAGTTCTACGATTTTCTCCAAGTAAACACGGAATCCAATAAAAGTGAAGTGATAGACAATGCAAGAAGAGAGGATGAAGAGGTAGATCAGCAGGAAGTCATATTGGAACATCAGAGCAAGGCTGGCGTTCCATTCATTGATTTTTTCTCAGCTTGA SEQ ID No.3 ATGTTTGATGGAGGAGAATATTCTCCTTCAGCAACAGAGCCTTTTAGCTGCTTGAACAGTGTCACAACCTCCAGAAAGAAGAAGAACAAGATTAAAAGGAGGTTTAGCGATGAGCAGATTAAATCATTGGAAACTATGTTCGAATCGGAAACAAGGCTTGAGCCTCGAAAGAAGATGCAGTTGGCAAGAGAGCTTGGGTTGCAACCACGACAGGTTGCGATATGGTTTCAGAATAAGAGAGCTAGATGGAAGTCTAAGCAACTCGAAAGAGACTACAGCATGCTACGAGCTAATTACAACAGCTTGGCTTCCCGGTTTGAGACTCTGAAGAAAGAGAAGCAAGCGTTGGCGATACAGTTGCAGAAGCTAAATGATCTGATGAAGAAGCCGGTAGAGGAAGGAGAGTGTTGTGGCCAAGGAGCTGCTGTGAACAGCAGTGAGGGCGAATCGGAGAATGGAGACGCAACCAAGGGTGAATCAGAAACGAAACCTAGATTGTCAATTGAACAACCAGAGCATGGATTAGGAGTCCTTTCAGATGAAGATAGCAGCATAAAGGTAGATTATTTTGAATTAGAAGAAGAACCGAACCTAATGAGTATGGTGGAACCGGCTGAAGGATCATTGACATCACAAGAAGATTGGGGGAGTATAGACTCCGATGGCCTCTTCGATCAATCAAGCAGTGGATATCAGTGGTGGGATTTCTGGGCTTGA SEQ ID No.4 MCTRGHWRPAEDEKLKELVEKYGPHNWNAIAEKLQGRSGKSCRLRWFNQLDPRINRSPFTEEEEERLLASHRIHGNRWAIIARFFPGRTDNAVKNHWHVIMARRYRERSRLHAKRAAQTLVNDNKLSSKQDHMHMDCETRNFSSFSKKYCEKYGQYPMVTHSYLPAFCKEFYNEDPSHCEDQSRPIEFYDFLQVNTDSNKSEVIDNARRDDEEVDQQEALENNQSKADVPFIDFFSVNGKSSS* SEQ ID No. 5 MCTRGHWRPAEDEKLKELVEKYGPHNWNAIAEKLHGRSGKSCRLRWFNQLDPRINRSPFTEEEEERLLASHRIHGNRWAVIARLFPGRTDNAVKNHWHVIMARRYRERSRLHAKRTAQ ALVNEQKFSSKQDMQINCETRSFSSFVKKYCEKFGQYPLITHSYLPAFWKEFYNDDLSNCEDQNRPIEFYDFLQVNTESNKSEVIDNARREDEEVDQQEVILEHQSKAGVPFIDFFSA* SEQ ID No. 6 MFDGGEYSPSATEPFSCLNSVTTSRKKKNKIKRRFSDEQIKSLETMFESETRLEPRKKMQLARELGLQPRQVAIWFQNKRARWKSKQLERDYSMLRANYNSLASRFETLKKEKQALAIQ LQKLNDLMKKPVEEGECCGQGAAVNSSEGESENGDATKGESETKPRLSIEQPEHGLGVLSDEDSSIKVDYFELEEEPNLMSMVEPAEGSLTSQEDWGSIDSDGLFDQSSSGYQWWDFWA* 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.

[0083] 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 knocking out the PtrMYB167 and PtrMYB90 genes and overexpressing the PtrHox52 gene in high-yield and drought-resistant aggregation breeding of Populus tomentosa, characterized by: To increase the biomass of Populus hairy-fruited tamarisk and enhance its drought resistance; The nucleotide sequence of the PtrMYB167 gene is shown in SEQ ID No. 1, the nucleotide sequence of the PtrMYB90 gene is shown in SEQ ID No. 2, and the nucleotide sequence of the PtrHox52 gene is shown in SEQ ID No.

3.

2. The application of knocking out the PtrMYB167 and PtrMYB90 genes and overexpressing the PtrHox52 gene in the high-yield and drought-resistant aggregation breeding of Populus tomentosa according to claim 1, characterized in that: The overexpression recombinant vector of the PtrHox52 gene was constructed by recombining the full-length CDS of the PtrHox52 gene into the plant expression vector pCAMBIA1300 and then digesting it with KpnI and SalI.

3. The application of knocking out the PtrMYB167 and PtrMYB90 genes and overexpressing the PtrHox52 gene in the high-yield and drought-resistant aggregation breeding of Populus tomentosa according to claim 1, characterized in that: The application was achieved through Agrobacterium-mediated genetic transformation of Populus tomentosa. First, the recombinant vector that knocked out PtrMYB167 and PtrMYB90 was transformed to obtain the ptrmyb167 / 090 mutant, and then the PtrHox52 overexpression vector was transformed to obtain transgenic plants with aggregated traits.

4. A method for increasing the biomass of Populus tomentosa while simultaneously enhancing its drought resistance, characterized in that: By knocking out the PtrMYB167 and PtrMYB90 genes and overexpressing the PtrHox52 gene, the biomass of Populus hairy-fruited can be increased and drought resistance enhanced. The nucleotide sequence of the PtrMYB167 gene is shown in SEQ ID No. 1, the nucleotide sequence of the PtrMYB90 gene is shown in SEQ ID No. 2, and the nucleotide sequence of the PtrHox52 gene is shown in SEQ ID No.

3. Step 1: Constructing a knockout vector: gRNAs targeting the PtrMYB167 and PtrMYB90 genes of Populus tomentosa were designed, recombined into a CRISPR / Cas9 plant expression vector, and transformed into Populus tomentosa to obtain ptrmyb167 / 090 mutant plants. Step 2: Constructing an overexpression vector: The full-length CDS of the PtrHox52 gene was recombined into the plant expression vector pCAMBIA1300 and transformed into the ptrmyb167 / 090 plants from Step 1 to obtain transgenic plants with aggregated traits. Step 3: Evaluation of growth and drought tolerance: Compared with wild-type Populus tomentosa, plants with aggregate traits showed increased biomass and improved survival rate after drought stress.

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