Application of populus trichocarpa PtrMYB167 and PtrHox52 genes in high-yield drought-tolerant pyramiding breeding of trees

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to application of populus trichocarpa PtrMYB167 and PtrHox52 genes in high-yield drought-tolerant pyramiding breeding of trees. The problem that a common forest tree improvement technology cannot simultaneously enhance drought tolerance and increase growth is solved. Comprising populus trichocarpa PtrMYB167 with nucleotide sequences shown as SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3, homologous genes PtrMYB90 and PtrHox52 of the populus trichocarpa PtrMYB167, and application of a recombinant vector containing the genes or fragments of the genes or engineering bacteria containing the genes or fragments of the genes in high-yield drought-tolerant pyramiding breeding of trees. According to the method disclosed by the invention, the polymerized character transgenic poplar plant with synchronously improved growth and drought tolerance is obtained, and a new thought and strategy are provided for cultivating high-quality, high-yield and high-resistance new forest germplasm.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and particularly relates to the application of Populus trichocarpa PtrMYB167 and PtrHox52 genes in high-yield and drought-resistant aggregate breeding of trees. Background Art

[0002] Forests are a key resource for human survival and provide sustainable biological resources for our lives and social development. Drought is the most frequent and most harmful environmental stressor in plant life activities. Under drought stress, slowing down the growth and development rate 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 tolerance. Polymer breeding can integrate multiple excellent genes into a single variety, thereby obtaining new germplasm resources with multiple resistance or excellent traits. It is an effective breeding strategy for creating excellent germplasm. At present, polybreeding technology mainly includes three types of approaches: traditional polybreeding, polybreeding using molecular marker-assisted selection, and polybreeding achieved through genetic transformation. Among them, genetic transformation polybreeding uses gene editing technology to modify the relevant genes that control the corresponding traits. It 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 forest trees. Generally speaking, improvements that enhance drought resistance are often accompanied by the negative effect of reduced growth.

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

[0005] The present invention was developed to address the inability of conventional forest improvement techniques to simultaneously enhance drought tolerance and increase biomass. A brief overview of the present invention is provided below to provide a basic understanding of certain aspects of the present invention. It should be understood that this overview is not an exhaustive overview of the present invention. It is not intended to identify key or important aspects of the present invention, nor is it intended to limit the scope of the present invention.

[0006] The technical solution of the present invention:

[0007] The present invention relates to the application of the PtrMYB167 and PtrHox52 genes of Populus trichocarpa in the high-yield and drought-resistant aggregate breeding of trees, and the application of the PtrMYB167 and its homologous genes PtrMYB90 and PtrHox52 genes with nucleotide sequences shown in SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3, recombinant vectors containing the genes or fragments thereof, or engineered bacteria containing the genes or fragments thereof in the high-yield and drought-resistant aggregate breeding of trees.

[0008] Preferably, the amino acid sequences of the proteins encoded by the PtrMYB167, PtrMYB90 and PtrHox52 genes of Populus trichocarpa are shown as SEQ ID No.4, SEQ ID No.5 and SEQ ID No.6.

[0009] Preferably: use of a recombinant vector for simultaneously knocking out the PtrMYB167 and PtrMYB90 genes of Populus trichocarpa in increasing the biomass of Populus trichocarpa.

[0010] Preferred: Use of the recombinant vector of the PtrHox52 gene in enhancing the drought tolerance of Populus trichocarpa.

[0011] Preferably, knocking out PtrMYB167 and PtrMYB90 and overexpressing PtrHox52 genes significantly increases the biomass of poplar trees and significantly enhances drought tolerance.

[0012] Preferably, the recombinant vector is a gRNA that simultaneously knocks out the PtrMYB167 and PtrMYB90 genes of Populus trichocarpa and is recombined into the plant expression vector pEgP237.

[0013] Preferably, the recombinant vector is a plant expression vector pCAMBIA1300 in which the PtrHox52 gene of Populus trichocarpa is recombined.

[0014] Preferably: use of an engineered bacterium in which both the PtrMYB167 and PtrMYB90 genes of Populus trichocarpa are knocked out simultaneously in increasing tree biomass.

[0015] Preferred: Use of engineered bacteria expressing the PtrHox52 gene of Populus trichocarpa in enhancing drought tolerance of trees.

[0016] A method for increasing tree biomass while enhancing drought tolerance, comprising constructing a recombinant vector for simultaneously knocking out the PtrMYB167 and PtrMYB90 genes of Populus trichocarpa according to the above scheme, and transforming Populus trichocarpa to obtain ptrmyb167 / 090 transgenic plants;

[0017] Construct a recombinant vector containing the PtrHox52 gene of Populus trichocarpa as claimed in claim 1, transform the ptrmyb167 / 090 transgenic material, and obtain a transgenic plant with a polymeric trait

[0018] The present invention has the following beneficial effects:

[0019] The present invention obtains transgenic poplar plants with simultaneously improved growth and drought resistance, providing new ideas and strategies for cultivating high-quality, high-yield, and highly resistant new forest germplasm. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Figure 1 is the DNA level identification result of ptrmyb167 / 090 mutant plants; M is DNAMarker DL 2000bp, 1 is the positive control using the pEgP237-PtrMYB167 / 090 recombinant plasmid as the template, 2 to 5 are identification samples using the genomic DNA of the resistant plants as the template, 6 is the negative control using the WT genomic DNA as the template; 7 is the negative control using deionized water as the template.

[0021] Figure 2 Figure 1 shows the DNA level identification results of ptrmyb167 / 090 / 35S:PtrHox52 polyexpressing plants; M is DNA Marker DL 2000; 1 is a negative control using WT genomic DNA as a template; 2 is a positive control using pCAMBIA1300-PtrHox52 recombinant plasmid as a template; 3-5 are identification samples using genomic DNA from resistant plants as templates; 6 is a negative control using deionized water as a template;

[0022] Figure 3 The relative expression levels of PtrHox52 in the xylem of Ptrmyb167 / 090 / 35S:PtrHox52 co-expressing Populus trichocarpa plants are shown in Figure 2. ptrmyb167 / 090 represents a mutant Populus trichocarpa plant with both PtrMYB167 and PtrMYB090 knocked out. L2, L38, and L39 represent three different lines of co-expressing plants. Error bars represent standard errors calculated from at least three biological replicates. Asterisks represent t-test results. **P < 0.01.

[0023] Figure 4 The growth of P. trichocarpa plants under ptrmyb167 / 090 / 35S:PtrHox52 co-expression and control groups; the control groups include: WT wild type, OE-PtrHox52 overexpression and ptrmyb167 / 090 mutant P. trichocarpa. Scale bar: 10 cm.

[0024] Figure 5Figure 1 shows the growth phenotypic analysis results of Populus trichocarpa plants grown in the greenhouse for 60, 90, and 120 days after co-expression of ptrmyb167 / 090 / 35S:PtrHox52 and the control group. The control groups included WT wild-type plants, OE-PtrHox52 overexpressing plants, and ptrmyb167 / 090 mutant plants. A represents the statistical results of plant height; B represents the statistical results of ground diameter; C represents the statistical results of stem node number; and D represents the statistical results of the length of the 10th stem node. Error bars represent standard errors calculated from at least three biological replicates. Asterisks represent t-test results, **P < 0.01, ***P < 0.001.

[0025] Figure 6 Figure 3 shows the biomass of P. trichocarpa plants grown in a greenhouse for 120 days after co-expression of ptrmyb167 / 090 / 35S:PtrHox52 and control groups; the control groups included WT wild-type, OE-PtrHox52 overexpressing, and ptrmyb167 / 090 mutant P. trichocarpa plants. Error bars represent standard errors calculated from at least three biological replicates. Asterisks represent t-test results, **P < 0.01, ***P < 0.001.

[0026] Figure 7 The results of drought stress treatment on P. trichocarpa plants with ptrmyb167 / 090 / 35S:PtrHox52 co-expression and control groups are shown. The control groups include WT wild type, OE-PtrHox52 overexpression, and ptrmyb167 / 090 mutant P. trichocarpa. The scale bar is 10 cm.

[0027] Figure 8 Figure 3. Statistical results of plant survival rates after drought stress in poplar plants expressing ptrmyb167 / 090 / 35S:PtrHox52 and the control group. The control groups included WT wild type, OE-PtrHox52 overexpression, and ptrmyb167 / 090 mutant poplar. Error bars represent standard errors calculated from at least three biological replicates. Asterisks represent t-test results. **P<0.01, ***P<0.001. DETAILED DESCRIPTION

[0028] To make the objectives, technical solutions, and advantages of the present invention more clearly apparent, the present invention is described below using specific embodiments shown in the accompanying drawings. However, it should be understood that these descriptions are merely illustrative and are not intended to limit the scope of the present invention. In addition, in the following description, descriptions of well-known structures and technologies are omitted to avoid unnecessary confusion of the concepts of the present invention.

[0029] Specific implementation method 1: Combination Figures 1-8The present embodiment is described, and the application of the PtrMYB167 and PtrHox52 genes of Populus trichocarpa in the present embodiment in the high-yield and drought-resistant aggregate breeding of trees is described, and the application of the PtrMYB167 and its homologous genes PtrMYB90 and PtrHox52 genes having nucleotide sequences as shown in SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3, recombinant vectors containing the genes or fragments thereof, or engineered bacteria containing the genes or fragments thereof in the high-yield and drought-resistant aggregate breeding of trees is described.

[0030] Specific implementation method 2: Combination Figures 1-8 This embodiment is described. The application of the PtrMYB167 and PtrHox52 genes of Populus trichocarpa in this embodiment in the high-yield and drought-resistant aggregate breeding of trees is described. The amino acid sequences of the proteins encoded by the PtrMYB167, PtrMYB90 and PtrHox52 genes of Populus trichocarpa are shown in SEQ ID No.4, SEQ ID No.5 and SEQ ID No.6.

[0031] Specific implementation method three: Combination Figures 1-8 The present embodiment is described, and the application of the PtrMYB167 and PtrHox52 genes of Populus trichocarpa in the present embodiment in the high-yield and drought-resistant aggregate breeding of trees is described, and the application of the recombinant vector for knocking out the PtrMYB167 and PtrMYB90 genes of Populus trichocarpa in increasing the biomass of Populus trichocarpa is described.

[0032] Specific implementation method four: Combination Figures 1-8 The present embodiment is described, including the application of the PtrMYB167 and PtrHox52 genes of Populus trichocarpa in the present embodiment in the high-yield and drought-resistant aggregate breeding of trees, and the application of the recombinant vector of the PtrHox52 gene in enhancing the drought resistance of Populus trichocarpa.

[0033] Specific implementation method five: Combination Figures 1-8 The present embodiment is described. The application of the PtrMYB167 and PtrHox52 genes of Populus trichocarpa in the present embodiment in the high-yield and drought-resistant aggregate breeding of trees is described. By knocking out PtrMYB167 and PtrMYB90 and expressing the PtrHox52 gene, the biomass of the poplar is significantly increased, and the drought resistance is significantly enhanced.

[0034] Specific implementation method six: combination Figures 1-8 The present embodiment is described, in which the PtrMYB167 and PtrHox52 genes of Populus trichocarpa are used in high-yield and drought-resistant tree breeding. The recombinant vector is a vector that simultaneously knocks out the PtrMYB167 and PtrMYB90 genes of Populus trichocarpa and recombines the gRNA into the plant expression vector pEgP237.

[0035] Specific implementation method seven: combination Figures 1-8 This embodiment describes the application of the PtrMYB167 and PtrHox52 genes of Populus trichocarpa in high-yield and drought-resistant tree breeding. The recombinant vector is a recombinant vector of the PtrHox52 gene of Populus trichocarpa into the plant expression vector pCAMBIA1300.

[0036] Specific implementation method eight: combination Figures 1-8 The present embodiment is described, in which the PtrMYB167 and PtrHox52 genes of Populus trichocarpa are used in high-yield and drought-resistant tree breeding, and the PtrMYB167 and PtrMYB90 gene-knockout engineered bacteria are used in increasing tree biomass.

[0037] Specific implementation method nine: Combination Figures 1-8 The present embodiment is described, and the application of the PtrMYB167 and PtrHox52 genes of Populus trichocarpa in the present embodiment in the high-yield and drought-resistant aggregate breeding of trees, and the application of the engineered bacteria of the PtrHox52 gene of Populus trichocarpa in enhancing the drought resistance of trees is described.

[0038] The engineered bacteria include Escherichia coli and Agrobacterium.

[0039] Specific implementation method ten: Combination Figures 1-8 This embodiment is described. This embodiment provides a method for increasing tree biomass while enhancing drought tolerance. The method comprises constructing a recombinant vector containing the PtrMYB167 and PtrMYB90 genes of Populus trichocarpa that are simultaneously knocked out as described in claim 1, and transforming Populus trichocarpa to obtain ptrmyb167 / 090 transgenic plants.

[0040] A recombinant vector containing the PtrHox52 gene of Populus trichocarpa as claimed in claim 1 is constructed and used to transform the ptrmyb167 / 090 transgenic material to obtain transgenic plants with aggregated traits.

[0041] The plant expression vectors used in the construction of the recombinant vector are pEgP237 and pCAMBIA1300.

[0042] The transformation method is Agrobacterium-mediated Populus trichocarpa genetic transformation method.

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

[0044] Example 1 Creation of Populus trichocarpa ptrmyb167 / 090 mutant plants

[0045] (1) Construction of pEgP237 plant expression vector

[0046] On the website (http: / / crispr.hzau.edu.cn / CRISPR2 / ), according to the gRNA selection principle, a 20bp long sequence with high homology and strong specificity was selected in the exon region of the gene as the gRNA for knocking out PtrMYB167 and the homologous gene PtrMYB090. The sequence is: GAATCATTGGCATGTCATCA. The selected gRNA was primer annealed. The sequence is shown in Table 1

[0047] Table 1 Primer sequences for gRNA annealing

[0048]

[0049] Note: The underlined part represents the BsaI restriction site.

[0050] gRNA renaturation reaction system:

[0051] Ex-taqBuffer 2μL

[0052] pEgP237-PtrMYB167 / 090-F 9 μL

[0053] pEgP237-PtrMYB167 / 090-F 9 μL

[0054] gRNA renaturation reaction conditions:

[0055]

[0056] The product was diluted 100-fold and stored at -20°C until use.

[0057] The renatured gRNA was constructed on the pEgP237 vector. First, the pEgP237 vector was single-enzyme digested, and the reaction system and conditions were as follows:

[0058] Enzyme digestion system:

[0059]

[0060]

[0061] Enzyme digestion conditions:

[0062] 37℃ for 2 hours

[0063] After enzyme digestion, separate the target band by electrophoresis and recover the target band using a gel recovery kit. Use T4 ligase to load the renatured product into the pEgP237 vector. The specific reaction system and conditions are as follows:

[0064] T4 connection system:

[0065]

[0066] Connection conditions:

[0067] 4℃ overnight

[0068] Thaw the TOP10 competent cells on ice. Add 5μ of the ligation product to the competent cells and mix thoroughly. Heat shock at 42°C for 1 minute, place on ice for 3 minutes, add resistance-free LB medium, and activate at 37°C and 220 rpm for one hour. Collect the cells at low speed and spread them on solid LB medium (containing 50 mg / L kanamycin) and culture at 37°C overnight. The next day, perform PCR identification on the obtained single clones. The primers are shown in Table 2:

[0069] Table 2 Primer sequences used for single clone detection of pEgP237 plant expression vector

[0070]

[0071] Positive single colonies were selected and cultured in 5 mL of LB medium (kanamycin, 50 mg / L) at 220 rpm and 37°C overnight. The next day, the strains were preserved and sequenced. Sequencing analysis confirmed that the gRNA had been successfully inserted into the vector, and the pEgP237 plant expression vector for generating ptrmyb167 / 090 mutant plants had been successfully constructed.

[0072] (2) Freeze-thaw transformation of Agrobacterium

[0073] ① Take out the Agrobacterium competent cells and place them on ice. Add about 100 ng of plasmid to the Agrobacterium competent cells, mix well and place them in an ice bath for 30 minutes. Immediately place them in liquid nitrogen and freeze them for 1 minute. Then immediately place them in a 37°C water bath for 3 minutes.

[0074] ②Add 500 μL liquid YEP and incubate at 28°C, 180-200 rpm for 2-3 hours.

[0075] ③ Centrifuge for 15 seconds, remove the supernatant, retain 300-400 μL of the supernatant, resuspend, spread on YEP (kanamycin + gentamicin + rifampicin) solid culture medium, and culture at 28°C for about 48 hours.

[0076] ④ Perform PCR using a single clone as a template to confirm the positive clone. The specific method is as described in Example 1.

[0077] ⑤Pick the positive clones, inoculate them into 5mL YEP (kanamycin + gentamicin + rifampicin) liquid culture medium, and culture them at 180rpm and 28℃ overnight.

[0078] ⑥ To preserve positive clones, add 500 μL bacterial solution and 500 μL 50% glycerol, mix well, freeze quickly in liquid nitrogen, and store at -80°C.

[0079] (3) Agrobacterium-mediated genetic transformation of Populus trichocarpa

[0080] ① Take the constructed Agrobacterium culture liquid and streak it on YEP (kanamycin + gentamicin + rifampicin) plates, and incubate the streaked plates at 28°C for two days.

[0081] ② Pick a single clone and inoculate it into 20mL YEP (kanamycin + gentamicin + rifampicin) liquid culture medium, culture overnight at 28℃ and 180rpm until OD 600 =0.9. Take 1 mL of the cultured bacterial solution and add it to 50 mL of YEP (kanamycin + gentamicin + rifampicin) liquid culture medium. Cultivate at 28°C, 200 rpm until OD 600 =0.6, centrifuge the cells, and resuspend them in suspension solution until OD 600 =0.4, reserve.

[0082] ③ Cut the stem segments of wild-type tissue culture seedlings of Populus trichocarpa into small segments of about 1 cm, place them in the bacterial solution, and infect for 20 minutes.

[0083] ④ Remove the stem segments, place them on co-cultivation medium, and culture in the dark for 2 days.

[0084] ⑤ Two days later, the co-cultured stem segments were washed with cephalosporin water (250 mg / L) for 5 minutes, and then washed twice with sterile water for 3 minutes each time.

[0085] ⑥ Place the cleaned stem segments on selective culture medium (cephalosporin 250 mg / L + kanamycin 25 mg / L) and culture under light.

[0086] ⑦ After the resistant buds grow, insert them into the rooting medium of kanamycin resistance (20 mg / L) to screen positive plants.

[0087] (4) Identification of transgenic plants

[0088] ① Cut leaves from resistant plants for DNA extraction. For specific steps, refer to the instructions of the Plant DNA Extraction Kit (TIANGEN).

[0089] ② The above DNA was used as a template and PCR amplification was performed with M13F and pEgP237-PtrMYB167 / 090-R as primers. 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 ), indicating that the recombinant plasmid had been successfully integrated into the genome of Populus trichocarpa.

[0090] ③ In order to further determine the specific editing status of the ptrmyb167 / 090 mutant plants, DNA was extracted from the third leaf of the mutant plants and used as an amplification template to amplify the PtrMYB167 and PtrMYB090 genes, including gRNA. The amplification primers are shown in Table 3. The theoretical amplified sequence of the PtrMYB167 gene is 1012 bp (SEQ ID NO. 7), and the theoretical amplified sequence of the PtrMYB90 gene is 974 bp (SEQ ID NO. 8). They were constructed into the pMD18-T vector and transformed into Escherichia coli. 30 single clones were selected from each plant and sent to the company for sequencing and comparison of the results. The results are shown in Table 4. Compared with the wild-type sequence, the PtrMYB090 gene of the ptrmyb167 / 090-L4 mutant plant had two editing situations, with one base inserted in one DNA chain and nine bases deleted in the other chain. Its PtrMYB167 gene had one editing situation, and one chain was frameshifted due to the deletion of one base, resulting in the inability to express the PtrMYB167 protein normally. The PtrMYB090 gene of the ptrmyb167 / 090-L5 mutant plant had only one editing situation, with one base inserted in one DNA chain, while the PtrMYB167 gene had two editing situations, with one chain missing 13 bases and the other chain missing 5 bases. The editing situations of the above two strains proved that the ptrmyb167 / 090 mutant plant was successfully obtained.

[0091] Table 3 Primer sequences for identifying PtrMYB90 / 167 gene editing

[0092]

[0093] Table 4 gRNA editing status of ptrmyb167 / 090 gene mutants

[0094]

[0095] Note: The underlined part represents the insertion or replacement of bases, and “-” represents the deletion of a base.

[0096] SEQ ID NO.7:

[0097]

[0098] SEQ ID NO.8:

[0099] GTGCTATAGCTATAGCTAGGGTTCATATATTCAAGTACTATAGAAATATCGTGTTTTTGGTTCTTGGGATTGGATTCTTGATTGTTGTATTTAGAAGATTAAAAAAAATGTGTACTAGAGGCCATTGGAGGCCTGCAGAAGATGAGAAGCTTAAGGAGTTGGTTGAGAAGTATGGCCCTCATAATTGGAACGCGATCGCCGAAAAGCTTCATGGAAGATCAGGTGAAAATTTAAACCTAAAGAGAAACTAATTTGTATCCTCACAAATATTTTCATTTCAATATTTTTTTTATGTTGTTATTTAGAAACAACTAGGGTTTGGTACTGTATGCAAAAGTTGAGATATACTTATAACTAATGGAAATTAAAAGGGAAGCTAACTATGACTGTGACTAATAACTAACAAAACGAAGTTTGAACAAATTGAATCAGGGAAGAGTTGTAGGTTGAGATGGTTTAATCAGTTGGATCCAAGAATCAATAGAA GTCCTTTTACCGAAGAGGAAGAAGAAAGACTTCTTGCTTCTCACCGGATCCATGGGAATAGATGGGCAGTTATTGCAAGGCTTTTCCCAGGTCGCACCGATAATGCAGTGAAGAATCATTGGCATGTCATCATGGCAAGAAGATATAGAGAAAGGTCTAGGCTTCATGCAAAAAGAACTGCTCAAGCTTTGGTAAACGAGCAAAAATTCTCTTCAAAACAAGACATGCAGATAAATTGTGAGACGAGGAGCTTTTCTTCATTTGTCAAGAAATATTGTGAGAAATTCGGCCAATATCCTTTGATCACTCACAGCTACTTACCGGCCTTTTGGAAAGAGTTCTACAATGACGATCTAAGTAATTGTAAAGGTAACTAGATCGAGCTAGCATCATGGTGATGTGCTATATATACAGTTCGGGTTCTAGTTTTACAATATTGATAATAATTTCTTAGCTCTTATTGCTCATGTTCCTTACATACATGG

[0100] Example 2: Creation of Populus trichocarpa ptrmyb167 / 090 / 35S:PtrHox52 Poly-expressing Plants

[0101] The full-length CDS sequence of the PtrHox52 gene was constructed into a plant expression vector. Primers were designed and commissioned to be synthesized by a biological company. The primer sequences for constructing the pCAMBIA1300 plant expression vector are shown in Table 5.

[0102] Table 5 Primer sequences used to construct the pCAMBIA1300 plant expression vector

[0103]

[0104] Note: The underlined part represents the restriction site, and the restriction site is preceded by a protective base.

[0105] The target sequence was obtained by polymerase chain reaction (PCR) using the above primers, and the target band was separated by agarose gel electrophoresis of the obtained PCR product. The target fragment was recovered by gel recovery kit.

[0106] The recovered target gene DNA fragment and the pCAMBIA1300 plant expression vector were double-digested. The specific reaction system and reaction conditions are as follows:

[0107] Target DNA fragment digestion system:

[0108]

[0109] pCAMBIA1300 vector enzyme digestion system:

[0110]

[0111] Enzyme digestion conditions:

[0112] 37℃ for 2 hours

[0113] After enzyme digestion, separate the target bands by electrophoresis and recover them using a gel recovery kit. Use T4 ligase to insert the target gene into the plant expression vector pCAMBIA1300. The specific reaction system and conditions are as follows:

[0114] T4 connection system:

[0115]

[0116] Connection conditions:

[0117] 4℃ overnight

[0118] Thaw the TOP10 competent cells on ice. Add 5μ of the ligation product to the competent cells and mix thoroughly. Heat shock at 42°C for 1 minute, place on ice for 3 minutes, add resistance-free LB medium, and activate at 37°C and 220 rpm for one hour. Collect the cells at low speed and spread them on solid LB medium (containing 50 mg / L kanamycin) and culture at 37°C overnight. The next day, perform PCR identification on the obtained single clones. The primers are shown in Table 6:

[0119] Table 6 Primer sequences for constructing monoclonal detection using pC1300 plant expression vector

[0120]

[0121] PCR products were analyzed by electrophoresis to identify positive clones. Single positive clones were selected and cultured in 5 mL of LB medium (kanamycin, 50 mg / L) at 220 rpm and 37°C overnight. The next day, the strain was preserved and sequenced using primer 35SP1.

[0122] Sequencing analysis revealed that the inserted fragment of 717 bp (SEQ ID NO. 3) in the vector was consistent with the sequence information of Populus trichocarpa PtrHox52 provided by the phytozome website, indicating that we have successfully obtained the pCAMBIA1300 plant expression vector fused with the Populus trichocarpa PtrHox52 gene.

[0123] SEQ ID NO.3:

[0124] ATGAGGAAGCGACAAGTGGTGGTAAGAAGATCAGAAGAACCTTCAAGAAGCTCAACGACTTCTTCTTTCACTATCAGGAATGTTAAATACGGAGAGTGCCAAAAGAATCATGCTGCTGGAGTTGGAGGTTACGCTGTTGATGGG TGCAGAGAGTTCATGGCAAGTGGTGAAGAAGGCACAGCTGCTGCACTCACTTGTGCGGCTTGTGGTTGCCATAGGAACTTCCACAGAAGGGAAGTGGAAACAGAGGTGGCATGTGATTGTTCTTCACCTTCTTCAAATGGTAAT

[0125] (2) Freeze-thaw transformation of Agrobacterium

[0126] The specific method is as shown in Example 1 (2) Agrobacterium-mediated genetic transformation of Populus trichocarpa

[0127] The specific method is as described in Example 1. The difference from Example 1 is that the transformed material is tissue culture seedlings of ptrmyb167 / 090 mutant plants, and the resistance to hygromycin is screened using a selection medium (cephalosporin 250 mg / L + hygromycin 5 mg / L) and a resistance rooting medium (hygromycin 5 mg / L).

[0128] (3) Identification of transgenic plants

[0129] ① Cut leaves from resistant plants for DNA extraction. For specific steps, refer to the instructions of the Plant DNA Extraction Kit (TIANGEN).

[0130] ② Using the above DNA as a template, PCR amplification was performed using primers specific for the F and R ends of hygromycin (see Table 7 for primer sequences). The recombinant plasmid served as a positive control, while wild-type DNA and water served as negative controls. DNA analysis confirmed the correct band positions, indicating that the recombinant plasmid had successfully integrated into the Populus trichocarpa genome.

[0131] Table 7 Primer sequences used for DNA level identification of transgenic plants

[0132]

[0133] ③ The resistant plants were grown in a greenhouse, with transgenic plants in which both PtrMYB167 and PtrMYB090 genes were knocked out as controls. After culturing for 4 months, xylem tissues of the resistant and control plants were collected for RNA extraction. The specific steps were referred to the instructions of the Plant RNA Extraction Kit (Omega). The concentration and quality of the obtained RNA were determined using NanoDrop.

[0134] ④ Refer to the instructions and use the OligdT primer to reverse transcribe the obtained RNA to obtain cDNA.

[0135] ⑤ The obtained cDNA was diluted 10-fold and subjected to RT-qPCR. The internal control was PtrActin7. The primer sequences are shown in Table 8.

[0136] Quantitative PCR reaction system 15 μL:

[0137]

[0138] ⑥Use 2 △△Ct The quantitative results were calculated.

[0139] Table 8 Primer sequences used for identification of transcriptional levels in transgenic plants

[0140]

[0141]

[0142] Transcriptional analysis revealed that the transcriptional level of the PtrHox52 gene was significantly increased in resistant plants compared to control plants. Based on these results, ptrmyb167 / 090 / 35S:PtrHox52 poly-expressing plants were successfully generated through Agrobacterium-mediated genetic transformation. Three independent transgenic lines were designated L2, L38, and L39. L39 had the highest expression fold change and was therefore selected for subsequent growth phenotype and drought tolerance analysis.

[0143] Example 3: Growth and drought tolerance analysis of poplar ptrmyb167 / 090 / 35S:PtrHox52 co-expressing plants

[0144] (1) The polyexpressing plant L39 obtained in Example 2 and a control group were simultaneously planted in a greenhouse for growth phenotype analysis. The control group consisted of a transgenic plant with both PtrMYB167 and PtrMYB090 knockout, a plant overexpressing PtrHox52, and a wild-type plant. The polyexpressing plants and the control group were grown in the greenhouse for 4 months.

[0145] Growth phenotyping analysis includes the following indicators:

[0146] Tree height measurement: The height from the soil surface to the terminal bud was measured for the aggregate-expressing plants and the control group at 60, 90, and 120 days of growth, with at least 10 biological replicates for the aggregate-expressing plants and the control group.

[0147] Basal stem measurement: The diameter of the aggregate-expressing plants and the control group at the soil surface was measured using a vernier caliper at 60, 90, and 120 days of growth, with at least 10 biological replicates for the aggregate-expressing plants and the control group.

[0148] Number of stem nodes: The number of stem nodes of the poly-expressing plants and the control group were counted at 60, 90, and 120 days of growth, and at least 5 biological replicates were performed for the poly-expressing plants and the control group.

[0149] Internode length: The length of the 10th internode was counted for the poly-expressing plants and the control group at 60, 90, and 120 days of growth, respectively. At least 10 biological replicates were performed for the poly-expressing plants and the control group.

[0150] Observations revealed that plants co-expressing ptrmyb167 / 090 / 35S:PtrHox52 exhibited a significant growth advantage. Statistical analysis revealed that OE-PtrHox52 overexpressing plants showed no significant growth differences compared to wild-type plants. However, ptrmyb167 / 090 mutant plants significantly surpassed wild-type plants in plant height, ground diameter, number of nodes, and tenth node length. These indicators were slightly improved in plants co-expressing ptrmyb167 / 090 / 35S:PtrHox52 compared to wild-type plants, but not exceeding those in ptrmyb167 / 090 mutant plants. These results suggest that plants co-expressing ptrmyb167 / 090 / 35S:PtrHox52 maintain a favorable growth advantage.

[0151] (2) Biomass statistics of the polymer expression plants obtained in Example 2

[0152] Whole-plant biomass analysis was performed on plants expressing ptrmyb167 / 090 / 35S:PtrHox52, as well as controls (transgenic plants with both PtrMYB167 and PtrMYB090 knockout, plants overexpressing PtrHox52, and wild-type plants) grown in the greenhouse for 120 days. Biomass was divided into three parts: leaves and stems as the aboveground portion, intact roots as the underground portion, and peeled stems as the wood portion. The total weight of each part was weighed and recorded as the fresh weight. The materials were then air-dried or oven-dried at 40-60°C. After complete evaporation of moisture, they were weighed and recorded as the dry weight of each part. Biomass analysis showed that three biological replicates were sufficient for both the ptrmyb167 / 090 / 35S:PtrHox52 knockout plants and the control group. The results showed that compared with the wild type, the fresh and dry weights of the aboveground and underground parts and stems of the ptrmyb167 / 090 mutant plants were significantly higher. The fresh and dry weights of the aboveground parts of the ptrmyb167 / 090 / 35S:PtrHox52 co-expressing plants were slightly higher than those of the wild type. Furthermore, OE-PtrHox52 overexpressing plants showed no significant difference in biomass compared to the wild type. In summary, ptrmyb167 / 090 / 35S:PtrHox52 co-expressing plants can still maintain the advantageous trait of high biomass.

[0153] (3) Drought tolerance analysis of the polyexpression plants obtained in Example 2

[0154] The experimental subjects were ptrmyb167 / 090 / 35S:PtrHox52 co-expressing plants and a control group (transgenic plants with simultaneous knockout of PtrMYB167 and PtrMYB090, PtrHox52 overexpressing plants, and wild-type plants) grown in a greenhouse for 120 days. Nine plants in each group were selected for drought stress treatment after sufficient watering and then withheld water. The soil moisture content of each plant was measured every day during the drought stress period. When the soil moisture content of the plants reached about 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 the survival rate of the OE-PtrHox52 overexpressing plants after rehydration was significantly higher than that of the wild-type, and the survival rate of the ptrmyb167 / 090 / 35S:PtrHox52 co-expressing plants was between that of the OE-PtrHox52 and ptrmyb167 / 090 transgenic plants. Based on the above results, after overexpressing PtrHox52 on the basis of ptrmyb167 / 090 transgenic plants, the adaptability of the poly-expressing plants to drought stress was significantly enhanced.

[0155] Serial number

[0156] SEQ ID No.1

[0157] ATGTGCACTAGAGGACATTGGAGGCCTGCTGAGGATGAGAAACTTAAGGAATTGGTTGAAAAGTATGGTCCTCATAATTGGAACGCCATTGCCGAAAAGCTTCAAGGAAGATCAGGGAAGAGTTGTAGGTTGAGATGGTTTAATCAGCTGGATCCAAGAATCAATAGAAGCCCGTTTACAGAAGAGGAAGAAGAAAGACTACTTGCTTCCCACAGGATTCATGGGAATAGATGGGCAATTATTGCAAGATTTTTCCCTGGTCGCACCGATAATGCAGTGAAGAATCATTGGCATGTCATCATGGCAAGAAGATATAGAGAGAGGTCTAGACTTCATGCAAAAAGGGCTGCTCAAACTTTGGTAAAT GATAACAAATTATCCTCAAAACAAGATCACATGCACATGGATTGTGAGACGAGGAATTTTTCTTCATTTTCCAAGAAATATTGTGAAAAATATGGCCAATATCCTATGGTTACTCACAGCTACTTACCGGCCTTTTGCAAAGAGTTCTACAATGAAGATCCAAGTCATTGTGAAGATCAAAGTCGGCCGATTGAGTTTTATGATTTTCTCCAAGTAAACACTGACTCCAATAAAAGTGAAGTGATAGACAATGCAAGAAGAGATGATGAGGAGGTAGATCAGCAGGAAGCCTTGGAAAATAATCAGAGCAAGGCTGATGTTCCATTCATTGATTTTTTCTCTGTTAATGGCAAATCCTCATCATAA

[0158] SEQ ID No. 2

[0159] ATGTGTACTAGAGGCCATTGGAGGCCTGCAGAAGATGAGAAGCTTAAGGAGTTGGTTGAGAAGTATGGTCCTCATAATTGGAACGCGATCGCCGAAAAGCTTCATGGAAGATCAGGGAAGAGTTGTAGGTTGAGATGGTTTAATCAGTTGGATCCAAGAATCAATAGAAGTCCTTTTACCGAAGAGGAAGAAGAAAGACTTCTTGCTTCTCACCGGATCCATGGGAATAGATGGGCAGTTATTGCAAGGCTTTTCCCAGGTCGCACCGATAATGCAGTGAAGAATCATTGGCATGTCATCATGGCAAGAAGATATAGAGAAAGGTCTAGGCTTCATGCAAAAAGAACTGCTCAAG CTTTGGTAAACGAGCAAAAATTCTCTTCAAAACAAGACATGCAGATAAATTGTGAGACGAGGAGCTTTTCTTCATTTGTCAAGAAATATTGTGAGAAATTCGGCCAATATCCTTTGATCACTCACAGCTACTTACCGGCCTTTTGGAAAGAGTTCTACAATGACGATCTAAGTAATTGTGAAGATCAAAATCGGCCCATTGAGTTCTACGATTTTCTCCAAGTAAACACGGAATCCAATAAAAGTGAAGTGATAGACAATGCAAGAAGAGGAGGATGAAGAGGTAGATCAGCAGGAAGTCATATTGGAACATCAGAGCAAGGCTGGCGTTCCATTCATTGATTTTTTCTCAGCTTGA

[0160] SEQ ID No. 3

[0161] ATGTTTGATGGAGGAGAATATTCTCCTTCAGCAACAGAGCCTTTTAGCTGCTTGAACAGTGTCACAACCTCAGAAAGAAGAAGAACAAGATTAAAAGGAGGTTTAGCGATGAGCAGATTAAATCATTGGAAACTATGTTCGAATCGGAAACAAGGCTTGAGCCTCGAAAGAAGATGCA GTTGGCAAGAGAGCTTGGGTTGCAACCACGACAGGTTGCGATATGGTTTCAGAATAAGAGAGCTAGATGGAAGTCTAAGCAACTCGAAAGAGACTACAGCATGCTACGAGCTAATTACAACAGCTTGGCTTCCCGGTTTGAGACTCTGAAGAAAGAGAAGCAAGCGTTGGCGATACAGT TGCAGAAGCTAAATGATCTGATGAAGAAGCCGGTAGAGGAAGGAGAGTGTTGTGGCCAAGGAGCTGCTGTGAACAGCAGTGAGGGCGAATCGGAGAATGGAGACGCAACCAAGGGTGAATCAGAAACGAAACCTAGATTGTCAATTGAACAACCAGAGCATGGATTAGGAGTCCTTTTCA GATGAAGATAGCAGCATAAAGGTAGATTATTTTGAATTAGAAGAAGAACCGAACCTAATGAGTATGGTGGAACCGGCTGAAGGATCATTGACATCACAAGAAGATTGGGGGAGTATAGACTCCGATGGCCTCTTCGATCAATCAAGCAGTGGATATCAGTGGTGGGATTTCTGGGCTTGA

[0162] SEQ ID No. 4

[0163] MCTRGHWRPAEDEKLKELVEKYGPHNWNAIAEKLQGRSGKSCRLRWFNQLDPRINRSPFTEEEERLLASHRIHGNRWAIIARFFPGRTDNAVKNHWHVIMARRYRRSRLHAKRAAQTLVN DNKLSSKQDHMHMDCETRNFSSFSKKYCEKYGQYPMVTHSYLPAFCKEFYNEDPSHCEDQSRPIEFYDFLQVNTDSNKSEVIDNARRDDEEVDQQEALENNQSKADVPFIDFFSVNGKSSS*

[0164] SEQ ID No.5

[0165] MCTRGHWRPAEDEKLKELVEKYGPHNWNAIAEKLHGRSGKSCRLRWFNQLDPRINRSPFTEEEERLLASHRIHGNRWAVIARLFPGRTDNAVKNHWHVIMARRYRERSRLHAKRTAQ ALVNEQKFSSKQDMQINCETRSFSSFVKKYCEKFGQYPLITHSYLPAFWKEFYNDDLSNCEDQNRPIEFYDFLQVNTESNKSEVIDNARREDEEVDQQEVILEHQSKAGVPFIDFFSA*

[0166] SEQ ID No.6

[0167] MFDGGEYSPSATEPFSCLNSVTTSRKKKNKIKRRFSDEQIKSLETMFESETRLEPRKKMQLARELGLQPRQVAIWFQNKRARWKSKQLERDYSMLRANYNSLASRFETLKKEKQALAIQ LQKLNDLMKKPVEEGECCGQGAAVNSSEGESENGDATKGESETKPRLSIEQPEHGLGVLSDEDSSIKVDYFELEEEPNLMSMVEPAEGSLTSQEDWGSIDSDGLFDQSSSGYQWWDFWA*

[0168] 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 permutations and combinations. Therefore, the present invention will no longer describe the technical solutions after permutations and combinations one by one, but it should be understood that the technical solutions after permutations and combinations have been disclosed by the present invention.

[0169] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. Application of PtrMYB167 and PtrHox52 genes of Populus trichocarpa in high-yield and drought-resistant aggregate breeding of trees, characterized in that: The application of the Populus trichocarpa PtrMYB167 and its homologous genes PtrMYB90 and PtrHox52 genes as shown in nucleotide sequences such as SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3, recombinant vectors containing the genes or fragments thereof, or engineered bacteria containing the genes or fragments thereof in high-yield and drought-resistant aggregate breeding of trees.

2. The use of the PtrMYB167 and PtrHox52 genes of Populus trichocarpa in high-yield and drought-resistant aggregate breeding of trees according to claim 1, characterized in that: The amino acid sequences of the proteins encoded by the PtrMYB167, PtrMYB90 and PtrHox52 genes of Populus trichocarpa are shown in SEQ ID No.4, SEQ ID No.5 and SEQ ID No.

6.

3. The use of the PtrMYB167 and PtrHox52 genes of Populus trichocarpa in high-yield and drought-resistant aggregate breeding of trees according to claim 1, characterized in that: Application of recombinant vector for simultaneously knocking out PtrMYB167 and PtrMYB90 genes in increasing biomass of Populus trichocarpa.

4. The use of the PtrMYB167 and PtrHox52 genes of Populus trichocarpa in high-yield and drought-resistant aggregate breeding of trees according to claim 1, characterized in that: Application of the recombinant vector of PtrHox52 gene in enhancing drought tolerance of Populus trichocarpa.

5. The use of the PtrMYB167 and PtrHox52 genes of Populus trichocarpa in high-yield and drought-resistant aggregate breeding of trees according to claim 1, characterized in that: Knocking out PtrMYB167 and PtrMYB90 and overexpressing PtrHox52 genes significantly increased the biomass of poplar and significantly enhanced its drought resistance.

6. The use of the PtrMYB167 and PtrHox52 genes of Populus trichocarpa in high-yield and drought-resistant aggregate breeding of trees according to claim 3, characterized in that: The recombinant vector is a plant expression vector pEgP237 in which the gRNA for simultaneously knocking out the PtrMYB167 and PtrMYB90 genes of Populus trichocarpa is recombined.

7. The use of the PtrMYB167 and PtrHox52 genes of Populus trichocarpa in high-yield and drought-resistant aggregate breeding of trees according to claim 4, characterized in that: The recombinant vector is obtained by recombining the Populus trichocarpa PtrHox52 gene into a plant expression vector pCAMBIA1300.

8. The use of the PtrMYB167 and PtrHox52 genes of Populus trichocarpa in high-yield and drought-resistant aggregate breeding of trees according to claim 1, characterized in that: The application of engineered bacteria with simultaneous knockout of PtrMYB167 and PtrMYB90 genes in increasing tree biomass.

9. The use of the PtrMYB167 and PtrHox52 genes of Populus trichocarpa in high-yield and drought-resistant aggregate breeding of trees according to claim 1, characterized in that: Application of engineered bacteria expressing the PtrHox52 gene of Populus trichocarpa in enhancing drought tolerance of trees.

10. A method for increasing tree biomass and drought tolerance, characterized in that: Constructing a recombinant vector containing the PtrMYB167 and PtrMYB90 genes of Populus trichocarpa that are simultaneously knocked out as described in claim 1, and transforming Populus trichocarpa to obtain ptrmyb167 / 090 transgenic plants; A recombinant vector containing the PtrHox52 gene of Populus trichocarpa as claimed in claim 1 is constructed and transformed into ptrmyb167 / 090 transgenic material to obtain transgenic plants with aggregated traits.

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