Paghb7a gene related to salt tolerance and drought tolerance of poplar and use thereof

Abstract

Provided is a PagHB7a gene related to the salt tolerance and drought tolerance of poplar. The gene is used to create new germplasms of salt-tolerant and drought-tolerant 84K poplar (Populus alba×Populus glandulosa). Being a candidate gene related to the salt tolerance and drought resistance of poplar, the PagHB7a gene provides an important gene resource for breeding new varieties of salt-tolerant and drought-resistant poplar, and can be directly used for molecular-assisted breeding of stress-resistant poplar. In addition, a primer pair for amplifying the candidate gene and a transgenic modification method for poplar are provided, which can evaluate the stress resistance of poplar on the molecular level; on this basis, varieties with remarkable stress resistance can be accurately and efficiently screened, thus effectively shortening the breeding period of poplar.

Description

PagHB7a, a gene associated with salt and drought tolerance in poplar trees, and its application.

[0001] Cross-referencing

[0002] This application claims priority to Chinese Patent Application No. 2024105698494, filed on May 9, 2024, entitled “A gene PagHB7a related to salt and drought tolerance in poplar and its application thereto”, the entire disclosure of which is incorporated herein by reference. Technical Field

[0003] This invention belongs to the field of poplar technology, specifically relating to a gene PagHB7 related to poplar stress resistance and its application in breeding salt-tolerant and drought-resistant transgenic poplars. Background Technology

[0004] Poplar trees are excellent afforestation trees and also beautiful urban greening species. However, in arid, semi-arid, and severely saline-alkali areas, salinity and drought severely restrict the growth, development, and spatial distribution of poplar trees. In particular, frequent extreme weather events exacerbate the harmful effects of abiotic stress on plant growth. Therefore, identifying key genes and regulatory networks for stress resistance and using strategies such as molecular design breeding to cultivate new poplar varieties with strong stress resistance is of great significance for increasing timber production in marginal arid and saline-alkali lands.

[0005] The PagHB7a gene belongs to the HD-Zip I subfamily, and previous studies have shown that genes in the HD-Zip I subfamily play important roles in drought stress resistance. In Arabidopsis thaliana, the expression of the HD-Zip I subfamily genes AtHB6 and AtHB7 is induced by ABA application or water deficiency. [1,2 , 3] In addition, overexpression of the HB7 gene can enhance the drought resistance of poplar trees. [4] Previous RNA-seq studies in our laboratory have shown that two AtHB7 homologs in 84K poplar (named PagHB7a and PagHB7b) are significantly induced by salt, with PagHB7a showing a higher fold induction. [5] Therefore, studying the role of the PagHB7a gene in poplar stress resistance can lay a theoretical and genetic resource foundation for molecular breeding of poplar stress resistance.

[0006] References

[0007] [1] E., et al. The Arabidopsis homeobox gene ATHB-7 is induced by water deficit and by abscisic acid. Plant J, 1996, 10: 375–381.

[0008] [2] Himmelbach A, et al. Homeodomain protein ATHB6 is a target of the protein phosphatase ABI1 and regulates hormone responses in Arabidopsis. EMBO J, 2002, 21: 3029–3038.

[0009] [3] Lechner E, et al. MATH / BTB CRL3 receptors target the homeodomain-leucine zipper ATHB6 to modulate abscisic acid signaling. Dev Cell, 2011, 21: 1116–1128.

[0010] [4] Baofeng Zhang, et al. A COMPASS histone H3K4 trimethyltransferase pentamer transactivates drought tolerance and growth / biomass production in Populus trichocarpa. New Phytol, 2023, 32(1): 1.

[0011] [5] Jin-Gui Liu, et al. Genome-wide transcriptional adaptation to salt stress in Populus. BMC Plant Biol, 2019, (1): 367. Summary of the Invention

[0012] To address the aforementioned problems, this invention provides a PagHB7a gene related to the salt tolerance and drought resistance of poplar, and utilizes this gene to create a new 84K poplar germplasm exhibiting both salt tolerance and drought resistance. The PagHB7a gene is a candidate gene related to the salt tolerance and drought resistance of poplar. This invention provides an important gene resource for breeding new stress-resistant poplar varieties and can be directly applied to poplar assisted breeding. Furthermore, primer pairs for amplifying the candidate gene and methods for poplar genetic improvement are provided, enabling the evaluation of poplar stress resistance at the molecular level. Based on this, tree species with significant stress resistance can be precisely and efficiently screened, effectively shortening the poplar breeding cycle, thus completing this invention.

[0013] The first aspect of this invention aims to provide a PagHB7a gene related to the salt tolerance and drought resistance of poplar trees, the nucleotide sequence of which is shown in SEQ ID NO.21.

[0014] The second aspect of this invention aims to provide a protein encoded by the PagHB7a gene, which is related to the salt tolerance and drought resistance of poplar trees, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.22.

[0015] The third aspect of this invention aims to provide a breeding method for transgenic 84K poplar with enhanced salt tolerance and drought resistance, characterized in that the method specifically includes the following steps:

[0016] Step 1. Construct the PagHB7a gene into the pPZP211 overexpression vector to obtain the recombinant plasmid;

[0017] Step 2. Transform the Agrobacterium with the recombinant vector into Agrobacterium and activate it to obtain Agrobacterium bacterial culture;

[0018] Step 3. Infect 84K poplar leaf tissue with Agrobacterium bacterial solution;

[0019] Step 4. Through co-culture and selective culture of leaf tissues, adventitious buds are differentiated and rooted to form complete plants.

[0020] The present invention has the following beneficial effects:

[0021] (1) The PagHB7a gene provided by this invention is significantly associated with salt tolerance and drought resistance. Although overexpression of PagHB7a enhances the salt tolerance and drought resistance of poplar, there is no significant difference in growth between transgenic poplar and wild-type 84K poplar (WT) under normal conditions. This is of great significance for enhancing the stress resistance of poplar and mitigating the damage caused by stress to forestry yield and quality.

[0022] (2) The primer pairs for quantitative real-time PCR related to salt tolerance and drought resistance of poplar provided by this invention can accurately evaluate the salt tolerance and drought resistance of poplar at the molecular level, accurately and efficiently screen salt-tolerant and drought-resistant poplar germplasm during the seedling stage, shorten the poplar breeding cycle, and provide an effective means for molecular-assisted breeding of poplar. Attached Figure Description

[0023] Figure 1A shows the expression level of PagHB7a under salt treatment detected by RNA-seq. H1, H3, H6, and H12 represent 1, 3, 6, and 12 hours, and D1, D5, and D10 represent 1, 5, and 10 days.

[0024] Figure 1B shows the expression levels of PagHB7a in the root, stem, leaf, and petiole tissues of 84K poplar as detected by RT-qPCR;

[0025] Figure 1C shows the expression level of PagHB7a detected by RT-qPCR at different salt treatment times (0-48h);

[0026] Figure 2A shows the transcriptional level of PagHB7a in overexpressing plants (OE-22, OE-26, OE-27) detected by RT-qPCR, with the error bars representing the standard deviation;

[0027] Figure 2B shows the mutations at the sgRNA target sites in PagHB7a knockout plants (KO-2, KO-6);

[0028] Figure 3A shows the morphological phenotypes of PagHB7a overexpression (OE-22, OE-26) and knockout (KO-2, KO-6) transgenic plants before and after treatment with 100mM NaCl for 22 days;

[0029] Figure 3B shows the height of WT, PagHB7a overexpression (OE-22, OE-26) and knockout (KO-2, KO-6) transgenic plants;

[0030] Figure 3C shows the growth rates of WT, PagHB7a overexpression (OE-22, OE-26), and knockout (KO-2, KO-6) transgenic plants (growth rate = (plant height on day 21 - plant height before salt treatment) / plant height before salt treatment). Error bars represent standard deviations. An asterisk indicates a significant difference from the WT value (Student's t-test): *p<0.05; **p<0.01;

[0031] Figure 4A shows the morphological phenotypes of WT, PagHB7a transgenic plants overexpressing OE-22 and knockout KO-2 before and after salt treatment;

[0032] Figure 4B shows the analysis of malondialdehyde (MDA), hydrogen peroxide (H2O2), peroxidase (POD), and catalase (CAT) content in WT, PagHB7a transgenic plants overexpressing OE-22 and knockout KO-2 before and after salt treatment.

[0033] Figure 5 shows the phenotypes of PagHB7a transgenic and WT plants under drought stress. Detailed Implementation

[0034] The present invention will now be described in detail through specific embodiments, and the features and advantages of the present invention will become clearer and more explicit with these descriptions.

[0035] The first aspect of this invention provides a PagHB7a gene related to the salt tolerance and drought resistance of poplar, the nucleotide sequence of which is shown in SEQ ID NO.21. It can be obtained by downloading the 84K poplar genome file (https: / / figshare.com / articles / dataset / 84K_genome_zip / 12369209) and searching for Pag.A14G001024.1.v3.1 in the 84K.v3.1.cds.fasta file. The PagHB7a gene is located on chromosome 14, with start position 7754287 and end position 7754643; or start position 7754760 and end position 7755116; see the 84K.v3.1.gff file for details.

[0036] A second aspect of this invention provides the protein encoded by the PagHB7a gene, which is associated with the salt tolerance and drought resistance of poplar, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.22. It can be obtained by downloading the 84K poplar genome file (https: / / figshare.com / articles / dataset / 84K_genome_zip / 12369209) and searching for Pag.A14G001024.1.v3.1 in the 84K.v3.1.proteins.fasta file.

[0037] A third aspect of this invention provides a breeding method for transgenic 84K poplar with enhanced salt tolerance and drought resistance, the method specifically comprising the following steps:

[0038] Step 1. Construct the PagHB7a gene into the pPZP211 overexpression vector to obtain the recombinant plasmid;

[0039] The PagHB7a gene was obtained by quantitative real-time PCR, and the primer sets P1 and P2 used for quantitative real-time PCR are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0040] Step 1 includes the following steps:

[0041] Step 1-1. Digest the selected overexpression vector pPZP211 with enzymes;

[0042] Steps 1-2. Extract RNA from 84K Yang, and use cDNA transcribed from RNA as a template for PCR amplification. The primer sets for the PCR amplification are P5 and P6, as shown in SEQ ID NO.5 and SEQ ID NO.6, respectively.

[0043] Steps 1-3. PagHB7a was constructed into the pPZP211 overexpression vector to obtain the recombinant vector, which was then transformed into Escherichia coli DH5α competent cells and identified by PCR to obtain positive clones.

[0044] Steps 1-4. Culture the colonies of positive clones and extract the recombinant plasmids.

[0045] Step 2. Transform the Agrobacterium with the recombinant vector into Agrobacterium and activate it to obtain Agrobacterium bacterial culture;

[0046] The Agrobacterium species is GV3101;

[0047] Step 3. Infect 84K poplar leaf tissue with Agrobacterium bacterial solution;

[0048] Step 4. Through co-culture and selective culture of leaf tissues, adventitious buds are differentiated and rooted to form complete plants.

[0049] Positive plants were obtained through PCR identification, that is, transgenic 84K poplar plants with salt tolerance and drought resistance were obtained.

[0050] Compared to WT, the positive plants were transgenic 84K poplar plants with salt tolerance and drought resistance, exhibiting better growth and higher growth rate under salt stress and / or drought conditions. Under non-salt stress and / or drought conditions, there was no significant difference in growth status and growth rate between transgenic 84K poplar and WT.

[0051] Example

[0052] Example 1

[0053] Poplar trees (Poplar alba × Poplar glandulosa) from 84K were subjected to stress treatment with 100mM NaCl. Root samples were collected at eight time points (0, 1, 3, 6, 9, 12, 24, and 48 hours) for RNA extraction. Based on the PagHB7a gene sequence of Poplar 84K, primers for quantitative real-time PCR (PCR) were designed as shown in Table 1 (P1 and P2). The expression level of the PagHB7a gene under different stress times was determined using PCR. The results showed that the expression level of the PagHB7a gene initially increased and then decreased with increasing salt treatment time (Figures 1A and 1C).

[0054] The expression level of PagHB7a gene in the roots, stems, leaves and petioles of 84K poplar was detected by real-time PCR. It was found that under normal growth conditions, the expression level of PagHB7a gene was very low, the highest expression level was in the leaves, followed by the petioles, and the expression level was very low in the roots and stems (Figure 1B).

[0055] Table 1 Primer set used for quantitative real-time PCR of PagHB7a gene

[0056] RT-qPCR experiments were performed using 2×ChamQ SYBR Color qPCR Master Mix. The reaction volume (20 μL) is as follows:

[0057] Table 2 RT-qPCR reaction system

[0058] Example 2 Cloning and Overexpression Vector Construction of PagHB7a Gene

[0059] The selected overexpression vector was pPZP211, purchased from BioWind. The vector is 9015 bp in length and contains a strong 35S promoter (CaMV35S). It exhibits resistance to spectinomycin (Spe) in bacterial culture and kanamycin (Kana) in transgenic plant screening. It can be digested with XbaI and BamHI.

[0060] RNA was extracted from *Populus 84K* using a plant RNA extraction kit from Yeasen Biotechnology. The RNA was reverse transcribed into cDNA using the HiScript III 1st Strand cDNA Synthesis Kit (Nanjing Novizan Biotechnology Co., Ltd.). Primers were designed using the Primer blast tool (NCBI, https: / / blast.ncbi.nlm.nih.gov), taking into account various primer design principles, as shown in Table 3, P5 and P6. Using the primers shown in Table 3, and with the obtained genomic cDNA as a template, PCR amplification was performed using Phanta Max Super-Fidelity DNA Polymerase from Nanjing Vazyme, as shown in Table 3.

[0061] Table 3 Primer sets used to clone the PagHB7a CDS sequence

[0062] The PCR reaction mixture (50 μL) is as follows:

[0063] Table 4 PCR reaction system

[0064] PCR amplification program: 95℃ for 5 min; (95℃ for 15 s; 57℃ for 15 s; 72℃ for 22 s (amplification efficiency 30 s / kb)) 36 cycles; 72℃ for 5 min; 4℃ to infinity.

[0065] (1) Purification of vector and PCR product after enzyme digestion

[0066] Since both PCR and enzyme digestion reactions contain multiple components, to avoid these components affecting the ligation reaction and reducing ligation efficiency, the digested vector and PCR products must be purified to obtain pure DNA fragments. After successful agarose gel electrophoresis, the products were purified using the Kangwei Century DNA Clean-up Kit. Following purification, the purity and concentration of the purified DNA product were determined using instruments.

[0067] (2) Connection transformation

[0068] 1) Connecting expression vectors

[0069] PagHB7a was constructed into the pPZP211 overexpression vector using the LightNing DNA Assembly Mix Plus seamless cloning kit from Jiangsu Yugong Life Science & Technology Co., Ltd., to obtain the recombinant vector. Ligation was performed at 50℃ for 20-30 min, and the ligation product was used to transform *E. coli* DH5α competent cells.

[0070] 2) Transformation of E. coli DH5α competent cells

[0071] Take 20 μl of DH5α competent cells, add 2 μl of ligation product, gently tap to mix, and incubate on ice for half an hour. After the ice bath, heat shock in a 42°C water bath for 45 seconds, then rapidly cool on ice for 3 minutes, avoiding shaking to prevent reducing transformation efficiency. Add 450 μl of antibiotic-free sterile LB liquid medium, mix well by pipetting, and incubate at 37°C with shaking at 200 rpm for 50-60 minutes to allow the cells to recover.

[0072] After resuscitation, the bacterial cells were collected by centrifugation at 5000 rpm for 2 min, and 100 μl of supernatant was used to resuspend the bacterial cells. The cells were then spread onto LB solid agar plates containing spectinomycin (10 mg / ml) and incubated upside down at 37°C for 14-16 h.

[0073] (3) Identification of positive clones by bacterial culture PCR

[0074] Using a sterilized pipette tip, single colonies were picked sequentially from the plate and added to 250 μl of LB liquid medium containing spectinomycin. The culture was incubated at 37°C with shaking at 200 rpm for approximately 3 hours to serve as amplification templates. Separately, positive and negative controls were set up using the purified gene PCR product and ddH2O (double-distilled water) as templates, and PCR amplification was performed using Taq Plus Master Mix from Nanjing Vazyme.

[0075] The PCR reaction system is as follows:

[0076] Table 5 PCR reaction system

[0077] The reaction conditions were set as follows: 94℃ for 5 min; (94℃ for 30 s; 58℃ for 30 s; 72℃ for 70 s) for 35 cycles; 72℃ for 10 min; 4℃ for ∞.

[0078] The PCR products were detected by 1% agarose gel electrophoresis. Colonies that amplified a band of the same size as the positive control were considered positive clones.

[0079] (4) Extraction of positive clone plasmids

[0080] PCR-positive clones were aspirated into 6 ml of LB liquid medium containing spectinomycin and incubated overnight at 37°C with shaking at 200 rpm. Plasmids were extracted using a plasmid miniprep kit from Beijing Kangwei Reagent Co., Ltd., and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. Vector construction was completed after sequence alignment was confirmed to be correct.

[0081] Example 3 Construction of PagHB7a gene knockout vector

[0082] (1) Target design

[0083] Download the 84K Yang genome file (https: / / figshare.com / articles / dataset / 84K_genome_zip / 12369209). Locate Pag.A14G001024.1.v3.1 in the 84K.v3.1.cds.fasta file to obtain the CDS sequence of PagHB7a, as shown in SEQ ID NO.21. Log in to the CRISPR online target site design service (http: / / crispr.dbcls.jp / ), enter the PagHB7a CDS sequence, select Western balsam poplar (Populus trichocarpa) genome, JGI2.0 (Jan, 2010) in the Specificity check column, and click design to obtain several target sequences. Select two targets with high specificity, proximity to the gene's early stage, location within exons, and high GC content. The results are as follows:

[0084] Table 6 PagHB7a gene knockout target sequence

[0085] (2) Design vector primers and expression cassette amplification primers

[0086] This experiment has two target sites, requiring the use of two gRNA vectors: pYLgRNA-AtU3d and pYLgRNA-AtU3b. The Cas9 binary vector used is pYLCRISPR / Cas9-DN, which is mainly used in dicotyledonous plants and provides kanamycin resistance in both bacterial culture and transgenic plant screening. The CRISPR / Cas9 system vector used in this experiment was prepared according to the following literature (Xingliang Ma, et al. A Robust CRISPR / Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants. Molecular plant, 2015, 8(8): 1274-1284.).

[0087] The vector primers and expression cassette amplification primers are designed as follows:

[0088] Table 7. Primer sets used for vector design and expression cassette amplification.

[0089] (3) Target linker primer design

[0090] The target adapter primers were designed based on the gRNA cassette vector as follows:

[0091] Table 8 Primer sets used for target linkers

[0092] (4) Carrier construction

[0093] 1) Target connector preparation

[0094] Using the 10 μM working solution provided by the primer design company, add 5 μl each of the 5' and 3' primers of the target linker to 40 μl of ddH2O, mix well by pipetting, and dilute to 1 μM. Place in a PCR instrument, incubate at 90°C for 35 s, and then cool to room temperature.

[0095] 2) Target site and gRNA expression cassette

[0096] The target linkers 1 and 2 were ligated to the pYLgRNA-AtU3d and pYLgRNA-AtU3b vectors, respectively, using a cleavage-while-ligating method. The reaction system is as follows:

[0097] Table 9. Reaction system for linking target to gRNA expression cassette

[0098] PCR program: (37℃ for 5 min; 20℃ for 5 min) 5 cycles; 4℃∞.

[0099] 3) First round of amplification of gRNA expression cassette

[0100] To obtain stable, specific target products and avoid amplifying empty vector products, two rounds of nested PCR were used to amplify the expression cassettes. Each gRNA expression cassette underwent two PCR reactions, as shown in the following systems:

[0101] Table 10 First-round amplification reaction system for gRNA expression cassette 1

[0102] Table 11 First-round amplification reaction system of gRNA expression cassette 2

[0103] PCR reaction program: 95℃ for 3 min; (95℃ for 16 sec; 60℃ for 16 sec; 72℃ for 30 sec) 27 cycles; 72℃ for 5 min; 4℃ to infinity.

[0104] 4) Second round of amplification of gRNA expression cassette

[0105] Template preparation: Take 1 μl of each of the products from the first round of PCR reaction 1 and reaction 2, add 9 μl of ddH2O to dilute, then take 1 μl of each and mix them to make 2 μl of template, so that you have two templates at this time.

[0106] Location-specific primer preparation: For the two target sites, two primers, PR1 (Ba'+Bb) and PR2L (Bb'+BL), need to be prepared and mixed into a 10× working solution. Taking PR1 as an example, take 3 μL of each of the 10 μM Ba' and Bb primers and add 14 μL of ddH2O to make a 1.5 μM 10× working solution.

[0107] Next, a second round of amplification reaction will be carried out, with the following system:

[0108] Table 12 Second round of amplification of gRNA expression cassettes

[0109] 5) Ligation of gRNA expression cassette with pYLCRISPR / Cas9-DN

[0110] The gRNA expression cassette was ligated to the pYLCRISPR / Cas9-DN vector using a cut-and-ligate method. The reaction system is as follows:

[0111] Table 13 Ligation reaction system of gRNA expression cassette and pYLCRISPR / Cas9-DN vector

[0112] PCR reaction: 37℃ for 10 min.

[0113] Immediately after the reaction is complete, add 1 μL of T4 DNA ligase and 1 μL of 10 x T4 DNA ligase buffer. Place in a PCR instrument and run the following reaction program: (37℃ for 120 s, 10℃ for 3 min, 20℃ for 5 min) for 15 cycles; 37℃ for 2 min; 16℃ for ∞.

[0114] 5 μl of the ligation product was transformed into competent E. coli DH5α cells. After identifying positive clones by PCR, plasmids were extracted by shaking the culture, following the same method as in Example 2. The PCR reaction system is as follows:

[0115] Table 14 PCR Reaction System

[0116] Example 4 Genetic transformation of the PagHB7a gene

[0117] (1) Special culture medium formulation

[0118] Table 15 1 / 2 MS medium (1L)

[0119] Sterilize at 121℃ for 20 minutes under high temperature and pressure.

[0120] Table 16 AS medium (1L)

[0121] Adjust the pH to 5.2 with NaOH and HCl, then autoclave at 121℃ for 20 minutes.

[0122] Selective differentiation medium: After the differentiation medium is sterilized by high temperature and high pressure, it should be cooled slightly before adding 3 ml of 100 mg / ml cephalosporin and 400 μl of 100 mg / ml kanamycin before use.

[0123] Selective rooting medium: After the rooting medium has been sterilized by high temperature and high pressure, it should be cooled slightly before use. Then add 3 ml of 100 mg / ml cephalosporin and 400 μl of 100 mg / ml kanamycin.

[0124] (2) Transformation of Agrobacterium tumefaciens with recombinant plasmids

[0125] 1) Take about 1 μg of PagHB7a overexpression vector plasmid and knockout vector plasmid and add them to 100 μL of Agrobacterium GV3101 competent cells, and mix gently.

[0126] 2) Place on ice for 5 minutes, freeze in liquid nitrogen for 1 minute, immediately place in a 37°C water bath for 5 minutes, and then in an ice bath for 5 minutes.

[0127] 3) Add 700 μL of antibiotic-free YEP liquid medium, mix well, and incubate at 28°C with shaking for 2–3 h to revive. After revival, centrifuge at 4000 rpm for 2 min, discard part of the supernatant, and mix 100 μL of supernatant with the bacterial culture. Spread the mixture evenly onto YEP solid medium containing 50 mg / L gentamicin, 50 mg / L rifampin, and 50 mg / L spectinomycin using sterilized and cooled glass beads. Incubate upside down at 28°C for 72–90 h.

[0128] 4) After single colonies have grown, use a sterile pipette tip to pick up several single-clone plaques and place them in a 2 mL centrifuge tube containing 250 μL of YEP liquid medium (1:1000 with added Rif, Gm, and Spe / Kana). Incubate at 30°C and 200 rpm for 2 hours. After incubation, aspirate the bacterial solution for PCR identification, using the same method as described in Example 2. Correctly identified positive bacterial solutions are then flash-frozen in liquid nitrogen with 50% glycerol and stored at -80°C for subsequent genetic transformation experiments.

[0129] (3) Activation of Agrobacterium

[0130] Take the Agrobacterium tumefaciens solution containing the recombinant plasmid from the -80℃ freezer, streak it onto YEP solid medium (1:1000 with added Rif, Gm, Spe / Kana), seal the plate, and incubate upside down in a 30℃ dark incubator for 2-3 days.

[0131] Using a sterile pipette tip, pick a single colony from the plate and inoculate it into 3 mL of YEP liquid medium (1:1000 with added Rif, Gm, and Spe / Kana). Incubate overnight at 30°C and 200 rpm with shaking. Transfer 1 mL of the bacterial culture to a 250 mL sterile Erlenmeyer flask containing 100 mL of YEP liquid medium (1:1000 with added Rif, Gm, and Spe / Kana). Incubate at 30°C and 200 rpm with shaking for 4-5 hours, until the OD600 reaches 0.3-0.5.

[0132] In a clean bench, transfer 100 mL of bacterial culture into two 50 mL sterile centrifuge tubes and centrifuge at 2560 g for 20 min at 4 °C.

[0133] Collect the bacterial cells. In a clean bench, discard the supernatant and resuspend the bacterial cells in 100 mL 1 / 2 MS in a sterile wide-mouth bottle. This bacterial suspension will be used for subsequent infection experiments.

[0134] (4) Leaf disc infection

[0135] In a clean bench, cut off the entire tissue culture seedling, select the healthy leaves from the middle part, and cut each leaf into 6mm×6mm pieces with a scalpel blade. Take 3-4 seedlings and transfer all the cut leaves to a sterile wide-mouth bottle containing bacterial solution. Place the bottle on a shaker at 28℃ and 160rpm for 15-20 minutes.

[0136] (5) Co-cultivation

[0137] In a clean bench, use tweezers to remove the leaves from the bacterial solution, absorb excess bacterial solution with sterile filter paper, lay the leaves flat on a co-culture plate with the underside facing down, and incubate in the dark at 25°C for 2 days.

[0138] (6) Selection of culture

[0139] After dark culture, select appropriate plant resistance based on the vector and prepare differentiation medium containing the corresponding antibiotics.

[0140] In a clean bench, the leaf discs were transferred to selective culture medium to induce callus formation. Every ten days, the leaf discs were transferred to fresh differentiation medium, and this process was repeated for 3-4 weeks until white or pale yellow callus tissue grew at the edge of the leaf discs. The entire process was carried out in a 25°C light incubator.

[0141] (7) Inducing rooting from buds

[0142] After growing on the differentiation selective medium for 1 to 2 months, adventitious buds can differentiate. When the adventitious buds grow to about 1 cm, they can be cut off and transferred to the rooting selective medium to root and grow into complete plants.

[0143] Example 5: Identification of PagHB7a gene overexpression and knockout plants

[0144] (1) DNA level identification

[0145] ① Crude extraction of PagHB7a transgenic 84K Yang gDNA:

[0146] 1) Take a leaf sample to be extracted, place it in a 2mL centrifuge tube and add a steel ball. After quick freezing with liquid nitrogen, use a tissue homogenizer to homogenize for 5 minutes.

[0147] 2) After the mixture is broken into powder, add 500 μL of TBS buffer to the centrifuge tube, mix well, and then heat in a 65°C water bath for 10 min.

[0148] 3) Centrifuge at 12000 rpm for 15 min, transfer the supernatant to a new centrifuge tube, add an equal volume of isopropanol, and allow to settle at room temperature for 30 min. A white flocculent precipitate will be visible.

[0149] 4) Centrifuge at 12000 rpm for 15 min, slowly discard the supernatant, and add 1 ml of 75% ethanol to wash the precipitate.

[0150] 5) Centrifuge at 7500 rpm for 10 minutes, slowly pour off the supernatant and use a pipette to remove any excess liquid.

[0151] 6) Place in a 40℃ oven for 2 hours.

[0152] 7) Add 50 μL of ddH2O to the centrifuge tube to dissolve the precipitate, and store it in a -20°C freezer.

[0153] ②PCR identification

[0154] 1) Identification of plants overexpressing the PagHB7a gene

[0155] Using crudely extracted gDNA from transgenic plants as templates, identification was performed using Taq Plus Master Mix. The PCR reaction system was the same as in Table 4. The PCR products were directly run by electrophoresis to check if the bands were correct. If correct, the plant was a PagHB7a overexpressing transgenic plant.

[0156] 2) Identification of PagHB7a gene knockout plants

[0157] As described above, the transgenic plants were first preliminarily screened to identify whether they contained recombinant plasmids. The PCR reaction system was the same as in Table 13. The gDNA of the positive lines was sent to Wuhan Boyuan Biotechnology Co., Ltd. for CRISPR editing mutation detection, and two independent biallelic edited mutants were successfully obtained. As shown in Figure 2B, both mutant lines showed nucleotide deletions or insertions near two target sites, leading to premature termination of PagHB7a protein expression.

[0158] (2) RT-qPCR was used to detect the transcriptional level of PagHB7a in overexpressing plants.

[0159] ① Extract RNA from the tissues or plants to be tested, and reverse transcribe it to obtain template cDNA, using the same method as in Example 2.

[0160] ②RT-qPCR experiments were performed using 2×ChamQ SYBR Color qPCR Master Mix, following the same method as in Example 1, and the reaction system was as shown in Table 1.

[0161] ③ Analyze the RT-qPCR results. As shown in Figure 2A, the overexpression lines with high expression levels (OE-22, OE-26 and OE-27) were obtained, with expression levels 175.62 times, 69.06 times and 54.03 times that of WT, respectively.

[0162] Example 6 Salt tolerance analysis of PagHB7a gene overexpression and knockout plants

[0163] (1) Long-term salt treatment

[0164] Plants with uniform growth after 25 days of tissue culture were washed with warm water to remove the agar from their roots and transferred to a soil culture medium containing a 1:1.5 mixture of substrate and vermiculite. The light conditions were 8 hours of darkness followed by 16 hours of light. The plants were then cultured at 25°C under the same light conditions for 30 days, followed by 22 days of irrigation with 100 mM NaCl solution.

[0165] As shown in Figure 3A, compared with WT, the PagHB7a overexpressing plants (OE-22, OE-26) showed better growth under salt stress, while most leaves of the PagHB7a knockout plants (KO-2, KO-6) had withered. As shown in Figure 3B, before salt treatment, the PagHB7a overexpressing plants were shorter than the WT and PagHB7a knockout plants; after salt treatment, the height of the PagHB7a overexpressing plants gradually became the same as that of the WT and PagHB7a knockout plants. As shown in Figure 3C, the growth rate of WT was 36.28%, OE-22 was 51.23%, and PagHB7a overexpressing plants were 41.76%, indicating a higher growth rate. Conversely, the growth rate of KO-2 was 33.67%, and that of KO-6 was 33.56%, indicating a lower growth rate of PagHB7a knockout plants.

[0166] (2) Short-term salt treatment

[0167] Plants with uniform growth after 25 days of tissue culture were cleaned of agar from their roots with warm water. The plants were then secured near the rootstock using planting cotton and a floating board, and placed in containers filled with half Hoglund's medium. They were cultured in a constant temperature incubator at 25°C under the same light conditions, with the medium changed every 3-5 days. Wild-type 84K poplar, PagHB7a gene overexpressing plants, and knockout plants grown hydroponically for 20 days were subjected to short-term salt treatment with half Hoglund's medium containing 150 mM NaCl. Physiological parameters were measured 2 days after salt treatment.

[0168] As shown in Figure 4A, after treatment with 150mM NaCl, compared with WT, the leaves of the PagHB7a gene knockout plant KO-2 showed more severe wilting, while the PagHB7a gene overexpression plant OE-22 showed better growth and less wilting damage to its leaves.

[0169] Physiological indicators were measured, as shown in Figure 4B. Under salt stress, the content of reactive oxygen species (ROS) such as H2O2 and MDA in the PagHB7a gene overexpressing plant OE-22 was lower than that in the root mean tachycardia (WT), while the activities of antioxidant enzymes such as SOD and POD were higher than those in the WT. This indicates that the activity of antioxidant enzymes increased under salt stress, effectively mitigating the excessive accumulation of ROS and reducing the degree of plant damage. In contrast, the PagHB7a knockout plant KO-2 showed the opposite trend (Figure 5B).

[0170] Example 7: Drought resistance analysis of PagHB7a gene overexpression and knockout plants

[0171] Plants that have grown uniformly in tissue culture bottles for 25 days were washed with warm water to remove the agar from their roots. They were then transferred to a soil culture medium containing a 1:1.5 mixture of substrate and vermiculite and cultured in the soil for 30 days. After that, watering was stopped and the plants were allowed to dry.

[0172] As shown in Figure 5, after 10 days of drought, the WT and PagHB7a gene knockout plants (KO-2, KO-6) withered, while the PagHB7a gene overexpression plants (OE-22, OE-26) wilted slightly but did not wither.

[0173] SEQ ID NO.21

[0174] The PagHB7a CDS sequence is as follows:

[0175] SEQ ID NO.22

[0176] The amino acid sequence of PagHB7a is as follows: "*" is the stop codon.

[0177] The present invention has been described in detail above with reference to specific embodiments and / or exemplary examples, as well as the accompanying drawings. However, these descriptions should not be construed as limiting the present invention. Those skilled in the art will understand that various equivalent substitutions, modifications, or improvements can be made to the technical solutions and embodiments of the present invention without departing from the spirit and scope of the invention, and all such modifications and improvements fall within the scope of the present invention. The scope of protection of the present invention is defined by the appended claims. Industrial applicability

[0178] This invention provides a PagHB7a gene associated with salt tolerance and drought resistance in poplar trees, and utilizes this gene to create new germplasm of salt-tolerant and drought-resistant 84K poplar (Poplar alba × Poplar glandulosa). The PagHB7a gene is a candidate gene associated with salt tolerance and drought resistance in poplar trees. This invention provides an important gene resource for breeding new salt-tolerant and drought-resistant poplar varieties and can be directly applied to molecular-assisted breeding of stress-resistant poplars. Furthermore, this invention also provides primer pairs for amplifying the candidate gene and a method for transgenic genetic improvement of poplar trees, enabling the evaluation of poplar stress resistance at the molecular level. Based on this, tree species with significant stress resistance can be screened accurately and efficiently, effectively shortening the poplar breeding cycle and possessing good economic value and application prospects.

Claims

1. A PagHB7a gene associated with salt tolerance and drought resistance in poplar trees, characterized in that, Its nucleotide sequence is shown in SEQ ID NO.

21.

2. A protein encoded by the PagHB7a gene, which is associated with salt tolerance and drought resistance in poplar trees, characterized in that... The amino acid sequence of the encoded protein is shown in SEQ ID NO.

22.

3. A breeding method for transgenic Populus 84K with enhanced salt tolerance and drought resistance, characterized in that, The method specifically includes the following steps: Step 1. Construct the PagHB7a gene into the pPZP211 overexpression vector to obtain the recombinant plasmid; Step 2. Transform the Agrobacterium with the recombinant vector into Agrobacterium and activate it to obtain Agrobacterium bacterial culture; Step 3. Infect 84K poplar leaf tissue with Agrobacterium bacterial solution; Step 4. Through co-culture and selective culture of leaf tissues, adventitious buds are differentiated and rooted to form complete plants.

4. The method according to claim 3, characterized in that, Step 1 includes the following steps: Step 1-1. Digest the selected overexpression vector pPZP211 with enzymes; Steps 1-2. Extract RNA from 84K Yang, and use the cDNA transcribed from the RNA as a template for PCR amplification; Steps 1-3. PagHB7a was constructed into the pPZP211 overexpression vector to obtain the recombinant vector, which was then transformed into Escherichia coli DH5α competent cells and identified by PCR to obtain positive clones. Steps 1-4. Culture the colonies of positive clones and extract the recombinant plasmids.

5. The method according to claim 4, characterized in that, In steps 1-2, the primer sets for PCR amplification are P5 and P6, as shown in SEQ ID NO.5 and SEQ ID NO.6, respectively.

6. The method according to claim 3, characterized in that, In step 3, the Agrobacterium is GV3101.

7. The method according to claim 3, characterized in that, In step 4, positive plants were obtained by PCR identification. The positive plants were transgenic Populus 84K, which is both salt-tolerant and drought-resistant.

8. The method according to claim 3, characterized in that, Compared with wild-type 84K poplar, the positive plants are salt-tolerant and drought-resistant transgenic 84K poplar plants, which have better growth status and higher growth rate under salt stress and / or drought conditions. Under non-salt stress and / or drought conditions, there is no significant difference in growth status between transgenic 84K poplar and WT.

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