Pagafp2a gene related to salt tolerance and drought tolerance of poplar trees and use thereof

Abstract

Provided is PagAFP2a gene related to salt tolerance and drought tolerance of poplar trees. Under salt stress, PagAFP2a gene-knockout mutant poplar trees have a significantly enhanced antioxidant enzyme activity compared to that of WT (non-transgenic poplar trees), have significantly reduced reactive oxygen and malondialdehyde contents and relative conductivity, and have significantly enhanced resistance. However, the growth of the mutant poplar trees under normal culture conditions is not significantly different from that of WT. PagAFP2a gene-knockout mutants can significantly enhance salt tolerance and drought tolerance of poplar trees.

Description

A PagAFP2a gene associated with salt and drought tolerance in poplar and its application

[0001] Cross-references

[0002] This application claims priority to Chinese Patent Application No. 2024107011439, filed on May 31, 2024, entitled “A PagAFP2a gene related to salt and drought tolerance of 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 plant molecular breeding technology, specifically relating to a gene PagAFP2a that regulates the stress resistance of poplar trees and its application. Background Technology

[0004] As the global climate continues to worsen, forest plants are becoming increasingly vulnerable to abiotic stresses in nature. [1] Among these, abiotic stresses such as drought and salinity are the main environmental factors that adversely affect global forest growth, timber yield, and quality. [2] With the continuous increase in the area affected by drought and secondary salinization of the land, the sustainable development of forestry production has been severely constrained.

[0005] Poplar is an important ecological and economic tree species, widely used in plantations and vegetation restoration. [3] It also provided ample raw materials for the paper and timber industries. [4] In arid, semi-arid, and severely saline-alkali areas, salinity and drought severely restrict the growth, development, and spatial distribution of poplar trees. With the continuous development of molecular breeding technology, utilizing genetic engineering to improve the stress resistance of forest trees provides an economical and effective way to develop the productivity potential of saline-alkali and arid zones. Currently, the breeding of new stress-resistant poplar germplasm urgently needs to be carried out. Simultaneously, functional identification of genes playing important regulatory roles in the response to salt and drought stress will be of great significance for constructing the molecular regulatory network of poplar's response to abiotic stress.

[0006] Under adverse conditions, plants can respond to and adapt to external stresses through a network of factors involved in stress signaling and gene expression regulation. [5] AFP is a highly conserved family of plant-specific proteins. The roles of AFP genes in plant responses to abiotic stresses have been reported; for example, in Arabidopsis, the AtAFP4 gene acts as a negative regulator in ABA or salt signaling pathways. [6] Mutations in Arabidopsis AFP1 or AFP2a lead to increased sensitivity to ABA and salt. [7]The rice AFP homolog (modd) negatively regulates ABA signaling and drought stress response by modulating the activity and stability of OsbZIP46, a member of the ABF subfamily. [8] These reports indicate that the AFP gene participates in abiotic stress responses in plants by negatively regulating the ABA signaling pathway. However, the effects of the AFP gene on poplar stress resistance have not yet been reported. Therefore, studying the role of the AFP gene in poplar salt and drought tolerance could provide important target genes for breeding new fast-growing and stress-resistant poplar varieties.

[0007] References

[0008] [1]Lobell DB,Schlenker W,Costa-Roberts J.Climate trends and global crop production since 1980[J].Science,2011,333(6042):616-620.

[0009] [2]Kataria S, Verma S K. Salinity stress responses and adaptive mechanisms in major glycophytic crops: the story so far[M]. 2018.

[0010] [3]Ai J,Tschirner U(2010) Fiber length and pulping characteristics of switchgrass,alfalfa stems,hybrid poplar and willow biomasses. Bioresource Technology 101(1):215-221.

[0011] [4]Luo ZB,Janz D,Jiang X,Gobel C,Wildhagen H,Tan Y,Rennenberg H,Feussner I,Polle A(2009b)Upgrading root physiology for stress tolerance by ectomycorrhizas:insights from metabolite and transcriptional profiling into reprogramming for stress anticipation.Plant Physiology 151(4):1902-1917.

[0012] [5]Hirayama,T.,and Shinozaki,K.(2010).Research on plant abiotic stress responses in the post-genome era:past,present and future.Plant J.61:1041–1052.

[0013] [6]Huang MD,et al.Overexpression of TMAC2,a novel negative regulator of abscisic acid and salinity responses,has pleiotropic effects in Arabidopsis thaliana.Plant Mol Biol.2007.

[0014] [7]Garcia ME,et al.Asmall plant-specific protein family of ABI five binding proteins(AFPs)regulates stress response in germinating Arabidopsis seeds and seedlings.Plant Mol Biol.2008.

[0015] [8]Tang N, et al. MODD Mediates Deactivation and Degradation of OsbZIP46 to Negatively Regulate ABASignaling and Drought Resistance in Rice. Plant Cell. 2016. Summary of the Invention

[0016] To address the aforementioned problems, this invention provides a PagAFP2a gene that regulates the response of poplar trees to drought and salinization stress. Poplar trees with PagAFP2a gene knockout mutants exhibit significantly enhanced antioxidant enzyme activity under salt stress compared to WT plants (non-transgenic poplar), with significantly reduced reactive oxygen species, malondialdehyde (MDA) content, and relative conductivity, demonstrating significantly enhanced resistance. However, their growth under normal culture conditions shows no significant difference compared to WT plants. The PagAFP2a gene knockout mutant can significantly enhance the salt and drought tolerance of poplar trees, thus completing this invention.

[0017] The first aspect of this invention aims to provide a PagAFP2a gene related to the salt tolerance and drought tolerance of poplar trees, the nucleotide sequence of which is shown in SEQ ID NO.21.

[0018] A second aspect of the present invention aims to provide a protein encoded by the PagAFP2a gene, which is associated with salt and drought tolerance in poplar trees, the amino acid sequence of which is shown in SEQ ID NO.22.

[0019] A third aspect of this invention aims to provide an overexpression recombinant vector containing the aforementioned gene PagAFP2a and recombinant Agrobacterium. Preferably, the base vector of the recombinant expression vector is the pPZP211 vector, and the Agrobacterium is GV3101.

[0020] A fourth aspect of this invention aims to provide a gene knockout recombinant vector containing the aforementioned gene PagAFP2a and recombinant Agrobacterium. Preferably, the gene knockout recombinant vector is a CRISPR / Cas9 binary vector, and the Agrobacterium is GV3101.

[0021] The fifth aspect of this invention aims to provide a breeding method for transgenic poplar with enhanced salt and drought tolerance, the method specifically comprising the following steps:

[0022] Step 1. Design the PagAFP2a gene target, construct the gene target into a CRISPR / Cas9 binary vector, and transform Agrobacterium;

[0023] Step 2. The PagAFP2a gene CRISPR / Cas9 gene knockout vector constructed in Step 1 was introduced into 84K poplar leaves;

[0024] Step 3. Adventitious buds are obtained through co-culture and selection culture, and rooted plants are cultured into complete plants. Positive plants are identified, and finally PagAFP2a gene knockout mutant plants are obtained.

[0025] The present invention has the following beneficial effects:

[0026] (1) This invention provides the application of the PagAFP2a gene in regulating the response of poplar to drought and salinization stress. The PagAFP2a gene knockout mutant can significantly enhance the salt tolerance and drought tolerance of poplar, which is of great significance for the selection of superior stress-resistant poplar plants and the innovation of poplar germplasm resources.

[0027] (2) The experimental results provided by this invention show that the antioxidant enzyme activity of the PagAFP2a gene knockout mutant poplar under salt stress is significantly enhanced compared with that of the WT plant, while the content of reactive oxygen species, malondialdehyde and relative conductivity are significantly reduced, and its resistance is also significantly enhanced. However, its growth under normal culture conditions is not significantly different from that of WT. The above results show that the PagAFP2a gene knockout mutant poplar can significantly enhance the salt and drought resistance of poplar, but does not affect the normal growth of poplar and timber yield. This provides a new poplar germplasm that can improve the stress resistance of poplar without affecting the timber yield, and provides a guarantee for improving the yield and quality of timber under high salt and drought stress. Attached Figure Description

[0028] Figure 1 shows the high expression of the PagAFP2a gene under salt (Figure 1A) and drought (Figure 1B) stress;

[0029] Figure 2A shows the phenotypes of WT plants and transgenic plants overexpressing the PagAFP2a gene, specifically plants OE5, OE8, OE9, OE10, and OE22; Figure 2B shows the expression levels of WT plants OE5, OE8, OE9, OE10, and OE22; Figure 2C shows the plant height of WT plants OE5, OE8, OE9, OE10, and OE22; Figure 2D shows the stem diameter of WT plants OE5, OE8, OE9, OE10, and OE22.

[0030] Figure 3A shows a schematic diagram of PagAFP2a gene target knockout; Figure 3B shows a schematic diagram of the target region editing method in PagAFP2a gene knockout plants; Figure 3CWT plants and PagAFP2a knockout plants KO8, KO11, KO24, KO26, and KO27 phenotypes.

[0031] Figure 4 shows the phenotypes of WT plants and PagAFP2a overexpressing transgenic plants OE5, OE10 and PagAFP2a knockout plants KO11, KO26 and KO27 under short-term salt stress.

[0032] Figure 5 shows the phenotypes of WT plants and PagAFP2a overexpressing transgenic plants OE5, OE10 and PagAFP2a knockout plants KO11, KO26 and KO27 under long-term salt stress.

[0033] Figure 6 shows the phenotypes of WT plants and PagAFP2a overexpressing transgenic plants OE5, OE10, OE8, OE9 and PagAFP2a knockout plant KO11 under drought stress.

[0034] Figure 7A shows the catalase content analysis of WT plants, PagAFP2a overexpressing transgenic plants OE5, OE10, and PagAFP2a knockout plants KO11, KO27 under short-term salt stress; Figure 7B shows the peroxidase activity analysis of WT plants, PagAFP2a overexpressing transgenic plants OE5, OE10, and PagAFP2a knockout plants KO11, KO27 under salt stress; Figure 7C shows the hydrogen peroxide content analysis of WT plants, PagAFP2a overexpressing transgenic plants OE5, OE10, and PagAFP2a knockout plants KO11, KO27 under salt stress. Figure 7D shows the superoxide anion content analysis of WT plants and PagAFP2a overexpressing transgenic plants OE5, OE10 and PagAFP2a knockout plants KO11, KO27 under salt stress; Figure 7E shows the malondialdehyde (MDA) content analysis of WT plants and PagAFP2a overexpressing transgenic plants OE5, OE10 and PagAFP2a knockout plants KO11, KO27 under salt stress; Figure 7F shows the relative conductivity analysis of WT plants and PagAFP2a overexpressing transgenic plants OE5, OE10 and PagAFP2a knockout plants KO11, KO27 under salt stress.

[0035] Figure 8A shows a comparison of the relative leaf water content of WT, PagAFP2a overexpressing transgenic line, and PagAFP2a knockout plant after 20 days of 100mM salt treatment in Example 8; Figure 8B shows a comparison of plant height of WT, PagAFP2a overexpressing transgenic line, and PagAFP2a knockout plant after 15 and 20 days of 100mM salt treatment; Figure 8C shows the relative leaf water content of WT, PagAFP2a overexpressing transgenic line, and PagAFP2a knockout plant after 15 and 20 days of 100mM salt treatment. Figure 8D shows a comparison of stem diameter between gAFP2a transgenic lines and PagAFP2a knockout plants after 15 and 20 days of 100mM salt treatment, comparing stem diameter (WT), overexpression of PagAFP2a transgenic lines, and PagAFP2a knockout plants. Figure 8E shows a comparison of stem diameter (WT), overexpression of PagAFP2a transgenic lines, and PagAFP2a knockout plants after 15 and 20 days of 100mM salt treatment. Detailed Implementation

[0036] 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.

[0037] The first aspect of this invention provides a PagAFP2a gene associated with salt tolerance and drought resistance in poplar trees, the CDS 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.A08G001603.1.v3.1 in the file 84K.v3.1.cds.fasta. The PagAFP2a gene is located on chromosome 8, with start position 20846996 and end position 20847640; and start position 20850297 and end position 20850718.

[0038] A second aspect of the present invention provides the protein encoded by the PagAFP2a gene, which is associated with the salt and drought tolerance of poplar, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.22.

[0039] A third aspect of the present invention provides an overexpression recombinant vector containing the gene PagAFP2a and recombinant Agrobacterium; preferably, the base vector of the expression recombinant vector is the pPZP211 vector, and the Agrobacterium is GV3101.

[0040] A fourth aspect of the present invention provides a gene knockout recombinant vector containing the above-mentioned gene PagAFP2a and recombinant Agrobacterium; preferably, the gene knockout recombinant vector is a CRISPR / Cas9 binary vector and the Agrobacterium is GV3101.

[0041] The fifth aspect of this invention provides a breeding method for transgenic poplar with enhanced salt and drought tolerance, the method specifically comprising the following steps:

[0042] Step 1. Design the PagAFP2a gene target, construct the gene target into a CRISPR / Cas9 binary vector, and transform Agrobacterium;

[0043] The gene knockout target sequences are shown as AFP2a-1 and AFP2a-2, respectively.

[0044] Step 2. The PagAFP2a gene CRISPR / Cas9 gene knockout vector constructed in Step 1 was introduced into 84K poplar leaves;

[0045] Step 3. Adventitious buds are obtained through co-culture and selection culture, and rooted plants are cultured into complete plants. Positive plants are identified, and finally PagAFP2a gene knockout mutant plants are obtained.

[0046] Positive plants were obtained by PCR identification, and the positive plants were transgenic poplar plants that were salt-tolerant and drought-tolerant.

[0047] Compared to WT plants, the transgenic poplar plants with enhanced salt and drought tolerance showed better growth under salt stress and / or drought conditions.

[0048] Example

[0049] Example 1: Obtaining the stress-response gene PagAFP2a from 84K Populus alba × Populus glandulosa

[0050] The AFP2a (Pag.A08G001603.1) gene sequence of 84K Populus alba × Populus glandulosa (available by downloading the file 84K.v3.1.cds.fasta from the publicly available URL (https: / / figshare.com / articles / dataset / 84K_genome_zip / 12369209) and searching for Pag.A08G001603.1.v3.1) is located on chromosome 8, with start position 20846996 and end position 20847640; and start position 20850297 and end position 20850718, totaling 1065 bp. Sequences such as SEQ ID NO.1 and SEQ ID NO. were designed. NO.2 shows a pair of real-time PCR primers for the PagAFP2a gene in Populus 84K. The results confirmed that the PagAFP2a gene is highly expressed in Populus 84K in response to salt and drought stress (Figure 1), suggesting that the PagAFP2a gene plays a role in the response of Populus 84K to salt and drought stress.

[0051] RT-qPCR experiments were performed using 2×ChamQ SYBR Color qPCR Master Mix. The reaction volume (20 μL) is as follows:

[0052] Table 1 RT-qPCR reaction system

[0053] Table 2 Primer set used for quantitative real-time PCR of PagAFP2a gene

[0054] Table 3 Primer sets used to clone the PagAFP2a CDS sequence

[0055] Example 2: Cloning of the PagAFP2a gene

[0056] Design primers flanking the gene coding sequence as shown in SEQ ID NO.5 and SEQ ID NO.6.

[0057] 1. Extraction of total RNA from poplar trees

[0058] The RNA extraction reagent was prepared according to the instructions of the MolPure Plant RNAKit kit from Yisheng Biotechnology Co., Ltd., and the obtained RNA was stored at -80℃ for later use. The specific steps are as follows:

[0059] (1) Quickly wrap the removed fresh 84K poplar plant tissue in tin foil and freeze it in liquid nitrogen.

[0060] (2) Grind the sample into powder thoroughly with liquid nitrogen in a mortar for removing RNase.

[0061] (3) Transfer 100 mg of powder to a 1.5 mL RNase-free centrifuge tube containing 1 mL of lysis buffer LB, and immediately vortex to mix for 30 seconds until homogenized. Centrifuge at 4°C for 12000 rpm for 10 min.

[0062] (4) Transfer 480 μL of the supernatant to a new 1.5 mL RNase-free centrifuge tube. Add 240 μL of anhydrous ethanol and mix well.

[0063] (5) Add the above pretreatment mixture to the RNA adsorption column, centrifuge at 12000 rpm for 65 s, and discard the waste liquid.

[0064] (6) Add 350 μL of protein removal solution PL, let stand at room temperature for 65 s, centrifuge at 12,000 rpm for 30 sec, and discard the waste liquid.

[0065] (7) Place the RNA adsorption column back into the collection tube, add 50 μL of DNase I working solution to the center of the adsorption column membrane, and incubate at room temperature for 15 min.

[0066] (8) Add 350 μL of protein removal solution PL, 12000 rpm, 25℃, 30 sec, then discard the waste liquid.

[0067] (9) Place the RNA adsorption column back into the collection tube, add 500 μL of wash buffer W* (ensure anhydrous ethanol has been added), centrifuge at 12000 rpm at room temperature for 30 seconds, and discard the waste liquid. Repeat once more.

[0068] (10) Centrifuge the empty column at 12,000 rpm at room temperature for 125 s to remove residual wash solution W*.

[0069] (11) Place the RNA adsorption column into a new 1.5 mL RNase-free centrifuge tube, add 30-50 μL RNase-free H2O to the center of the adsorption column, incubate at room temperature for 2 min, centrifuge at 12,000 rpm for 1 min, and collect the filtrate.

[0070] 2. Reverse transcription to synthesize cDNA

[0071] The procedure was performed according to the instructions for the HiScript III 1st Strand cDNASynthesis Ki reverse transcription kit from Nanjing Vazyme Biotechnology Co., Ltd., and the specific steps are as follows:

[0072] (1) RNA template denaturation

[0073] ① Add RNA sample (total amount not exceeding 1 μg) according to RNA concentration.

[0074] ②Use RNase-free dd H2O to bring the total volume to 8 μl.

[0075] ③ The reaction temperature is 65℃, and the reaction time is 5 minutes.

[0076] ④ Quickly place it on ice to cool rapidly, and let it stand on ice for 2 minutes.

[0077] (2) Remove genomic DNA

[0078] ① Add 2 μl of 5x g DNA wiper Mix to the above reaction tube.

[0079] ② The reaction conditions were 42℃ and 120s.

[0080] (3) cDNA synthesis

[0081] ① Add the following reaction mixture to the previous reaction system:

[0082] Table 4 Reverse transcription synthesis CDS reaction system

[0083] ② The reaction conditions were 53℃ for 45 min, 85℃ for 5 s.

[0084] ③ The obtained product can be directly used for PCR reaction.

[0085] 3. PCR amplification

[0086] Primers were designed flanking the gene coding sequence as shown in SEQ ID NO.3 and SEQ ID NO.4. The obtained 84K Yang genomic cDNA was used as a template for PCR amplification to obtain the CDS sequence of the target gene, which was then sequenced. The amplification system is as follows:

[0087] Table 5 RT-PCR reaction system

[0088] PCR amplification program: 95℃ for 5 min; (95℃ for 15 s; 62℃ for 15 s; 72℃ for 70 s) for 36 cycles; 72℃ for 6 min; 4℃ for ∞.

[0089] Example 3: Construction of PagAFP2a overexpression vector

[0090] (1) Vector enzyme digestion

[0091] The overexpression vector used in this invention is pPZP211 overexpression vector, which is 9015bp in total and contains a 35S strong promoter (CaMV35S). The resistance in bacterial culture is spectinomycin (Spe), and the resistance in transgenic plant screening is kanamycin (Kana). Double digestion with XbaI and BamHI restriction sites is selected.

[0092] The enzyme digestion reaction system is as follows:

[0093] Table 6 Enzyme digestion reaction system

[0094] The reaction conditions were: 37℃ water bath for 7-9 hours. The enzyme-digested plasmids were recovered by gel extraction and used as the vector backbone for later use, and stored at -20℃.

[0095] (2) Recovery and purification of vector backbone and target fragment

[0096] Since both PCR and enzyme digestion reactions contain multiple components, in order to avoid the influence of each component on the ligation reaction and reduce the ligation efficiency, the PCR products and enzyme digestion products must be purified by gel recovery to obtain pure DNA fragments.

[0097] The carrier backbone was recovered using the Kangwei Century Gel Extraction Kit.

[0098] PCR products were purified using the Kangwei Century DNAClean-up Kit. After purification, the purity and concentration of the purified DNA products were determined using instruments.

[0099] (3) Connection transformation

[0100] ① Connecting expression vectors

[0101] The PagAFP2a gene was constructed into the pPZP211 overexpression vector using the LightNing DNAAssembly 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.

[0102] ② Transform E. coli DH5α competent cells

[0103] 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.

[0104] 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.

[0105] (4) Colony PCR identification of positive clones

[0106] Using a sterile pipette tip, single colonies from the plate were sequentially picked up and transferred to PCR tubes as amplification templates. Positive and negative controls were set up using the purified gene PCR product and ddH2O (double-distilled water) as templates, respectively.

[0107] The PCR reaction system is as follows:

[0108] Table 7 PCR Reaction System

[0109] 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 ∞.

[0110] The product was detected by 1% agarose gel electrophoresis. A positive clone was one that could amplify a band of the same size as the positive control.

[0111] (5) Extraction of positive clone plasmids

[0112] PCR-positive clones were picked and cultured overnight at 37°C with shaking at 200 rpm in LB broth containing spectinomycin. Plasmid extraction was performed using a plasmid mini-prep kit from Kangwei Reagent Co., Ltd. The plasmids were then sent to the company for sequencing. Vector construction was completed after successful sequence alignment. The successfully constructed recombinant plasmid was then transformed into Agrobacterium GV3101.

[0113] Example 4: Construction of PagAFP2a gene knockout vector

[0114] (1) Target design

[0115] The gDNA sequence of PagAFP2a was obtained from the 84K poplar database using TBtools. The CRISPR online target site design service (http: / / crispr.dbcls.jp / ) was accessed, and the gDNA sequence of PagAFP2a was entered. In the Specificity check section, Western balsam poplar (Populus trichocarpa) genome, JGI2.0 (Jan, 2010) was selected. Clicking "design" generated several target sequences. Two highly specific target sites were chosen, located near the gene's early stage, situated in exons, and exhibiting high CG content. The results are as follows:

[0116] Table 8 Gene knockout target sequences

[0117] (2) Design vector primers and expression cassette amplification primers

[0118] This experiment targets two sites and requires two gRNA vectors: pYLgRNA-AtU3d and pYLgRNA-AtU3b. The Cas9 binary vector used is pYLCRISPR / Cas9-DN, which is primarily used in dicotyledonous plants and selects for Kana resistance in plants.

[0119] The vector primers and expression cassette amplification primers are designed as follows:

[0120] Table 9 Primer sets used for expression cassette amplification

[0121] (3) Target linker primer design

[0122] The target adapter primers were designed based on the gRNA cassette vector as follows:

[0123] Table 10 Primer sets used for target linkers

[0124] (4) Carrier construction

[0125] ① Target junction preparation

[0126] Using the 10 μM working solution provided by the primer design company, 5 μl each of the F and R primers (primer numbers P17, P18, P19, P20) for target linker 3b and 3d were added to 40 μl of ddH2O and diluted to 1 μM by pipetting and mixing. The mixture was then placed in a PCR instrument and incubated at 90°C for 35 seconds, followed by cooling to room temperature.

[0127] ② The connection between the target site and the gRNA expression cassette

[0128] 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:

[0129] Table 11. Reaction system for linking target to gRNA expression cassette

[0130] PCR program: (37℃ for 5 min; 20℃ for 5 min) 5 cycles; 4℃∞.

[0131] ③ First round of amplification of gRNA expression cassette

[0132] 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:

[0133] Table 12 First-round amplification reaction system for gRNA expression cassette 1

[0134] Table 13 First-round amplification reaction system for gRNA expression cassette 2

[0135] 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.

[0136] ④ Second round of amplification of gRNA expression cassette

[0137] 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.

[0138] Position-specific primer preparation: For the two target sites, two primers are required: PR1 (Uctcg-B1'+gRctga-B2) (primer sequences P13 and P14) and PR2L (Uctga-B2'+gRcggt-BL) (primer sequences P15 and P16). Mix them into a 10× working solution. Taking PR1 as an example, take 3 μL of each of the 10 μM Uctcg-B1' and gRctga-B2 primers and add 14 μL of ddH2O to obtain a 1.5 μM 10× working solution.

[0139] Next, a second round of amplification reaction will be carried out, with the following system:

[0140] Table 14 Second round of amplification of gRNA expression cassettes

[0141] PCR reaction program: 95℃ for 3 min; (95℃ for 16 sec; 60℃ for 30 sec; 72℃ for 35 sec) 20 cycles; 72℃ for 3 min; 4℃ for ∞.

[0142] Take 4 μl of the amplification product and check the bands for correctness using a 1.5% gel electrophoresis. Mix the two products with correct band sizes and purify them using a DNAClean-up Kit. After purification, determine the concentration using a spectrophotometer.

[0143] ⑤ The gRNA expression cassette was ligated to pYLCRISPR / Cas9-DN using a cut-and-ligate method.

[0144] The pYLCRISPR / Cas9-DN vector was linked, and the reaction system is as follows:

[0145] Table 15 Ligation reaction system of gRNA expression cassette and pYLCRISPR / Cas9-DN vector

[0146] PCR reaction procedure: 37℃ for 10 min.

[0147] Immediately after the reaction, add 1 μL of T4 DNA ligase and 1 μL of 10x 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℃ at ∞. Take 5 μL of the ligation product and transform it into competent E. coli DH5α cells, using the same method as in Example 2.

[0148] Example 5: Genetic transformation of PagAFP2a, a stress-resistance response gene in 84K poplar

[0149] (1) Transformation of Agrobacterium tumefaciens with recombinant plasmid

[0150] ① Take 0.01-1 μg of pPZP211 overexpression recombinant plasmid and pYLCRISPR / Cas9-DN recombinant plasmid and add them to 100 μL of Agrobacterium GV3101 competent cells, and mix gently.

[0151] 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.

[0152] ③ Add 700 μL of antibiotic-free YEP liquid medium, mix well, and incubate at 28℃ with shaking for 2–3 h for recovery. After recovery, centrifuge at 4000 rpm for 2 min, discard part of the supernatant, and keep 100 μL of supernatant to mix 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, and incubate upside down at 28℃ for 72–90 h.

[0153] 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 rifampin, gentamicin Gm, and spectinomycin / kanamycin 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.

[0154] (2) Activation of Agrobacterium

[0155] Take the Agrobacterium tumefaciens culture (GV3101 culture) 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.

[0156] 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.

[0157] 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.

[0158] 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.

[0159] (3) Leaf disc infection

[0160] 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.

[0161] (4) Co-cultivation

[0162] 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.

[0163] (5) Selection of culture

[0164] After dark culture, select appropriate plant resistance based on the vector and prepare differentiation medium containing the corresponding antibiotics.

[0165] 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.

[0166] (6) Inducing rooting from buds

[0167] 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.

[0168] Example 6: Identification of PagAFP2a overexpression and gene knockout plants

[0169] (1) Crude extraction of DNA from WT and PagAFP2a transgenic poplar

[0170] 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.

[0171] ② After the mixture is broken into powder, add 500 μL TBS buffer to the centrifuge tube, mix well, and then heat in a 65°C water bath for 10 minutes.

[0172] 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.

[0173] ④ Centrifuge at 12000 rpm for 15 min, slowly discard the supernatant, and add 1 ml of 75 wt% ethanol to wash the precipitate.

[0174] ⑤ Centrifuge at 7500 rpm for 10 min, slowly pour off the supernatant and remove the excess liquid with a pipette.

[0175] ⑥ Place in a 40℃ oven for 2 hours.

[0176] ⑦ Add 50 μL of ddH2O to the centrifuge tube to dissolve the precipitate, and store at -20°C.

[0177] (2) PCR identification

[0178] ①Identification of PagAFP2a overexpressing plants

[0179] Using crudely extracted transgenic plant DNA as a template, identification was performed using Taq Plus Master Mix. The reaction volume (20 μL) is as follows:

[0180] Table 16 PCR Identification Reaction System

[0181] PCR reaction program: 95℃ for 5 min; (95℃ for 15 sec; 58℃ for 2 min; 72℃ for 30 sec) 35 cycles; 72℃ for 5 min; 4℃ to infinity. Run the PCR products directly into electrophoresis to check if the bands are correct. If correct, it indicates a PagAFP2a overexpressing transgenic plant.

[0182] Transgenic plants overexpressing PagAFP2a, namely OE5, OE8, OE9, OE10, and OE22, were obtained through screening.

[0183] The relative expression levels of OE5, OE8, OE9, OE10, and OE22 in the plant were 224.5 times, 37.6 times, 19.6 times, 111.7 times, and 74.4 times that of WT, respectively, as shown in Figure 2B.

[0184] The plant heights of WT, OE5, OE8, OE9, OE10, and OE22 were 24.8cm, 19.75cm, 26.1cm, 25.5cm, 27.2cm, and 22.3cm, respectively, as shown in Figure 2C.

[0185] The stem diameters of plants WT, OE5, OE8, OE9, OE10, and OE22 were 2.37 mm, 1.745 mm, 2.1 mm, 2.18 mm, 2.23 mm, and 1.885 mm, respectively, as shown in Figure 2D.

[0186] ②Identification of PagAFP2a gene knockout plants

[0187] As described in step ① above, the transgenic plants are first screened to identify whether they contain recombinant plasmids. The reaction system (20 μL) is as follows:

[0188] Table 17 PCR Identification Reaction System

[0189] The DNA of the selected plants was sent to a sequencing company to compare the sequencing results and check whether any editing occurred near the target site.

[0190] The PagAFP2a gene knockout plants KO8, KO11, KO24, KO26, and KO27 were obtained through screening. A schematic diagram of PagAFP2a target site knockout is shown in Figure 3A, a schematic diagram of the target site region editing method in the PagAFP2a gene knockout plants is shown in Figure 3B, and photographs of the PagAFP2a gene knockout plants KO8, KO11, KO24, KO26, and KO27 are shown in Figure 3C.

[0191] Example 7: Expression level analysis of PagAFP2a-overexpressing transgenic poplar

[0192] 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.

[0193] ②RT-qPCR experiments were performed using 2×ChamQ SYBR Color qPCR Master Mix, following the same method as in Example 1. The reaction system is shown in Table 1.

[0194] Example 8: Salt tolerance analysis of PagAFP2a overexpression and gene knockout plants

[0195] (1) Short-term salt treatment

[0196] Plants that had grown well for 25 days under tissue culture conditions were washed 2-3 times with warm water to remove the agar from the roots. 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 solution changed every 3-5 days. Then, WT plants grown for 18 days under normal hydroponic conditions, PagAFP2a overexpressing plants OE5 and OE10, and gene knockout plants KO11 and KO27 were subjected to short-term salt treatment with half Hoglund's medium containing 150 mM NaCl. Plant growth was photographed at 0, 1, 6, and 48 hours after salt treatment, and physiological indicators were measured for each plant after 48 hours of salt treatment.

[0197] The results showed that after treatment with 150 mM salt, compared with WT, the leaves of transgenic lines OE5 and OE10, which overexpressed PagAFP2a, were more severely wilted, while the PagAFP2a knockout plants KO11 and KO27 showed better growth and less wilting, indicating that knocking out PagAFP2a can significantly improve the resistance of poplar to salt stress (Figure 4).

[0198] Physiological index measurements revealed that under salt stress, the antioxidant enzyme activities (such as CAT and POD) of PagAFP2a-overexpressing plants (OE5 and OE10) were significantly lower than those of WT (Figures 7A and 7B), while the relative conductivity and reactive oxygen species (such as H2O2 and O2) were also significantly lower. - The content of MDA (malondialdehyde) was significantly higher than that of WT (Figures 7C, 7D, 7E, and 7F), while the PagAFP2a gene knockout plants showed the complete opposite trend, that is, their antioxidant enzyme activity was significantly increased under salt stress, which effectively slowed down the excessive accumulation of reactive oxygen species and reduced the degree of plant damage.

[0199] (2) Long-term salt treatment

[0200] Plants in good condition after 25 days of tissue culture were transferred to flowerpots. Soil culture conditions were maintained with a substrate-vermiculite ratio of 1:1.5. The plants were cultured normally for 25 days under constant temperature and light conditions in a 25℃ incubator. Then, a salt treatment was applied by watering with a 100mM NaCl solution. Plant growth was photographed at 0, 15, and 20 days after salt treatment. Growth was statistically analyzed for plants at 15 and 20 days of salt treatment, and the relative leaf water content was measured for plants at 20 days of salt treatment.

[0201] The results showed that after 15 and 20 days of treatment with 100 mM salt, compared with WT, the leaves of the overexpressing PagAFP2a transgenic line were severely dried out, while the PagAFP2a knockout plants were less wilted, indicating that knocking out PagAFP2a can significantly improve the tolerance of poplar to salt stress (Figure 5).

[0202] After 20 days of treatment with 100 mM salt, compared with WT, the relative water content of leaves in the overexpressing PagAFP2a transgenic lines was significantly reduced, while that in the PagAFP2a knockout plants was significantly increased (Fig. 8A). After 15 and 20 days of treatment with 100 mM salt, compared with WT, the plant height, stem diameter, plant height growth rate, and stem diameter growth rate of the overexpressing PagAFP2a transgenic lines were significantly reduced, while those of the PagAFP2a knockout plants were significantly increased (Fig. 8B, Fig. 8C, Fig. 8D, Fig. 8E).

[0203] Example 9: Drought tolerance analysis of PagAFP2a gene overexpression and gene knockout plants

[0204] Tissue culture seedlings that had grown uniformly in the culture bottles for 20 days were transferred to a soil culture medium containing a 1:1.5 mixture of substrate and vermiculite. The seedlings were cultured in the soil for 25 days, with 400 mL of water added to the trays daily. Once the seedlings had recovered well, healthy and robust seedlings were selected for drought treatment. Watering was stopped and their growth status was recorded by taking photos.

[0205] The results showed that after 7 days of drought stress, the overexpressing PagAFP2a transgenic lines exhibited severe wilting and leaf drying, while the WT plants showed slight yellowing and wilting. Simultaneously, drought treatment of the WT and PagAFP2a knockout plants KO11 revealed that after drought stress, the lower and middle leaves of the WT plants severely dried, wilted, and fell off, while only the lower leaves of the knockout plants showed drying, and the overall damage to the plants was less severe (Figure 6). The experiment indicates that overexpression of the PagAFP2a gene in 84K poplar weakens the plant's drought tolerance. When plants are under drought stress, WT plants adapt to drought better than overexpressing plants; the PagAFP2a knockout plants exhibit better drought tolerance than WT plants.

[0206] SEQ ID NO.21

[0207] The PagAFP2a CDS sequence is as follows:

[0208] SEQ ID NO.22

[0209] The amino acid sequence of PagAFP2a is as follows: "*" is the stop codon.

[0210] 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

[0211] This invention provides a PagAFP2a gene associated with the salt and drought tolerance of poplar. Poplars with PagAFP2a gene knockout mutants exhibit significantly enhanced antioxidant enzyme activity under salt stress compared to WT (non-transgenic poplar), and significantly reduced reactive oxygen species, malondialdehyde content, and relative conductivity, resulting in significantly enhanced resistance. However, their growth under normal culture conditions is not significantly different from that of WT. The PagAFP2a gene knockout mutant can significantly enhance the salt and drought tolerance of poplars, and has good economic value and application prospects.

Claims

1. A PagAFP2a gene associated with salt tolerance and drought tolerance of Populus, characterized in that, The nucleotide sequence of the protein is shown as SEQ ID NO.

21.

2. A protein encoded by the PagAFP2a gene related to salt tolerance and drought tolerance of Populus, characterized in that, The amino acid sequence of the protein is shown as SEQ ID NO.

22.

3. A recombinant vector comprising the PagAFP2a gene overexpression according to claim 1, characterized by, The basic vector of the expression recombinant vector is pPZP211 plasmid.

4. A recombinant Agrobacterium comprising the PagAFP2a gene of claim 1, characterized in that, The agrobacterium is GV3101.

5. A recombinant vector comprising the PagAFP2a gene knockout according to claim 1, characterized by, The recombinant vector is a CRISPR / Cas9 binary vector.

6. A recombinant Agrobacterium comprising the PagAFP2a gene knockout of claim 1, wherein, The agrobacterium is GV3101.

7. A breeding method of a transgenic poplar with enhanced salt tolerance and drought tolerance, characterized by, The method specifically comprises the following steps: Step 1. Designing a PagAFP2a gene target, constructing the gene target to a CRISPR / Cas9 binary vector, and transforming agrobacterium; Step 2. Introducing the PagAFP2a gene CRISPR / Cas9 gene knockout vector constructed in step 1 into the leaves of 84K poplar; Step 3. Obtaining adventitious buds by co-culturing and selecting culture, rooting culture into complete plants, and identifying positive plants, and finally obtaining PagAFP2a gene knockout mutant plants.

8. The method of claim 7, wherein, In step 1, the gene knockout target sequences are shown as AFP2a-1 and AFP2a-2, respectively.

9. The method of claim 7, wherein, In step 3, the positive plants are obtained by PCR identification, and the positive plants are salt-tolerant and drought-tolerant transgenic poplar plants.

10. The method of claim 7, wherein, Compared with WT plants, the salt-tolerant and drought-tolerant transgenic poplar plants grow better under salt stress and / or drought conditions.

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