Application of transcription inhibition factor PuZAT68 gene in regulating and controlling drought resistance of populus japonica

Abstract

The invention provides application of a transcription inhibition factor PuZAT68 gene in regulation and control of drought resistance of populus japonica, and belongs to the technical field of gene engineering and plant breeding. The nucleotide sequence of the transcription inhibition factor PuZAT68 gene is shown as SEQ ID NO.3, and the amino acid sequence of the transcription inhibition factor PuZAT68 gene is shown as SEQ ID NO.4. It is found that a PuZAT68 inhibition expression plant shows the growth state obviously superior to that of wild type WT; the PuZAT68 overexpression plant shows an obvious growth inhibition phenomenon, physiological index determination of different plants finds that inhibition of PuZAT68 expression can reduce production of reactive oxygen species (ROS), damage of oxidative stress to cell membranes can be effectively relieved, more osmotic regulation substances are accumulated, the osmotic regulation capability of cytoplasm is remarkably enhanced, and the osmotic regulation effect is improved. The plant growth regulator plays a better role in drought resistance, so that the plant can still maintain relatively normal growth under drought stress.

Description

Technical Field

[0001] The invention belongs to the fields of genetic engineering and plant breeding, and particularly relates to the application of a transcription inhibitor PuZAT68 gene in regulating the drought resistance of Populus ussuriensis. Background Art

[0002] The impacts of abiotic stresses such as drought, salinity, osmotic stress, and high temperatures on plant growth and development, as well as the stability and sustainability of agricultural ecosystems, are becoming increasingly significant as global climate change intensifies. According to statistics, approximately 30% of the world's soils are currently suffering from drought stress. Global water shortages are becoming increasingly severe, leading to a continuous expansion of arid areas and exponentially increasing economic losses. According to relevant data, among all abiotic stress factors, drought stress has the greatest impact on agricultural productivity. As a vital component of terrestrial ecosystems, forests not only play a key role in maintaining ecosystem balance and providing ecological services, but also provide humanity with a rich resource of timber and forest products. However, drought stress can slow tree growth, reduce yield and quality, and even lead to forest decline and death, causing significant losses to the forestry industry and the ecological environment. Therefore, in-depth research on the drought resistance mechanisms of forests and the cultivation of drought-resistant trees and crops are of vital importance for future ecosystems and human development.

[0003] Populus ussuriensis, an economically important forest species, encounters numerous challenges during its growth, with drought being a common one. When plants experience drought stress, they regulate the expression of related genes through signal transduction, triggering changes. Therefore, improving the drought tolerance of Populus ussuriensis is crucial for enhancing its productivity and adapting to climate change. Summary of the Invention

[0004] The purpose of the present invention is to study the application of the transcription inhibitor PuZAT68 gene in regulating the drought resistance of Populus ussuriensis.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides the use of the transcription repressor PuZAT68 gene in regulating the drought resistance of Populus ussuriensis. The nucleotide sequence of the transcription repressor PuZAT68 gene is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.4:

[0007] The method for improving the drought resistance of Populus ussuriensis by utilizing the PuZAT68 gene is as follows: the drought resistance of Populus ussuriensis is enhanced by inhibiting the expression of the PuZAT68 gene.

[0008] Preferably, the PuZAT68 protein encoded by the PuZAT68 gene comprises two QALGGH-type domains, two C2H2-type zinc finger domains, and an EAR motif at its C-terminus.

[0009] Preferably, the specific operation of using the PuZAT68 gene to regulate the drought resistance of Populus ussuriensis is: constructing a PuZAT68 inhibition expression vector, transferring it into Populus ussuriensis plants by Agrobacterium infection, and obtaining Populus ussuriensis inhibition expression resistant plants PuZAT68-IE.

[0010] Preferably, the original vector of the PuZAT68 inhibition expression vector is pFGC5941, the restriction sites at the left end of the forward primer are XhoI and AscI, and the restriction sites at the left end of the reverse primer are XbaI and SmaI. The constructed PuZAT68 inhibition expression vector is named pFGC5941-PuZAT68-RNAi.

[0011] The present invention provides a transcriptional repressor PuZAT68 gene.

[0012] The present invention also provides the use of the PuZAT68 gene in screening or identifying Populus cathayana strains with high drought resistance.

[0013] The present invention uses Populus ussuriensis as experimental material to conduct an in-depth study on the drought resistance function of the PuZAT68 transcription factor of the C1-2i subfamily of C2H2-type zinc finger proteins. Through bioinformatics analysis, the PuZAT68 protein exhibits significant water-solubility characteristics and is a hydrophilic protein. Domain analysis shows that the protein contains two conserved C2H2-type zinc finger domains belonging to the C1-2i subfamily. Through BLAST comparison, it was found that PuZAT68 has close homology with drought-responsive zinc finger proteins that have been studied, such as the Arabidopsis AtZAT12 gene and the wheat TaZFP21 gene. Based on structural conservation and functional relevance, we speculate that the PuZAT68 gene may play a relevant functional role in the drought response mechanism of Populus ussuriensis. The present invention verifies that PuZAT68 is localized in the cell nucleus through subcellular localization experiments. Its amino acid sequence has a typical EAR domain. Yeast transcription activation experiments were performed on the front and back regions of the EAR domain, and the results verified that none of the regions of PuZAT68 had transcriptional activation activity. To further analyze the transcriptional regulatory characteristics of this gene, the transient transfection protoplast LUC / GUS enzyme activity experiment found that PuZAT68 significantly inhibited the expression of the relative gene GUS, showing transcriptional inhibition activity similar to the transcription inhibition control, indicating that PuZAT68 is a transcription repressor located in the cell nucleus.

[0014] The present invention successfully constructed and obtained genetic transformation strains of PuZAT68 overexpression (PuZAT68-OE) and suppressed expression (PuZAT68-IE). Under experimental conditions of 6% PEG6000 simulating drought stress, phenotypic observations showed that PuZAT68-IE plants exhibited a growth state significantly better than the wild-type WT; while PuZAT68-OE plants showed obvious growth inhibition. Subsequently, by measuring the physiological indicators of different genetic transformation strains, it was found that PuZAT68 suppressed expression can reduce the production of reactive oxygen species (ROS) by activating the antioxidant enzyme system, effectively alleviating the damage to the cell membrane caused by oxidative stress, thereby accumulating more osmotic regulatory substances and significantly enhancing the osmotic regulatory capacity of the cytoplasm. This makes it play a better role in drought resistance and enables the plant to maintain relatively normal growth under drought stress.

[0015] PuZAT68, a transcriptional repressor of the C2H2-type zinc finger family, may play a key regulatory role in plant stress response mechanisms. It may selectively bind to specific cis-acting elements in target gene promoters, downregulating the expression of a series of genes negatively correlated with stress adaptation, thereby creating an environment conducive to drought tolerance. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is the PuZAT68 gene CDS sequence clone;

[0017] Figure 2 This is the comparison result of PuZAT68 clone sequence;

[0018] Figure 3 This is the comparison result of the PuZAT68 amino acid sequence;

[0019] Figure 4 This is the PuZAT68 protein structure prediction result;

[0020] Figure 5 Comparison of conserved regions and phylogenetic tree analysis of PuZAT68 homologous genes;

[0021] Figure 6 Quantitative expression analysis of PuZAT68 under 6% PEG6000 stress;

[0022] Figure 7 PCR detection of proPuZAT68 transgenic Populus ussuriensis, -: WT; +: plasmid; 1-10: 10 transgenic lines;

[0023] Figure 8GUS staining of the proPuZAT68::GUS line (A: histochemical staining of unstressed transgenic plants; B: histochemical staining of transgenic plants after 6% PEG6000 stress; the length of the scale bar is 1 cm);

[0024] Figure 9 A is the result of PCR detection of PuZAT68 restriction enzyme cutting site, and B is the result of PCR detection of pUC19-PuZAT68-GFP bacterial solution;

[0025] Figure 10 The results of PuZAT68 subcellular localization detection are shown. pUC19-GFP is used as a control, and mCherry is localized in the cell nucleus. The scale bar is 5 μm.

[0026] Figure 11 for PuZAT68 transcriptional activity analysis and protein structure;

[0027] Figure 12 is the relative GUS activity of PuZAT68 protein, * represents significant (*P < 0.05) and ** represents extremely significant (**P < 0.01);

[0028] Figure 13 Schematic diagram of the restriction enzyme cutting sites of plasmid pFGC5941;

[0029] Figure 14 For PCR identification of the recombinant plasmid pBI121-PuZAT68-GFP, 1-11: pBI121-PuZAT68-GFP PCR product;

[0030] Figure 15 PCR detection results of pFGC5941-PuZAT68-Cis-Anti bacterial solution, M: DNAMarker DL2000; 1-6: pFGC5941-PuZAT68-Cis-Anti PCR products;

[0031] Figure 16 This is the initial screening of pBI121-PuZAT68-GFP transgenic resistant seedlings. Scale bar: 1 cm;

[0032] Figure 17 Detection of DNA levels in PuZAT68-OE transgenic plants, M: DNAMarker DL2000; -: WT; +: plasmid; 1-12: 12 transgenic lines;

[0033] Figure 18 Analysis of PuZAT68 expression in WT and overexpressing transgenic lines;

[0034] Figure 19This is the initial screening of pEGC5941-PuZAT68-RNAi transgenic resistant seedlings. Scale: 1 cm;

[0035] Figure 20 Detection of DNA levels in PuZAT68-IE transgenic plants, M: DNAMarker DL2000; -: WT; +: plasmid; 1-12: 12 transgenic lines;

[0036] Figure 21 Analysis of PuZAT68 expression in WT and suppressed expression transgenic lines;

[0037] Figure 22 The phenotypic observations of stems, leaves and roots of WT and transgenic lines before and after 6% PEG6000 stress are shown in the figure. The scale is 1 cm.

[0038] Figure 23 The root length and root dry weight of WT and transgenic plantlets were measured before and after 6% PEG6000 stress;

[0039] Figure 24 The relative electrical conductivity and MDA content of the roots of WT and transgenic plantlets before and after 6% PEG6000 stress were measured;

[0040] Figure 25 NBT staining of leaves and roots of WT and transgenic plantlets before and after 6% PEG6000 stress, scale bar is 1 cm;

[0041] Figure 26 DAB staining of leaves and roots of WT and transgenic plantlets before and after 6% PEG6000 stress, scale bar is 1 cm;

[0042] Figure 27 The H2O2 content in the roots of WT and transgenic plantlets before and after 6% PEG6000 stress was determined. DETAILED DESCRIPTION

[0043] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0044] Example 1 Screening and identification of PuZAT68 gene

[0045] RNA was extracted from different root sites of wild-type Populus ussuriensis tissue culture seedlings using the CTAB method. The qualified RNA was then reverse-transcribed into cDNA using the Novozymes reverse transcription kit. Using the reverse-transcribed Populus ussuriensis cDNA as a template, a high-fidelity DNA polymerase was used to clone the target gene PuZAT68. Specific primers were designed as follows:

[0046] PuZAT68-F(SEQ ID NO.1):5'-ATGAAGAGAGATAGAGAACAGGCA-3'

[0047] PuZAT68-R(SEQ ID NO.2):5'-GATAAAACATCTCAACATGGGA-3'

[0048] The PCR amplification system was as follows: PCR template, 10 μL; PuZAT68-F, 6 μL; PuZAT68-R, 6 μL; 2 mM dNTPs, 40 μL; Buffer, 100 μL; reaction enzyme, 4 μL; deionized water, 34 μL.

[0049] The PCR reaction procedure was referred to the instruction manual of KOD FX DNA Polymerase high-fidelity enzyme.

[0050] Gel imaging system shows ( Figure 1 ): The target band is around 590bp. After recovering the product, it was preliminarily determined to be consistent with the target gene band of this study.

[0051] After recovering the target fragment using the gel recovery kit, the concentration of the recovered product was tested. Once the test was acceptable, the recovered DNA fragment was ligated with the pCloneEZ-Blunt-TOPO vector (refer to the instructions for the ligation system of the Sino-US and Clone Smarter TOPO cloning kits). The rapid ligation was performed at a constant temperature of 25°C for 5 minutes, and the E. coli transformation experiment was carried out (heat shock method):

[0052] (1) In a clean bench, add the ligation product to 100 μL of DH5α, thoroughly mix the ligation product with the semi-thawed competent cells, and place the centrifuge tube on ice for 30 minutes;

[0053] (2) Heat shock the tube in a 42°C water bath for 90 seconds. Immediately transfer the tube to ice and let it sit for 2 minutes to terminate the heat shock reaction.

[0054] (3) Under sterile conditions, add 600 μL of antibiotic-free LB liquid medium. Incubate the mixture in a 37°C shaker for 60 min. Meanwhile, prepare an LB plate containing Kan and equilibrate it to room temperature for later use.

[0055] (4) Centrifuge the cultured bacterial solution at 4000 rpm for 5 min, carefully remove most of the supernatant, and retain approximately 200 μL of culture medium for resuspension of the bacterial cells;

[0056] (5) The resuspended bacterial solution was evenly transferred to a LB solid medium plate containing 50 mg / L Kan;

[0057] (6) Use a sterilized and cooled spreading stick to evenly spread the bacterial solution on the plate. Then incubate the plate upside down at 37°C for 12-16 hours until single colonies are formed.

[0058] The next day, using aseptic operation techniques, single clones were picked from the plates containing transformed E. coli and inoculated into an appropriate amount of LB liquid culture medium containing Amp. After culturing for 6 hours, PCR detection was performed using the bacterial solution as a template. After electrophoresis detection of the amplified products, the bacterial solution with the correct band position was screened and sent to a biological company for sequencing. The sequencing results were compared and analyzed with the sequence of Potri.001G235800 (PtrZAT68). By parallel comparison of the sequencing results, it was found that there were 30 different bases between the sequences of Populus ussuriensis and Populus trichocarpa, and the sequence similarity rate reached 94.9%. Figure 2 The results showed that the cloning was successful and there were 12 different sites between the amino acid sequence and the amino acid sequence of Populus trichocarpa, such as Figure 3 .

[0059] Bioinformatics analysis of PuZAT68

[0060] The PuZAT68 gene and its protein were comprehensively analyzed using multi-platform bioinformatics tools. First, the PuZAT68 gene sequence was analyzed online using The Sequence Manipulation Suite and converted into an amino acid sequence. The results showed that the protein encoded by the gene is composed of 192 amino acids, and the chemical formula of the protein is C 929 H 1520 N 282 O 276 S 12 The calculated molecular mass is 21440.73Da, the total number of atoms is 3019, and the theoretical isoelectric point is 9.62. Amino acid composition analysis shows that among the 20 amino acids, leucine (Leu) has the highest content (12.1%), while tyrosine (Tyr) has the lowest content (1%).

[0061] The PuZAT68 protein was analyzed using a protein hydrophobicity prediction tool. The results showed that the arginine at position 5 of the sequence exhibited the strongest hydrophilicity (-2.60), the leucine at position 170 exhibited the greatest hydrophobicity (2.20), and the number of negative value regions (hydrophilic amino acids) was significantly dominant. Therefore, it can be inferred that the PuZAT68 protein is a hydrophilic protein.

[0062] The conserved domains of PuZAT68 protein were predicted by CD-Search tool of NCBI conserved domain database, and the map was analyzed ( Figure 4) showed that the protein has typical C2H2-type zinc finger domains at amino acid positions 55-80 and 102-127, respectively, as shown in SEQ ID NO. 4. This characteristic domain distribution pattern provides sufficient basis for classifying PuZAT68 protein as a C2H2-type zinc finger protein family.

[0063] Using the WoLF PSORT online tool (https: / / wolfpsort.hgc.jp / form.html), we performed a predictive analysis of its subcellular localization. Bioinformatics identification of nuclear localization signals revealed the presence of multiple basic amino acid-rich regions within the protein sequence. This characteristic amino acid composition is highly consistent with a classic nuclear localization signal. The results predict that the protein has a 100% probability of being localized in the cell nucleus.

[0064] Table 1

[0065] queryProtein WoLFPSORT prediction nucl:14

[0066] PSORT features and traditional PSORTll prediction

[0067] 14 Nearest Neighbors

[0068] id site distance identity comments RBE_ARATH nucl 375.51 <![CDATA[ 18.5022％ ]]> <![CDATA[[ Arath ][ Uniprot ]SWISS-PROT45:Nuclear.Evidence:IDA Pubmed: 14681191 ]]> HBPB_WHEAT nucl 392.542 <![CDATA[ 12.3494％ ]]> <![CDATA[[ Uniprot ]SWISS-PROT45:Nuclear.]]> ATH7_ARATH nucll 409.923 <![CDATA[ 13.1783％ ]]> <![CDATA[[ Uniprot ]SWISS-PROT45:Nuclear.]]> HKN1_MAIZE nucl 444.867 <![CDATA[ 11.6992％ ]]> <![CDATA[ Uniprot ]:SWISS-PROT45:Nuclear.]]> TGA2_ARATH nucl 446.726 <![CDATA[ 13.9394％ ]]> <![CDATA[[ Arath ][ Uniprot ]SWISS-PROT45:Nuclear.Evidence:IDA Pubmed: 11479572 ]]> TGA6_ARATH nucl 453.589 <![CDATA[ 14.8485％ ]]> <![CDATA[[ Uniprot ]SWISS-PROT45:Nuclear.]]> OCS1_MAIZE nucl 453.83 <![CDATA[ 12.1212％ ]]> <![CDATA[[ Uni p rot ]SWISS-PROT45:Nuclear.]]> AGL3_ARATH nucl 456.8 <![CDATA[ 11.6279％ ]]> <![CDATA[[ Uniprot ]SWISS-PROT45:Nuclear.]]> AG15_ARATH nucl 464.405 <![CDATA[ 14.5522％ ]]> <![CDATA[[ Uniprot ]SWISS-PROT45:Nuclear.]]> SEP2_ARATH nucl 467.922 <![CDATA[ 15.2％ ]]> <![CDATA[[ Uniprot ]SWISS-PROT45:Nuclear.]]> ATH5_ARATH nucl 467.943 <![CDATA[ 13.7821％ ]]> <![CDATA[[ Uniprot ]SWISS-PROT45:Nuclear.]]> TGAA_TOBAC nucl 470.712 <![CDATA[ 11.1421％ ]]> <![CDATA[[ Uniprot ]SWISS-PROT45:Nuclear.]]> CAL_ARATH nucl 484.905 <![CDATA[ 14.6245％ ]]> <![CDATA[[ Uniprot ]SWISS-PROT45:Nuclear.]]> HAT5_ARATH nucl 485.509 <![CDATA[ 11.3971％ ]]> <![CDATA[[ Uniprot ]SWISS-PROT45:Nuclear.]]>

[0069] By searching for homologous proteins of PuZAT68 protein and performing multiple sequence alignment, we analyzed its conserved regions. The results showed that the protein not only contains two QALGGH-type domains, but also two C2H2-type zinc finger domains, and an EAR motif at its C-terminus. Further phylogenetic analysis using the neighbor-joining method to construct a phylogenetic tree revealed that PuZAT68 is most closely related to ZAT12 in Arabidopsis thaliana, and both belong to the C1-2i subfamily ( Figure 5 The present invention successfully obtained the complete coding sequence (CDS) of PuZAT68 gene from Populus ussuriensis, encoding 192 amino acids.

[0070] The complete coding sequence of the PuZAT68 gene is (SEQ ID NO.3):

[0071] ATGAAGAGAG ATAGAGAACAGGCAGAGTTAGACTTGGCCAAATGCTTGATGCTACTTTCTAAAGTTGGCAAAGCTGATCACGAGATACTAACTAATTATAGATCAGCAGCAGCAGCAGCACCGGCAGG GGCCGGGGCCGGTCGCTC ATTTTCATGCAAAACATGCGACAAGAATTTCCCTTCATTTCAAGCATTAGGAGGCCACCGGGCAAGTCACAAGAAAACAAAACTCATGGAATCAACTGGGAACTTGTTGAAGCTGCCTAATTCGCCTTCAAAGCCAAAAACTCACCAGTGCTCCATTTGCGGCCTTGAATTTCCTCTCGGGCAAGCACTTGGAGGTCACATGAGGAGACATCGGGCACCTCATAATGTTGATACCACCAGTACTAGTAGTAAAGATCATGAATTGGCTGTGACTCAACCGCCTTTCTTACCCGCTGTCCCAGTTTTGAAGAGATCCAATAGCAGCAAGAGAGTTCTGTGCTTGGATTTGAGCTTGGCCCTGCCTATGTATCAGAATGACTCGGAGTTGCAGTTGGAGAAGGTCGCTCGTCCCATGTTGAGATGTTTTATC TAA

[0072] The amino acid sequence encoded by the PuZAT68 gene is (SEQ ID NO.4):

[0073] MKRDREQAEIDLAKCLMLLSKVGQADHEILTNYRSAAAAAAAATAGAGAGAGRSF SCKTCNKNFPSFQ ALGGHRASHKKP KLKESTGNLLKLPNSPSKPKTH QCSICGLEFPLGQALGGHMRRHRAP HNVDTTSTSSKDHELAAVTQPPFLPAVPVLKRSNSSKRVLCLDLSLALPMYQNDSELQLEKVDRPMLRCFI*

[0074] Sequence comparison revealed that the CDS sequences of PuZAT68 and the Populus trichocarpa homologous gene PtrZFP68 differ by 30 bases, yet the overall sequence similarity remains at 94.9%, indicating a high degree of evolutionary conservation. PuZAT68 was thoroughly analyzed using a variety of bioinformatics analysis tools and online prediction platforms. Structural prediction revealed that the PuZAT68 protein contains a typical C2H2-type zinc finger domain at amino acid positions 55-80 and 102-127, respectively. Further analysis revealed the presence of a conserved QALGGH sequence between amino acid positions 68-73 and 115-120, representing the functional core motif of the C2H2-type zinc finger structure. Furthermore, a signature sequence, LCLDLSLALP, was identified between amino acid positions 160-180 of PuZAT68, closely matching the known repressor domain of EAR transcription factors. Based on the above results, it is speculated that PuZAT68 may play an important regulatory role in abiotic stress through its DNA binding activity mediated by zinc finger structure and transcriptional repression function mediated by EAR domain. Its specific molecular mechanism of action needs to be verified by subsequent experiments.

[0075] Example 2 Analysis of PuZAT68 gene expression pattern

[0076] Three-week-old wild-type Populus ussuriensis seedlings of consistent growth status were used as experimental subjects and subjected to simulated drought stress with 6% PEG6000. Observation and detection were performed at seven time points (0, 6, 12, 24, 48, 96, and 144 hours). RT-qPCR was used to measure the expression of PuZAT68 in roots, stems, and leaves of Populus ussuriensis at different time points to assess their response to simulated drought stress.

[0077] Specific primers were designed based on the sequence of the PuZAT68 gene. PuActin from Populus ussuriensis was used as the internal reference gene in RT-qPCR. The primer sequences designed for quantitative detection of the PuZAT68 gene are as follows:

[0078] PuActin-F(SEQ ID NO.5):5'-AGGGTATGCCCTTCCACAT-3'

[0079] PuActin-R(SEQ ID NO.6):5'-TCAAGGGCAACATATGCAAG-3'

[0080] DL-PuZAT68-F(SEQ ID NO.7):5'-CATAATGTTGATACCACCAG-3'

[0081] DL-PuZAT68-R(SEQ ID NO.8):5'-CTTCTCCAACTGCAACTCCG-3'

[0082] The cDNA obtained by reverse transcription was diluted 10 times as a quantitative PCR template. The reaction was completed using a fluorescent quantitative PCR instrument. The reaction system is shown in Table 2:

[0083] Table 2

[0084] DL-PuZAT68-F 0.6μL DL-PuZAT68-R 0.6μL PCR template 100ng Reaction enzyme 10 μL Deionized water to20μL

[0085] The real-time fluorescence quantitative PCR reaction procedures and conditions are as follows: HiScript IIQ RT SuperMix for qPCR Reverse Transcription Kit Instructions. -△△CT Formula calculation and analysis were performed, and GraphPad Prism10 software was used to draw a bar graph to show the relative expression levels of the target gene under different treatment conditions.

[0086] like Figure 6 After simulated drought stress, PuZAT68 expression levels in leaves showed minimal changes, while in roots and stems, expression levels varied significantly. Specifically, PuZAT68 expression peaked at 12 hours in both roots and stems, with expression in roots significantly higher than in stems. These results indicate that under 6% PEG6000-simulated drought stress, roots are more sensitive to stress, with PuZAT68 expression primarily induced by drought in the roots.

[0087] Cloning of PuZAT68 promoter and construction of expression vector

[0088] DNA extraction from Populus ussuriensis

[0089] (1) Using sterile tweezers in a clean bench, carefully remove the leaf of a tissue cultured Populus ussuriensis seedling and place it in a clean 1.5 mL centrifuge tube. After the leaf is fully frozen in liquid nitrogen, take out the grinding rod that has been pre-cooled to a low temperature. Use this grinding rod to grind the leaf to ensure that the leaf tissue is fully broken up.

[0090] (2) Add 700 μL of preheated CTAB (cetyltrimethylammonium bromide) to the powdered centrifuge tube and mix gently;

[0091] (3) Place the mixture in a 65°C constant temperature water bath and oscillate every 10 minutes for a total of 30 minutes.

[0092] (4) Centrifugation at 12000 rpm for 10 min at room temperature;

[0093] (5) Prepare a 2 mL centrifuge tube containing an equal volume of phenol-chloroform mixture (350 μL each) and transfer the supernatant obtained after the above centrifugation into the tube; mix thoroughly until the solution is no longer separated, centrifuge at 12,000 rpm for 5 min, and repeat this step once;

[0094] (6) Add 700 μL of chloroform to a 2 mL centrifuge tube for secondary extraction. Add the supernatant from the previous centrifugation step to the tube, mix thoroughly until the solution is no longer separated, and then centrifuge at 12,000 rpm for 5 min.

[0095] (7) Add 800 μL of anhydrous ethanol and 100 μL of 3 M sodium acetate solution to a 1.5 mL centrifuge tube. Use a pipette to accurately draw the supernatant obtained in the previous step and slowly add it to the centrifuge tube. After mixing, place the centrifuge tube at room temperature for 20 minutes to utilize the properties of anhydrous ethanol and sodium acetate solution to promote the complete precipitation of DNA.

[0096] (8) Place the centrifuge tube after standing still in a centrifuge and set the centrifugal speed to 12,000 rpm for 20 minutes. Under the action of high-speed centrifugation, the DNA molecules in the solution will gradually aggregate and form a white precipitate, while other impurities will remain in the supernatant, thus achieving the initial separation of DNA and impurities;

[0097] (9) After centrifugation, discard the supernatant and wash the precipitate with 75% ethanol. Repeat the washing operation twice to remove impurities on the surface of the precipitate. After washing, carefully remove the small amount of residual liquid in the centrifuge tube with a pipette (to prevent the residual liquid from affecting subsequent experiments). After drying at room temperature, add 30 μL ddH2O to dissolve the precipitated DNA.

[0098] (10) Take 2 μL of the DNA extraction product and perform 1.2% agarose gel electrophoresis. The integrity of the bands was observed using a gel imaging system. The DNA quality was then measured using a nucleic acid analyzer, with A260 / A280 = 2.0. The DNA was then stored in a -20°C refrigerator until use.

[0099] According to the PtrZAT68 gene sequence, the primer sequences were designed as follows:

[0100] ProPuZAT68-F (SEQ ID NO.9):5'-CTTGATGGCTTTATAGTCTA-3';

[0101] ProPuZAT68-R (SEQ ID NO.10):5'-TGCTGCTGCTGATCTATAATTAG-3';

[0102] The PuZAT68 promoter was cloned using the extracted wild-type Populus ussuriensis DNA as a template. The reaction system is shown in Table 3.

[0103] Table 3 KOD amplification system

[0104]

[0105]

[0106] PCR reaction conditions refer to the instructions of KOD FX DNA Polymerase. The cloning vector was subjected to a BP recombination reaction. A PCR instrument was set to 25°C for 5 minutes. The recombinant product was then transformed into E. coli. The bacterial solution was evenly spread onto an LB plate containing Kan. After ensuring uniformity, the plate was sealed in a clean bench and incubated upside down in a 37°C incubator for 12 hours. After 12 hours of incubation, a single clone was selected for shaking culture. After 4-6 hours, the bacterial solution was tested by PCR. Bacterial solutions with a band size of approximately 2000 bp were selected and sent to a sequencing company for verification. Clones with verified correct sequences were used in subsequent experiments.

[0107] Plasmids were extracted from the successfully sequenced bacterial suspension and the pGWB3-GUS vector. The extracted plasmids were subjected to an LR replacement reaction at 25°C for 3 hours. After incubation at 37°C for 10 minutes, 1 μL of Proteinase K Solution (2 μg / μL) was added to terminate the reaction. E. coli transformation was then performed. The culture, resuspended after centrifugation, was evenly plated on LB selection medium supplemented with 50 mg / L Kan and 3 mg / L Hydroxyprogesterone (Hydroxyprogesterone). After incubation at 37°C overnight, single colonies were picked the next day and cultured. After 8 hours of incubation, the culture was analyzed by PCR. Positive recombinants were screened by PCR. Cultures containing the target band located around 2000 bp were sent to a sequencing company for verification. If the sequence was correct, the pGWB3-PuZAT68pro-GUS vector was successfully constructed.

[0108] Plasmid transformation was performed using GV3101 competent cells. For specific experimental methods, refer to the instructions for the GV3101 Agrobacterium competent cells from Sangon. In the final step of the experiment, a spreading rod was used to evenly spread the treated, resuspended bacterial suspension onto the surface of LB solid medium supplemented with antibiotics (50 mg / L Kan and 50 mg / mL L-Rif), providing optimal conditions for subsequent bacterial growth and screening. The coated medium was placed in an incubator at 28°C and incubated upside down for two days. At this temperature, bacteria were able to grow and multiply on the LB solid medium containing antibiotics. The presence of antibiotics acted as a selection agent, allowing only bacteria carrying the corresponding resistance genes to survive and form colonies on this medium. Single colonies were randomly selected and transferred to LB medium supplemented with dual antibiotics (50 mg / L Kan and 50 mg / mL L-Rif) and cultured with shaking at 28°C for two days until the culture became turbid. The turbid bacterial solution was subjected to PCR amplification and 1.2% agarose gel electrophoresis analysis. The correct bacterial solution with a single amplified band that was consistent with the target band was selected. Agrobacterium containing the pGWB3-ProPuZAT68-GUS plasmid was used as the transformation vector. A sterilized 10μL pipette tip was used to streak the plate in a clean bench and cultured at 28°C for 2 days. A single colony was picked to prepare the engineered bacteria for transformation. After the engineered bacteria were prepared, the Agrobacterium-mediated leaf disc method was used to genetically transform Populus ussuriensis. The bacterial solution concentration OD 600 =0.6, infection for 15 minutes.

[0109] Method for stable transformation of Populus ussuriensis:

[0110] (1) Select wild-type Populus ussuriensis tissue culture seedlings grown for 3 weeks in sterile culture, cut off the fifth dark green healthy leaf (with complete petiole) and stem segment from the morphological upper end using sterile scissors, and place them in sterilized 1 / 2MS liquid culture medium to prepare 40 leaves and an appropriate amount of stem segments;

[0111] (2) Use a sterile blade to cut vertically along the base of the petiole and cut about 1 cm of the leaf along the main vein, keeping the cut surface flat. The stem segment needs to be removed at the axillary bud, leaving only the stem node structure. All operations must avoid damaging the plant;

[0112] (3) Use sterile tweezers to transfer the leaves to the prepared engineered bacterial solution and infect them with gentle shaking at room temperature for 25 min to ensure that the bacterial solution completely infiltrates the leaves and stems;

[0113] (4) After infection, immediately and thoroughly remove any bacterial liquid remaining on the surface of the leaves and stem segments using sterile absorbent paper. The treated plant material was then spread onto a WPM co-culture plate with the leaves facing downward and the stem segments closely arranged. The plate was then placed in a dark, constant-temperature culture environment for 48 hours of co-culture.

[0114] (5) After the co-cultivation, sterilization was performed. The leaves and stems on the co-cultivation plate were washed five times (2 min each time) with two bottles of 200 mL sterile water sterilized by high temperature and high pressure until the sterile water was no longer turbid, indicating that the initial sterilization was completed.

[0115] (6) Wash the sample eight times (8 min each) with two 200 mL bottles of 1 / 2 MS liquid culture medium sterilized with high temperature and high pressure containing Cef (200 mg / L) until the washing solution is completely clear.

[0116] (7) After washing, use sterile paper to absorb excess water from the leaves and stems, spread the leaves and stems onto screening medium containing various hormones for screening purposes, and perform preliminary screening under dark conditions;

[0117] (8) Regularly observe and promptly remove materials with bacterial growth, and replace fresh culture medium every 7 days; when resistant callus tissue forms at the wound and differentiates into adventitious buds, transfer the culture medium to light conditions for cultivation;

[0118] (9) After 3 weeks of light culture, the resistant buds with good growth and no foreign bacteria were cut with sterile scissors and transferred to WPM rooting medium with Kan, Cef, and Tim. After 3 weeks of screening, the surviving and healthy plants were considered as transgenic resistant seedlings for subsequent molecular testing.

[0119] Genomic DNA was extracted from the initially screened plants for molecular testing. The pGWB3-PuZAT68pro-GUS plasmid containing the target gene fragment was used as a positive control. This plasmid has the complete PuZAT68 promoter and GUS gene sequence, which can serve as a clear control for the presence of the target gene. At the same time, DNA from wild-type Populus ussuriensis plants was selected as a negative control. Wild-type plants have not undergone genetic manipulation and can serve as an important reference for excluding nonspecific amplification. DNA detection was performed using a forward primer for the PuZAT68 promoter (ProPuZAT68-F) and a reverse primer designed on the GUS sequence (GUS-R (SEQ ID NO.11): 5'-GATGCTCCATCACTTCCTG-3'). The PCR product was analyzed by agarose gel electrophoresis. The results are as follows: Figure 7 As shown, the PCR product of the proPuZAT68::GUS strain displayed a single specific band on the gel, and the position of this band was consistent with that of the positive control. However, the PCR product of the wild-type control did not show the target amplified band at the corresponding position, verifying the specificity of the test results. Through the above molecular identification, 10 independent transgenic lines were ultimately obtained.

[0120] The PlantCare online website was used to analyze and predict the cis-acting elements contained in the promoter region of PuZAT68, as shown in Table 4.

[0121] Table 4

[0122]

[0123] The PuZAT68 promoter sequence contains a rich and diverse set of regulatory elements. These elements include a series of conserved expression elements widely found in the plant kingdom. These conserved expression elements play fundamental and important roles in plant growth and development, regulating physiological and biochemical processes to ensure normal plant growth, development, and reproduction. Specifically, these elements include the MBS, MYC, and MYB elements associated with drought response; the TC-rich repeats, cis-acting elements involved in defense and stress responses; the WUN motif, a cis-acting element associated with wound response; the AuxRR core, an auxin-responsive element; the CGTCA motif, a cis-acting regulatory element involved in MeJA responsiveness; and the CAT-box element associated with meristems. These predicted results suggest that PuZAT68 gene expression may be induced by drought and growth-related signals.

[0124] After 14 days of normal culture, the proPuZAT68::GUS transgenic lines obtained earlier were subjected to 6% PEG6000 for 12 hours to simulate drought stress. Untreated transgenic lines served as controls. After the stress treatments were complete, GUS staining was performed to visualize GUS gene expression in the plants.

[0125] GUS staining experiment

[0126] (1) Treatment: DNA-positive proPuZAT68::GUS transgenic seedlings were selected and divided into two groups: the control group was cultured normally in 1 / 2MS medium for 14 days; the experimental group was cultured normally for 14 days and then treated with 6% PEG6000 to simulate drought stress for 12 hours.

[0127] (2) Staining: Take out the two groups of plants that have been cultured, and use sterilized paper to remove the residual culture medium on the roots to maintain the integrity of the plant structure. Immerse the whole plant in a 50 mL centrifuge tube containing GUS staining solution and perform vacuum treatment (5 min vacuum for 5 min, repeat 2-3 times), and stain overnight at 37°C in the dark until the staining effect appears.

[0128] (3) Decolorization: Recover the GUS dye solution (store it at 4°C to avoid bacterial growth in the dye solution), and slowly add Carnoy's fixative along the tube wall for decolorization. The decolorization solution can be replaced every 30 minutes for the first two decolorizations, and every 1 hour for the subsequent decolorizations. Repeat the operation 4 to 6 times until the plant is completely decolorized. The operation needs to be gentle to avoid damage to plant tissue.

[0129] (4) Transparency: Place the completely decolorized plants in the transparent agent, carefully move the plants to the scanner and use paper to absorb the water on the plants. After placing them in an orderly manner, use the HP scanner to scan and save the image.

[0130] The staining results are as follows Figure 8 As shown in Figure 2 , under normal culture conditions, both the roots and leaves of the plants exhibited staining. However, the staining was relatively light, indicating that under normal physiological conditions, while the GUS gene is expressed to a certain extent, the expression level is relatively low. However, after stress treatment, the staining in the roots and leaves of the promoter-transgenic plants showed a significant trend toward deeper staining. This change intuitively demonstrates that stress treatment can induce upregulation of GUS gene expression, resulting in a significant increase in GUS protein levels in the plants. These results demonstrate that the PuZAT68 promoter is capable of initiating expression of the GUS reporter gene, and its expression activity is significantly upregulated by drought stress. Under normal growth conditions, the promoter maintains basal expression; upon exposure to simulated drought stress, promoter activity is activated, driving enhanced expression of downstream genes. This suggests that the PuZAT68 promoter may participate in plant drought stress response mechanisms and play an important role in regulating the expression of stress-resistance-related genes.

[0131] PuZAT68 subcellular localization

[0132] Ligation of PuZAT68 and pUC19-GFP and plasmid extraction

[0133] The restriction endonuclease sites of the PuZAT68 gene coding region (CDS) were predicted using Bioedit sequence analysis software. Based on the characteristics of the multiple cloning site of the pUC19-GFP vector, specific primers containing SalI and XbaI recognition sites were designed, and the corresponding restriction site sequences were introduced at both ends of the primers.

[0134] pUC19-PuZAT68-BamHI homology F (SEQ ID NO.12):

[0135] 5'-GGGGACTCTAGAGGATCCATGAAGAGAGATAGAGAACAG-3';

[0136] pUC19-PuZAT68-BamHI homologous R (SEQ ID NO.13):

[0137] 5'-GTCGACCTCGAGGGATCCGATAAAACATCTCAACATGG-3';

[0138] High-fidelity enzymes were used for PCR amplification to avoid base mutations, and PCR products were detected by agarose gel electrophoresis (the detection results were as follows Figure 9 A) After confirming the correct band position and size, which is around 750 bp, gel recovery was performed. After double enzyme digestion of the pUC19-GFP plasmid (reaction system as shown in Table 5), the product was purified:

[0139] Table 5 Enzyme digestion reaction system

[0140]

[0141] The purified pUC19-GFP plasmid and the PuZAT68 gene fragment were subjected to an overnight ligation reaction using T4 ligase (PCR was set at a temperature of 16° C. for 24 h, and the reaction system was as shown in Table 6).

[0142] Table 6 Homologous recombination reaction system

[0143]

[0144] The ligation product was transformed into E. coli. The resuspended bacterial solution after centrifugation was evenly spread on an LB plate containing Amp. Random single colonies were identified by colony PCR. The results of agarose gel electrophoresis (such as Figure 9 B) Confirm the correct band position, around 750 bp, and send it to a sequencing company for verification. After sequencing results are confirmed by BLAST comparison, positive clones are stored in glycerol (1:1 volume with sterile 50% glycerol), snap-frozen in liquid nitrogen, and stored in a -80°C freezer. Simultaneously, the sequenced pUC19-PuZAT68-GFP plasmid is extracted (concentration must reach 2 μg).

[0145] High concentrations of pUC19-GFP (control) and pUC19-PuZAT68-GFP plasmids were mixed with the vector plasmid pUC19-mCherry for nuclear localization at a volume ratio of 1:1, and then transfected into the protoplasts of Populus alba. The transfected protoplasts were wrapped with tin foil in a 30°C constant temperature oven to provide dark conditions. They were cultured under this condition for 18 hours, and then the distribution of fluorescence signals was observed using a laser confocal microscope. The microscopic examination results showed that green fluorescence signals of pUC19-GFP could be detected in all parts of the protoplast cells; while the green fluorescence signal of pUC19-PuZAT68-GFP was only present in the cell nucleus (such as Figure 10 This result indicates that PuZAT68 is localized in the cell nucleus.

[0146] PuZAT68 transcriptional activity analysis

[0147] According to the sequence characteristics of the PuZAT68 gene, the restriction enzyme cutting sites of its nucleotide sequence were predicted using Bioedit biological software. Combined with the distribution of multiple cloning sites in the pGBKT7 yeast expression vector, EcoRI and SalI were selected as restriction enzyme cutting sites, and specific primers containing the corresponding restriction enzyme recognition sites were designed.

[0148] EcoRI and SalI were selected as restriction sites, and the primer sequences were designed as follows:

[0149] EcoRI-PuZAT68-F(SEQ ID NO.14):

[0150] 5'-GGAATTCATGAAGAGAGATAGAGAACAGGC-3';

[0151] SalI-PuZAT68-R (SEQ ID NO.15):

[0152] 5'-GCGTCGACGATAAAACATCTCAACATGGGACG-3'.

[0153] Using the previously obtained PuZAT68 clone as a template, PCR amplification using high-fidelity enzymes was performed to obtain PCR products of the full-length gene and various fragments. The products were verified by agarose gel electrophoresis and then recovered. The pGBKT7 vector was double-digested with EcoRI and SalI to form a linearized vector. The recovered fragments were ligated with T4 ligase. The ligated products were then transformed into E. coli. After centrifugation, the resuspended bacterial suspension was evenly spread on LB plates containing Kan and incubated at 37°C for 16 hours. Single colonies were randomly selected from the transformation plates and cultured in liquid medium with shaking. PCR analysis was then performed on the bacterial suspension. A specific band, approximately 750 bp in length, was obtained, consistent with the target band. Sequencing confirmed the successful construction of the pGBKT7 vector.

[0154] The full-length PuZAT68 gene and its deletion fragments were constructed into the pGBKT7 vector to verify their transcriptional activation activity. Using the pGBKT7 vector plasmid (BD) as a negative control and the BD-P53 plasmid as a positive control, the recombinant plasmids were transformed into Y2H Gold yeast cells. The transformants were plated on three solid media: yeast-deficient medium SD / -Trp, SD / -Trp / X-α-Gal, and triple-deficient medium SD / -Trp / -His / -Ade, and their growth was observed. If the transcription factor has transcriptional activation activity, the transformants will grow normally on all three media, and the colonies on SD / -Trp / X-α-Gal medium will turn blue. If the transcription factor does not have transcriptional activation activity, the transformants will only grow on SD / -Trp medium, and the colonies on SD / -Trp / X-α-Gal medium will remain white, but no growth will occur on SD / -Trp / -His / -Ade medium. Figure 11 ).

[0155] The results showed that PuZAT68 failed to form colonies on the three-deficient medium. However, all transformants maintained white colonies on SD / -Trp / X-α-Gal medium, similar to the negative control, demonstrating that PuZAT68 lacks transcriptional activation activity. Analysis of the PuZAT68 amino acid sequence revealed the presence of an EAR transcriptional repression motif at amino acids 161-170 (LCLDLSLALP). Given the presence of an EAR domain within the PuZAT68 protein, we conducted segmented functional validation experiments, targeting the anterior and posterior regions of the EAR domain in yeast transcriptional activation assays. These experiments tested whether each segment of the PuZAT68 protein possessed the ability to activate transcription. Neither the anterior nor posterior regions of the EAR domain allowed normal growth in SD / -Trp / -His / -Ade medium, indicating that PuZAT68 lacks transcriptional activation activity and that regions outside the EAR domain also lack transcriptional activation activity.

[0156] Furthermore, two proteins with clear transcriptional activity were selected as controls. ARF5M protein is known to have transcriptional activation function and was used as a positive control, providing a clear transcriptional activation control standard in the experiment; while HOS15 protein has transcriptional repression activity and was used as a negative control. Specifically, the PEG-mediated protoplast transformation method was used to co-transfect 35S::BD, 35S::PuZAT68-BD, 35S::HOS15-BD, and 35S::ARF5M-BD into Populus alba protoplasts with D3-GAL-GUS and pGREEN 0800II-LUC plasmids, respectively, to ensure a conversion rate of more than 80%. Luciferase (LUC) and β-glucuronidase (GUS) activities were measured, and the relative expression activity of the GUS reporter gene was calculated using the LUC value as an internal reference.

[0157] Experimental results ( Figure 12 ) showed that compared with the BD empty vector, BD-PuZAT68 significantly inhibited the expression of the reporter gene GUS, and its relative activity dropped to 0.54. Its inhibitory effect was stronger than that of the HOS15 protein. This trend is similar to that of the inhibition control, so it can be inferred that PuZAT68 has transcriptional repression activity and is a transcriptional repressor. PuZAT68 may perceive drought signals through the stress response elements in its promoter and act as a transcriptional repressor in the cell nucleus. This protein may enhance the drought resistance of Populus ussuriensis by negatively regulating the expression of downstream target genes (such as growth-related genes or stress resistance negative regulatory factors) to increase the survival rate of plants under adverse conditions.

[0158] Example 3 Study on the drought resistance function of PuZAT68 gene

[0159] 1. Construction of PuZAT68 overexpression vector

[0160] Vector map analysis identified the multiple cloning site region of the pBI121-GFP expression vector. Bioedit, a bioinformatics software, was used to predict restriction endonuclease sites within the full-length sequence of the target gene, PuZAT68. Based on the predicted results, SmalI was selected as the restriction site, and corresponding restriction primers were designed accordingly. During primer design, special attention was paid to removing the translation stop codon in the reverse primer. The specific primer sequences are:

[0161] SmalI-PuZAT68-F(SEQ ID NO.16):

[0162] 5'-ACGGGGGACTCTAGAGGATCCCCATGAAGAGAGATAGAGAACAG-3'

[0163] SmalI-PuZAT68-R(SEQ ID NO.17):

[0164] 5'-TCGGTACCCTCGAGGTCGACCCCGATAAAACATCTCAACATGG-3'

[0165] Using the pCloneEZ-T-PuZAT68 positive plasmid as the template, the target fragment was amplified using specific primers. The PCR amplification product was detected by agarose gel electrophoresis, the target band was excised using a UV gel imaging system, and the target fragment was purified using a gel recovery kit. The recovered product was simultaneously digested with the pBI121-GFP vector by SmalI and incubated at 37°C for 3 hours. After digestion, the digested product was purified and then subjected to homologous recombination. The recombinant plasmid was transformed into competent Escherichia coli cells using the heat shock method. After overnight incubation at 37°C, single colonies were selected and screened using LB medium containing Kan. Positive single colonies were tested by bacterial culture PCR.

[0166] The sequence obtained was compared with the known sequence to confirm the successful construction of the pBI121-PuZAT68-GFP overexpression vector. The bacterial suspension that had been successfully sequenced was selected and added with 50% glycerol at a 1:1 volume ratio and stored at -80°C for subsequent experiments.

[0167] 2. Construction of PuZAT68 inhibition expression vector

[0168] We compared the amino acid sequence of Populus ussuriensis ZAT68 and selected a sequence in the non-conserved region for amplification. The specific sequence is as follows: (SEQ ID NO. 18)

[0169] ATAGAGAACAGGCAGAGTTAGACTTGGCCAAATGCTTGATGCTACTTTCTAAAGTTGGCAAAGCTGATCACGAGATACTAACTAATTATAGATCAGCAGCAGCAGCAGCACCGGCAGG

[0170] According to the basic principle of RNAi interference, the forward sequence and reverse sequence of the target fragment were connected to both sides of the gene intron (chsAintron) of the interference vector pFGC5941 ( Figure 13 ), in the relatively non-conserved region of the PuZAT68 sequence, we selected the optimal restriction sites XhoI and AscI at the left end of the forward primer, and the optimal restriction sites XbaI and SmaI at the left end of the reverse primer. The second half of the gene was homologously recombined into the vector using the homologous recombination method. The specific primer sequences are as follows:

[0171] PuZAT68-CisF (SEQ ID NO.19):5'-CCCTCGAGATAGAGAACAGGCAGAGTTA-3';

[0172] PuZAT68-CisR (SEQ ID NO.20):5'-TTGGCGCGCCCCTGCCGGTGCTGCTGCT-3;

[0173] PuZAT68-AntiF (SEQ ID NO.21):5'-CGCGGATCCCCTGCCGGTGCTGCTGCT-3';

[0174] PuZAT68-AntiR (SEQ ID NO.22):5'-TCCCCCGGGATAGAGAACAGGCAGAGTTA-3';

[0175] pFGC5941 vector-Cis F (SEQ ID NO. 23): 5'-CCTTCGCAAGACCCTTCCTC-3';

[0176] pFGC5941 vector-Cis R (SEQ ID NO. 24): 5′-ACCTTCCCACAATTCGTCGG-3′;

[0177] pFGC5941 vector-anti F (SEQ ID NO. 25): 5'-GCATGCTATGCATTCAAT-3';

[0178] pFGC5941 vector-anti R (SEQ ID NO. 26): 5'-CGTGCACAACAGAATTGAAAGC-3'.

[0179] Using the pCloneEZ-T-PuZAT68 plasmid as a template, the forward and reverse sequences of the PuZAT68 silencing fragment were amplified to generate PuZAT68-Cis and PuZAT68-Anti products. After electrophoresis to confirm their positions, the products were recovered and purified from the gel. The concentration of the purified products was measured and set aside for use. The forward silencing fragment and the vector were double-digested with XhoI and AscI, and the digested products were purified. A ligation reaction was then performed, and the ligation products were transformed into competent E. coli cells. Single clones were randomly selected and tested by PCR to confirm the insertion of the target fragment. The positive strains screened were sent to a sequencing company for sequencing and alignment. After ensuring the sequence was correct, the plasmid was extracted from the pFGC5941-PuZAT68-Cis strain and stored in a -80°C freezer.

[0180] The pFGC5941-PuZAT68-Cis plasmid obtained in the previous step was double-digested with XbaI and SmaI. The digestion product was purified and ligated with the reverse silencing fragment gel-recovered product using homologous recombination. The ligation product was transformed into 50 μL of competent E. coli cells. The next day, a single, well-formed colony was picked using a sterile pipette tip and shake-cultured. PCR was performed on the culture to screen for positive clones. Positive clones were sequenced to confirm sequence accuracy. This yielded the pFGC5941-PuZAT68-RNAi recombinant bacterial solution and plasmid containing the target interference fragment.

[0181] The constructed pBI121-PuZAT68-GFP (PuZAT68-OE) and pFGC5941-PuZAT68-RNAi (PuZAT68-IE) plasmids were transformed into GV3101 Agrocybe competent cells. Single clones were randomly selected and placed in 550 μL of liquid culture medium containing the corresponding resistance to screen for positive clones. The screening results of pBI121-PuZAT68-GFP are shown in Figure 2. Figure 14 The results of pFGC5941-PuZAT68-RNAi screening are as follows Figure 15 As shown, Agrobacterium strains with positive results were screened and stored for use.

[0182] 3. Screening and detection of 35Spro::PuZAT68 transgenic plants

[0183] The Agrobacterium liquid with positive results was streaked onto a LB solid culture dish containing Kan and Rif resistance for activation, and the plate was inverted and cultured in a 28°C oven for 3 days. After a single colony was formed, a single clone was picked and placed in LB liquid culture medium (containing the corresponding antibiotics) and shaken to prepare an engineered strain. Using plant tissue culture technology, the transgenic leaves and stem segments were co-cultured. After Agrobacterium infection, co-cultivation, sterilization and selection of culture medium, transgenic resistant seedlings (such as Figure 16 ), during the entire process, Kan antibiotics were used for screening, and clustered buds were subcultured, ultimately obtaining the overexpression resistant Populus ussuriensis plantlet PuZAT68-OE. DNA identification was performed:

[0184] Detection of Transgenic Populus ussuriensis

[0185] DNA was extracted from each transgenic resistant plant that was initially screened. Based on the vector sequence characteristics, the overexpression vector-specific detection primer GFP (146)-R was designed and used in conjunction with the cloning primer PuZAT68-F to identify the overexpression transgenic plant DNA. The GFP-R primer sequence is as follows: GFP (146)-R (SEQ ID NO. 27): 5'-CAGATGAACTTCAGGGTCAGC-3'. The above-mentioned specific primers were used to detect the DNA level of the transgenic overexpression plants. During the detection process, a strict control system was set up, with the wild-type Populus ussuriensis as the negative control (this control has not been subjected to transgenic manipulation and its DNA should not contain the target transgenic fragment); the pBI121-PuZAT68-GFP plasmid was used as the positive control (this plasmid carries the relevant sequence of the target gene and can be used as the target reference for detection). The PCR reaction was carried out using 2×EsTaq MasterMix, with the extracted DNA (100 ng / μL) as the template. The system is shown in the table below:

[0186] Table 7

[0187]

[0188] Overexpression plant detection uses a 10μL reaction system, an annealing temperature of 60°C, and an extension time of 10 seconds. For suppressed expression plants, to ensure the accuracy of the test results, two independent tests are required, and the extension time for both tests is 5 seconds. After PCR amplification is completed, the amplified products are analyzed by agarose gel electrophoresis. The electrophoresis pattern is used to determine which strains have successfully amplified the target band, approximately 750bp, and the strains that have successfully tested are saved.

[0189] Analysis of expression levels in transgenic plants

[0190] Total RNA was extracted from transgenic lines identified by DNA sequencing and quality tested to ensure integrity and purity. The total RNA was then reverse-transcribed into cDNA, which was diluted 10-fold. RT-qPCR analysis was performed using the quantitative primers DL-PuZAT68-F / R and the internal reference primers PuActin-F / R.

[0191] DNA level identification, agarose gel electrophoresis results showed that the positive plasmid and PuZAT68-OE strains amplified clear bands at around 750bp, while the wild type (WT) had no band at the target band. These results proved the effectiveness of the test and showed that the transgenic plants were successfully constructed. A total of 12 transgenic Populus ussuriensis seedlings were obtained (see the results). Figure 17 ).

[0192] Twelve positive transgenic plants verified by DNA level were selected as the experimental group, and wild-type Populus ussuriensis plants were used as negative control. RT-qPCR was performed to detect the relative expression levels. The test results are as follows: Figure 18 As shown in the figure, among the 12 transgenic lines, the expression levels of the target genes in four lines (OE1, OE5, OE6, and OE8) were significantly upregulated, among which the expression level of the OE1 line was the highest (about 21 times that of the control), the expression levels of the OE5 and OE8 lines were about 15 times that of the control, and the expression level of the OE6 line was about 14 times that of the control.

[0193] 4. Screening and detection of pFGC5941-PuZAT68-RNAi transgenic plants

[0194] According to the above method, the expression-inhibiting resistant clustered seedlings (such as Figure 19 ) and PuZAT68-RNAi (PuZAT68-IE) P. ussuriensis were identified at the DNA level, and 12 transgenic P. ussuriensis seedlings with suppressed expression were obtained (the results are shown in Figure 20 Agarose gel electrophoresis showed that the positive control plasmid exhibited a clear, bright, specific amplification band at approximately 250 bp. The PuZAT68-IE transgenic line exhibited a distinct amplification band at the same position, while the wild-type (WT) strain showed no band at the target band, confirming the validity of the test results.

[0195] The transgenic plants with suppressed expression verified by DNA level were selected as the experimental group, and the wild-type Populus ussuriensis plants were used as the control. RT-qPCR was performed to detect the relative expression levels. The test results are shown in Figure 21 The results showed that among the 12 transgenic lines, the expression levels of the target genes in four lines (IE5, IE7, IE12, and IE11) were significantly reduced, among which lines 5, 7, and 12 had the most significant inhibitory effects, with their gene expression levels being about 0.1 times that of the wild-type plants.

[0196] 5. Analysis of Drought Resistance of PuZAT68 Transgenic Seedlings

[0197] Wild-type Populus ussuriensis that had grown for 3 weeks and were in good growth condition, as well as PuZAT68 overexpression and inhibition expression transgenic seedlings were selected as experimental materials. Three independent strains were set up for each genotype to ensure biological repetition. The third leaf was cut from the morphological upper end of the plant, and the length of the retained stem segment was strictly controlled to keep it consistent. On the basis of retaining two leaves, sterile cuttings were carried out. Specifically, the treated stem segment was inserted into a new 1 / 2MS rooting medium. The cuttings were cultured under normal culture conditions for 7 days. When the adventitious roots grew to about 2 mm in length, three genotype strains with similar growth conditions were selected: wild type (WT), PuZAT68 overexpression (PuZAT68-OE) and PuZAT68 inhibition expression (PuZAT68-IE). The screened strains were divided into two groups on average. For the experimental group, some lines were seeded in a simulated drought stress medium containing 6% PEG6000 to simulate the effects of drought on plants. The remaining lines were seeded in 1 / 2 MS medium as a control group for comparative analysis. Both groups of plants were then cultured for an additional two weeks. During the culture process, the growth of plants in each treatment group was regularly observed and recorded, including plant height, leaf color and morphology, as well as changes in phenotypic characteristics such as leaf wilting and stem bending. After the culture period, drought-related physiological indicators were measured in the WT, PuZAT68-OE, and PuZAT68-IE lines under normal culture conditions and drought stress. These indicators, including leaf malondialdehyde (MDA) content and antioxidant enzyme activity, provide insights into the physiological responses of different genotypes to drought stress.

[0198] (1) Growth trait analysis

[0199] The growth of wild-type (WT), PuZAT68-overexpressing (PuZAT68-OE), and PuZAT68-inhibiting (PuZAT68-IE) strains was observed under normal culture conditions and after treatment with 6% PEG6000, with particular attention paid to root phenotypic differences. The roots of the tissue-cultured seedlings were removed from the culture medium and separated from the aerial parts. The residual culture medium on the root surface was carefully cleaned to ensure the roots were clean. The cleaned roots were then placed in an 80°C oven for 12 hours, and root length and dry weight were subsequently measured.

[0200] Under normal culture conditions, the three experimental strains (WT, PuZAT68-OE, and PuZAT68-IE) showed similar growth states and no significant phenotypic differences. After being treated with 6% PEG6000 to simulate drought stress, the growth of all plants was inhibited to varying degrees, but significant differences were observed between different genotypes (e.g. Figure 22Further measurements and analysis revealed that the knockdown strain exhibited a superior root structure, with significantly increased root number and longer root length compared to the wild type. Furthermore, the aboveground growth of the strain was significantly better than that of the wild type. The overexpression strain showed the opposite effect, with reduced adventitious root length and root dry weight.

[0201] The statistical results show that ( Figure 23 Under normal culture conditions, root biomass showed no significant difference among the strains. However, under 6% PEG6000 stress, the biomass of all strains decreased to varying degrees. The degree of inhibition in the strain with PuZAT68 knockdown was significantly lower than that in the wild type, with increased adventitious root length and root dry weight compared to the WT and PuZAT68 overexpression strains. The PuZAT68 overexpression strain (PuZAT68-OE) was most severely inhibited, with decreased adventitious root length and root dry weight compared to the WT.

[0202] (2) Relative conductivity (EL) analysis

[0203] (2.1) Pre-fill a 50 mL centrifuge tube with ddH2O and let it sit overnight to complete ion equilibration.

[0204] (2.2) Root samples were collected from wild-type and transgenic Populus ussuriensis tissue culture seedlings treated with 6% PEG6000 drought stress and untreated. 0.2 g of root was accurately weighed and placed in a 50 mL centrifuge tube containing 20 mL of ddH2O. A blank control group (containing only 20 mL of ddH2O) was set up as a negative control.

[0205] (2.3) Gently shake at 20 rpm for 10 min at room temperature to ensure that the plant tissue is completely immersed in water;

[0206] (2.4) Vacuum the centrifuge tube for 15 minutes;

[0207] (2.5) Use a conductivity meter to measure the conductivity of each sample. The initial measurement value is recorded as C1, and the blank control conductivity is recorded as CK1. Set three technical replicates;

[0208] (2.6) After the sample has been treated in a boiling water bath for 20 min, it is immediately rinsed with running cold water until cooled to room temperature. The secondary conductivity measurement is performed and the measured values ​​are recorded. The secondary conductivity measurement value is recorded as C2, and the blank control measurement value is recorded as CK2.

[0209] (2.7) The calculation formula is: relative conductivity C (%) = (C1-CK1) / (C2-CK2)×100%.

[0210] The results show that ( Figure 24Left) Under normal culture conditions, root plasma membrane permeability in strains overexpressing and knocking down PuZAT68 showed no significant differences compared to wild-type Populus ussuriensis plants. However, under 6% PEG6000-induced drought stress, root relative conductivity increased in all experimental strains, with the increase in PuZAT68-IE plants being smaller than that in the wild-type (WT) and the increase in PuZAT68-OE plants being greater. This suggests that knocking down this gene effectively mitigates membrane lipid peroxidation, maintains plasma membrane integrity, and thus enhances plant tolerance to drought stress.

[0211] (3) Malondialdehyde (MDA) determination

[0212] The malondialdehyde (MDA) content in the roots of WT and transgenic lines after normal culture and 6% PEG6000-simulated drought stress was determined using a plant malondialdehyde (MDA) test kit.

[0213] like Figure 24 As shown on the right, under normal culture conditions, the MDA content in the roots of PuZAT68-OE, PuZAT68-IE, and wild-type P. ussuriensis plants was essentially identical, with no significant differences among the experimental lines, indicating that the cell membrane system maintained a relatively stable physiological state. However, under 6% PEG6000-simulated drought stress, MDA content in the roots of all plants increased significantly, but with significant differences among the different genotypes. The increase in MDA content in the knockdown lines was significantly less than that in the wild type, with root MDA content increasing from 19.9-21.3 nmol / g to 25-27.7 nmol / g. The increase in MDA content in the overexpression lines was significantly greater than that in the wild type, with root MDA content increasing from 19-20.8 nmol / g to 36.8-41.3 nmol / g, indicating that they were the most severely damaged. This suggests that inhibiting the expression of this gene can effectively alleviate the degree of cell membrane lipid peroxidation, maintain the integrity of the plasma membrane, and thus significantly improve the plant's tolerance to drought stress. This difference may be closely related to the function of the PuZAT68 gene in regulating the activity of antioxidant enzyme systems and the synthesis of membrane protective substances.

[0214] (4) DAB staining, NBT staining

[0215] Wild-type and transgenic lines under normal culture and 6% PEG6000 drought stress treatment were selected, and root samples were cut at the same position. The roots were stained using DAB staining and NBT staining, and the corresponding images were obtained.

[0216] like Figure 25 As shown in the figure, under normal culture conditions, there was no significant difference in NBT staining of root and leaf tissues of each strain, indicating that their O2- The content of NBT was basically the same. However, under simulated drought stress, the roots and leaves of the plants showed different degrees of blue-purple staining. The staining intensity of all strains was significantly enhanced, but the staining depth of PuZAT68-IE strain was significantly lower than that of PuZAT68-OE strain. The leaves of PuZAT68-OE strain showed a large area of ​​dark staining, and the staining was more obvious in the roots. This shows that after drought stress, the O 2- The contents of O increased, but the O produced in PuZAT68-IE plants 2- Relatively few.

[0217] like Figure 26 As shown in the figure, under normal culture conditions, there was no significant difference in DAB staining among the strains, indicating that the H2O2 content was similar under this state. However, after 6% PEG6000 simulated drought stress treatment, the DAB staining of the root and leaf tissues of the PuZAT68-IE strain was significantly weaker than that of the WT and PuZAT68-OE strains. The PuZAT68-OE strain was the darkest in the dye solution, indicating that under drought stress, the PuZAT68-IE strain had less H2O2 accumulation and lower degree of cell membrane lipid peroxidation, which is consistent with the O2 content in the NBT staining experiment. 2- The accumulation pattern shows a positive correlation.

[0218] (5) Determination of hydrogen peroxide (H2O2) content

[0219] A hydrogen peroxide (H2O2) test kit (Nanjing Jiancheng Bioengineering Institute) was used to determine the hydrogen peroxide content in the roots of WT and each transgenic line after normal culture and 6% PEG6000 simulated drought stress.

[0220] like Figure 27 As shown in the figure, under normal culture conditions, the H2O2 content in the roots of the plants was almost the same between the WT and PuZAT68-OE lines; however, under 6% PEG6000 simulated drought stress, the H2O2 content in the roots of the WT, PuZAT68-OE and PuZAT68-IE lines increased. Among them, the H2O2 content in the roots of the overexpression plants increased most significantly, while the H2O2 content in the roots of the expression-inhibited plants increased relatively less. These results indicate that the inhibition of PuZAT68 gene expression can reduce the production of reactive oxygen species to a certain extent, which will reduce the production of H2O2 and O 2- The accumulation of nutrients can improve the drought resistance of Populus cathayana.

[0221] To investigate the impact of PuZAT68 gene expression on drought response in Populus ussuriensis, the present invention constructed the plant expression vectors pBI121-PuZAT68-GFP and pFGC5941-PuZAT68-RNAi, and genetically transformed them to generate stable transgenic lines PuZAT68-OE and PuZAT68-IE. Phenotypic observation and root development analysis of the three lines were performed under simulated drought stress with 6% PEG6000. The results showed that the PuZAT68-IE line exhibited superior growth, with greater adventitious roots, longer root length, and greater root dry weight after simulated drought stress. Compared to the WT and PuZAT68-OE lines, the PuZAT68-IE line exhibited superior drought resistance.

[0222] The present invention measured the physiological indicators of the PuZAT68 transgenic strain and found that under 6% PEG6000 simulated drought stress treatment, the relative conductivity, malondialdehyde content, and H2O2 content of the PuZAT68-IE transgenic strain were significantly lower than those of the wild-type (WT) and PuZAT68 overexpression (PuZAT68-OE) transgenic strains. Physiological studies have confirmed that the degree of cell membrane damage can be assessed using two physiological indicators: conductivity and malondialdehyde content. Lower ion permeability indicates less cell membrane damage and better osmotic regulation ability. These results suggest that inhibiting the expression of the PuZAT68 gene may respond to drought stress by increasing osmotic regulatory substances and improving ROS scavenging ability, thereby enhancing the plant's drought resistance.

[0223] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. Application of the transcriptional repressor PuZAT68 gene in regulating the drought resistance of Populus ussuriensis, characterized in that: The nucleotide sequence of the transcription repressor PuZAT68 gene is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.4: The method for regulating the drought resistance of Populus ussuriensis by utilizing the PuZAT68 gene is as follows: the drought resistance of Populus ussuriensis is enhanced by inhibiting the expression of the PuZAT68 gene.

2. The use according to claim 1, characterized in that The PuZAT68 protein encoded by the PuZAT68 gene contains two QALGGH-type domains, two C2H2-type zinc finger domains, and an EAR motif at its C-terminus.

3. The use according to claim 2, characterized in that The specific operation of using the PuZAT68 gene to regulate the drought resistance of Populus ussuriensis is as follows: constructing a PuZAT68 inhibition expression vector, transferring it into Populus ussuriensis plants by Agrobacterium infection, and obtaining the Populus ussuriensis inhibition expression resistant plant PuZAT68-IE.

4. The use according to claim 3, characterized in that The original vector of the PuZAT68 inhibition expression vector is pFGC5941, the restriction enzyme sites at the left end of the forward primer are XhoI and AscI, and the restriction enzyme sites at the left end of the reverse primer are XbaI and SmaI. The constructed PuZAT68 inhibition expression vector is named pFGC5941-PuZAT68-RNAi.

5. The transcription repressor PuZAT68 gene for use according to any one of claims 1 to 4, characterized in that: The nucleotide sequence of the transcription repressor PuZAT68 gene is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.

4.

6. Use of the PuZAT68 gene according to claim 5 in screening, identifying or breeding Populus cathayana strains with high drought resistance.

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