Method for identifying drought-resistant key AGO gene in haloxylon ammodendron and haloxylon ammodendron

Abstract

The invention provides a method for identifying drought-resistant key AGO genes in haloxylon ammodendron and haloxylon ammodendron, and relates to the technical field of gene identification. According to the method for identifying drought-resistant key AGO genes in haloxylon ammodendron and haloxylon ammodendron, 45 HaAGO genes in haloxylon ammodendron and 30 HpAGO genes in haloxylon ammodendron are respectively identified through hmmscan screening, homologous comparison and CDD / PFAM structural domain verification; then, through protein physicochemical property analysis, phylogenetic tree construction, conservative motif and gene structure analysis, chromosome localization, promoter cis-acting element prediction and protein interaction network analysis, molecular characteristics and evolution laws of an AGO gene family are determined, and finally, in combination with transcriptome sequencing data under a wet / drought habitat, the AGO gene family is determined. And screening out the gene which is obviously up-regulated under drought stress. The invention provides a systematic method for mining drought-resistant gene resources of haloxylon ammodendron and haloxylon ammodendron, and the screened key gene can be used as a target for drought-resistant molecular breeding and has important significance on ecological restoration of northwest desert regions.

Description

Technical Field

[0001] This invention relates to the field of gene identification technology, specifically a method for identifying key drought-resistant AGO genes in Haloxylon ammodendron and Haloxylon buergerianum. Background Technology

[0002] Haloxylon ammodendron and Haloxylon buergerianum are key sand-fixing tree species in the oasis-desert ecotone of arid regions and important constructive species in desert plant communities. They play an irreplaceable role in maintaining the stability of fragile ecosystems. Because Haloxylon ammodendron plants exhibit excellent adaptability in extreme arid environments, they have become ideal model systems for studying the molecular and biochemical mechanisms of plant drought resistance. Therefore, in-depth revelation of their drought resistance adaptation mechanisms has important theoretical and practical significance for ecological restoration and vegetation reconstruction in desert areas.

[0003] Argonaute (AGO) proteins, as highly conserved RNA-induced silencing complexes, play an important role in sRNA-mediated RNA silencing pathways. They are composed of variable N-terminal, PAZ, MID, and PIWI domains and participate in gene expression during transcription. In recent years, research on plant AGO genes has been increasing, mainly focusing on millet, cotton, and Arabidopsis thaliana. AGO genes play an important role in plant growth, development, and abiotic stress by binding to specific AGO proteins through different types of sRNA (miRNA, siRNA, phasiRNA, etc.).

[0004] In plant growth and development (taking Arabidopsis, rice, and maize as examples), AGO proteins regulate plant organ formation and reproductive development through sRNA-mediated gene silencing or activation. For instance, in Arabidopsis, AtAGO1, by binding to 21-nt miRNA and a small amount of siRNA, affects leaf polarity development, adventitious root formation, and lateral organ development. AtAGO1 can also interact with SVI / SNF by binding to sRNA, thereby regulating hormone and stress response gene expression through chromatin binding. AtAGO9 binds to 24-nt siRNA, ensuring the genetic stability of female gametes. AtAGO5 interacts with miR156 to inhibit senescence-induced flowering and regulate the expression of SPL transcription factors, thereby controlling flowering time. In maize, ZmAGO7 mediates ta-siRNA production through miR390, affecting leaf morphology and playing a role in male sterility and fertility restoration. AGO genes also participate in abiotic stress in plants. In apples, studies have found that multiple MdAGO genes are upregulated under stress conditions such as salt stress, low temperature, and high temperature. Similarly, polyethylene glycol stress treatment can induce upregulation of ZmAGO1a gene expression in maize. Another rice study found that overexpression of OsAGO2 not only increases grain size but also enhances salt tolerance by positively regulating the abscisic acid (ABA) response. Summary of the Invention

[0005] (a) Technical problems to be solved To address the shortcomings of existing technologies, this invention provides a method for identifying key drought-resistant AGO genes in Haloxylon ammodendron and Haloxylon buergerianum. This invention employs bioinformatics methods to identify AGO gene family members in Haloxylon ammodendron and Haloxylon buergerianum, and further analyzes their chromosomal location, gene structure, motifs, cisfunctional elements, phylogenetic trees, and protein-protein interactions. Furthermore, by combining cisfunctional elements with transcriptome data, several key genes related to drought resistance were screened, providing technical support for the mining of drought-resistant gene resources and drought-resistant molecular breeding in Haloxylon ammodendron and Haloxylon buergerianum.

[0006] (II) Technical Solution To achieve the above objectives, the present invention provides the following technical solution: a method for identifying key drought-resistant AGO genes in Haloxylon ammodendron and Haloxylon buergerianum, comprising the following steps: (1) Identification of AGO gene family members: ① Obtain the whole genome sequences of Haloxylon ammodendron and Haloxylon buergerianum, and retrieve the hmm files of the conserved domains of PAZ (PF02170.25) and PIWI (PF02171.20) from the Uniprot database; ② The whole genome sequence was screened using the hmmscan tool, and candidate genes containing PAZ and PIWI domains were retained; ③ Using the Arabidopsis thaliana AtAGO1-AtAGO10 protein sequences (accession numbers: AT1G48410.1, AT1G31280.1, AT1G31290.1, AT2G27040.1, AT2G27880.1, AT2G32940.1, AT1G69440.1, AT5G21030.1, AT5G21150.1, AT5G43810.1) as references, BLASTP homology alignment was performed (E-value < 1e-5). ④ The candidate genes were verified by CDD database, and sequences containing only a single structural domain were removed, finally obtaining members of the Haloxylon ammodendron HaAGO family and Haloxylon spp. HpAGO family; (2) Multidimensional bioinformatics analysis: ①Analysis of protein physicochemical properties and subcellular localization: The number of amino acids, molecular weight, isoelectric point, instability coefficient, lipid coefficient and hydrophilicity were calculated using the ProtParam tool in ExPASy; subcellular localization was predicted using the WoLFPSORT online tool. ② Phylogenetic tree construction: AGO protein sequences of Haloxylon ammodendron, Haloxylon spp. and 7 reference plants (Arabidopsis thaliana, apple, grape, tomato, poplar, maize, and rice) were collected. After multiple sequence alignment using Muscle 5.1 software and redundancy removal using TrimAl 1.4 software, the maximum likelihood method (bootstrap value set to 1000) of Fasttree 2.1.11 software was used to construct the phylogenetic tree. FigTree 1.4.4 software was used to optimize the tree structure and classify subfamilies. ③ Conserved motif, gene structure, and chromosome location analysis: MEME 4.11.2 software was used to analyze conserved motifs (maximum number of motifs set to 10, E-value < 1e-10); exon and intron location information were extracted from the genome gff3 annotation file, and gene structure was visualized using the "GeneStructureView" function of TBtools 2.337 software; gene chromosome location visualization was achieved using the "GeneLocationVisualizefromGTF / GFF" plugin of TBtools. ④ Promoter cis-acting element analysis: The 2000bp sequence upstream of the start codon of the AGO gene was extracted and submitted to the PlantCAR database for cis-acting element analysis. The element types (light response, hormone response, stress response, tissue development related) and quantities were counted and visualized using TBtools2.337 software. ⑤ Protein interaction network analysis: Using Arabidopsis thaliana as a reference species, AGO protein sequences were submitted to the String database to obtain interaction relationship data. Interaction network diagrams were drawn using Cytoscape 3.9.1 software and core node genes were identified. (3) Gene expression analysis and screening of key drought-resistant genes: ① Sample collection and transcriptome sequencing: Two habitat quadrats, humid low-salt (HS / HB) and arid low-salt (LS / LB), were set up in the Aibi Lake Wetland National Nature Reserve (44°31'5″N~45°9'35″N, 82°33'47″E~83°53'21″E). Assimilation branches of Haloxylon ammodendron and Haloxylon spp. were collected (3 biological replicates). After being flash-frozen in liquid nitrogen, total RNA was extracted using the TRIzol method. After RNA quality testing (RNA Integrity Number ≥ 8.0), high-throughput sequencing was performed using the Illumina NovaSeq platform. ② Transcriptome data processing and expression level calculation: The quality of raw reads was assessed using FastQC 0.11.9 software, and adapters and low-quality sequences (Q<20) were removed using Trimmomatic 0.39 software; clean reads were aligned to the Haloxylon ammodendron / Haloxylon spp. reference genome using HISAT 22.2.1 software (alignment rate ≥85%); the number of reads for each gene was counted using featureCounts 2.0.1 software, and gene expression levels were normalized using the FPKM method; ③ Screening and Validation of Differentially Expressed Genes: Differentially expressed genes under drought vs. humid habitat conditions were screened using the DESeq2 package in R (screening conditions: |Log2FC|≥1, padj<0.05). The five HaAGO genes and five HpAGO genes with the highest fold change were selected, and specific primers were designed (primer length 18-22bp, Tm value 58-62℃). qRT-PCR validation was performed using the TaKaRa SYBR PremixExTaqⅡ kit (reaction program: 95℃ pre-denaturation 30s; 95℃ denaturation 5s, 60℃ annealing 30s, 40 cycles; melting curve analysis: 95℃ 15s, 60℃ 1min, 95℃ 15s). Actin gene was used as an internal control, and the relative expression level was calculated using the 2^(-ΔΔCt) method to verify the reliability of the transcriptome data. ④ Identification of Key Drought-Resistant Genes: Combining the results of differential expression analysis (significant upregulation) and qRT-PCR validation (consistent with RNA-seq trends, R... 2 ≥0.85) and cis-acting element analysis (including drought response element MBS) were used to identify key AGO genes for drought resistance in Haloxylon ammodendron and Haloxylon buergerianum.

[0007] Preferably, in step (1) ④, the final identified Haloxylon ammodendron AGO gene family contains 45 members (HaAGO1-HaAGO45), and the Haloxylon spp. AGO gene family contains 30 members (HpAGO1-HpAGO30).

[0008] Preferably, in step (2) ②, the phylogenetic tree analysis divides 45 HaAGO genes into 5 subfamilies and 30 HpAGO genes into 4 subfamilies. Genes in the same subfamilies have similar conserved motif composition and gene structure.

[0009] Preferably, in step (2) ④, among the 52 cis-acting elements identified, light-responsive elements account for the highest proportion (44% of the HaAGO family and 42% of the HpAGO family, mainly Box-4, TCT-motif, and GT1-motif). Among the stress-responsive elements, ARE (anaerobic response) and MBS (drought response) are the most numerous (117 and 42 in the HaAGO family, respectively; 67 and 22 in the HpAGO family, respectively).

[0010] Preferably, in step (3) ④, the identified key drought-resistant AGO genes are: HaAGO15, HaAGO17, HaAGO30, HaAGO33, and HaAGO42 in Haloxylon ammodendron, and HpAGO2, HpAGO3, HpAGO15, HpAGO24, and HpAGO30 in Haloxylon ammodendron; the expression level of the key genes in arid habitats is more than twice that in humid habitats (Log2FC≥1), and the correlation coefficient between the relative expression level verified by qRT-PCR and the RNA-seq result is ≥0.9.

[0011] Preferably, the key AGO genes (HaAGO15, HaAGO17, HaAGO30, HaAGO33, HaAGO42, HpAGO2, HpAGO3, HpAGO15, HpAGO24, HpAGO30) are cloned and constructed into vectors, and then transformed into Haloxylon ammodendron or Haloxylon esculenta callus tissue using Agrobacterium-mediated transformation to obtain transgenic plants with enhanced drought resistance.

[0012] (III) Beneficial Effects This invention provides a method for identifying key AGO genes for drought resistance in Haloxylon ammodendron and Haloxylon persicum. It has the following beneficial effects: This invention identifies members of the Argonaute gene family based on whole-genome data of Haloxylon ammodendron and Haloxylon buergerianum, analyzing their evolutionary relationships, chromosomal locations, gene structures and conserved motifs, cis-regulatory elements, etc. Furthermore, it analyzes the expression of AGO genes in these two species under long-term drought stress, and screens candidate key genes related to drought stress response by combining cis-regulatory elements with transcriptome data, providing theoretical guidance for subsequent gene function verification and gene involvement in plant drought resistance.

[0013] (1) The identification method provided by the present invention identifies and analyzes the AGO gene family of Haloxylon ammodendron and Haloxylon buergerianum, and identifies 45 HaAGO gene family members and 30 HpAGO gene family members in the genomes of Haloxylon ammodendron and Haloxylon buergerianum, respectively. (2) Through evolutionary analysis, the AGO gene family of Haloxylon ammodendron is divided into five subgroups, and Haloxylon ammodendron is divided into four subtribes. These AGO genes are distributed on six chromosomes, and their gene structures and conserved motifs are different. (3) The results of cis-acting element analysis showed that 52 cis-acting elements were identified in the promoters of the AGO gene family of Haloxylon ammodendron and Haloxylon buergerianum. According to their functions, they can be divided into four categories: plant hormone response elements, stress response elements, tissue development related elements and light response elements. Among them, the number of light response elements was the most abundant, followed by hormones and stress, indicating that they play an important role in the abiotic stress and hormone response of Haloxylon ammodendron and Haloxylon buergerianum.

[0014] (4) The key drought-resistant genes discovered in this invention provide new targets for plant drought-resistant genetic engineering and lay the foundation for subsequent gene function verification and drought-resistant molecular breeding. Attached Figure Description

[0015] Figure 1 This is a statistical chart of the physicochemical properties of members of the AGO gene family of Haloxylon ammodendron and Haloxylon buergerianum in this invention; Figure 2 This invention provides a phylogenetic tree for the AGO gene family of Haloxylon ammodendron and Haloxylon spp., where A is a phylogenetic tree of AGO gene family members from Haloxylon ammodendron, Haloxylon spp., Arabidopsis thaliana, apple, grape, tomato, poplar, and maize, and B is a phylogenetic tree of AGO gene family members from Haloxylon ammodendron, Arabidopsis thaliana, apple, grape, tomato, poplar, and maize. Figure 3 This invention presents the conserved motif analysis and gene structure diagram of the Argonaute gene family in Haloxylon ammodendron and Haloxylon buergerianum. A and C represent the phylogenetic tree and gene structure analysis of the HaAGO gene, B shows the distribution of conserved motifs in the HaAGO protein, and D shows the distribution of conserved motifs in the HpAGO protein. Figure 4 This is a chromosomal mapping of the AGO gene in Haloxylon ammodendron and Haloxylon buergerianum according to the present invention. A represents the distribution of the HaAGO gene on the chromosome, and B represents the chromosomal distribution of the HpAGO gene. Figure 5 This is a predicted diagram of cis-regulatory elements for the promoters of the *Haloxylon ammodendron* and *Haloxylon spp.* A represents the analysis of cis-regulatory elements in the HaAGO promoter, and B represents the analysis of cis-regulatory elements in the HpAGO promoter. From left to right in the diagram, the diagram shows: the distribution of cis-regulatory elements in different subfamily genes, the number of different cis-regulatory elements in each AGO gene, and the total number of cis-regulatory elements in various AGO proteins. Figure 6 This is a network diagram of AGO protein interactions between Haloxylon ammodendron and Haloxylon perlatum in this invention. A represents HaAGO protein interactions, and B represents HpAGO protein interactions. Figure 7 The images show heatmaps of HaAGO gene expression levels and qRT-PCR verification data for Haloxylon ammodendron and Haloxylon buergerianum. A represents the HaAGO gene expression level heatmap under humid and arid habitats, B represents the HpAGO gene expression level heatmap under humid and arid habitats, C represents the relative expression levels of the five HaAGO genes between RNA-seq and qRT-PCR, and D represents the relative expression levels of the five HpAGO genes between RNA-seq and qRT-PCR. Detailed Implementation

[0016] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0017] Example 1: like Figure 1-7 As shown, this embodiment of the invention provides a method for identifying key drought-resistant AGO genes in Haloxylon ammodendron and Haloxylon buergerianum; Physicochemical analysis of the HaaGO and HpAGO gene families: Log in to ExPASy and use the ProtParam tool, input the protein sequence of each HaAGO and HpAGO gene, calculate the number of amino acids, molecular weight and isoelectric point, and then use the WoLFPSORT online tool to predict subcellular localization.

[0018] The instability coefficients of the AGO proteins in *Haloxylon ammodendron* ranged from 39.59 to 53.00, with only HaAGO42 having an instability coefficient below 40. The instability coefficients of the Argonaute proteins in *Haloxylon spp.* ranged from 34.09 to 52.82, with HpAGO9 and HpAGO10 having instability coefficients below 40. This indicates that most Argonaute proteins in both species are unstable. The adipose coefficients of both *Haloxylon ammodendron* (75.24–89.41) and *Haloxylon spp.* (71.49–90.35) were less than 100, while the total average hydrophilicities were -0.491 to -0.149 and -0.51 to -0.24, respectively, indicating that the Argonaute proteins in both species are hydrophilic.

[0019] Furthermore, subcellular localization analysis showed that the Argonaute proteins of *Haloxylon ammodendron* and *Haloxylon spp.* were mostly located in the nucleus (35 and 25, respectively), followed by the cytoplasm (5 and 3, respectively). This provides important information for understanding the functional locations of Argonaute proteins. Figure 1 ).

[0020] Example 2: Phylogenetic tree construction: One monocotyledonous plant (maize) and seven dicotyledonous plants (Haloxylon ammodendron, Haloxylon ammodendron var. chinensis, poplar, apple, grape, Arabidopsis thaliana, and tomato) were selected, and their Argonaute members were used to construct a phylogenetic tree to identify the homology of Argonaute genes in monocotyledonous and dicotyledonous plants. Multiple sequence alignment was performed using muscle (5.1.linux64) software. After alignment, TrimAl 1.4 software was used to remove insertion or deletion regions in the alignment results to obtain high-quality aligned sequences and avoid interference from missing regions in the phylogenetic tree construction. Then, fasttree (2.1.11) software was used to construct the phylogenetic tree using the maximum likelihood method to generate the phylogenetic tree file. Finally, FigTree 1.4.4 software was used to open the generated phylogenetic tree file, and the tree structure, branch length, etc. were adjusted and beautified to more clearly show the evolutionary relationship of the Argonaute gene family of Haloxylon ammodendron and Haloxylon ammodendron var. chinensis.

[0021] Based on phylogenetic tree analysis, the 45 identified HaAGO and 30 HpAGO genes were divided into 5 and 4 subfamilies, respectively. Figure 2 Members located in the same branch are presumed to have closer evolutionary relationships and similar functions.

[0022] Example 3: Conserved motif, gene structure, and chromosome localization analysis: MEME 4.11.2 software was used. The Argonaute protein sequences of Haloxylon ammodendron and Haloxylon buergerianum were input, and the output files included motif sequences, E values, and motif location files. The exon and intron location information of each HaAGO and HpAGO gene were extracted from the Haloxylon ammodendron and Haloxylon buergerianum genome annotation files and compiled into the "AGO_gene_structure.gff" file. The TBtools (2.337) software was used for visualization. The Haloxylon ammodendron and Haloxylon buergerianum gff3 annotation files and gene ID files were put into the GeneLocationVisualizefromGTF / GFF plugin in TBtools (2.337) to perform chromosome localization and visualization.

[0023] The results showed that the number of exons in HaAGO genes ranged from 3 to 28, with most genes containing 21 to 27 exons, while the number of exons in HpAGO genes ranged from 2 to 24, with most genes containing 16 to 23 exons. Figure 3 ).

[0024] Ten conserved motifs were identified in both HaAGO and HpAGO proteins, each possessing its own specific conserved domain. Most HaAGO family members within the same group or subgroup share similar conserved motifs, but some differences exist between groups. Furthermore, members within the same group or subgroup possess specific conserved motifs. All members of the HaAGO family contain the same motifs (motifs 2, 4, 5, 6, 8, and 10), indicating that these six motifs are the core motifs of the HaAGO gene family. Motifs 8 and 3 in the HpAGO family are distributed across all members, suggesting that motifs 8 and 3 are its core motifs. Figure 3 ).

[0025] 45 HaAGO and 30 HpAGO genes are unevenly distributed across the six chromosomes of the H. ammodendron and H. persicum genomes. Figure 4 Chr04 contains 10 HaAGOs (HaAGO13–HaAGO22, 22.22%), while Chr03, Chr08, and Chr09 each contain 9 HaAGO genes. However, Chr08 contains 8 HpAGOs (HpAGO17–HpAGO24, 26.67%), while Chr04 and Chr09 each contain 6 HpAGOs.

[0026] Example 4: Promoter cis-acting element analysis: The 2000 bp sequence upstream of the start codon was extracted from the HaAGO and HpAGO genomes and submitted to the PlantCAR database for analysis of cis-acting elements and their potential functions, and visualized using TBtools (2.337) software.

[0027] The results showed that HaAGO and HpAGO contained 52 elements related to stress (19% / 22%), light response (44% / 42%), hormones (24% / 22%), and growth and development (13% / 13%). Figure 5Among them, three light-response element motifs—Box-4 (97 / 66), TCT-motif (55 / 41), and GT1-motif (49 / 24)—were highly enriched in the upstream regions of the HaAGO and HpAGO genes. Nine hormone-response elements were also included: ABRE, AuxRR-core, TGA-element, GARE-motif, P-box, CGTCA-motif, TCA-element, TGACG-motif, and TATC-box, which are associated with auxin, ABA, ethylene, gibberellin, salicylic acid, and methyljasmonic acid responses, respectively. The promoter region also contained various stress-response elements (including hypoxia, drought, low temperature, and anaerobic induction, defense, and stress responses), associated with the sequences LTR, TC-richrepeats, W-box, GC-motif, ARE, MBS, and MBSI. Among them, ARE (117 / 67) is the most abundant in the HaAGO and HpAGO promoter regions, followed by MBS (42 / 22).

[0028] These results indicate that the Argonaute gene in both Haloxylon ammodendron and Haloxylon buergerianum plays a crucial role in hormonal regulation and stress response. Furthermore, the differences in the number of stress-response and hormonal-response elements between the two species may be one reason for their differences in adapting to prolonged drought.

[0029] Example 5: Protein interaction network analysis: Using Arabidopsis thaliana as a reference, protein sequences of members of the Haloxylon ammodendron and Haloxylon lancifolium families were submitted to the String website to obtain the node relationships between proteins. Protein interaction network diagrams were then drawn using Cytoscape 3.9.1 software. Figure 6 ).

[0030] The network diagram reveals strong interactions among some Argonaute proteins, forming a complex interaction network. In *Haloxylon ammodendron*, HaAGO16 is connected to multiple other genes (HaAGO1, HaAGO6, HaAGO18, and HaAGO25), indicating its central position in the Argonaute protein interaction network. HaAGO1 also interacts with multiple genes and is an important member of the network. In *Haloxylon spp.*, HpAGO6 and HpAGO2 are relatively central, interacting with multiple other genes. The interaction networks of Argonaute proteins in *Haloxylon ammodendron* and *Haloxylon spp.* show some differences in core nodes and interaction patterns. Analysis of these interaction networks contributes to a deeper understanding of the functions and synergistic mechanisms of Argonaute genes in these two plants, providing an important foundation for further research on their molecular regulation in desert adaptation.

[0031] Experimental example: Analysis of Argonaute gene expression in Haloxylon ammodendron and Haloxylon buergerianum under drought stress: Assimilated branches of Haloxylon ammodendron and Haloxylon buergerianum were collected in mid-June 2024 in Aibi Lake Wetland National Nature Reserve (44°31'5″N~45°9'35″N, 82°33'47″E~83°53'21″E). Starting from the Dongdaqiao Management Station of Aibi Lake Wetland Nature Reserve, two 0.05- ... A 1km × 2.0km transect (transect 1: Haloxylon ammodendron distribution area; transect 2: Haloxylon persicum distribution area) was established, and 50m × 50m quadrats were set up under two different soil moisture conditions (H. ammodendron: A: moist low salinity (HS), B: dry low salinity (LS); H. persicum: C: moist low salinity (HB), D: dry low salinity (LB). The collected assimilated branches were flash-frozen with liquid nitrogen and sent to the company for transcriptome sequencing.

[0032] This study used transcriptome data from *Haloxylon ammodendron* and *Haloxylon spp.* under drought stress to assess the response of HaAGOs and HpAGOs genes to drought. Specifically, assimilating branches of *Haloxylon ammodendron* and *Haloxylon spp.* were collected, total RNA was extracted using the TRIzol method and its quality was assessed, and high-throughput RNA sequencing was performed using the Illumina NovaSeq platform to obtain raw reads. After quality control of the data using FastQC and Trimmomatic, clean reads were aligned to the reference genome assembled in this study using HISAT2. FeatureCounts was used to count the number of reads for each gene, and expression levels were normalized using the FPKM method. Homology and domain analysis (using BLASTP and HMMER) were performed to identify all members of the target family from the annotated gene set. Expression data of family members in various samples were then collected and clustered and analyzed using R software. The analysis revealed that genes HaAGO15, HaAGO17, HaAGO30, HaAGO33, HaAGO42, HpAGO2, HpAGO3, HpAGO15, HpAGO24, and HpAGO30 showed the highest fold change in expression under arid conditions compared to humid environments, and all exhibited an upregulated trend. This indicates that these genes play an important regulatory role in the adaptation of Haloxylon ammodendron and Haloxylon buergerianum to long-term arid environments. Figure 7 ).

[0033] To verify the reliability of the RNA-seq data, qRT-PCR was performed on these 10 Argonaute genes. The experimental results confirmed that the qRT-PCR results were highly consistent with the RNA-seq data. Figure 7 This indicates that the transcriptome data is reliable.

[0034] These upregulated AGO genes may participate in plant responses to drought stress by regulating the expression of genes related to stress signaling pathways, osmotic regulation, or antioxidant defense. However, their specific functional mechanisms require further functional studies to verify.

[0035] This invention screened and identified the Argonaute gene family of *Haloxylon ammodendron* and *Haloxylon spp.* using bioinformatics techniques. Further analysis of transcriptome data revealed the expression trends of HaAGOs and HpAGOs gene family members under long-term drought stress. A visualization heatmap of Argonaute gene expression in *Haloxylon ammodendron* and *Haloxylon spp.* was analyzed, identifying five highly critical genes in each: HaAGO15, HaAGO17, HaAGO30, HaAGO33, HaAGO42, and HpAGO2, HpAGO3, HpAGO15, HpAGO24, and HpAGO30. By identifying key drought-resistant genes closely related to the long-term drought stress response of *Haloxylon ammodendron* and *Haloxylon spp.*, these genes may be potential candidate genes for studying the molecular mechanisms of stress resistance in these plants, and also serve as genetic resources for breeding new drought-resistant varieties.

[0036] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for identifying key AGO genes against drought in Haloxylon ammodendron and Haloxylon persicum, comprising, characterized in that, The method comprises the following steps: (1) AGO gene family member identification: ① Obtain the whole genome sequence of Haloxylon ammodendron and H. persicum, and obtain the hmm file of PAZ (PF02170.25) and PIWI (PF02171.20) conserved domains from the Uniprot database; ② The whole genome sequence is screened by using the hmmscan tool, and the candidate genes containing the PAZ and PIWI domains are reserved; ③ BLASTP homologous comparison is carried out by using the protein sequences of Arabidopsis thaliana AtAGO1-AtAGO10; ④ The candidate genes are verified by CDD database, and the sequences containing only a single domain are removed, and finally the members of the HaAGO family of Haloxylon ammodendron and the HpAGO family of H. persicum are obtained; (2) Multi-dimensional bioinformatics analysis: ① Protein physicochemical property and subcellular localization analysis: the number of amino acids, molecular weight, isoelectric point, instability coefficient, fat coefficient and hydrophilicity are calculated by the ProtParam tool of ExPASy; The subcellular localization is predicted by using the online tool WoLFPSORT; ② Phylogenetic tree construction: the AGO protein sequences of Haloxylon ammodendron, H. persicum and seven reference plants are collected, after multi-sequence alignment by Muscle5.1 software and redundancy removal by TrimAl1.4 software, the maximum likelihood method of Fasttree2.1.11 software is used to construct the phylogenetic tree, and the tree structure is optimized and divided into subfamilies by using FigTree1.4.4 software; ③ Analysis of conserved motif, gene structure and chromosome location: the conserved motif is analyzed by using MEME4.11.2 software; the exon and intron position information is extracted from the genome gff3 annotation file, and the gene structure is visualized by using the "Gene Structure View" function of TBtools2.337 software; the gene chromosome location visualization is realized by using the "Gene Location Visualize from GTF / GFF" plug-in of TBtools; ④ Analysis of promoter cis-acting elements: the sequence of 2000 bp upstream of the start codon of the AGO gene is extracted, and is submitted to the PlantCAR database for analysis of cis-acting elements, and the element type and number are counted and visualized by using TBtools2.337 software; ⑤ Protein-protein interaction network analysis: taking Arabidopsis thaliana as a reference species, the AGO protein sequence is submitted to the String database to obtain the interaction relationship data, and the interaction network diagram is drawn by using Cytoscape3.9.1 software and the core node genes are identified; (3) Gene expression analysis and drought-resistant key gene screening ① Sample collection and transcriptome sequencing: wet low-salt and drought low-salt habitat quadrats are set in the Ebinur Lake Wetland National Nature Reserve, and assimilation branches of Haloxylon ammodendron and H. persicum are collected, and total RNA is extracted by using the TRIzol method after quick freezing in liquid nitrogen; after RNA quality detection, high-throughput sequencing is carried out by using the Illumina NovaSeq platform; ii. Transcriptome data processing and expression calculation: FastQC0.11.9 software was used for raw reads quality assessment, and Trimmomatic0.39 software was used to remove adapters and low-quality sequences; HISAT22.2.1 software was used to align clean reads to the reference genome of Haloxylon ammodendron / white Haloxylon ammodendron; featureCounts2.0.1 software was used to count reads of each gene, and FPKM method was used to standardize gene expression; iii. Differential expression gene screening and verification: DESeq2 package of R language was used to screen differential expression genes under dry vs. wet habitats; 5 HaAGO genes and 5 HpAGO genes with the highest differential fold were selected, specific primers were designed, and TaKaRa SYBR Premix Ex Taq II kit was used for qRT-PCR verification; Actin gene was used as an internal reference, and 2^(-ΔΔCt) method was used to calculate the relative expression, and the reliability of transcriptome data was verified; iv. Determination of drought-resistant key genes: combined with the results of differential expression analysis, qRT-PCR verification and cis-acting element analysis, the drought-resistant key AGO genes of Haloxylon ammodendron and white Haloxylon ammodendron were determined.

2. The method for identifying key AGO genes for drought resistance in Haloxylon ammodendron and H. persicum according to claim 1, characterized in that: In step (1) iv, the final identified HaAGO gene family of Haloxylon ammodendron contains 45 members, and the HpAGO gene family of white Haloxylon ammodendron contains 30 members.

3. The method of identifying key AGO genes for drought resistance in Haloxylon and white Haloxylon according to claim 1, characterized in that: In step (2) ii, phylogenetic tree analysis divides the 45 HaAGO genes into 5 subfamilies and the 30 HpAGO genes into 4 subfamilies, and genes in the same subfamily have similar conserved motif composition and gene structure.

4. The method of identifying key AGO genes for drought resistance in Haloxylon and white Haloxylon according to claim 1, characterized in that: In step (2) iv, among the 52 identified cis-acting elements, light-responsive elements account for the highest proportion, and ARE and MBS are the most numerous in stress-responsive elements.

5. The method of identifying key AGO genes for drought resistance in Haloxylon and white Haloxylon according to claim 1, characterized in that: In step (3) iv, the identified drought-resistant key AGO genes are: HaAGO15, HaAGO17, HaAGO30, HaAGO33, HaAGO42 in Haloxylon ammodendron, and HpAGO2, HpAGO3, HpAGO15, HpAGO24, HpAGO30 in white Haloxylon ammodendron; the expression of the key genes in dry habitats is more than 2 times of that in wet habitats, and the correlation coefficient of the relative expression verified by qRT-PCR and the RNA-seq result is ≥0.

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6. The method for identifying key AGO genes for drought resistance in Haloxylon and white Haloxylon according to any one of claims 1-5, characterized in that: After gene cloning, vector construction, and Agrobacterium-mediated transformation of Haloxylon ammodendron or white Haloxylon ammodendron callus, transgenic plants with enhanced drought resistance are obtained.

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