Huperzia serrata hsNAC12 gene and application thereof

Abstract

The application belongs to the technical field of plant genetic engineering, and particularly relates to a Huperzia serrata HsNAC12 gene and application thereof, and the nucleotide sequence of the gene is shown as SEQ ID NO. 1. Through research NAC family genes, the drought resistance molecular mechanism of Huperzia serrata is analyzed. By using a model plant Arabidopsis thaliana, drought-resistant plants are bred through transgenic breeding, which has practical significance for protection of medicinal plant resources, stress resistance breeding and sustainable development of natural products. The transgenic breeding method has low cost, high efficiency, simple and clear operation, and is beneficial to be widely used in production.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of plant genetic engineering, and particularly relates to a Huperzia serrata HsNAC12 gene and application thereof. BACKGROUND

[0002] Huperzia serrata (Huperzia serrata) is a herbaceous plant of the genus Huperzia in the family Huperziaceae. It is called Qiancengta in the National Chinese Herbal Medicine Compilation, and has various local names such as snake foot grass, gold not exchange, and Qianjincao in She medicine. It is a kind of fern with important medicinal value. Modern research shows that Huperzia serrata contains active ingredients huperzine A (Huperzine A) which is widely used in the treatment of Alzheimer's disease. Huperzine A has the characteristics of low toxicity, high efficiency, reversibility and high selectivity. In 1993, the FDA of the United States approved huperzine A tablets as cholinergic agents. At present, the huperzine A drugs on the market include huperzine A and bilive, which still use wild Huperzia serrata as raw materials. However, Huperzia serrata has high requirements for growth environment, small plant size, long growth cycle (annual biomass growth <5 cm), and often low yield. Large-scale artificial cultivation of Huperzia serrata is an inevitable choice to alleviate the contradiction between supply and demand of raw materials.

[0003] Huperzia serrata likes shade and is not resistant to high temperature and drought. By exploring the functions of Huperzia serrata non-biological stress-related genes, a new way is provided for the molecular genetic improvement of Huperzia serrata. Drought stress is one of the main non-biological stress factors faced by Huperzia serrata. Huperzia serrata is extremely sensitive to drought stress (lethal soil moisture content <15%). At present, people often use the way of stress-resistant breeding to screen drought-tolerant individuals, collect spores or branch propagation, and directionally cultivate drought-tolerant strains, or screen resistant seedlings through tissue culture technology combined with drought stress experiment.

[0004] In the paper Identification and Expression Analysis of Huperzia serrata DOF Gene Family (Li Haibo, Xie Liubo, Zhang Kai, Shi Jidong, Cao Yu, Li Muzi, Huayangguang, Wang Dekei), a HsDOF gene family was identified, and the physicochemical properties, conserved domains, subcellular localization of the proteins were analyzed, and the expression patterns of the HsDOP family genes in different tissues and under high temperature and drought stress were analyzed using RNA-seq data. This provides a good research foundation for further study of Huperzia serrata breeding against non-biological stress factors. However, the paper only focuses on the DOF gene family, and there are many gene families in Huperzia serrata that resist drought, and other gene families have not been tried to study their effects on Huperzia serrata's resistance to drought stress. Moreover, the paper does not specifically describe the way of transgenic breeding of Huperzia serrata, and how to select the best variety. SUMMARY

[0005] In order to overcome the problem that Huperzia serrata is not drought-tolerant, leading to low yield of product huperzine A and inability to breed effective drought-tolerant transgenic Huperzia serrata, the present application carries out simulated drought treatment on Huperzia serrata, reveals expression difference characteristics through dynamic monitoring of transcriptome at multiple time points, screens regulatory genes, and obtains transgenic plants through model plants, and provides a Huperzia serrata HsNAC12 gene and application thereof in drought stress tolerance.

[0006] In a first aspect, the present application provides a Huperzia serrata HsNAC12 gene, the nucleotide sequence of which is shown as SEQ ID NO. 1.

[0007] In a second aspect, the present application further provides a recombinant expression vector containing the HsNAC12 gene.

[0008] The recombinant expression vector containing the HsNAC12 gene is a plasmid. 35S-GFP

[0009] In a third aspect, the present application provides a genetically engineered bacterium containing the expression vector.

[0010] The genetically engineered bacterium is Escherichia coli.

[0011] In a fourth aspect, the present application further provides application of the HsNAC12 gene in improving plant resistance to adversity stress.

[0012] Specifically, the adversity stress is drought stress.

[0013] In a fifth aspect, the present application provides a method for improving plant drought stress tolerance, and the method comprises expressing or overexpressing the HsNAC12 gene in the plant.

[0014] Specifically, the model organism for expressing or overexpressing the HsNAC12 gene is Huperzia serrata and / or Arabidopsis thaliana.

[0015] The present application uses Arabidopsis thaliana as a model organism, and Arabidopsis thaliana has the characteristics of simple genome, complete sequencing, convenient genetic operation, short growth cycle and high reproduction efficiency, which can provide support for transgenic research of Huperzia serrata.

[0016] Specifically, the Arabidopsis thaliana is selected from part of 5-6 weeks old and healthy growth.

[0017] The Arabidopsis thaliana is in the best flower bud development period, also known as initial flowering period, at 5-6 weeks old, and has sufficient nutrient reserves and high tissue vitality, and the transformation efficiency can be maximized.

[0018] ​Specifically, the Agrobacterium tumefaciens is used for transforming the Arabidopsis.

[0019] The Agrobacterium tumefaciens can efficiently infect the reproductive tissues of Arabidopsis, and directly transform the developing female gametophytes by the dipping method, so that the transformation efficiency is high and the operation is simple. Moreover, the T-DNA of the Agrobacterium tumefaciens can be stably integrated into the genome, which is beneficial to the expression of exogenous genes. Meanwhile, the method has low cost, high throughput, and is highly compatible with the biological characteristics of Arabidopsis.

[0020] Specifically, the T0 generation seeds of the Arabidopsis are sterilized with 75% ethanol for 5 minutes, and then washed with sterile deionized water for 4-5 times.

[0021] The disinfection can effectively surface sterilize and reduce the risk of pollution. Meanwhile, 5 minutes is a single circle window period for ethanol disinfection of Arabidopsis seeds, and it is a mild disinfection method, which can reduce seed damage. The washing with sterile water is thorough, and can avoid the inhibition of germination caused by residues. The method is simple in operation, suitable for high-throughput processing, and has specific protective effect on transgenic seeds.

[0022] The beneficial effects of the present application mainly include:

[0023] (1) Most of the genes in the present application NAC show different degrees of expression induction under drought stress. Among them HsNAC12 the expression level is continuously up-regulated (up to 5.3 times), which indicates that the gene may play an important role in the drought resistance of Huperzia serrata. The present application has important significance for analyzing the drought resistance molecular mechanism of Huperzia serrata. Not only has important theoretical value for revealing the adversity adaptation evolution of ferns (a key group of early terrestrial plants), but also provides a research basis for molecular targets for artificial cultivation, stress-resistant germplasm creation and secondary metabolism engineering of Huperzia serrata;

[0024] (2) After the transgenic Arabidopsis of the present application is cultured under drought stress for 10 days, the leaves of the wild type plants are seriously softened and necrotic, while most of the leaves of the overexpression plants are still full and tough. This shows that the present application accumulates theoretical basis for the research on the drought resistance mechanism of Huperzia serrata through the functional analysis of the candidate gene. At the same time, it has practical significance for the protection of medicinal plant resources, stress-resistant breeding and sustainable development of natural products;

[0025] (3) The transgenic breeding method of the present application has low cost, high efficiency, simple and clear operation, and is beneficial to be widely used in production process. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 is a transcriptome sample correlation analysis diagram. Note: principal component 1 (PC1) and principal component 2 (PC2) correspond to X axis and Y axis respectively, and the numerical value in the bracket is the variance contribution rate of each principal component.

[0027] Figure 2 Figure 8 is a graph of the expression pattern of HsNAC gene under drought stress.

[0028] Figure 3 Figure 9 is a graph of the cDNA quality detection.

[0029] Figure 4 Figure 10 is a graph of the HsNAC12 fragment, Note: M: DL2000.

[0030] Figure 5 Figure 11 is a graph of the subcellular localization of HsNAC12 protein.

[0031] Figure 6 Figure 12 is a graph of the molecular detection of transgenic Arabidopsis.

[0032] Figure 7 Figure 13 is a graph of the growth status of Arabidopsis seedlings under different PEG6000 treatments.

[0033] Figure 8 Figure 14 is a graph of the root length statistics of Arabidopsis seedlings under different PEG6000 treatments, Note: the scale is 1 cm.

[0034] Figure 9A Figure 15 is the change of POD (A) activity of transgenic Arabidopsis under drought stress.

[0035] Figure 9B Figure 16 is the change of SOD (B) activity of transgenic Arabidopsis under drought stress.

[0036] Figure 9C Figure 17 is the change of CAT (C) activity of transgenic Arabidopsis under drought stress.

[0037] Figure 10 Figure 18 is a graph of the phenotype change of transgenic Arabidopsis under drought stress.

[0038] Figure 11 Figure 19 is a graph of the MDA content of transgenic Arabidopsis under drought stress.

[0039] Figure 12 Figure 20 is a graph of the expression level of stress resistance related genes of transgenic Arabidopsis under drought stress. DETAILED DESCRIPTION

[0040] The present application is illustrated by way of example and not limitation based on the following detailed description. Other advantages and novel features of the present application will be readily apparent to those skilled in the art from the following details, and others can be obvious upon an understanding of the disclosure or can be learned from practice of the application. The present application can be practiced by other than the described embodiments without departing from the spirit or scope of the application. Description herein is directed to certain embodiments with additional embodiments apparent from the disclosure and described herein. The embodiments from the disclosure may, however, be implemented in various ways and of should not be construed to limit the application to the examples described herein; instead, these examples are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the application to those skilled in the art. As used herein, the indefinite articles "a" and "an" are intended to mean zero or one; the definite article "the" is intended to mean zero or one; and the conjunctive term "or" is intended to mean at least one. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description of the application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. All publications, patent applications, patents, figures, and other references mentioned herein are incorporated by reference in their entirety for the teachings relevant to the sentence and / or paragraph in which the reference is presented.

[0041] Example 1: Transcriptome analysis of Huperzia serrata under drought stress

[0042] Experimental materials

[0043] 1.1 Plant material

[0044] 3-year-old Huperzia serrata plants. From Huperzia serrata resource garden.

[0045] 1.2 Reagents

[0046] PEG6000, Horgland, plant RNA extraction kit, Gold View nucleic acid dye, agarose Biosharp ).

[0047] 1.3 Instruments

[0048] pipette Thermo science ), high-speed desktop centrifuge Thermo Fisher ), PCR instrument (Novogene ), water bath DK-8D ), dry thermostat, gel electrophoresis instrument, ultraviolet gel imaging system major science ), ultraviolet gel cutter (Shengwo), spectrophotometer (Shanghai Yidian).

[0049] Experimental method

[0050] 2.1 Pretreatment of plant material

[0051] This study aimed to investigate the early molecular response mechanism of *Huperzia spp.* to simulated drought conditions. *Huperzia spp.* plants with consistent growth stages were selected, and after root cleaning, they were transplanted into a hydroponic system and cultured in Hoagland total nutrient solution (the solution was changed every 48 hours). The incubator environmental parameters were set to a daily temperature of 22-25°C and a relative humidity of 70%. After 21 days of acclimatization, drought stress was simulated using a 10% PEG-6000 solution. Root, stem, and leaf tissue samples were collected at three time points: 4h, 8h, and 24h after the stress treatment began (including an untreated control group; each treatment had three biological replicates). Samples were immediately frozen in liquid nitrogen and stored at -80°C. Library construction was performed using third-generation sequencing (PacBio SequeI platform), and this library was used as a reference for second-generation sequencing (Illumina platform) of samples from each drought treatment time point to correct transcripts.

[0052] 2.2 RNA Extraction and Library Construction

[0053] This embodiment follows standard operating procedures and uses a dedicated kit from Tiangen Biotech Co., Ltd. to extract whole RNA from Huperzia spp. To ensure the reliability of the transcriptome data, multi-dimensional quality control was implemented: RNA integrity was assessed by 1% agarose gel electrophoresis, purity parameters were determined using a spectrophotometer, and quantitative PCR was performed using a fluorescence quantitative analyzer (QQI). Qubit 2.0 Accurately calculate nucleic acid concentration and use a biological analyzer ( Agilent 2100 ) Detection of RNA integrity value ( RIN For the mRNA enrichment process, poly-T oligonucleotide-modified magnetic beads were used to screen 5 μg of total RNA twice, specifically capturing poly(A) tail RNA. The enriched mRNA was then subjected to controlled fragmentation at high temperature in a buffer system containing divalent metal ions. Subsequently, following the instructions of the mRNA-Seq library preparation kit, double-stranded cDNA was synthesized via reverse transcription, ultimately constructing a PE library with an insert fragment of 300 ± 50 bp. All sequencing experiments were technically implemented and data output was completed by Nanjing Jisi Huiyuan Technology Co., Ltd.

[0054] 2.3 Unigenes Annotation and Functional Classification

[0055] Unigene sequences were aligned with databases such as NR, Swiss-Prot, GO, COG, KOG, and KEGG using BLAST (v2.2.26). Based on the predicted ORF sequences, gene function annotation was further completed using HMMER software in conjunction with the Pfam database.

[0056] 2.4 GO and KEGG enrichment analysis of DEGS

[0057] Salmon algorithm was used to quantify the transcript abundance, and TPM (Transcripts Per Million) was used to represent the expression intensity of unigenes. The edgeR software package was used to screen the differentially expressed genes, and the screening criteria were set as follows: the absolute value of the logarithmic expression difference was greater than 1 (|log2FC|>1), and the significance threshold p<0.05. For the differentially expressed genes obtained by screening, the functional enrichment analysis was carried out in two dimensions: GO annotation level: the significant differentially expressed genes were mapped to the GO database for functional mapping, and the hypergeometric distribution test was used to identify the enrichment degree of each functional term (term), and the statistically significant p value was calculated; KEGG pathway level: metabolic pathways were used as analysis units, and the hypergeometric test was also used to determine the metabolic pathways in which the differentially expressed genes were significantly aggregated, and the corresponding p value was calculated. Finally, the ggplot2 graphics toolkit was used to visualize the enrichment analysis results of GO and KEGG as scatter distribution plots.

[0058] 2.5 qRT-PCR verification of transcriptome data

[0059] To verify the reliability of the transcriptome data, quantitative real-time PCR (qRT-PCR) technology was used for detection. RNA reverse transcription experiment synthesized cDNA. Based on the transcriptome sequencing results, the GAPDHB gene of Huperzia serrata (Table 1) was used as an internal reference, and specific primers were designed by NCBI Primer Blast tool. PCR amplification was completed in a 7500 QPCR instrument (reaction system and program were shown in Tables 2 and 3) using a Beijing Zison Gold reagent kit, and each group of experiments was set up for three biological replicates. The relative expression of genes was calculated by the 2-ΔΔCt method.

[0060] Table 1 qRT-PCR primers used for verification of transcriptome

[0061]

[0062] Table 2 qRT-PCR reaction system

[0063]

[0064] Table 3 qRT-PCR reaction program

[0065]

[0066] 3. Results and analysis

[0067] 3.1 Transcriptome analysis of Huperzia serrata

[0068] 3.1.1 Overview of transcriptome data

[0069] Third-generation full-length sequencing: Single molecule real-time sequencing by PacBio SMRT Resq sequencer was used to sequence the mixed samples of roots, stems and leaves of each treatment time period, obtaining 966182 CCS (Complementary DNA Sequences) sequences, of which 523067 were full-length non-chimeric sequences. Circular Consensus FLNC, full length readsnon-chimeric

[0070] Second-generation sequencing: With the help of Illumina's double-end sequencing technology, the root, stem and leaf samples of Huperzia serrata were sequenced under drought stress (10% PEG6000) and heat stress (42°C) for 4h, 8h and 24h, with 3 biological replicates at each time point, totaling 27 samples. The total sequencing data of all samples was 227.18 Gb Clean Data, with each sample Clean Data reaching 7.35 Gb and above, and the Q30 base percentage exceeding 88.86%. Lordec software was used to correct the third-generation long reads through large-scale and high-precision iteration of second-generation short reads. The high-quality transcripts were clustered and de-redundant using cd-hit software, and the same transcripts were clustered into one category, obtaining a total of 43189 genes. The parameter was set to -c similarity (Clustering threhold) 0.95, and Table 4 shows the clustering results.

[0071] Table 4 Transcript clustering results table

[0072]

[0073] 3.1.2 Transcript sample correlation analysis

[0074] In experimental designs involving biological replicates, the quality assessment of sample reproducibility is a key step to ensure the reliability of transcriptome data analysis. Principal component analysis (PCA) is a classic dimensionality reduction method based on orthogonal linear transformation, whose core mechanism is to extract the main variance information in high-dimensional data and map it to a low-dimensional orthogonal coordinate system. In this study, PCA was performed on the transcriptome sequencing data of all 27 samples to systematically analyze the overall variation characteristics and clustering rules among different experimental groups through the spatial distribution pattern of principal component vectors. Principal component analysis showed that the first principal component (PC1) and the second principal component (PC2) were the two principal components with the highest data variability, with cumulative contribution rates of 18.96% and 11.68%, respectively. The biological replicate samples of the untreated group and each treatment group showed obvious clustering characteristics, indicating low intra-group differences and further verifying the high reliability of the sequencing data in this study (e.g. Figure 1 ).

[0075] 3.1.3 Unigenes annotation and functional classification

[0076] ​​To obtain comprehensive genetic annotation, we analyzed the total of 43189 genes by BLASTN (E value 1e-10), NCBI non-redundant protein sequences (Nr), Eukaryotic Orthologous Groups (KOG), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Ontology (GO). 16,998, 28,345, 24,002, 36,359, and 35,954 single genes were successfully annotated in COG, GO, KOG, KEGG, and NR databases, respectively (Table 5). The KEGG database had the most single gene annotations, with 36,359 (84.19%).

[0077] Table 5 Functional annotation results of Unigenes in each database

[0078]

[0079] 3.2 DEGS differential expression analysis in H. heterophyllum leaves under drought stress

[0080] Under drought stress treatment, a large number of genes in H. heterophyllum leaves were differentially expressed, with 680, 165, and 468 genes differentially expressed in PEG treatment for 4 h, 8 h, and 24 h, respectively. Among them, compared with the blank sample, there were 342 up-regulated genes and 338 down-regulated genes in the 4 h treatment group, 94 up-regulated genes and 61 down-regulated genes in the 8 h treatment group, and 197 up-regulated genes and 271 down-regulated genes in the 24 h treatment group.

[0081] This indicates that the gene expression in H. heterophyllum leaves under drought stress shows significant time dependence. H. heterophyllum may adopt a "rapid perception-delayed execution" strategy, starting signal transduction in the early stage (4 h) and activating effector genes in the late stage (24 h). Rapid signal response is dominant at 4 h, the treatment may enter a regulatory buffer period at 8 h, and adaptive mechanisms are activated and growth is inhibited at 24 h. Subsequent functional enrichment and experimental verification are needed to identify key genes and their regulatory networks, providing a basis for analyzing the drought resistance mechanism of H. heterophyllum (e.g. Figure 5 ).

[0082] 3.3 GO enrichment analysis of DEGS under drought stress

[0083] GO enrichment analysis showed that the differentially expressed genes (DEGs) at different time points of drought stress were mainly involved in biological processes such as oxidation-reduction, metabolic regulation, stress response, biological regulation, and translation, and were enriched in structural components such as nucleus, cytoplasm, and membrane system, as well as molecular functions such as DNA / protein binding and catalytic activity. The DEGs of the 4h and 8h treatment groups were highly similar in core GO term categories, but the number of genes differed significantly (about 4 times as many DEGs in the 4h group as in the 8h group). By 24h, the key GO term categories remained consistent, but the number of DEGs increased to 3 times that of the 8h group. Notably, the DEGs of the 4h group were significantly enriched in transcriptional regulation, DNA template binding, and transcription factor activity-related functions, while the DEGs of the 8h group were more involved in oxidation-reduction, ribosome assembly, and biological adhesion processes. GO enrichment analysis of leaf differentially expressed genes (DEGs) under drought stress showed that plants achieve stress adaptation through phased transcriptional reprogramming: the gene response was most intense in the early stage (4h) (about 4 times as many DEGs as in the 8h group), significantly enriched in regulatory processes such as DNA binding (e.g. NAC, WRKY ), transcription factor activity, and initiation of signal transduction networks; the number of DEGs decreased sharply in the middle stage (8h), with functions shifting to oxidation-reduction balance (SOD, APX) and ribosome biosynthesis, prioritizing the maintenance of basic metabolic homeostasis; the number of DEGs increased to 3 times that of the 8h group in the long-term (24h), with enriched pathways similar to those in the early stage but focusing on osmotic regulation and ion transport, and system reconstruction of cellular homeostasis. The core GO terms at each time point were highly conserved (e.g. "response to stimulus", "biological regulation"), but the gene expression scale showed a bimodal fluctuation (4h > 24h > 8h), suggesting that plants employ a three-stage strategy of "rapid perception - resource redistribution - system adaptation" to cope with drought stress.

[0084] 3.4 KEGG enrichment analysis of DEGS under drought stress

[0085] Based on the functional analysis of DEGs, KEGG pathway enrichment analysis was conducted. The results showed that the core metabolic pathways of leaf response to drought stress were consistent in time dynamics, but the gene enrichment degree differed significantly. Combined with gene enrichment degree and statistical significance (p value) screening, the cornin biosynthesis, thiamin metabolism, and photosynthesis antenna protein-related pathways were significantly enriched at 4h, 8h, and 24h, suggesting that they may play a key role in drought adaptation of S. sarmentosum. In addition, some pathways (such as specific secondary metabolic pathways) showed time-specific enrichment characteristics.

[0086] Example 2: Expression pattern analysis of drought stress-related genes in S. sarmentosum HsNAC

[0087] To determine the expression patterns of drought stress-related genes in S. sarmentosum NAC ​The expression patterns of the gene family under high temperature stress were analyzed by RNA-seq of H. fordii treated at different times. Most of the genes NAC The expression of the gene family was induced to different degrees under drought stress. HsNAC29, HsNAC34, HsNAC12, HsNAC15, HsNAC25 The expression level of the gene was significantly increased under drought stress, and the expression level at 8h reached the peak of the expression level among the three time periods (4h, 8h, 24h), and fell at 24h, but still maintained at a significantly up-regulated level. Among them HsNAC12 The expression level of the gene was significantly increased under drought stress, and the expression level at 8h reached the peak of the expression level among the three time periods (4h, 8h, 24h), and fell at 24h, but still maintained at a significantly up-regulated level. Among them HsNAC8, HsNAC33, HsNAC24, HsNAC18 The expression level of the gene was significantly increased under drought stress, and the expression level at 8h reached the peak of the expression level among the three time periods (4h, 8h, 24h), and fell at 24h, but still maintained at a significantly up-regulated level. Among them HsNAC8 The expression level of the gene was significantly increased under drought stress, and the expression level at 8h reached the peak of the expression level among the three time periods (4h, 8h, 24h), and fell at 24h, but still maintained at a significantly up-regulated level. Among them HsNAC27, HsNAC37, HsNAC11 The expression level of the gene was significantly increased under drought stress, and the expression level at 8h reached the peak of the expression level among the three time periods (4h, 8h, 24h), and fell at 24h, but still maintained at a significantly up-regulated level. Among them HsNAC33 HsNAC17 HsNAC24 The expression level of the gene was significantly increased under drought stress, and the expression level at 8h reached the peak of the expression level among the three time periods (4h, 8h, 24h), and fell at 24h, but still maintained at a significantly up-regulated level. Among them HsNAC11 The gene showed the most significant down-regulation of expression (as Figure 2 ).

[0088] Example 3: HsNAC12 Cloning and functional analysis of the gene

[0089] 1. Plant material

[0090] The experimental material H. fordii used in this experiment was taken from the H. fordii planting resource garden.

[0091] 2. Experimental method

[0092] 2.1 Extraction of total RNA from H. fordii and synthesis of cDNA

[0093] 2.1.1 Extraction of total RNA

[0094] Take the new leaves of H. fordii, freeze them in liquid nitrogen, and extract the total RNA by TRIzol method, the specific operation is as follows:

[0095] (1) Pre-cooling treatment: sterilize the mortar and pestle without RNAse at high temperature and pre-cool them for standby use.

[0096] ​​(2) Sample crushing: Take fresh leaves and grind them quickly into fine powder in a mortar pre-cooled by liquid nitrogen. Transfer them to a 1.5 mL RNase-free centrifuge tube containing 1 mL TRIzol lysis buffer and vortex to mix.

[0097] (3) Phase separation: Add 200 μL of chloroform and shake vigorously for 30 s, let stand at room temperature for 30 min to separate the phases, and centrifuge at 12,000 × g for 10 min at 4 ℃.

[0098] (4) RNA precipitation: Aspirate the supernatant into a new tube, add pre-cooled anhydrous ethanol at a volume ratio of 1:1, incubate at -20℃ for 30 min, then centrifuge for 10 min under the same conditions and discard the supernatant.

[0099] (5) Removal of impurities: The precipitate was washed with 1 mL of 70% ethanol, centrifuged at 12,000×g for 1 min at 4℃, washed repeatedly, and dried at room temperature in a clean bench for 5 min.

[0100] (6) RNA dissolution: Add 20 μL of RNase-free water to reconstitute the precipitate, store on ice, and perform UV spectrophotometric concentration detection (NanoDrop assay). 260 / A 280 ratio).

[0101] 2.1.2 RNA reverse transcription

[0102] RNA was reverse transcribed into cDNA using a reverse transcription kit from TransGen, as follows.

[0103] (1) First-strand cDNA synthesis and gDNA removal: Prepare the following reaction solutions on ice (as shown in Table 6).

[0104] Table 6 Reverse Transcription System

[0105]

[0106] (2) Mix gently and incubate at 2°C for 30 minutes.

[0107] (3) Heating at 85℃ for 5 seconds inactivates EasyScript*RT / RI and gDNA Remover.

[0108] (4) Store the cDNA at -20°C.

[0109] 2.2 Target gene amplification

[0110] according to HsNAC12 PCR primers were designed using CDS (Table 7), and PCR amplification was performed. The primers and PCR procedures are shown in Tables 8 and 9 below.

[0111] Table 7 PCR primer sequences

[0112]

[0113] Table 8 PCR reaction system of target gene

[0114]

[0115] Table 9 PCR reaction program

[0116]

[0117] After the band of the target fragment was amplified, the target band was recovered by gel recovery, and the recovered product was used as a template to perform PCR fragment amplification using the second batch of primers (as shown in Table 10) used to construct the p35S:GFP- CaMV35S:GUS vector. The system and PCR program were the same as above. HsNAC12

[0118] Table 10 PCR primer sequence

[0119]

[0120] 2.3 Construction of cloning vector

[0121] (1) Preparation of linearized cloning vector, the 35S-GFP plasmid was digested using BamHI, and the system was as shown in Table 11.

[0122] Table 11 Digestion system

[0123]

[0124] (2) The gel recovery product of the above step was connected with the linearized p35S:GFP through the In-Fusion kit, and the connection system was as shown in Table 12.

[0125] Table 12 Linearized plasmid and gene connection system

[0126]

[0127] (3) The above reaction system was placed in a metal bath or a water bath at 50°C for 20 min, and then cooled on ice. Then, direct transformation or storage in a -20°C refrigerator was performed.

[0128] 2.4 Transformation of E. coli

[0129] ​The plasmid transformation and positive clone screening process is as follows: mix the recombinant plasmid with 50 μL DH5α competent cells, then ice bath for 25 minutes, heat shock treatment at 42°C for 30 seconds, and then ice bath for 2 minutes. Add 500 μL preheated LB liquid medium (37°C), incubate at 37°C, 200 rpm for 1 hour. Centrifuge at 4000 rpm for 1 minute, discard most of the supernatant, retain about 100 μL bacterial solution, evenly spread on LB solid plate containing 50 mg / L Kana, and incubate at 37°C for 12-16 hours. The next day, pick single colonies for colony PCR identification (reaction system see Table 13), and transfer positive clones to 10 mL LB liquid medium containing 50 mg / L Kana, and incubate at 37°C, 200 rpm for 12 hours. After secondary PCR verification, select positive strains for testing HsNAC12 -F primer sequencing, the rest of the bacterial solution is stored at 4°C. After sequence alignment by DNA MAN software, the positive bacterial solution is stored at -80°C with 15% glycerol. Take 5 mL of correct bacterial solution for plasmid extraction, and store the obtained plasmid at -20°C.

[0130] Table 13 Bacterial solution PCR reaction system

[0131]

[0132] 2.5 HsNAC12 Bioinformatics analysis

[0133] The bioinformatics analysis process is as follows: sequence analysis of genes and their encoded proteins is carried out by integrating NCBI and other bioinformatics platforms HsNAC12 For the analysis of the potential function of the gene, the PlantTFDB 5.0 database and the TAIR database are used for functional annotation analysis of the gene. Based on MEGA11 software, multiple sequence alignment is carried out, and a phylogenetic tree is constructed by DNAMAN 9.0.

[0134] 3. Results and analysis

[0135] 3.1 RNA and cDNA quality detection

[0136] The RNA quality is determined by spectrophotometer, the concentration is between 100-300 ng / μL, OD 260 / 280 is between 1.8-2.2, indicating that the RNA purity is high. After reverse transcription of RNA into cDNA, PCR verification is carried out using the GAPDHB primer of Ginkgo biloba, the electrophoresis map of the product is clear Figure 3 , the position is consistent with the theoretical length, indicating that the cDNA quality is good and can be used for the next step test.

[0137] 3.2 Gene cloning and sequence analysis

[0138] The cDNA of Huperzia serrata was used as template, and the specific primers were used for PCR amplification with KOD enzyme. The products after reaction were detected by gel electrophoresis, and a band with a length of 1000-1500 bp was obtained, which was consistent with the theoretical length of 1230 bp of the specific primer amplification product. HsNAC12

[0139] In this embodiment, two rounds of PCR amplification combined with molecular cloning technology were used for gene recombination experiment. The first round of PCR product was separated by 1.5% agarose gel electrophoresis, and the target fragment was purified by gel recovery kit. The purified product was used as template, the second set of specific primers with homologous arms were replaced, and the second amplification was carried out under the same thermal cycling parameters. The target band was purified again by gel electrophoresis. Linearized vector and purified product were directionally connected by In-Fusion® HD cloning kit to construct recombinant plasmid. The transformation experiment used DH5α competent cell system: the strain stored at-80℃ was thawed in ice bath, and the standard transformation process was operated, and was coated on LB solid medium containing Kana antibiotic, and was incubated at 37℃ for 16h.

[0140] In the screening stage of recombinant cloning, single clone colonies were randomly picked for colony PCR verification (primers HsNAC12 -F / R), and the positive clones were sequenced HsNAC12 -F primer) to confirm the correctness of the sequence, and the recombinant plasmid sample was prepared by plasmid extraction kit and stored at-20℃ for standby. The open reading frame of the target gene was determined to be 1230 bp by bioinformatics analysis, and its coding product was a protein composed of 410 amino acid residues (such as Figure 4 ). The gene was named HsNAC12, The nucleotide sequence is shown as SEQ ID NO. 1.

[0141] Example 4: Genetic transformation of Huperzia serrata HsNAC12 gene

[0142] 1. Experimental materials

[0143] 1.1 Plant materials and strains

[0144] (1) Wild type Arabidopsis thaliana seeds and wild type Nicotiana benthamiana seeds were obtained from the laboratory.

[0145] (2) Strains: DH5α E. coli competent cells, GV3101 Agrobacterium competent cells (purchased from Shanghai Weidi Biotechnology Co., Ltd.).

[0146] (3) Vectors: p35S: GFP (laboratory preservation).

[0147] 1.2 Culture medium and drugs​

[0148] (1) LB liquid medium: Tryptone 10 g, Yeast Extract 5 g, NaCl 10 g, distilled water to 1 L, pH 7.0.

[0149] (2) LB solid medium: LB liquid medium: Tryptone 10 g, Yeast Extract 5 g, add Agar 15 g, NaCl 10 g, distilled water to 1 L, pH 7.0.

[0150] (3) MS medium: MS 4.74 g / L, Sucrose 20 g / L, Agar 7.8 g / L.

[0151] (4) 1 / 2MS medium: 1 / 2MS 2.47 g / L, Sucrose 20 g / L, Agar 7.8 g / L.

[0152] (5) Tobacco resuspension solution: 1 M MES-KOH (PH 5.6) 1 mL, 1 M Mgcl2 21 mL, 1 M AS 10 μL, distilled water to 100 mL.

[0153] (6) Arabidopsis resuspension solution: MS medium 2.215 g, Sucrose 50 g, Silwet-77 200 μL, distilled water to 1 L.

[0154] (7) Screening medium: MS medium (containing KANA 50 μg / mL, Rif 30 μg / mL).

[0155] (8) Kanamycin (Kana, 100 mg / mL): In a clean bench, 1 g of Kana was dissolved in 8 mL of sterile water, and after fully mixed and dissolved, it was diluted to 10 mL. After sterilization by 0.22 μm filter membrane, it was divided into 1.5 mL centrifuge tubes and stored at -20°C.

[0156] (9) Rifampicin (Rif, 50 mg / mL): In a clean bench, 0.5 g of Rif powder was poured into a 50 mL centrifuge tube, 8 mL of dimethyl sulfoxide was added, and the powder was shaken to completely dissolve. After dilution to 10 mL, it was filtered by 0.22 μm filter membrane and divided into 1.5 mL centrifuge tubes and stored at -20°C.

[0157] (10) 50% Glycerol: In a clean bench, 20 mL of glycerol and 20 mL of sterile water were thoroughly mixed, and after high-temperature high-pressure sterilization, they were stored at 4°C.

[0158] 2. Experimental method

[0159] 2.1 HsNAC12 Subcellular localization of proteins

[0160] 2.1.1 Transformation of Agrobacterium tumefaciens with recombinant plasmid

[0161] (1) Gradient thawing of GV3101 frozen competent cells (-80°C) was performed: after equilibration at room temperature to a semi-melted state of ice crystals, the cells were transferred to an ice water bath to maintain a phase transition equilibrium at 0°C.

[0162] (2) Genetic transformation was performed using a micro-heat shock method: 1 μL of recombinant plasmid (verified by sequencing) was gently mixed with 100 μL of GV3101 suspension, and subjected to a series of consecutive treatments: ice bath equilibration (5 min), liquid nitrogen instantaneous freezing (5 min), 37°C heat shock (5 min), and ice bath recovery (5 min).

[0163] (3) 500 μL of LB liquid medium (pH 7.0) was added to the transformation system, and the cells were cultured at 28°C on a constant temperature shaker at 200 rpm for 2.5 h to restore the membrane potential.

[0164] (4) After centrifugation at 6000 x g and room temperature for 1 min, the bacterial pellet was resuspended in 250 μL of supernatant, and evenly spread on LB selection medium (containing kanamycin 50 mg / L + rifampicin 30 mg / L). The plate was incubated at 28°C in the dark for 48-72 h to obtain single colonies.

[0165] (5) Morphologically typical colonies were picked for HsNAC12 F-primer PCR verification, and positive clones were mixed with sterile glycerol (final concentration 25%) in equal volumes and stored at -80°C for long-term freezing.

[0166] 2.1.2 Tobacco injection infection

[0167] (1) The GV3101 recombinant strain was cultured on a double-antibiotic LB plate (Kana 50 mg / L + Rif 30 mg / L) using a zonal streaking method. After incubation at 28°C in the dark for 48 h, a single colony was inoculated into LB liquid medium containing the same concentration of antibiotics, and incubated at 28°C on a constant temperature shaker (200 rpm) for 12-16 h to reach the stable phase.

[0168] (2) 1 mL of bacterial solution at the late logarithmic phase was transferred to 20 mL of fresh LB medium (containing the corresponding antibiotics and 200 μM acetylchalcone / vir gene inducer), and incubated at 28°C in the dark for 3.5 h to reach the mid-logarithmic phase (OD 600 = 0.5).

[0169] (3) Collect the bacteria by centrifugation at 4000xg (room temperature, 10 min), and resuspend the bacteria to a final concentration OD 600 = 1.0 in pre-cooled plant transformation buffer (10 mM MES, 10 mM MgCl2, 150 μM AS, pH 5.6).

[0170] (4) Simultaneously prepare p35S::GFP the empty vector control bacterial solution and the nuclear localization marker reference strain H2b-mcherry , and normalize them to the same optical density.

[0171] (5) Mix the target strain and the nuclear localization reference strain at a ratio of 1:1 (v / v), and inject them into the lower epidermis cells of N. benthamiana Nicotiana benthamiana using the pressure infiltration method (1 mL sterile syringe without needle injection), and mark the injection site.

[0172] (6) After infection, the tobacco is cultured at 22°C for 72 h under a light cycle, the injection area is cut to make a live section, and a confocal microscope is used for subcellular localization imaging.

[0173] 2.2 Arabidopsis thaliana transformation of the target gene and identification of positive plants

[0174] (1) Preparation of buffer. Put 4.43 g MS and 50 g sucrose into a 1 L beaker, add 800 mL of deionized water, stir to dissolve, and adjust the pH to 5.8.

[0175] (2) Preparation of bacterial solution. Pick 5-7 single colonies of Agrobacterium from LB solid medium, transfer them to 100 mL of freshly prepared LB liquid medium containing antibiotics (containing kanamycin and rifampicin), and incubate them in a 28°C rotary incubator for 16-18 hours. When the OD value of the bacterial solution is 2.0, the bacteria are ready for use.

[0176] (3) Put the bacterial solution into a 50 mL centrifuge tube, centrifuge at 3000 rpm / min for 4 min, discard the supernatant. Then add an equal amount of buffer and mix slowly. Measure the OD value until the OD value is around 0.6-0.8 for use in the transfection experiment.

[0177] (4) Add 200 μL Silwet L-77 to the remaining transformation buffer, resuspend the bacterial particles with the buffer, and then use it to transform A. thaliana 5-6 weeks old healthy A.thaliana are used for transformation.

[0178] (5) One day before transformation, remove the fruit pods and pour sufficient nutrient solution. During the transformation process, the Arabidopsis thaliana plants are placed in a A.tumefaciensIncubate the plants in the dark for 24 h, then continue normal culture. Harvest the seeds, which are T0 generation transgenic seeds, and dry them in a 28°C incubator for 1 week before screening.

[0179] (6) Sterilize the T0 generation transgenic seeds with 75% ethanol for 5 minutes, then wash them 4-5 times with sterile deionized water, and store them at 4°C. After 3 days of low-temperature treatment, evenly sow the seeds in 1 mL of MS solid medium containing 25 mg of hygromycin resistance using a pipette gun. The transgenic positive plants grow normally and are green, but the leaves of non-transgenic plants turn yellow and cannot grow normally. Gently pull the transgenic positive plants out of the medium with tweezers and transplant them into pots. After 7 days of growth, cut the leaves for PCR identification. After identification of positive plants, screen them for 3 generations in succession to obtain homozygous seeds.

[0180] 2.3 Phenotype observation of transgenic Arabidopsis thaliana

[0181] Sow wild-type and transgenic Arabidopsis thaliana seeds on MS medium. After 3 days of vernalization, place the plates vertically in a constant-temperature light incubator. After 7 days of growth, select Arabidopsis thaliana seedlings with uniform growth in a clean bench, and transplant them into 4 pots each containing an equal amount of nutrient soil and vermiculite mixed at a ratio of 3:1. Sow 1 pot of wild-type and 3 pots of transgenic Arabidopsis thaliana seedlings, and place them in the same tray for standby. Transplant the 7-day-old Arabidopsis thaliana seedlings into the pots, sow 3 seedlings per pot, and water them every 2 days. After 21 days of normal culture, take photos, stop watering for 10 days, and then observe the growth state of the plants and take photos.

[0182] 2.3.1 Effect of drought stress on the growth of transgenic Arabidopsis thaliana seedlings

[0183] Sow wild-type and transgenic Arabidopsis thaliana seeds on MS medium. After 3 days of vernalization, place the plates vertically in a constant-temperature light incubator. After 7 days of growth, select Arabidopsis thaliana seedlings with uniform growth in a clean bench, and transplant them into 4 pots each containing an equal amount of nutrient soil and vermiculite mixed at a ratio of 3:1. Sow 1 pot of wild-type and 3 pots of transgenic Arabidopsis thaliana seedlings, and place them in the same tray for standby. Transplant the 7-day-old Arabidopsis thaliana seedlings into the pots, sow 3 seedlings per pot, and water them every 2 days. After 21 days of normal culture, take photos, stop watering for 10 days, and then observe the growth state of the plants and take photos.

[0184] 2.4 Phenotype differences and physiological index determination of transgenic Arabidopsis thaliana under drought stress

[0185] 2.4.1 Determination of antioxidant enzymes SOD, POD, and CAT

[0186] The tissue homogenate or cell lysate was centrifuged at 4°C, 12,000 x g for 15 min before the supernatant was taken as the sample to be tested, and the protein concentration was determined by BCA method and diluted to an appropriate concentration. The SOD activity was detected by hydroxylamine method, and the absorbance at 550 nm was measured after 40 min of reaction at 37°C. The enzyme activity was calculated according to the inhibition rate of superoxide anion radical on the oxidation of hydroxylamine; the POD activity was based on the guaiacol method, and the absorbance rate of H2O2 oxidizing guaiacol at 470 nm was measured; the CAT activity was detected by ultraviolet spectrophotometry, and the absorbance decrease value of H2O2 decomposition at 240 nm was recorded. The enzyme activity was converted to specific activity (U / g prot) per unit protein concentration according to the kit formula. Duplicate holes and blank controls were set up throughout the experiment, and the reaction temperature, time and spectrophotometer calibration were strictly controlled to ensure the reliability of the data.

[0187] 2.4.2 MDA assay

[0188] Based on the thiobarbituric acid (TBA) colorimetric method. The tissue homogenate or cell lysate was centrifuged at 4°C, 10,000 x g for 10 min, and the supernatant was collected as the sample to be tested (if the kit requires special pretreatment, adjust accordingly). During the experiment, the sample was mixed with TBA reagent in proportion, and the MDA and TBA were condensed to generate a red product in a boiling water bath for 60 min. After cooling, the precipitate was removed by centrifugation, and the absorbance was measured at 532 nm, with 600 nm as the reference to correct the turbidity interference. The MDA content was calculated according to the standard curve or the molar extinction coefficient (ε = 155 mM -1 cm -1 ) provided by the kit, and the final result was expressed as the content per unit protein concentration (nmol / g prot). The experiment was carried out in the dark to avoid photosensitive degradation. Each group was set up with duplicate holes, blank tubes and standard controls, and the water bath time and temperature were strictly consistent to ensure the specificity of the reaction. The samples were avoided from repeated freezing and thawing.

[0189] 2.5 Fluorescent quantitative PCR of related stress resistance genes in transgenic Arabidopsis

[0190] In order to explore HsNAC12 the downstream genes that this transcription factor may regulate, quantitative PCR was performed on the key genes involved in drought stress regulation in transgenic Arabidopsis. The above positive transgenic Arabidopsis and wild-type Arabidopsis were grown to 20 d, and 0.1 g of their leaves was taken to extract RNA and reverse transcribe cDNA as the template for fluorescent quantitative PCR. By consulting the literature, six key genes involved in drought response metabolic pathways were selected, including ATRD, ATSOD1, ATCAT1, ATWRKY66, ATCHR16, ATMYB32 The sequences of these genes were downloaded to design quantitative PCR primers, and the Actin gene of Arabidopsis was used as an internal reference gene for real-time fluorescent quantitative PCR. The primer sequences are shown in Table 14.

[0191] Table 14 Primers for Arabidopsis thaliana real-time PCR

[0192]

[0193] 2.6 Data Processing

[0194] Data processing and analysis were performed using Excel 2019 software, and graphing was performed using Prism software.

[0195] 3. Results and Analysis

[0196] 3.1 Subcellular localization

[0197] The built p35S:GFP-HsNAC12 , p35S: GFP Empty vector and nuclear localization marker were introduced into Agrobacterium strain GV3101, respectively. Positive strains were then cultured and the resulting OD values ​​were obtained after treatment. 600 A bacterial suspension of 1.0 was subsequently injected into tobacco leaves and cultured for 3 days. Observation was performed using a laser confocal microscope at excitation wavelengths of 488 nm and 561 nm. HsNAC12 Fluorescence signal of protein in tobacco leaf cells. The results are shown in the figure (e.g.) Figure 5 ), you can see HsNAC12 The green fluorescent signal of the protein and the red fluorescent signal of the nuclear localization marker overlap in the cell nucleus, thereby determining... HsNAC12 The protein is located in the cell nucleus.

[0198] 3.2 Arabidopsis transformation of the target gene

[0199] 3.2.1 Identification of positive transgenic plants

[0200] Will HsNAC12 Seeds of transgenic Arabidopsis thaliana were collected in 1.5 mL centrifuge tubes, dried in an oven, and stored under dry conditions. A portion of these seeds were sterilized and cultured on 0.9% agar medium for 4 days. After transferring to MS medium and culturing for 7–10 days, Arabidopsis thaliana seedlings with 4 leaves were selected and transplanted into nutrient soil. The seedlings were cultured at 24°C under 16 hours of light and 8 hours of darkness for 2–3 weeks. DNA was extracted from four transgenic Arabidopsis thaliana plants for PCR verification. The results are shown in the figure (e.g., ...). Figure 6 The transgenic plants all amplified bands consistent with the expected results, indicating that the transformation result was positive. OE1-3 were used for subsequent experiments.

[0201] wild type and 3 HsNAC12The seeds of transgenic Arabidopsis were grown on MS medium containing 0%, 10% PEG6000 and 30% PEG6000 for 14 days, respectively. The seeds of wild type Arabidopsis germinated normally and the seedlings grew vigorously on the medium without PEG, and the leaves were fresh green. With the increase of PEG concentration, the growth of seedlings was inhibited, and the wild type grew more weakly (such as Figure 7 , Figure 8 ). On the medium containing 30% PEG, the growth of three transgenic lines was better than that of WT, and the root length was longer, and the WT showed obvious leaf wilting.

[0202] 3.3 Effects of drought stress on the phenotype difference of transgenic Arabidopsis and the activities of antioxidant enzymes SOD, POD and CAT

[0203] In order to further verify the drought resistance function of the HsNAC12 gene, the wild type and transgenic Arabidopsis plants grown for 30 days under normal conditions were subjected to natural drought treatment. After 10 days of drought treatment, the phenotype changes were observed. The results showed that before drought treatment, the wild type and transgenic Arabidopsis grew well. After 10 days of drought treatment, the leaves of wild type and transgenic Arabidopsis plants wilted, but the leaves of wild type plants softened and necrotized seriously, while the leaves of most transgenic plants were still full and tough (such as Figure 10 ).

[0204] As the core defense enzyme system for scavenging reactive oxygen species (ROS), POD plays a key role in osmotic regulation and oxidative balance in abiotic stress response by catalyzing the dismutation reaction of superoxide anion (O2 - ·) to hydrogen peroxide (H2O2). The experimental data showed that there was no significant difference in the POD enzyme activity between wild type (WT) and transgenic tobacco plants under non-stress environment. When subjected to drought stress, the POD activity of both types of plants was induced to increase, and the increase of the enzyme activity of transgenic lines was significantly higher than that of the wild type. The POD activity of OE2 was significantly higher than that of OE1 and OE3, and the POD activity of OE1 and OE3 was about 2 times higher than that of WT (such as Figure 9A ).

[0205] As a key antioxidant enzyme system, SOD effectively reduces the membrane lipid peroxidation damage caused by ROS by mediating the catalytic conversion of hydrogen peroxide and phenolic substances, and plays an important regulatory function in maintaining the redox state of cells. Under non-stress conditions, the SOD enzyme activity of the wild type control (WT) and the transgenic lines showed no statistical difference, and the transgenic lines OE1 and OE2 showed a slight activity advantage, but did not reach a statistically significant level. When the system was subjected to drought stress, the SOD activity of the transgenic lines showed a significant up-regulation, and the enzyme activity level was significantly different from that of the WT group (p<0.05), and the OE2 strain showed the highest activity, which was 2.7 times higher than that of the wild type (as shown in Figure 9B ).

[0206] Catalase (CAT) plays a regulatory role in plant drought physiology. This enzyme effectively reduces the oxidative damage caused by the accumulation of intracellular ROS by catalyzing the enzymatic conversion of hydrogen peroxide (H2O2), and plays a key role in maintaining oxidative homeostasis. Experimental data show that under non-stress conditions, the CAT activity of the wild type control (WT) and the transgenic lines showed no statistical difference; drought stress treatment triggered the inducible up-regulation of CAT activity in all lines, and the enzyme activity of the transgenic line OE2 increased most significantly, and its activity level reached 3 times that of the wild type control, showing excellent antioxidant regulation ability, which is consistent with the higher activity of POD and SOD in this strain. The CAT activity of OE1 and OE3 is second, but it is significantly higher than that of the WT group (as shown in Figure 9C ).

[0207] 3.4 Effect of drought stress on malondialdehyde (MDA) in transgenic Arabidopsis thaliana

[0208] In plant physiological processes, when the plant is under stress conditions, the cell membrane system will trigger the membrane lipid peroxidation reaction, and the formation of malondialdehyde (MDA) marks the completion of this process. This lipid peroxidation product has high reactivity and can cross-link with biological macromolecules such as proteins and nucleic acids, resulting in loss of enzyme activity and damage to genetic material structure. By quantitatively detecting the accumulation level of MDA, the degree of damage to plant cells can be effectively evaluated, and important biomarker evidence can be provided for analyzing plant stress resistance mechanisms. From Figure 11 It can be seen that the MDA content of the transgenic lines is generally lower than that of the wild type (1.3 times), and the MDA content of the three transgenic plants is relatively close (7.3±1.2), which indicates that the transgenic lines exhibit better drought tolerance.

[0209] 3.5 Real-time fluorescence quantitative PCR of related drought resistance genes in transgenic Arabidopsis thaliana

[0210] To systematically analyze HsNAC12This study investigates the regulatory network of transcription factors in plant drought response using molecular biology techniques to explore the drought resistance mechanism of transgenic Arabidopsis thaliana. Based on previous phenotypic analysis, it was found that overexpression of… HsNAC12 The OE strain exhibited significantly enhanced drought tolerance compared to the wild-type (WT). To further elucidate its molecular mechanism, this study selected six key functional genes closely related to redox homeostasis, osmotic regulation, and stress signal transduction. AtWRKY66, AtMYB32, AtSOD1, AtCHR16, AtCAT1, AtRD29A The spatiotemporal expression profiles of the drought-treated group and the control group were analyzed using quantitative real-time reverse transcription PCR (qRT-PCR). The experimental results showed (e.g.) Figure 12 Under drought stress, the expression levels of six target genes in the OE lines showed a significant upregulation trend, with fold induction rates 1.5-8.8 times higher than those in the WT lines. Specifically: Antioxidant-related genes: Superoxide dismutase gene AtSOD1 (Average upregulation 6.9-fold) and catalase gene AtCAT1 Co-activation (upregulated 8.8-fold). Osmotic regulatory genes: Dehydration response genes AtRD29A Strong induction (upregulated 5.4-fold); transcriptional regulators: AtWRKY66 (Increased by 1.68 times) and AtMYB32 (Upgraded by 2.4 times) As a secondary regulatory node, it may participate in amplification. HsNAC12 Mediated stress signal cascades (e.g.) Figure 12 ).

Claims

1. A Huperzia spp. HsNAC12 gene, characterized in that, The nucleotide sequence of this gene is shown in SEQ ID NO.

1.

2. A recombinant expression vector, characterized in that, It contains the HsNAC12 gene of Huperzia spp. as described in claim 1.

3. A genetically engineered bacterium, characterized in that, It contains the recombinant expression vector as described in claim 2.

4. The application of the Huperzia spp. HsNAC12 gene as described in claim 1 in improving the drought stress resistance of Arabidopsis thaliana.

5. A method for improving plant drought stress tolerance, characterized in that, The HsNAC12 gene as described in claim 1 is expressed or overexpressed in Arabidopsis thaliana.

6. The method as described in claim 5, characterized in that, The Arabidopsis thaliana used is selected from parts that are 5-6 weeks old and growing healthily.

7. The method as described in claim 6, characterized in that, The Arabidopsis thaliana was transformed using Agrobacterium rhizogenes.

8. The method as described in claim 6, characterized in that, The Arabidopsis thaliana T0 generation seeds were disinfected with 75% ethanol for 5 minutes, and then washed 4-5 times with sterile deionized water.

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