Application of switchgrass pvhipp23 gene, recombinant vector containing the gene and recombinant bacteria

Abstract

Switchgrass PvHIPP23 The application of the gene, the recombinant vector containing the gene and the recombinant bacteria belongs to the technical field of genetic engineering. In order to improve the salt tolerance of switchgrass in a targeted manner and overcome the technical obstacles that the genetic resources of the HIPPs family protein cannot be effectively utilized for genetic improvement due to the lack of salt tolerance regulation mechanism cognition in the prior art, the present application clones and genetically transforms the switchgrass PvHIPP23 protein, and finds that PvHIPP23 The overexpression of the gene in switchgrass can improve the salt tolerance of the plant, and provides an important foundation for the molecular breeding and screening of germplasm resources of switchgrass.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to switchgrass. PvHIPP23 The application of genes, recombinant vectors containing these genes, and recombinant bacteria. Background Technology

[0002] Heavy metal-associated isoprenylated plant proteins (HIPPs) are a family of proteins unique to vascular plants. Their amino acid sequences exhibit highly conserved structural features, including a heavy metal-associated domain (HMA) capable of binding heavy metal ions and an isoprenylation site (CaaX motif) at the C-terminus. Studies have shown that HIPPs may act as metal chaperone proteins involved in the chelation, compartmentalization, and transport of heavy metal ions within cells, or as isoprenylated proteins anchored to the intracellular membrane system through lipid modification, thereby mediating protein-protein interactions and signal transduction processes.

[0003] In recent years, increasing evidence has shown that HIPPs family proteins play a crucial regulatory role in plant responses to abiotic stresses, particularly heavy metal stress, drought stress, and salinity stress. For example, in model plants such as Arabidopsis thaliana and rice, some HIPP family members have been shown to enhance plant resistance by regulating reactive oxygen species homeostasis, influencing ion balance, or participating in stress-related hormone signaling pathways. However, current research on the function of HIPPs family genes is relatively limited, mainly focusing on a few model plants such as Arabidopsis thaliana and rice, as well as some economic crops, while systematic research and functional identification in important forage and energy plants are very limited.

[0004] Willow branch millet ( Panicum virgatum Switchgrass (L.) is recognized as a second-generation bioenergy model plant and a high-quality forage resource, possessing excellent characteristics such as high biomass yield, tolerance to poor soil, and strong stress resistance. However, the molecular regulatory mechanisms of its tolerance to abiotic stresses such as salt remain unclear. Although the genome sequencing of switchgrass has been completed and gene family analysis is gradually underway, there are currently no publicly available functional studies internationally regarding the roles of HIPPs family members in stress adaptation. This research gap not only limits our understanding of the theory of stress resistance regulation in switchgrass but also hinders the process of variety improvement through molecular means to enhance its salt tolerance for cultivation on marginal land.

[0005] Therefore, based on the current technological background, it is urgent to conduct systematic functional studies on the HIPPs family genes of switchgrass to reveal their potential roles under adverse conditions such as salt stress response, thereby providing a theoretical basis and candidate gene resources for the genetic improvement of switchgrass's stress resistance. Summary of the Invention

[0006] To improve the salt tolerance of switchgrass and overcome the technical obstacle of not being able to effectively utilize its genetic resources for genetic improvement due to the lack of understanding of the salt tolerance regulation mechanism of HIPPs family proteins, this invention cloned and genetically transformed the switchgrass PvHIPP23 protein, revealing its functional role in switchgrass' response to salt stress.

[0007] To solve the above-mentioned technical problems and achieve the corresponding technical effects, the present invention provides the following technical solution: The first objective of this invention is to provide a switchgrass. PvHIPP23 The application of genes, namely, the use of switchgrass PvHIPP23 Genes were used to improve the salt tolerance of switchgrass, which... PvHIPP23 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0008] The second objective of this invention is to provide a switchgrass. PvHIPP23 The application of genes, specifically using switchgrass. PvHIPP23 Genetically bred switchgrass varieties with improved salt tolerance, the switchgrass PvHIPP23 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0009] A third object of the present invention is to provide an application of a recombinant vector containing switchgrass. PvHIPP23 Genes, the switchgrass PvHIPP23 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the application is to use the recombinant vector to improve the salt tolerance of switchgrass.

[0010] A fourth object of the present invention is to provide an application of a recombinant vector containing switchgrass. PvHIPP23 Genes, the switchgrass PvHIPP23 The nucleotide sequence of the gene is shown in SEQ ID NO.1. The application is to use the recombinant vector to breed switchgrass varieties with improved salt tolerance.

[0011] In one embodiment of the present invention, the skeleton of the recombinant vector is a binary expression vector pUbi1301.

[0012] The fifth object of the present invention is to provide a product containing the above-mentioned willow branch millet. PvHIPP23The application of the recombinant bacteria of the gene or the above-mentioned recombinant vector, wherein the application is to use the recombinant bacteria to improve the salt tolerance of switchgrass.

[0013] The sixth object of the present invention is to provide a product containing the above-mentioned willow branch millet. PvHIPP23 The application of the recombinant bacteria of the gene or the above-mentioned recombinant vector, wherein the application is to use the recombinant bacteria to cultivate switchgrass varieties with improved salt tolerance.

[0014] In one embodiment of the present invention, the recombinant bacteria is Agrobacterium.

[0015] The seventh object of the present invention is to provide a method for improving the salt tolerance of switchgrass, comprising the following steps: cloning the above-mentioned switchgrass. PvHIPP23 Genes; switchgrass PvHIPP23 Genes were constructed into expression vectors to obtain recombinant vectors; the recombinant vectors were introduced into Agrobacterium to obtain recombinant Agrobacterium; switchgrass was infected with recombinant Agrobacterium and overexpressing switchgrass was obtained through screening. PvHIPP23 Positive plants of the gene.

[0016] In one embodiment of the present invention, the expression vector is pUbi1301.

[0017] The beneficial effects of this invention are: This invention systematically analyzed the HIPPs family proteins of switchgrass and identified a total of 79 HIPPs family proteins in switchgrass. Among them, Pavir.2NG597900.2 clustered with AtHIPP23, and was therefore named PvHIPP23.

[0018] This invention performs subcellular localization analysis on switchgrass PvHIPP23 protein, and the results show that PvHIPP23 protein is located in the cell nucleus and cytoplasmic membrane. PvHIPP23 The gene was expressed in different tissues of switchgrass, with the lowest expression level in leaves and the highest expression level in R1 stage inflorescences.

[0019] This invention constructs PvHIPP23 Three gene overexpression and RNAi interference vectors were obtained in total. PvHIPP23 Overexpression of switchgrass lines and 21 strains PvHIPP23-RNAi Interfering with switchgrass lines. PvHIPP23 Overexpression of the gene in switchgrass enhances the plant's salt tolerance. PvHIPP23-RNAi Interference reduces the salt tolerance of switchgrass plants.

[0020] This study is the first to systematically analyze and identify the HIPPs family proteins of switchgrass, filling a gap in the research on switchgrass HIPPs; it also reveals for the first time the salt tolerance function of PvHIPP23 in switchgrass, providing an important foundation for molecular breeding of switchgrass for stress resistance and screening of germplasm resources. Attached Figure Description

[0021] Figure 1 Phylogenetic tree analysis diagram of Arabidopsis thaliana HIPPs, rice HIPPs and switchgrass HIPPs; Figure 2 For different tissues of switchgrass PvHIPP23 Gene expression level analysis relative to leaf expression level; all data are expressed as mean ± standard deviation (mean ± SD), n = 3; * and *** represent significant differences, respectively. P <0.05 and P <0.001; Figure 3 Image showing the subcellular localization of PvHIPP23 in tobacco leaves; scale bar size is 50 μm. Figure 4 for PvHIPP23 The image shows the PCR detection results of gene fragment amplification and recombinant vector; where (A) is... PvHIPP23 Amplification of gene fragments; M: Marker, 1-4: PvHIPP23 Gene fragment; (B) is pUbi1301-PvHIPP23 Agrobacterium single colony PCR detection; M: Marker, 1-3: pUbi1301-PvHIPP23 Agrobacterium-positive clones; (C) is PvHIPP23 Gene RNAi Amplification of interfering sequences; M: Marker, 1 and 2: PvHIPP23 Gene RNAi Interference fragments; (D) and (E) are PvHIPP23- RNAi PCR detection of Agrobacterium single colony interference vector; (D) PCR detection of forward-ligated interference fragment; (E) PCR detection of reverse-ligated interference fragment; M: Marker, +: PvHIPP23-RNAi Interference vector positive plasmid, -:H2O, 1-6: PvHIPP23-RNAi Agrobacterium-positive clones of the interference vector; Figure 5 The flowchart shows the genetic transformation process of switchgrass; (A) is the induction of callus; (B) is the screening and culture of resistant callus; (C) is the differentiation and culture of resistant callus; (D) is the rooting culture of resistant callus; and (E) is the culture of greenhouse plants. Figure 6 for PvHIPP23 overexpression and PvHIPP23-RNAi PCR identification results of interference with switchgrass; where (A) and (B) are... PvHIPP23 Identification of overexpressing plants; M: DNA marker; +: PvHIPP23Overexpression vector positive plasmid; -: H2O; WT: wild-type switchgrass; OE1-OE3: PvHIPP23 Overexpression of switchgrass positive lines; (C)-(F) are PvHIPP23- RNAi Identification of interfering plants; (C) and (E) are PCR identifications of forward-ligated interfering fragments; (D) and (F) are PCR identifications of reverse-ligated interfering fragments; M: DNA marker; +: PvHIPP23-RNAi Interference vector positive plasmid; -: H2O; WT: wild-type switchgrass; RNAi-PvHIPP23 (1): PvHIPP23-RNAi Detection of forward connectivity interference fragments interfering with switchgrass; RNAi- PvHIPP23 (2): PvHIPP23-RNAi Detection of inverse connection interference fragments interfering with switchgrass; 1-33: PvHIPP23-RNAi Interfering with different strains of switchgrass; Figure 7 In transgenic lines PvHIPP23 The results of gene expression level analysis are shown in the figure; where (A) is... PvHIPP23 Overexpression lines PvHIPP23 Gene expression level; (B) is PvHIPP23-RNAi In the interference strains PvHIPP23 Gene expression levels; WT: wild-type switchgrass; OE1-OE3: PvHIPP23 Positive lines overexpressing switchgrass; RNAi: PvHIPP23- RNAi Positive lines of switchgrass that interfere with the growth of switchgrass; *, * and *** represent different transgenic lines and WT strains, respectively. PvHIPP23 The differences in gene expression levels were statistically significant, respectively. P <0.05, P <0.01 and P <0.001; Figure 8 for PvHIPP23 Figure showing the analysis results of expression induced by salt stress; under control conditions (day 0). PvHIPP23 Gene expression levels in the aboveground parts and roots were normalized to 1; data are represented as mean ± SD, n = 3; * and *** represent different treatment times and control conditions (day 0), respectively. PvHIPP23 The differences in gene expression levels were statistically significant, respectively. P <0.05, P <0.01 and P <0.001; Figure 9 for PvHIPP23Salt stress analysis of detached leaves of transgenic switchgrass; 0 mM, 200 mM, and 300 mM represent different concentrations of NaCl solution; WT: wild-type switchgrass; OE1-OE3: PvHIPP23 Overexpression of different switchgrass lines; RNAi-15, RNAi-20, and RNAi-21: PvHIPP23-RNAi Different strains of switchgrass were disturbed; the scale bar was 2 cm. Figure 10 for PvHIPP23 Phenotypic representation of salt stress in switchgrass plants overexpressing NaCl; 0 d and 46 d represent 0 days and 46 days of 350 mM NaCl stress, respectively; CK represents the blank control group, and NaCl represents the salt treatment group; WT: wild-type switchgrass; OE1-OE3: PvHIPP23 Overexpression of different strains of switchgrass; Figure 11 After salt stress PvHIPP23 The results of biomass and physiological indicators of switchgrass overexpression plants are shown in the figure. (A) represents the fresh weight of the aboveground parts and roots; (B) represents the dry weight of the aboveground parts and roots; (C) represents the relative water content of the leaves; (D) represents the chlorophyll content of the leaves. CK represents the blank control group, and NaCl represents the salt treatment group. Data are expressed as mean ± SD, n ≥ 3; * and *** represent... PvHIPP23 The differences between different strains of switchgrass overexpressing and WT were statistically significant. P <0.05, P <0.01 and P <0.001; Figure 12 for PvHIPP23-RNAi Phenotypic diagram of salt stress in switchgrass plants; 20 d and 66 d represent 20 days and 66 days of 350 mM NaCl stress, respectively; CK represents the blank control group, and NaCl represents the salt treatment group; WT: wild-type switchgrass; RNAi-2, RNAi-22, and RNAi-24: PvHIPP23-RNAi Interfering with different strains of switchgrass; Figure 13 After salt stress PvHIPP23-RNAi Figure 1 shows the results of biomass and physiological index detection of switchgrass plants after interference. (A) represents the fresh weight of the aboveground parts and roots; (B) represents the dry weight of the aboveground parts and roots; (C) represents the relative water content of the leaves; (D) represents the chlorophyll content of the leaves. CK represents the blank control group, and NaCl represents the salt treatment group. Data are expressed as mean ± SD, n ≥ 3. * and ** represent... PvHIPP23-RNAi The differences between different strains of switchgrass and WT were statistically significant. P <0.05 and P <0.01. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be noted that the embodiments mentioned below are only for explaining the invention and are not intended to limit the scope of the invention. The embodiments mentioned below are only some embodiments of the invention, not all embodiments. Those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the objectives of the invention. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content and scope of this invention to realize and apply the technology of this invention. In the art, embodiments obtained by other those skilled in the art without creative effort are all protected by this invention.

[0023] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials, reagents, culture media and instruments used are conventional materials, reagents, culture media and instruments in the art, which can be obtained by those skilled in the art through commercial channels.

[0024] The plant materials used in this invention are as follows: Using seeds of the lowland cultivar Alamo (from Ernst Conservation Seeds, USA) as material, callus induction and differentiation and plant culture were performed. Stem tissues from switchgrass plants at the E2 stage were collected, and total RNA was extracted. Leaf tissues from E2 stage plants were collected for DNA and RNA extraction into transgenic plants. Switchgrass seedlings were used as material for gene expression analysis in response to salt stress. PvHIPP23 Transgenic switchgrass plants (overexpression lines and interference lines) were used for salt stress experiments on detached leaves and plants. Tobacco plants at the 2-3 leaf stage were used for subcellular protein localization analysis.

[0025] The strains used in this invention are as follows: Escherichia coli ( Escherichia coli , E.coli Competent DH5α cells are used for transformation and propagation of recombinant plasmid vectors; Agrobacterium ( Agrobacterium tumefaciens EHA105 competent cells were used for binary expression vector transformation and genetic transformation of switchgrass callus, while GV2260 competent Agrobacterium cells were used for subcellular transformation of tobacco.

[0026] The reagents used in this invention are as follows:The Blunt Kination Ligation (BKL) Kit was purchased from TaKaRa; the pClone007 Blunt Simple Vector Kit was purchased from Beijing Qingke Xinyue Biotechnology Co., Ltd.; the introductory vectors pKannibal, pDONR207, and the binary expression vector pCAMBIA1305.2 were all purchased from Youbao Biotechnology Co., Ltd.; the pMDC83 vector was purchased from Shanghai Qincheng Biotechnology Co., Ltd.; the binary expression vector pUbi1301 was purchased from Beijing Xinno Jinda Biotechnology Co., Ltd.; the plant tissue RNA extraction kit was purchased from Beijing Huayueyang Biotechnology Co., Ltd.; the TKS Gflex DNA Polymerase kit, the PrimeScript RT reagent kit with gDNA Eraser reverse transcription kit, and the TB Green™ Premix Ex Taq™ II (Tli RNaseH) kit were also purchased. The Plus kits were all purchased from Beijing TaKaRa Co., Ltd.; the microcolumn DNA gel extraction kit was purchased from Beijing ZOMANBIO International Biotechnology Co., Ltd.; the AxyPrep plasmid DNA mini-extraction kit was purchased from Corning Life Sciences (Wujiang) Co., Ltd.; BP enzyme (Invitrogen, Gateway BPClonase), LR enzyme (Invitrogen, Gateway LR Clonase), T4 ligase, restriction endonuclease, and 10 × CutSmart Buffer were purchased from New England Biolabs (NEB); 1 × TAE solid powder, agarose solid powder, nucleic acid dye, DNA Marker, 6 × loading buffer, and 6 × gelloading dye were purchased from Beijing GenStar Biotechnology Co., Ltd.; yeast extract, tryptone, maltose, sucrose, agar, proline, plant gel, and MS medium (Phyto Technology Laboratories, catalog number 100000) were purchased from Beijing GenStar Biotechnology Co., Ltd. M519), 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzylaminopurine (6-BA), naphthaleneacetic acid (NAA), gibberellin (GA), acetylsyringone (As), glutamine (Gln), ampicillin (Amp), kanamycin (Kan), rifampin (Rif), gentamicin, spectinomycin, hygromycin (50 mg / mL), and termethin were all purchased from Sigma-Aldrich.Sodium hypochlorite, ethylenediaminetetraacetic acid (EDTA), tris(hydroxymethyl)aminomethane (Tris), hexadecanetrimethylammonium bromide (CTAB), 2-(N-morpholino)ethanesulfonic acid (MES), dimethyl sulfoxide (DMSO), magnesium chloride hexahydrate, sodium chloride, sodium hydroxide, concentrated hydrochloric acid, chloroform, isopropanol, 95% ethanol, 75% ethanol, anhydrous ethanol, glycerol, trichloroacetic acid (TCA), thiobarbituric acid (TBA), phosphoric acid, glacial acetic acid, sulfosalicylic acid, ninhydrin, toluene, and other reagents were purchased from Beijing Guangda Hengyi Technology Co., Ltd.; 100 × modified Hoagland's nutrient solution (NS1010-50L) was purchased from Beijing Kulaibo Technology Co., Ltd.

[0027] The culture medium involved in this invention is as follows: MB induction medium (1 L): 4.428 g MS, 30 g maltose, 5 mg 2,4-D, 1 mg 6-BA, dissolved in deionized water, pH adjusted to 5.8, brought to volume with deionized water, added 4 g plant gel, and autoclaved. MP maintenance medium (1 L): 4.428 g MS, 30 g maltose, 5 mg 2,4-D, 1 mg 6-BA, 2 g proline, dissolved in deionized water, pH adjusted to 5.8, brought to volume with deionized water, added 4 g plant gel, and autoclaved. MP liquid medium (1 L): 4.428 g MS, 30 g maltose, 5 mg 2,4-D, 1 mg 6-BA, 2 g proline, dissolved in deionized water, pH adjusted to 5.4, brought to volume with deionized water, and autoclaved. REG differentiation medium (1 L): 4.428 g MS, 30 g maltose, 1 mg 6-BA, 0.5 mg GA, 0.2 mg GA, 0.2 mg 6-BA ... mg NAA, dissolved in deionized water, pH adjusted to 5.8, brought to volume with deionized water, added 4 g plant gel, and autoclaved; MS rooting medium (1 L): 4.428 g MS, 30 g maltose, dissolved in deionized water, pH adjusted to 5.8, brought to volume with deionized water, added 4 g plant gel, and autoclaved; LB liquid medium (1 L): 5 g yeast extract, 10 g tryptone, 10 g NaCl, dissolved in deionized water, pH adjusted to 7.0, brought to volume with deionized water, and autoclaved; LB solid medium (1 L): 5 g yeast extract, 10 g tryptone, 10 g NaCl, dissolved in deionized water, pH adjusted to 7.0, brought to volume with deionized water, added 15 g Agar, and autoclaved; YEP liquid medium (1 L): 10 g yeast extract, 10 g tryptone, 5 g Dissolve NaCl in deionized water, adjust pH to 7.0, bring to volume with deionized water, and autoclave. YEP solid medium (1 L): 10 g yeast extract, 10 g tryptone, 5 g NaCl, dissolved in deionized water, adjusted pH to 7.0, brought to volume with deionized water, added 15 g Agar, and autoclave.

[0028] All primers used in this invention were designed using Primer 5 software and synthesized by Sangon Biotech (Shanghai) Co., Ltd. The sequence information of the primers used is shown in Table 1.

[0029] Table 1 Primer sequence information

[0030] The data statistics and analysis methods used in this invention are as follows: Excel and SPSS 23 were used for statistical analysis and graphing. The Student's t-test method was used for significant data differences, and one-way ANOVA and Duncan's method were used for multiple comparisons. P <0.05) detection method.

[0031] Example 1: Bioinformatics Analysis of PvHIPP23 To investigate the members of the switchgrass HIPPs protein family, the switchgrass genome database was searched in the Phytozome 14 database (https: / / phytozome-next.jgi.doe.gov / ). Panicum virgatum var. AP13HAP1 v6.1 was used, and the whole genome sequence of switchgrass was downloaded. Forty-five Arabidopsis HIPPs protein sequences were downloaded from the Arabidopsis information website Tair (https: / / www.arabidopsis.org / ), and analyzed using Blastp (E-value ≤1e -5 Local alignment of switchgrass protein sequences was performed. Hidden Markov model files (HMA domain: PF00403) corresponding to the HIPP domain were downloaded from the Pfam protein family database (http: / / pfam.xfam.org). HMMER 3.0 (http: / / hmmer.org / ) was used to search for HIPPs in the switchgrass genome database. The intersection of the two results was taken, and then Pfam, SMART, and Interpro were used to confirm the presence of the HMA domain. Finally, it was confirmed whether the C-terminus of the HIPPs protein sequences contained a conserved CaaX motif. Only sequences containing the HMA domain and the C-terminus with a conserved CaaX motif were retained. Fifty-nine rice OsHIPPs protein sequences were downloaded from the rice genome database (https: / / www.ricedata.cn / ). Phylogenetic analysis was performed on the obtained protein sequences of switchgrass PvHIPPs, 45 Arabidopsis thaliana AtHIPPs, and 59 rice OsHIPPs. Multiple sequence alignment was performed using MAFFT software, and phylogenetic trees were constructed using the Maximum Likelihood (ML) method in MEGA11 software with the Bootstrap parameter set to 1000. Finally, iTOL (https: / / itol.embl.de / ) was used for phylogenetic tree visualization and enhancement.

[0032] Using the methods described above, 79 HIPPs proteins were identified in the switchgrass database. These proteins all contain a conserved CysXXCys motif in their HMA domains and possess a C-terminal CaaX isopreneylylation motif. To reveal the evolutionary relationship between PvHIPPs and model plants Arabidopsis and rice HIPPs, a phylogenetic tree was constructed for 45 Arabidopsis HIPPs (AtHIPPs), 59 rice HIPPs (OsHIPPs), and 79 switchgrass HIPPs (PvHIPPs). The results are as follows: Figure 1 As shown in the figure, all HIPPs proteins were divided into 5 groups, and members of each group showed close evolutionary associations, suggesting that they may have functional similarities. Group I consists of 11 PvHIPPs, 13 AtHIPPs, and 17 OsHIPPs; Group II consists of 23 PvHIPPs, 7 AtHIPPs, and 21 OsHIPPs; Group III contains 16 PvHIPPs, 8 AtHIPPs, and 10 OsHIPPs; Group IV is the smallest group, containing only 2 PvHIPPs and 3 AtHIPPs; Group V is the largest group, containing 27 PvHIPPs, 14 AtHIPPs, and 11 OsHIPPs. Among them, Pavir.2NG597900.2 clustered with Pavir.2KG543800.1, OsHIPP45, and AtHIPP23. Based on Arabidopsis thaliana, Pavir.2NG597900.2 was named PvHIPP23. PvHIPP23 ( Pavir.2NG597900.2 The full-length CDS sequence of the gene is 477 bp (SEQ ID NO.1), encoding 158 amino acid residues (SEQ ID NO.2). The conserved structural domains of the protein predict that it contains one heavy metal associated domain (HMA) and one isoprene-modified CaaX motif (CSVM), making it a typical isoprene-modified protein.

[0033] SEQ ID NO.1: ATGGGGGGCACCTTGGAGTACCTATCGGGACTGCTGGGAGGGAGCGGAGGCCATGGCCACGAGAAGACGAAGAAGAGGAAGCAGCTGCAGACTGTGGAGCTCAAGGTCAGGATGGACTGCGAGGGCTGTGAGCTCAAGGTCAAGAGCGCCCTCTCCTCCATGAAAGGTGTCGAGTCGGTGGAGATAAACCGGAAGCAGCAGAAGGTGACGGTGGTCGGGTACGTGGAGGCCGGCAAGGTGCTGAAGAAGGCGCAGTCGACGGGGAAGAAGGCCGAGATCTGGCCCTACGTGCCCTACAGCCTGGTGAGCCAGCCGTACGTCGCCGGCACCTACGACAAGCGCGCCCCGCCGGGCTACGTCCGGAGCGCTGAGCCCGGCTACGCGCCCAGCGTCCAGCAGCAGCAGCTCGGCCGGCCGCATGACCACCTCACCGACATGTTCAACGACGAGAACCCGAATTCCTGCTCGGTCATGTGA; SEQ ID NO.2: MGGTLEYLSGLLGGSGGHGHEKTKKRKQLQTVELKVRMDCEGCELKVKSALSSMKGVESVEINRKQQKVTVVGYVEAGKVLKKAQSTGKKAEIWPYVPYSLVSQPYVAGTYDKRAPPGYVRSAEPGYAPSVQQQQLGRPHDHLTDMFNDENPNSCSVM。

[0034] Example 2: PvHIPP23 Gene expression analysis Root tissue, tissue from the second internode (from bottom to top), leaf tissue from the second fully expanded leaf (approximately 1 cm from the base), tillers, and tissue from R1 and R3 inflorescences of switchgrass plants at the E3 stage were collected and flash-frozen in liquid nitrogen for RNA extraction and RT-qPCR analysis. Total RNA extraction from plant tissues followed the method described in the Rapid Universal Plant RNA Extraction Kit from Beijing Huayueyang Biotechnology Co., Ltd. Reverse transcription of plant tissue RNA followed the method described in the TaKaRa PrimeScript RT reagent Kit with gDNA Eraser. Real-time quantitative PCR was performed using the method described in the TaKaRa TB Green™ Premix Ex Taq™ II (Tli RNaseH Plus) kit. PvHIPP23 The primers used for relative gene expression levels were qPvHIPP23-F and qPvHIPP23-R, and the primers for the internal reference gene were qUbiquitin-F and qUbiquitin-R.

[0035] Different tissues of switchgrass Alamo variety PvHIPP23 Gene expression levels were analyzed, and the results were as follows: Figure 2 As shown in the figure. PvHIPP23 The gene was expressed in leaves, internodes, roots, R1 stage inflorescences, R3 stage inflorescences, and tillers, with the lowest expression level in leaves, compared to the lowest level in leaves. PvHIPP23 Compared to other tissues, gene expression levels were lower. PvHIPP23 Gene expression levels were significantly increased ( P <0.05), where PvHIPP23 The gene expression level was highest in the R1 stage inflorescence, and then in the leaves. PvHIPP23 The gene expression level was 47.08 times higher than that of R3 stage inflorescences, roots, internodes, and tillers.

[0036] Example 3: Subcellular localization analysis of PvHIPP23 1. Construction of PvHIPP23 subcellular localization vector To determine the localization of PvHIPP23, a localized expression vector for PvHIPP23 was constructed. The specific steps are as follows: Based on the switchgrass database... PvHIPP23Full-length CDS was obtained, and gene primers gfp-PvHIPP23-F and gfp-PvHIPP23-R with BP adapters and no stop codons were designed. Using *Symplocos spp.* stem tissue cDNA as a template, PCR amplification was performed using high-fidelity DNA polymerase (PCR reaction system shown in Table 2, reaction procedure shown in Table 3). The PCR amplification products were detected by agarose gel electrophoresis, and gene fragments with correct bands were recovered from the gel, following the method of the ZOMANBIO microcolumn concentrated DNA gel recovery kit from Beijing Zhuangmeng International Biotechnology Co., Ltd. The recovered DNA was used as a template for a BP reaction (reaction system shown in Table 4). The reactants were added to the bottom of centrifuge tubes, mixed well, and incubated at 25°C for 2 h. The reaction products were then transformed into *E. coli* competent cells DH5α, and single colonies (15 cells per ... PCR identification was performed using mg / L gentamicin screening. After successful identification, the samples were sent to the company for sequencing using pDonr-F and pDonr-R primers. The correctly sequenced *E. coli* were then amplified, and plasmids were extracted using the AxyPrep plasmid DNA mini-extraction kit. The plasmid DNA was then mixed with... GFP The subcellular localization vector pMDC83 was mixed and subjected to an LR reaction (reaction system shown in Table 5). The reactants were added to the bottom of a centrifuge tube, mixed well, and incubated in a metal bath at 25°C for 2 h. The reaction product was then transformed into E. coli competent cells DH5α. Single colonies were selected (screened with 100 mg / L kanamycin) for PCR identification. After correct identification, the samples were sent to the company for sequencing. The sequencing primers were pMDC83-F and pMDC83-R. The plasmid with correct sequencing was transformed into Agrobacterium tumefaciens competent cells GV2260.

[0037] Table 2 PCR amplification reaction system

[0038] Table 3 PCR amplification reaction procedure

[0039] Table 4 BP Reaction System

[0040] Table 5 LR reaction system

[0041] 2. Instantaneous transformation of tobacco leaves Will contain PvHIPP23 Agrobacterium-mediated transient transformation of tobacco leaves to produce a product containing... GFP The pMDC83 empty vector was used as a control. The specific steps were as follows: Agrobacterium tumefaciens that was correctly identified by PCR was cultured overnight at 28°C; the bacterial culture was transferred to fresh YEP liquid medium, and acetylsuccinone was added to a final concentration of 100 μM; the OD of the bacterial culture was measured.600 The concentration was approximately 1.0-1.5%. Centrifuge at 6000 rpm for 15 min to collect bacteria; remove the supernatant, add tobacco conversion broth to the centrifuge tube, and resuspend to OD. 600 It is around 1.0; it will contain PvHIPP23-GFP Recombination carrier, AHL22-mRFP Nuclear location markers, P19 Add the tobacco conversion solution of the inhibitor to a new centrifuge tube at a volume ratio of 1:1:1, mix well, and place in the dark for 1-2 hours. Select tobacco leaves of suitable size and with plump leaves, and inject the mixture of tobacco conversion solution into the back of the tobacco leaves using a disposable syringe, and mark the leaves. Place the injected tobacco leaves in the dark for 2-3 hours, and then move them to light to grow for 2-3 days. Observe the protein expression in the tobacco under a Nikon A1 laser confocal microscope (excitation light 488 nm, scanning light 505-530 nm).

[0042] Subcellular localization results showed that, similar to the control GFP, the fluorescence signal of PvHIPP23-GFP also appeared in the cell nucleus and cytoplasmic membrane. Figure 3 This indicates that the PvHIPP23 protein is located in the cell nucleus and cell membrane.

[0043] Example 4: Preparation of transgenic switchgrass plants 1. PvHIPP23 Construction of gene overexpression vectors Design with enzyme cleavage sites PvHIPP23 Gene cloning primers PvHIPP23-MF and PvHIPP23-MR were used. Total RNA was extracted from the stems of wild-type Alamo switchgrass plants. Using the reverse-transcribed cDNA as a template, PCR amplification was performed using high-fidelity DNA polymerase, and the product was recovered from the gel. The T-vector was ligated to the gel-recovered DNA using the TaKaRa Blunt Kination Ligation (BKL) Kit method. The ligation product was transformed into *E. coli* competent cells DH5α. Single colonies were selected (screened with 100 mg / L ampicillin) for PCR identification. After successful identification, the colonies were sent for sequencing using primers M13-F and M13-R. The correctly sequenced plasmid and the binary expression vector plasmid pUbi1301 were double-digested with restriction endonucleases. Bam HI and Kpn I. The double enzyme digestion system is shown in Table 6. After mixing the reaction solution, incubate at 37℃ in a metal bath for 3 h, then add 8 μL of 6× gel loading dye to stop the enzyme digestion reaction. After digestion, the products are separated by gel electrophoresis, and the corresponding bands are recovered from the gel. PvHIPP23The gene fragment and plasmid pUbi1301 were ligated, and the reaction system is shown in Table 7. After reacting at room temperature for 10 min, the ligation product was transformed into E. coli competent cells DH5α. Single colonies were selected (screened with 100 mg / L kanamycin) for PCR identification. The samples were sent to the company for sequencing, and the vector sequencing primers were ZmUbi-F and Noster-R. The recombinant vector plasmid with correct sequencing was then analyzed. pUbi1301-PvHIPP23 Enzyme digestion verification was performed (single enzyme digestion reaction system is shown in Table 8, double enzyme digestion reaction system is shown in Table 6). After mixing the reaction solution, the reaction was carried out in a metal bath at 37℃ for 15 min. 8 μL of 6× gel loading dye was added to stop the enzyme digestion reaction, and electrophoresis was performed for verification. The recombinant plasmid with correct enzyme digestion verification was transformed into Agrobacterium competent cells EHA105. Single colonies were selected (screened with 100 mg / L kanamycin). After PCR identification, the colonies were preserved for later use.

[0044] Table 6. Double enzyme digestion reaction system

[0045] Table 7 T4 enzyme ligation reaction system

[0046] Table 8 Single enzyme digestion reaction system

[0047] The above experimental procedure successfully obtained the correct size (477 bp) through PCR amplification using specific primers. PvHIPP23 Gene fragments ( Figure 4 (A) in the middle. PvHIPP23 After gene fragments were sequenced and verified, they were ligated into the pUbi1301 binary expression vector, and the recombinant plasmid was transformed into Agrobacterium EHA105 competent cells. The PCR verification results of the transformed Agrobacterium are as follows: Figure 4 As shown in (B) above, a successful conversion was achieved. PvHIPP23 Overexpression of Agrobacterium.

[0048] 2. PvHIPP23 Construction of gene RNAi interference vector The design size is approximately 200 bp. PvHIPP23 Gene RNAi Interference fragments were identified, and PCR amplification primers Pvhipp23-MF and Pvhipp23-MR with restriction enzyme sites were designed. PvHIPP23 Using the gene fragment as a template, PCR amplification was performed using high-fidelity DNA polymerase, and the product was then recovered via gel extraction. The interference fragment recovered from the gel and the pKannibal introductory vector were subjected to a first double digestion with restriction endonucleases of [specific enzyme name missing]. Sal I and EcoRI was performed, and the interference fragment was forward ligated to the vector. The ligation product was transformed into E. coli competent cells DH5α, and single colonies were selected (screened with 15 mg / L gentamicin). After PCR confirmation, the plasmid was extracted and used... Sal I and Eco RI was used for restriction enzyme digestion verification. The interfering fragment recovered from the gel and the correctly verified plasmid were subjected to a second double digestion, using the following restriction endonuclease: Xba I and Hin dIII. Reverse ligation of the interfering fragment and the recombinant vector, transformation of the ligation product, PCR identification, extraction of the recombinant plasmid, and... Xba I and Hin dIII restriction enzyme digestion verification. The recombinant plasmid that was correctly digested was mixed with the binary expression vector pCAMBIA1305.2 and subjected to an LR reaction. The product was transformed into DH5α, and single colonies were selected (screened with 100 mg / L kanamycin). After PCR identification, the colonies were sent to the company for sequencing using ZmUbi-F and M13-R primers. The recombinant plasmid was transformed into Agrobacterium competent cells EHA105, and single colonies were selected (screened with 100 mg / L kanamycin). After PCR identification of positive clones, the cells were preserved for later use.

[0049] In the above experiment, with PvHIPP23 Using a gene fragment as a template, PCR amplification with specific primers yields an interference sequence fragment of the correct size. Figure 4 (C)). The interfering fragment was ligated to the vector plasmid through two ligation reactions. After correct sequencing, the recombinant vector was subjected to an LR reaction with the pCAMBIA1305.2 binary expression vector to obtain the ligated pCAMBIA1305.2. PvHIPP23-RNAi Interference vector. The recombinant vector was transformed into Agrobacterium EHA105 competent cells, and after verification by bacterial culture PCR, the interference vector was successfully constructed. PvHIPP23-RNAi Interference carrier ( Figure 4 (D) and (E) in the text.

[0050] 3. Genetic transformation of switchgrass Will contain PvHIPP23 Gene overexpression vectors and RNAi Agrobacterium interfering with the vector was used to transform switchgrass callus tissue to obtain transgenic switchgrass material (see flowchart). Figure 5 The specific method is as follows: (1) Preparation of Agrobacterium tumefaciens bacterial culture: Agrobacterium tumefaciens was inoculated into 5 mL of YEP liquid medium containing 100 mg / L kanamycin and 50 mg / L rifampin, and shaken overnight at 200 rpm in a shaker at 28°C; the bacterial culture was then transferred to 15 mL of YEP liquid medium containing 100 mg / L kanamycin and 50 mg / L rifampin, and cultured at 200 rpm in a shaker at 28°C until OD was reached.600 The bacterial culture was centrifuged at 3000 rpm for 10 min, the supernatant was discarded, and the cells were resuspended in MP liquid medium containing 100 μM acetylsyringone at pH 5.4 until the OD value was 0.8-1.0. 600 Set the temperature to 0.8-1.0 and incubate at 28°C for 30 minutes.

[0051] (2) Induction and culture of callus: Mature seeds of switchgrass (Alamo) were disinfected with 5% sodium hypochlorite for 1 h, washed 3-5 times with sterile ddH2O, soaked in sterile ddH2O and placed at 4℃ for 12 h, soaked in 5% sodium hypochlorite for 30 min and washed 3-5 times with sterile water, and then placed in MB induction medium and cultured in the dark at 26℃ for 4-6 weeks. The core callus was removed from the callus tissue and transferred to MP maintenance medium and cultured in the dark at 26℃ for 4 weeks. The callus tissue was cut into small pieces and transferred to new MP maintenance medium and cultured in the dark at 26℃ for 4 weeks.

[0052] (3) Genetic transformation of callus tissue: Select callus tissue with good growth and a size of about 2 × 2 mm. 2 Callus tissue from *Stichopus japonicus* was placed in a 50 mL sterile centrifuge tube, and 10 mL of 3% maltose solution containing 300 μM glutamine was added. The mixture was incubated on ice for 20 min, and the maltose solution was discarded. 10 mL of the prepared *Agrobacterium* culture was added to the centrifuge tube, along with 100 μM acetylsyleugenol and 300 μM glutamine. The mixture was vacuum-sealed for 10 min and incubated at 28 °C with gentle shaking for 20 min. The culture solution was discarded, and the infected callus tissue was placed on three layers of sterile filter paper and dried in a laminar flow hood for 2 h. The callus tissue was then transferred to two layers of sterile filter paper, and 500 μL of MP liquid medium containing 300 μM glutamine and 100 μM acetylsyleugenol was added. The mixture was incubated in the dark at 26 °C for 2 d.

[0053] (4) Screening of resistant callus: The callus was transferred to MP maintenance medium containing 250 mg / L termethin and cultured in the dark at 26°C for 1 week; the callus was transferred to MP maintenance medium containing 50 mg / L hygromycin and 150 mg / L termethin and cultured in the dark at 26°C for 2 weeks; the callus was transferred to MP maintenance medium containing 100 mg / L hygromycin and 150 mg / L termethin and cultured in the dark at 26°C for 2 weeks.

[0054] (5) Differentiation and transplanting of resistant callus: The resistant callus was transferred to REG differentiation medium containing 20 mg / L hygromycin and 150 mg / L termethin; the differentiated seedlings were transferred to MS rooting medium containing 50 mg / L hygromycin and 150 mg / L termethin to root; after hardening off in the culture room for 1-2 days, the seedlings were transplanted into flower pots containing a mixed nutrient soil of humus, black soil and vermiculite in a ratio of 1:1:1 and cultured in a greenhouse.

[0055] 4. Identification of positive transgenic switchgrass seedlings (1) DNA extraction from transgenic switchgrass plants Positive seedling identification of transgenic switchgrass plants was performed, using wild-type switchgrass plants as a control (WT). DNA was extracted from different transgenic lines using the CTAB method, with the following steps: [The text then abruptly shifts to a different topic:] ...containing 2 × 2 cm... 2 Add 100 μL of CTAB buffer to centrifuge tubes containing small amounts of switchgrass leaf tissue, and break up the sample; add 400 μL of CTAB buffer, incubate at 65°C for 30 min; cool to room temperature, add 500 μL of chloroform, mix well, and centrifuge at 12,000 rpm for 10 min; take the supernatant, add an equal volume of isopropanol, mix well, centrifuge at 12,000 rpm for 10 min, and discard the supernatant; add 1 mL of 75% ethanol, mix well, centrifuge at 12,000 rpm for 5 min, and discard the supernatant; centrifuge again at 12,000 rpm for 2 min, discard the supernatant, and air dry for 5 min; add 30-50 μL of ddH2O to dissolve the precipitate, and determine the DNA concentration.

[0056] (2) PvHIPP23 Identification of positive seedlings overexpressing switchgrass by PvHIPP23 Using switchgrass DNA overexpressed as a template and switchgrass DNA derived from the same callus but without transgenesis as a control, PCR amplification was performed using the upstream primer ZmUbi-F and the downstream primer PvHIPP23-R (reaction system shown in Table 9, reaction procedure shown in Table 10). The PCR products were then analyzed by agarose gel electrophoresis to identify the gene. PvHIPP23 Positive seedlings of switchgrass with overexpressed genes.

[0057] Table 9 PCR amplification reaction system

[0058] Table 10 PCR Amplification Reaction Procedure

[0059] (3) PvHIPP23-RNAi Interference with the identification of positive seedlings of switchgrass by PvHIPP23 - RNAi Using switchgrass plant DNA as a template and non-transgenic switchgrass plant DNA derived from the same callus as a control, the upstream primer ZmUbi-F of the interference vector was used. PvHIPP23 Gene RNAi The downstream primer Pvhipp23-R of the interfering fragment identifies the forward linker. RNAi Interference fragments, withPvHIPP23 Gene RNAi The downstream primer Pvhipp23-R of the interfering fragment and the downstream primer Noster-R of the interfering vector were used to identify the reverse linker. RNAi Interference fragments, and then identification PvHIPP23 - RNAi Positive seedlings that interfere with switchgrass plants.

[0060] For the obtained PvHIPP23 overexpression and PvHIPP23-RNAi Interference was used to culture switchgrass plants until they reached the E2 stage, at which point DNA was extracted from leaf tissue for further analysis. PvHIPP23 Identification of transgenic plants, results as follows Figure 6 As shown. A total of 3 were identified. PvHIPP23 Positive lines OE1, OE2, and OE3 overexpressing switchgrass ( Figure 6 (A) and (B) in the middle); 27 were identified. PvHIPP23-RNAi Interference was performed on switchgrass lines, and RNAi-1, RNAi-18, and RNAi-33 only detected positive interference fragments, but no negative interference fragments were detected. Therefore, a total of 24 lines were obtained. PvHIPP23-RNAi Positive strains of switchgrass that interfere with ( Figure 6 (C)-(F) in the middle.

[0061] (4) In transgenic switchgrass plants PvHIPP23 Analysis of gene expression levels For identification PvHIPP23 In transgenic switchgrass positive plants PvHIPP23 Gene expression levels were measured using switchgrass from the same period, non-transgenic, and callus-derived plants as a control, and were extracted from plants grown to the E2 stage. PvHIPP23 RNA from leaf tissues of different transgenic switchgrass lines. Gene primers qPvHIPP23-F and qPvHIPP23-R, and internal control primers qUbiquitin-F and qUbiquitin-R were used to detect RNA in the leaf tissues of transgenic switchgrass plants. PvHIPP23 Analysis of gene expression levels.

[0062] In the obtained transgenic switchgrass PvHIPP23 Expression level, such as Figure 7 As shown. The results indicate that 3 PvHIPP23 Overexpression lines OE1, OE2 and OE3 PvHIPP23 Gene expression levels were significantly higher than WT ( P <0.001), and PvHIPP23 The gene expression levels were 55.02, 14.80, and 27.62 times that of WT, respectively. Figure 7 (A) from the list. 24 results were obtained. PvHIPP23-RNAiAmong the interference lines, 21 interference lines PvHIPP23 Gene expression levels were significantly lower than WT ( P <0.05), among which RNAi-27 strain PvHIPP23 Gene expression levels were the lowest, only 0.14 times that of WT. Figure 7 (B)). The above results show that the present invention has successfully obtained... PvHIPP23 overexpression and PvHIPP23-RNAi Disturbing the switchgrass plants can continue. ​ Functional analysis and identification of genes.

[0063] Example 5: ​ Gene expression analysis in response to salt stress The lemma of *Alamo* seeds was removed, soaked in 5% NaClO for 1 h, washed 5 times with sterile water, and placed in a 4℃ refrigerator for 12 h. The next day, the seeds were soaked in 5% NaClO for 30 min and washed 5 times with sterile water. After sterilization, the *Alamo* seeds were placed in a 4℃ refrigerator for 3 days, and then germinated in petri dishes containing moist filter paper for 7 days. Germinated seedlings were transferred to 1 / 4 × Hoagland solution for 7 days, then transferred to 1 / 4 × Hoagland solution with a final concentration of 200 mM NaCl (with a 1 / 4 × Hoagland solution without NaCl as a blank control), and cultured in a 25℃ incubator under 16 h light / 8 h darkness conditions for 7 days. During the 7 days of salt stress treatment, samples were taken at different time points: seedlings were quickly washed 3 times with deionized water, excess surface moisture was absorbed with filter paper, seedlings were cut from the root base, and the roots and aboveground parts were placed separately in centrifuge tubes and flash-frozen in liquid nitrogen. Three biological replicates were used for each treatment time point. Samples were used for RNA extraction and RT-qPCR analysis. Gene primers used were qPvHIPP23-F and qPvHIPP23-R, and internal control primers were qUbiquitin-F and qUbiquitin-R.

[0064] The results showed that with increasing salt stress treatment time, the aboveground parts and roots of switchgrass seedlings... ​ Gene expression levels initially increased and then decreased. Under different treatment durations, the aboveground parts of switchgrass showed... ​ Gene expression levels are upregulated by salt stress, but ​ The gene upregulation was small, reaching a significant level only after 1 day of salt treatment. P <0.05), ​ Gene expression levels were only 2.87 times that of the control. After 0.5 days of salt treatment, in the roots of *Stichopus japonica*... ​ Gene expression is significantly induced ( P <0.05), after 1 day of salt treatment ​Gene expression levels reached their highest point, 22.52 times that of the control, followed by... ​ Decreased gene expression levels. In the aboveground parts and roots of switchgrass. ​ Gene expression was induced by salt stress and in the roots ​ Higher fold changes in gene expression indicate ​ The gene can respond to salt stress-induced reactions, and the response is more intense in the roots. ​ ).

[0065] Example 6: ​ Salt tolerance analysis of transgenic switchgrass 1. ​ Salt tolerance analysis of detached leaves of transgenic switchgrass right ​ Gene-overexpressing switchgrass plants (OE1, OE2, and OE3) and ​ Salt treatment was performed on detached leaves of interference plants (RNAi-15, RNAi-20, and RNAi-21), with wild-type switchgrass as the control (WT). The second fully expanded leaf from the top of the switchgrass plant at E2 stage was taken, washed three times with deionized water, dried to remove excess moisture, and cut into 4 cm segments. These segments were then immersed in NaCl solutions with final concentrations of 0 mM, 200 mM, and 300 mM, respectively. The solutions were placed at 25°C in the dark, with the leaves continuously turned and the solution changed every 3 days. Photographs were taken and recorded after differences in leaf appearance were observed.

[0066] The results are as follows ​ As shown, leaf stress in switchgrass plants differed after 30 days of salt treatment. The degree of stress on the leaves increased with increasing salt concentration, with the most severe stress observed in detached leaves at 300 mM NaCl concentration. Compared to the maximum stress level (WT), different salt concentrations... ​ The detached leaves of the overexpressing plants (OE1, OE2, and OE3) showed better chlorophyll retention, indicating that the leaves suffered less damage from salt stress. ​ The detached leaves of the disturbed plants (RNAi-15, RNAi-20, and RNAi-21) showed poor chlorophyll retention, indicating that the leaves suffered more severe salt stress damage. Compared with WT, ​ Plants with overexpressed genes exhibit greater salt tolerance in detached leaves, while ​ The disturbed plants showed poorer salt tolerance than the WT plants, suggesting that... ​ It can enhance the salt tolerance of switchgrass plants.

[0067] 2. ​ Salt tolerance analysis of transgenic switchgrass plants Will ​Transgenic and WT plants were pruned to the same height (approximately 15 cm) and transplanted into vermiculite for cultivation, continuously irrigated with 1 / 4 × Hoagland. Once the switchgrass plants reached the E2 stage, they were treated with 1 / 4 × Hoagland at a final concentration of 350 mM NaCl (1 / 4 × Hoagland without NaCl served as a blank control). ​ Overexpression in switchgrass plants (OE1, OE2, and OE3) and ​ Interference plants (RNAi-2, RNAi-22, and RNAi-24) were used, with wild-type plants at the same stage serving as the control (WT). Each line had three biological replicates. Once the plants exhibited the stress phenotype, the aboveground parts and roots were photographed. The relative water content and chlorophyll content of leaves in both the control and treated groups were measured. Plant height, length and width of the second fully expanded leaf (from the top), length of the second internode (from the bottom), stem diameter, and number of tillers were measured and statistically analyzed in both the control and treated groups. The fresh and dry weights of the aboveground parts and roots were also determined.

[0068] ​ Plants overexpressing switchgrass exhibited a stress phenotype 46 days after salt stress treatment. ​ Before salt treatment, the plant growth status of the blank control group (CK) and the salt-treated group (NaCl) was relatively consistent. After 46 days of salt treatment, the control group plants were able to grow normally, with good growth of the aboveground parts and roots, while the growth of the aboveground parts and roots of the salt-treated group plants was inhibited, and compared with the WT, ​ Overexpression results in better growth of the aboveground parts and roots of the plant. ​ Analysis of plant growth indicators (Table 11) showed that, in the control group, except for... ​ The leaf width of the overexpression line OE1 was significantly lower than that of WT, and the stem diameter of OE3 was significantly higher than that of WT. P <0.05) ​ Other parameters of the overexpressing plants (plant height, leaf length, internode length, and number of tillers) showed no significant differences compared to the WT group. Analysis of the salt-treated group showed that, compared to the WT group, ​ Overexpression showed an increasing trend in plant height and tiller number, with OE3 exhibiting significantly more tillers than WT. P <0.05), indicating ​ Overexpressing switchgrass plants showed greater salt tolerance than WT plants.

[0069] Table 11 Growth indicators of PvHIPP23-overexpressing switchgrass plants after salt stress

[0070] Note: CK represents the blank control group, and NaCl represents the salt treatment group; data are expressed as mean ± SD, n ≥ 3; the same letter indicates no significant difference within groups, and different letters indicate significant differences within groups. P <0.05.

[0071] The biomass of plants after salt treatment was analyzed, and the control group... ​ The fresh weight of the aboveground parts and roots of the gene-overexpressing plants showed an increasing trend, with the aboveground fresh weight of OE3 being significantly higher than that of WT. P <0.01); After salt treatment, the fresh weight of the aboveground parts and roots of the plants decreased. Among them, the fresh weight of the aboveground parts and roots of OE3 showed an increasing trend compared with WT, but the difference was not significant. ​ (A)). The dry weight of the aboveground parts and roots of the plant was analyzed. ​ (B) shows results similar to those of plant fresh weight, indicating that after salt treatment, ​ There was no difference in biomass between overexpression plants and WT plants. Physiological parameters of the plants were measured; in the control group... ​ There were no significant differences in relative leaf water content and chlorophyll content between the overexpressing plants and the WT plants; in the salt treatment group... ​ The relative water content of the leaves of the overexpressing plants was significantly higher than that of the WT plants. P <0.05)( ​ In (C) of the OE2 chlorophyll content showed an increasing trend, and OE2 was significantly higher than WT. P <0.05)( ​ (D)). The results show that, compared to WT, ​ Gene overexpression alleviated the inhibitory effect of salt stress on the relative water content and chlorophyll content in plant leaves, and enhanced the salt tolerance of switchgrass.

[0072] right ​ Interfering with salt stress treatment on switchgrass plants resulted in salt stress occurring 20 days after treatment. ​ ​ The stress phenotype of disturbed plants, characterized by yellowing leaves and curling of terminal leaves, was observed. ​ To achieve a more pronounced stress phenotype, salt stress treatment was continued. After 66 days of salt stress, compared with WT, ​ The growth of the above-ground parts and roots of the plant is severely inhibited by stress. ​ Growth indicators of the plants were measured, and the results showed that in the control group... ​ The plant height, leaf length, leaf width, internode length, stem diameter, and number of tillers of the disturbed plants were not significantly different from those of the WT plants (Table 12); in the salt treatment group, ​ The plant height, leaf length, leaf width, and internode length of the affected plants were all significantly lower than those of the WT plants. P<0.05), while the stem diameter and tiller number also showed a decreasing trend, with the stem diameter and tiller number of RNAi-2 and the tiller number of RNAi-22 all significantly lower than WT ( P <0.05 (Table 12). The above results indicate that... ​ Interference reduces the salt tolerance of plants. ​ The interference with the genes reduced the salt tolerance of switchgrass.

[0073] Table 12 Post-salt stress ​ Interfering with the growth indicators of switchgrass plants

[0074] Note: CK represents the blank control group, and NaCl represents the salt treatment group; data are expressed as mean ± SD, n ≥ 3; the same letter indicates no significant difference within groups, and different letters indicate significant differences within groups. P <0.05.

[0075] The biomass of the treated plants was measured, and in the control group... ​ The fresh weight of the aboveground parts of the disturbed plants was not significantly different from that of the whole weight (WT), while the fresh weight of the roots showed a decreasing trend, but the difference was not significant; in the salt treatment group, ​ ​ The fresh weight of the aboveground parts and roots of the interfered plants was lower than that of the WT. Except for RNAi-24, where the difference between the aboveground fresh weight and the WT was not significant, the fresh weight of the aboveground parts and roots of the other interfered lines was significantly lower than that of the WT. P <0.05)( ​ (A)). The dry weight of the aboveground parts and roots of the plants after salt treatment was measured, and the results were similar to those of the plant fresh weight. Except for the RNAi-24 strain, which showed no significant difference in aboveground dry weight compared to the WT strain, the dry weight of the aboveground parts and roots of the other interfering lines was significantly lower than that of the WT strain. P <0.05)( ​ (B) indicates that salt stress severely inhibits ​ It interferes with the growth of the above-ground parts and roots of the plant. Physiological indicators of the plants after salt treatment were measured. In the control group, there was no significant difference in the relative water content of the leaves of the interfered lines compared to the WT (weighted total oxygen). In the salt-treated group, compared to the WT, ​ The relative water content of the interfered plants decreased, with RNAi-22 and RNAi-24 showing significantly lower values ​​than WT ( ). P <0.05)( ​ (C)); In the control group and the salt-treated group, except that the chlorophyll content of RNAi-22 in the control group was significantly higher than that in the WT group (C)); P Except for <0.05), the chlorophyll content in the leaves of the other interfering lines was not significantly different from that of WT. ​(D) in the middle. In summary, ​ Gene overexpression enhanced the salt tolerance of switchgrass plants, while ​ The disruption of genes reduced the salt tolerance of switchgrass plants, indicating that... ​ Genes positively regulate the salt stress response process of switchgrass.

[0076] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A type of willow branch millet PvHIPP23 The application of genes is characterized by, The application is to use switchgrass PvHIPP23 Genes were used to improve the salt tolerance of switchgrass, which... PvHIPP23 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. A type of willow branch millet PvHIPP23 The application of genes is characterized by, The application utilizes switchgrass. PvHIPP23 Genetically bred switchgrass varieties with improved salt tolerance, the switchgrass PvHIPP23 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

3. An application of a recombinant vector, characterized in that, The recombinant vector contains switchgrass. PvHIPP23 Genes, the switchgrass PvHIPP23 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the application is to use the recombinant vector to improve the salt tolerance of switchgrass.

4. An application of a recombinant vector, characterized in that, The recombinant vector contains switchgrass. PvHIPP23 Genes, the switchgrass PvHIPP23 The nucleotide sequence of the gene is shown in SEQ ID NO.

1. The application is to use the recombinant vector to breed switchgrass varieties with improved salt tolerance.

5. The application according to claim 3 or 4, characterized in that, The recombinant vector has a skeleton of binary expression vector pUbi1301.

6. A type of millet containing the willow branch millet as described in claim 1 PvHIPP23 The application of the gene or the recombinant bacteria of the recombinant vector described in claim 3 is characterized in that, The application involves using the recombinant bacteria to improve the salt tolerance of switchgrass.

7. A type of millet containing the willow branch millet as described in claim 1 PvHIPP23 The application of the gene or the recombinant bacteria of the recombinant vector described in claim 3 is characterized in that, The application involves using the recombinant bacteria to cultivate switchgrass varieties with improved salt tolerance.

8. The application according to claim 6 or 7, characterized in that, The recombinant bacteria is Agrobacterium.

9. A method for improving the salt tolerance of switchgrass, characterized in that, The steps include: cloning the switchgrass described in claim 1. PvHIPP23 Genes; switchgrass PvHIPP23 Genes were constructed into expression vectors to obtain recombinant vectors; the recombinant vectors were introduced into Agrobacterium to obtain recombinant Agrobacterium; switchgrass was infected with recombinant Agrobacterium and overexpressing switchgrass was obtained through screening. PvHIPP23 Positive plants of the gene.

10. The method according to claim 9, characterized in that, The expression vector is pUbi1301.

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