Application of BpC1H46 gene in regulation of plant adversity stress

Abstract

The invention belongs to the technical field of plant genetic engineering, and particularly provides application of a BpC1H46 gene in regulation and control of plant adversity stress. The nucleotide sequence of the BpC1H46 gene is as shown in SEQ ID NO. 1. Cloning identification, expression characteristic analysis and genetic transformation verification are carried out on the gene, and it is clear that BpC1H46 has an important regulation effect in plant cold resistance, salt resistance and other stress resistance characters for the first time. Exploration and functional verification of the gene provide theoretical basis and technical support for molecular breeding of stress-resistant varieties of broussonetia papyrifera. A new stress-resistant variety cultivated by utilizing the gene can obviously enhance the adaptive capacity of plants to stress such as salt and alkali, low temperature and the like, expand the planting range of the plants in marginal land and improve the development potential of paper mulberry as high-protein feed, so that the problem of shortage of woody protein feed resources is favorably relieved, and the gene has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, specifically to a... BpC1H46 Application of genes in regulating plant abiotic stress. Background Technology

[0002] Feed is a crucial guarantee for achieving high-quality, efficient, ecological, and safe development of animal husbandry. China is a major livestock producer globally. With the rapid development of my country's animal husbandry, the demand for feed ingredients is constantly increasing, while the feed supply gap is widening, becoming a major problem restricting the healthy and sustainable development of my country's animal husbandry. Therefore, developing and utilizing local high-yield, high-protein, and resilient forage plant resources has become an important solution.

[0003] Paper mulberry (Broussonetia papyrifera), belonging to the genus Broussonetia in the family Moraceae, is also known as paper mulberry, paper bark, paper peach, kudzu tree, and paper milk tree. It boasts advantages such as wide adaptability, strong overall resistance to adverse conditions, rapid growth, wide distribution, easy propagation, short rotation period, and strong regeneration ability. It is not only an excellent tree species for protecting the ecological environment but also a high-protein plant, making it a promising source of high-protein woody feed. my country has approximately 1.5 billion mu (100 million hectares) of saline-alkali land, widely distributed and with complex composition. Salt-alkali stress is one of the major abiotic stresses inhibiting plant growth and development, second only to drought stress. Similarly, low temperature is also a natural factor severely affecting plant growth. Salt and low temperatures not only affect the growth and development of paper mulberry but also pose a serious threat to agricultural production and the ecological environment. The current varieties of paper mulberry have weak salt and alkali tolerance and cold resistance, which greatly limits their large-scale planting and promotion in my country's vast saline-alkali land and northern regions. Therefore, breeding new salt- and alkali-tolerant and cold-resistant paper mulberry varieties is an effective way to fully develop and utilize my country's saline-alkali land and other marginal lands, improve the ecological environment, increase the self-sufficiency rate of protein feed, and ensure the sustainable development of animal husbandry.

[0004] Research on the resistance of paper mulberry (Broussonetia papyrifera) has made some progress both domestically and internationally, particularly in the study of environmental adaptation mechanisms. As a multifunctional tree species, paper mulberry's characteristics in drought resistance, salt and alkali tolerance, and heavy metal stress demonstrate its good application potential in ecological environment restoration and forage resource development. However, the number of functional genes clearly involved in abiotic stress in paper mulberry that have been cloned is currently limited, restricting the progress of molecular breeding for stress resistance in paper mulberry. C1H46 The gene is one with an unknown function; therefore, this invention chooses to clone this gene with an unknown function. C1H46 The aim is to explore the role of genes in stress response. Summary of the Invention

[0005] Based on the above technical problems, the present invention provides a BpC1H46 Application of genes in regulating plant abiotic stress.

[0006] In a first aspect, the present invention provides a BpC1H46 The application of genes in regulating plant abiotic stress. BpC1H46 The nucleotide sequence is shown in SEQ ID NO.1. The above gene... BpC1H46 The amino acid sequence of the encoded protein is shown in SEQ ID NO.2.

[0007] Preferably, the regulation of plant abiotic stress is the regulation of plant resistance to salt stress or cold stress.

[0008] Preferably, by making the BpC1H46 Gene overexpression can enhance plant resistance to salt or cold stress.

[0009] Preferably, by making the BpC1H46 Gene overexpression promotes the development of the aboveground parts of the plant.

[0010] Preferably, by containing BpC1H46 The introduction of genetically modified biological materials into plants to enable the aforementioned BpC1H46 Gene overexpression.

[0011] More preferably, containing BpC1H46 Gene-containing biological materials are BpC1H46 Recombinant plasmids or recombinant bacteria.

[0012] More preferably, the vector for the recombinant plasmid is pEASY-blunt or pCAMBIA1300-GFP.

[0013] More preferably, the host of the recombinant bacteria is Escherichia coli Trans1-T1, Escherichia coli DH5α, or Agrobacterium GV3101.

[0014] Preferably, the plant is Arabidopsis thaliana or Broussonetia papyrifera.

[0015] In a second aspect, the present invention provides a method for improving the resistance of plants to salt stress or cold stress, comprising making the... BpC1H46 The process of gene overexpression.

[0016] In a third aspect, the present invention provides a method for cultivating plants with enhanced resistance to salt stress or cold stress, comprising the following steps: Construct containing the BpC1H46 Gene overexpression vectors; The vector was introduced into Agrobacterium competent cells and cultured to obtain engineered bacteria. The engineered bacteria are used to infect plant tissues or organs; Transgenic plants are obtained by regenerating and screening infected tissues or organs; Identification of the genetically modified plants BpC1H46 Gene expression and its resistance to salt or cold stress.

[0017] Preferably, construct the BpC1H46 Gene overexpression plasmids are plasmids that express the gene BpC1H46 Gene sequence linked to pEASY -blunt vector or pCAMBIA1300-GFP vector.

[0018] Preferably, the competent cells are Escherichia coli DH5α competent cells.

[0019] Preferably, the plant is paper mulberry or Arabidopsis thaliana.

[0020] More preferably, the plant tissue is an Arabidopsis inflorescence.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention is the first to identify and verify the function of the BpC1H46 gene derived from Broussonetia papyrifera in plant abiotic stress response. The function of this gene was previously unknown; this invention, through cloning, expression, and functional verification, clarifies its role in regulating plant tolerance to salt and cold stress, filling a gap in the study of the molecular mechanisms of Broussonetia papyrifera stress resistance.

[0022] This invention demonstrates that the BpC1H46 gene can significantly enhance plant growth performance under salt and low temperature stress by constructing an overexpression vector and heterologously expressing it in Arabidopsis thaliana, including increasing aboveground biomass and enhancing antioxidant enzyme activity, thereby improving the plant's ability to adapt to adversity.

[0023] This invention provides new genetic resources and molecular tools for plant stress resistance breeding. The BpC1H46 gene can be applied to the genetic improvement of various plants such as paper mulberry and Arabidopsis thaliana. Through transgenic or gene editing technologies, it can be used to cultivate new salt-tolerant and cold-resistant varieties, expanding the planting range of plants in saline-alkali land and cold regions.

[0024] The application of the BpC1H46 gene will help develop new Broussonetia papyrifera varieties adapted to adverse conditions, enhance their production potential in marginal lands such as saline-alkali land and cold regions, alleviate the shortage of protein feed resources in my country, and promote ecological restoration and sustainable agricultural and pastoral development. Furthermore, this gene can be introduced into other target plants through genetic engineering to improve their tolerance to abiotic stresses such as salinity, low temperature, and alkalinity.

[0025] The BpC1H46 gene may participate in stress response by regulating the antioxidant enzyme system, providing an experimental basis and theoretical support for further analysis of plant stress resistance signaling networks and gene regulatory pathways. Attached Figure Description

[0026] Figure 1 For amplification BpC1H46 Gene agarose gel electrophoresis image; Figure 2 For the recombinant vector pCAMBIA1300- BpC1H46 -Agarose gel electrophoresis image of GFP-transformed DH5α-positive Escherichia coli colonies verified by PCR; Figure 3 for Spe I and Sma Agarose gel electrophoresis image after double enzyme digestion; Figure 4 For the recombinant vector pCAMBIA1300- BpC1H46 -Agarose gel electrophoresis image of positive colonies of Agrobacterium GV3101 transformed with GFP, verified by PCR. Figure 5 for BpC1H46 Subcellular localization analysis diagram of the protein; Figure 6 for BpC1H46 Images of positive seedlings from transgenic Arabidopsis thaliana T1 generation seeds; Figure 7 For T1 generation BpC1H46 Gel electrophoresis image of PCR amplification and verification of transgenic Arabidopsis thaliana positive seedlings; in the image, lane M is the standard, and lanes 1-11 all amplified the target gene, verifying them as positive transformants. Figure 8 Under normal culture conditions BpC1H46 Phenotypic and statistical data of transgenic Arabidopsis and wild-type Arabidopsis; where A is the morphological diagram of Arabidopsis after 7 days of vertical culture, B is the bar chart of taproot length of Arabidopsis, C is the bar chart of lateral root number of Arabidopsis, and D is the phenotypic diagram of aboveground fresh weight of Arabidopsis; Col: wild-type Arabidopsis; 35S:BpC1H46:BpC1H46 Genetically modified Arabidopsis thaliana; Figure 9 Under salt stress treatment BpC1H46 Phenotypic and statistical data of transgenic Arabidopsis and wild-type Arabidopsis; where A is a root phenotype of Arabidopsis under salt stress treatments of 25 mM NaCl, 75 mM NaCl, and 100 mM NaCl, respectively; B is a bar chart comparing the taproot length of Arabidopsis under 100 mM NaCl salt stress treatment and normal culture conditions; C is a bar chart comparing the number of lateral roots of Arabidopsis under 100 mM NaCl salt stress treatment and normal culture conditions; D is a bar chart comparing the fresh weight of the aboveground parts of Arabidopsis under 100 mM NaCl salt stress treatment and normal culture conditions; Col: wild-type Arabidopsis; 35S: BpC1H46: BpC1H46 Genetically modified Arabidopsis thaliana; Figure 10 Under salt stress treatment BpC1H46 Phenotypic data and statistics of aboveground parts of transgenic Arabidopsis and wild-type Arabidopsis; where A is the phenotype of different lines under normal culture conditions, B is the phenotype of different lines after one week of 100 mM NaCl salt stress treatment, C is a bar chart comparing the SOD enzyme activity of Arabidopsis under 100 mM NaCl salt stress treatment and normal culture conditions, and D is a bar chart comparing the POD enzyme activity of Arabidopsis under 100 mM NaCl salt stress treatment and normal culture conditions; Col: wild-type Arabidopsis. 35S:BpC1H46:BpC1H46 Genetically modified Arabidopsis thaliana; Figure 11 Under cold stress treatment BpC1H46 Phenotypic and statistical data of transgenic Arabidopsis thaliana and wild-type Arabidopsis thaliana; where A is the root phenotype of Arabidopsis thaliana under 8℃ cold stress treatment, B is a bar chart comparing the taproot length of Arabidopsis thaliana under 8℃ cold stress treatment and normal culture conditions, C is a bar chart comparing the number of lateral roots of Arabidopsis thaliana under 8℃ cold stress treatment and normal culture conditions, and D is a bar chart comparing the fresh weight of the aboveground parts of Arabidopsis thaliana under 8℃ cold stress treatment and normal culture conditions; Col-0: wild-type Arabidopsis thaliana; BpC1H46-L1, BpC1H46-L2, BpC1H46-L3: BpC1H46 Genetically modified Arabidopsis thaliana; Figure 12 This study presents the aboveground phenotypes and data statistics of BpC1H46 transgenic Arabidopsis thaliana and wild-type Arabidopsis thaliana under cold stress treatment. Among them, A shows the phenotypes of different lines under normal culture conditions, B shows the phenotypes of different lines after one week of 8℃ cold stress treatment, C shows the bar chart comparing the SOD enzyme activity of Arabidopsis thaliana under 8℃ cold stress treatment and normal culture, and D shows the bar chart comparing the POD enzyme activity of Arabidopsis thaliana under 8℃ cold stress treatment and normal culture. Detailed Implementation

[0027] The following examples are provided to better understand the present invention, but do not limit the invention. Unless otherwise specified, the experimental methods in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent companies. All quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0028] Example 1 Paper mulberry BpC1H46 Identification of gene sequences Using a homologous gene cloning strategy, similar sequences were searched for in the transcriptome of *Broussonetia papyrifera*. A 1420 bp open reading frame was found in the transcriptome of *Broussonetia papyrifera*, which is similar to... BpC1H46 The gene homology was 94.79%, and it was named... BpC1H46 Its nucleotide sequence is shown in SEQ ID NO.1: BpC1H46 The gene encodes a protein containing 460 amino acids, the amino acid sequence of which is shown in SEQ ID NO.2. Its molecular weight is 51371.96 Da and its isoelectric point (PI) is 7.74. BpC1H46 This protein lacks a transmembrane domain and is not a membrane protein. Its amino acid sequence is shown in SEQ ID NO.2:

[0029] MVKIVEVYMVAPPESSQLASEAPKSLPLTFFDIRWLRLPPVQRLFFYEISTPNTHSTTSFFHSNVLPRLKQSLSLTFQYFLPLTGHLIWPESSHKPVIN*AEGDTVPLTVAESDADF CRLSGTGEFFVTAAYHPLIPDLAVSHERAAVLALQVTLFPNSSGFSIGIATHHAVVDGKTSTSFMKSWAHICRTLGANHVESSLPIELKPVFDIEAIKGAAGLGAIFASQLLSLDGG PNNRSLMSWKVRQAQPDLIRGTFHLTRAHIEKLRHLMNITMAKKNEKSGHQELFHASTFSLAYSYTFLCLIKAEGIADDKVITVFAVDCRSRLKPPIPATYFGNCIGSKVMIKGTKD LVGEDGFIMAVTAIGEALKSLEVDGGLDGAQNWVKRLIFYNSTTGRVYSISGSPRFEVYSTDFGWGRPRKVDVVSIDRTRAISFSDSRDSDGIEIGVVLKKQHMEAFGSLFAQGLDS* Example 2 Paper mulberry BpC1H46 Construction of gene recombination vectors Using paper mulberry as material, total RNA was extracted and reverse transcribed into cDNA. Corresponding primers were designed for gene amplification. After detection by agarose gel electrophoresis, the target band was recovered and compared with... pEASY -blunt vector ligation, transfected into E. coli Trans 1-T1 competent cells were sequenced and analyzed. Positive clones were picked and cultured, followed by plasmid extraction. Restriction sites were added, and the cells were simultaneously digested with the vector pCAMBIA1300-GFP. The pCAMBIA1300-GFP was then ligated with T4 ligase. BpC1H46 The target gene fragment was then transformed into E. coli DH5α competent cells. Positive clones were picked again for plasmid extraction, and finally transformed into Agrobacterium GV3101 for preservation of positive bacteria. The specific operation steps are as follows: 1. Total RNA extraction Total RNA was extracted from mulberry leaves following the procedures outlined in a plant total RNA extraction kit. All reagents and consumables used were RNase-free. RNA concentration was measured using a Nano-300 micro-spectrophotometer. 260 / A 280 All values ​​were around 2.0, indicating good quality and meeting the requirements for subsequent reverse transcription experiments. The plant total RNA extraction kit was purchased from Nanjing Novizan.

[0030] 2. Obtaining cDNA Following the instructions of the reverse transcription kit, the RNA extracted from the mulberry leaves was reverse transcribed into cDNA. The cDNA product was then used for amplification of the target gene. The reverse transcription kit was purchased from Novizan.

[0031] 3. BpC1H46 Cloning of genes (1) According to BpC1H46 Based on the full-length gene sequence and the restriction enzyme sites of the expression vector used, specific primers containing the entire ORF (Original Gene Formation) with the stop codon removed were designed. Restriction enzyme sites were added to the 5' and 3' ends of the primers, respectively. The primer sequences are as follows: BpC1H46-F-SmaI: 5'- TCTAGAATGGTGAAAATAGTTGAGGTCTATATG - 3', SEQ IDNO.3; BpC1H46-R-SpeI: 5'- GGTACCTGAATCAAGACCTTGAGCAAAC - 3', SEQ ID NO.4; (2) Using the first strand of cDNA obtained above for PCR and BpC1H46 The specific primers were used for amplification with 2×PhantaMax high-fidelity enzyme. The amplification reaction conditions were: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 sec, 58℃ annealing for 15 sec, 72℃ extension at 1 kb / min, and 35 cycles followed by a further extension at 72℃ for 5 min. The high-fidelity enzyme was purchased from Novizan.

[0032] (3) Detected by 1% agarose gel electrophoresis.

[0033] like Figure 1 As shown, the amplified fragments were all the same size as the target gene. The target gene fragments were extracted and recovered from the gel according to the instructions of the gel extraction kit. The gel extraction kit was purchased from Tiangen Biotech.

[0034] 4. Connection pEASY -blunt vector and Escherichia coli Trans Transformation of 1-T1 competent cells 4 μL of gel recovery product was mixed with 1 μL of cloning vector. pEASY After gently mixing the -blunt carrier, react at 25°C for 30 min. Then, transfer all the ligation products to 50 µL of ice-thawed E. coli in a clean bench. Trans In 1-T1 competent cells, gently stir the bottom of the tube to mix slowly, incubate on ice for 30 min, heat shock at 42℃ for 1 min, then quickly incubate on ice for 2 min; add 900 µL of LB liquid medium in a clean bench, and incubate at 37℃ with shaking at 200 rpm for 1 h; centrifuge at 5000 rpm for 1 min, discard most of the supernatant in a clean bench, retaining 150 µL of supernatant, gently resuspend the cells with a pipette, and spread them all onto LB solid medium containing 100 μg / mL kanamycin. Seal the culture dish with sealing film and incubate upside down in a 37℃ incubator for 14–16 h.

[0035] After colonies grew on the plates, a single, neatly edged white colony was picked using a sterile disposable needle and transferred to 5 mL of LB broth containing 100 μg / mL kanamycin. The culture was incubated at 37°C and 200 rpm for 12 h. PCR amplification and verification were performed using the aforementioned BpC1H46-F-SmaI and BpC1H46-R-SpeI primers. The PCR reaction program was as follows: 95°C pre-denaturation for 3 min; 95°C denaturation for 15 sec; 58°C annealing for 15 sec; 72°C extension at 1 kb / min; 35 cycles followed by a final extension at 72°C for 5 min; storage at 4°C. After positive clones were detected by 1% agarose gel electrophoresis, the samples were sent to a sequencing company for analysis. The sequencing results were compared with... BpC1H46 Gene sequences were compared, and recombinant plasmids were extracted from colonies with correct sequencing using a plasmid miniprep kit. pEASY - BpC1H46 The plasmid miniprep kit was purchased from Beijing Kangwei.

[0036] 5. Paper mulberry BpC1H46 Construction of gene overexpression vectors The recombinant plasmid was digested using a double enzyme digestion method. pEASY - BpC1H46 With the expression vector pCAMBIA1300-GFP, respectively, Spe I and Sma I was subjected to double enzyme digestion at 37°C for 2 hours in a PCR instrument, and the digestion products were detected by electrophoresis using a 1% agarose gel.

[0037] like Figure 3 As shown, the judgment is based on the size of the band. BpC1H46 The gene and the pCAMBIA1300-GFP vector have been correctly cut.

[0038] After gel extraction and recovery of the target band, the digested vector and target gene were mixed at a 3:1 mass ratio, T4 DNA ligase was added, and the mixture was incubated at 25°C for 2 hours to obtain the ligation product. The ligation product was then transformed into *E. coli* DH5α competent cells to obtain the recombinant vector pCAMBIA1300-GFP- BpC1H46 A single colony was selected for PCR verification of positive bacteria, and the electrophoresis results are as follows. Figure 2 As shown, single positive colonies consistent with the target gene were cultured in a shake culture and sequenced. The sequencing results were consistent with... BpC1H46 The gene sequence alignment results are completely consistent, indicating that pCAMBIA1300-GFP- BpC1H46 The overexpression vector was successfully constructed.

[0039] Plasmids were extracted from positive transformants and transformed into Agrobacterium GV3101 competent cells using a freeze-thaw method. Single colonies were picked for colony PCR detection. The target bands were consistent, as shown in the following figures. Figure 4 As shown, this demonstrates successful transformation into Agrobacterium, resulting in Agrobacterium-positive transformants. The culture was then shaken and stored for later use.

[0040] Example 3 Paper mulberry BpC1H46 Verification of gene function By subcellular localization, clearly BpC1H46 The specific location of the protein within the cell; after Arabidopsis thaliana has been cultured to the appropriate age, it is transformed by Agrobacterium-mediated transformation, the T3 genotype is observed, statistical analysis is performed, and its function is analyzed. The specific operation steps are as follows.

[0041] 1. BpC1H46 Subcellular localization First, the online prediction software https: / / wolfpsort.hgc.jp was used to... BpC1H46 Subcellular localization prediction of the protein showed that... BpC1H46 The protein may be located in chloroplasts. Subcellular localization was verified by injecting it into the lower epidermis of tobacco. The Agrobacterium-positive transformants constructed in Example 2 were activated to the OD level of the bacterial cells. 600 After reaching a concentration of 1.2-1.5, Agrobacterium tumefaciens was resuspended in tobacco suspension to an OD value of 1.2-1.5. 600 A 0.9–1.0 g / mL infection solution was prepared and allowed to stand at room temperature for 3 hours. The solution was then injected into the lower epidermal cells of the tobacco leaves from the underside using a disposable syringe, and the injection area was marked. After transient transformation of the tobacco epidermal cells and culturing for 72 hours, the lower epidermal cells from the marked locations were harvested and observed using a laser confocal microscope. BpC1H46 The expression and localization of the protein in tobacco epidermal cells. Results are as follows: Figure 5 As shown, green fluorescent protein GFP is expressed throughout the cell nucleus, therefore BpC1H46 The protein is located in the cytoplasm.

[0042] The preparation method of the above 100 mM tobacco suspension is as follows: Weigh 0.213 g of 2-morpholine ethanesulfonic acid and 0.09521 g of MgCl2 into 100 mL of sterile ddH2O using a precision balance, adjust the pH to 5.6-5.7, add 0.5 g of glucose and 100 μL of 100 mM MAS solution, and prepare fresh before use; 100 mM acetylsyl syringone solution: Weigh 392.4 mg of acetylsyl syringone powder and dissolve it in 12 mL of 95% ethanol. Add 8 mL of sterile ddH2O, mix thoroughly, sterilize with a 0.22 mm filter membrane in a clean bench, dispense into sterile EP tubes, and store at -20℃.

[0043] 2. Obtaining [the desired product] using Agrobacterium-mediated genetic transformation. BpC1H46 Transgenic Arabidopsis Place Arabidopsis seeds in 1.5 mL centrifuge tubes. In a clean bench, add 75% ethanol to the tubes and vortex for 1 min. After the seeds settle to the bottom, remove the ethanol and add 10% sodium hypochlorite, vortex for 10 min. Discard the sodium hypochlorite and rinse 3-5 times with sterile water to thoroughly remove it. Sow the seeds onto MS medium using a sterile pipette tip, seal with plastic wrap, and incubate at 4°C in the dark for 48 h. After incubation, place the culture dishes at 22°C with a 16 h light / 8 h dark cycle and a light intensity of 40 μmol·m². -2 •s -1 After two weeks of cultivation in the culture room, the healthy wild-type Arabidopsis thaliana is transplanted into nutrient soil and maintained regularly until it bolts and produces flower buds.

[0044] Select healthy Arabidopsis thaliana plants, remove the pods and flowers that have completed self-pollination, and water thoroughly the day before infection; resuspend the Agrobacterium cells in 1 / 2 MS liquid medium to achieve OD... 600 The concentration was approximately 0.8-1. 0.01% Silwet77 was added to obtain the Arabidopsis thaliana infection solution. The Arabidopsis inflorescences were completely immersed in the infection solution, gently shaken, and held for 30 seconds. Afterward, the infected inflorescences were wrapped with plastic wrap, kept moist, and cultured in the dark for 24 hours. Then, they were transferred to normal growth conditions and grown for 7-9 days before a second infection. The T0 generation was harvested after the seeds matured. BpC1H46 Genetically modified Arabidopsis thaliana seeds.

[0045] 3. Screening and preliminary examination of transformed plants The harvested T0 generation seeds were dried in a 38℃ oven, disinfected with 10% sodium hypochlorite and washed, and then sown in MS medium containing 100 mg / L hygromycin for the T1 generation. BpC1H46 Screening of transgenic Arabidopsis thaliana, the screening results are as follows Figure 6As shown, Arabidopsis thaliana plants that had normally rooted on the culture medium were transplanted into nutrient soil. After one week of growth, Arabidopsis DNA was extracted using the CTAB method. The extracted DNA was then used as a template. BpC1H46 PCR verification was performed using gene-specific primers BpC1H46-F-XbaI and BpC1H46-R-SpeI. Electrophoresis results are shown below. Figure 7 The results showed that the target fragment band was amplified in the sample, indicating that the T1 generation was successfully obtained. BpC1H46 Transgenic Arabidopsis thaliana. The T1 generation... BpC1H46 Transgenic Arabidopsis thaliana was further cultured, and the genetic segregation ratio was used to screen for homozygous positive T3 generation. BpC1H46 Transgenic Arabidopsis thaliana.

[0046] 4. BpC1H46 Effects of ectopic gene expression in Arabidopsis on root development Using wild-type Arabidopsis thaliana as the control group, BpC1H46 Transgenic Arabidopsis thaliana was used as the treatment group, which included wild-type Arabidopsis thaliana and... BpC1H46 Transgenic Arabidopsis seeds were sown on petri dishes containing MS medium and vernalized at 4°C for 3 days. Then, they were cultured at 22°C under a 16-hour day / 8-hour dark environment for 7 days. The seedlings were then transferred to square (13×13cm) MS medium and cultured vertically for 7 days. Finally, the seedlings were transferred to 13cm×13cm square plates and cultured vertically under normal light for 10 days. Both the control and treatment groups were triplets.

[0047] Root length was measured using ImageJ software, and preliminary data statistics and bar charts were generated using Excel 2019. One-way ANOVA and significance analysis were performed using STST, with p < 0.05.

[0048] The results are as follows Figure 8 As shown in Figures A through D, phenotypic observations of the taproot and lateral roots of each strain reveal... BpC1H46 Compared with wild-type Arabidopsis, transgenic Arabidopsis showed no significant differences in taproot length, lateral root number, or aboveground fresh weight, indicating that... BpC1H46 The gene had no significant effect on root morphogenesis under normal culture conditions.

[0049] 5. Heterologous overexpression BpC1H46 Effects on the stress resistance of Arabidopsis thaliana Because the technology for genetically modifying paper mulberry is still immature, the selected... BpC1H46 Experiments were conducted on transgenic Arabidopsis thaliana to investigate stress resistance. Through stress treatment experiments in petri dishes and nutrient soil, and by detecting stress-resistant enzyme activities, the effects of heterologous expression of this gene on stress resistance in Arabidopsis thaliana were explored, thereby elucidating... BpC1H46 Genes' stress-resistance related functions.

[0050] In salt stress experiments, wild-type and BpC1H46 After sterilization, transgenic Arabidopsis seeds were sown on MS petri dishes at 4°C in the dark for 2 days. The MS dishes were then cultured for 7 days under a 16-hour light / 8-hour dark environment at 22°C until germination. Germinated wild-type and transgenic seedlings were then transferred to 13 cm × 13 cm square MS petri dishes containing 25 mM NaCl, 75 mM NaCl, and 100 mM NaCl, respectively, and cultured vertically for 14 days. The root growth of Arabidopsis under these treatments was photographed and recorded. Simultaneously, the germinating wild-type and transgenic Arabidopsis seedlings were... BpC1H46 Transgenic Arabidopsis thaliana plants were transplanted into nutrient soil, with five plants per square pot arranged regularly. They were cultured at 23°C under 14 h light / 10 h dark conditions. After four weeks of normal culture, salt stress treatment was applied: 100 Mm NaCl was applied by watering on days 1, 4, and 7. The above-ground leaves were harvested the following day, flash-frozen in liquid nitrogen, and stored at -80°C. SOD and POD enzyme activities were measured according to the instructions of the SOD and POD kits. Both the control and treatment groups were triplet.

[0051] The results are as follows Figures 9-10 As shown, BpC1H46 Compared to wild-type Arabidopsis, transgenic Arabidopsis lines showed significantly increased taproot length and lateral root number, indicating that taproot and lateral root growth may be positively regulated by this gene. Furthermore, the root richness and aboveground growth of the transgenic lines were superior to the wild type, suggesting that this gene enhances resistance to salt stress. In addition, the POD enzyme levels in the transgenic lines were significantly higher than those in the wild type and the untreated transgenic lines, leading to the hypothesis that *Broussonetia papyrifera*... BpC1H46 Genes may respond to salt stress by regulating the POD enzyme.

[0052] In the cold stress experiment, wild-type and BpC1H46 After sterilization, transgenic Arabidopsis seeds were sown on MS petri dishes at 4°C in the dark for 2 days. The MS dishes were then cultured for 7 days at 22°C with 16 hours of light / 8 hours of darkness until germination. After two days of incubation at 4°C in the dark, the petri dishes were placed at 8°C with 16 hours of light / 8 hours of darkness. The growth of the Arabidopsis under cold stress was observed daily, and the growth was recorded and photographed. Simultaneously, the germinated wild-type Arabidopsis and... BpC1H46Transgenic Arabidopsis thaliana were planted in nutrient soil, with four plants transplanted into each square pot in a regular arrangement. They were cultured at 23℃ under 14 h light / 10 h dark conditions. After four weeks of normal culture, they were subjected to a one-week cold stress treatment: placed in an environment of 16 h light / 8 h dark and 8℃ for seven days. The following day, aboveground leaves were harvested, flash-frozen in liquid nitrogen, and stored at -80℃. SOD and POD enzyme activities were measured according to the instructions of the SOD and POD kits. Both the control and treatment groups were performed in triplicate.

[0053] The results are as follows Figures 11-12 As shown, wild-type Arabidopsis thaliana and BpC1H46 Compared to the wild type, the transgenic Arabidopsis thaliana showed a significant increase in taproot length and lateral root number, indicating that taproot and lateral root growth may be positively regulated by this gene. Furthermore, the transgenic lines exhibited superior root richness and aboveground vigor compared to the wild type. The larger and healthier leaves of the transgenic Arabidopsis thaliana suggest that this gene enhances resistance to cold stress. In addition, the transgenic lines showed significantly higher POD and SOD enzyme activities than the wild type and the untreated transgenic lines, suggesting that the gene is associated with the wild type. BpC1H46 The gene may respond to cold stress by co-regulating POD and SOD enzymes. This indicates that overexpression in Arabidopsis thaliana... BpC1H46 Genes can improve a plant's cold tolerance. BpC1H46 The gene is a cold stress response gene, consistent with the predicted results.

[0054] In summary, BpC1H46 It improved the plant's salt and cold resistance. BpC1H46 In transgenic lines under salt stress, the growth of the primary and lateral roots may be a pathway by which this gene positively regulates salt stress response, and it may further participate in salt stress tolerance by regulating POD enzymes rather than SOD enzymes; additionally... BpC1H46 The transgenic lines of the gene may primarily respond to cold stress through the growth of the taproot, while simultaneously increasing the activity of stress-related POD and SOD enzymes to respond to cold stress and enhance the plant's cold resistance.

Claims

1. A kind BpC1H46 The application of genes in regulating plant abiotic stress is characterized by, The BpC1H46 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The regulation of plant abiotic stress refers to the regulation of plant resistance to salt stress or cold stress.

3. The application according to claim 2, characterized in that, By making the BpC1H46 Gene overexpression can enhance plant resistance to salt or cold stress.

4. The application according to claim 3, characterized in that, By containing BpC1H46 The introduction of genetically modified biological materials into plants to enable the aforementioned BpC1H46 Gene overexpression.

5. The application according to claim 4, characterized in that, Include BpC1H46 Gene-containing biological materials are BpC1H46 Recombinant plasmids or recombinant bacteria.

6. The application according to claim 5, characterized in that, The vector for the recombinant plasmid is either pEASY-blunt or pCAMBIA1300-GFP.

7. The application according to claim 5, characterized in that, The host of the recombinant bacteria is Escherichia coli Trans1-T1, Escherichia coli DH5α, or Agrobacterium GV3101.

8. The application according to claim 1, characterized in that, The plant in question is either Arabidopsis thaliana or Broussonetia papyrifera.

9. A method for improving plant resistance to salt stress or cold stress, characterized in that, Included in plants, as described in claim 1 BpC1H46 The steps of gene overexpression.

10. A method for cultivating plants with enhanced resistance to salt stress or cold stress, characterized in that, Includes the following steps: Constructing the structure containing the contents of claim 1 BpC1H46 Gene overexpression vectors; The vector was introduced into Agrobacterium competent cells and cultured to obtain engineered bacteria. The engineered bacteria are used to infect plant tissues or organs; Transgenic plants are obtained by regenerating and screening infected tissues or organs; Identification of the genetically modified plants BpC1H46 Gene expression and its resistance to salt or cold stress.

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