Gene of leymus secalinus participating in adversity stress and application thereof

Abstract

The invention relates to the technical field of plant genetic engineering, in particular to a gene of leymus secalinus participating in adversity stress and application of the gene. The gene LsHSFA participating in adversity stress in leymus secalinus is found for the first time, the nucleotide sequence of the gene LsHSFA is shown as SEQ ID NO.1, and the amino acid sequence of the coded protein is shown as SEQ ID NO.2. Through cloning and identification of the adversity stress participating gene of the leymus secalinus, expression analysis of the gene, genetic transformation of the gene and other means, the function of the gene is analyzed and verified, and the result shows that the adversity stress participating gene of the leymus secalinus has important influence on promotion of upper development of the leymus secalinus and improvement of heat resistance, salt resistance, drought resistance and other stress resistance; the stress-resistant gene of the leymus secalinus is utilized to positively regulate and control the abiotic stress function, so that the leymus secalinus can cope with various stress adversities better, the leymus secalinus can adapt to more complex and harsh ecological environments, degenerated and desertified grassland is improved, and potential application prospects are provided for improved variety breeding and excellent population construction of the leymus secalinus in the future.

Description

Technical Field

[0001] The invention relates to the technical field of plant genetic engineering, and in particular to a gene of Leymus japonicus involved in adverse stress and an application thereof. Background Art

[0002] Leymus chinensis Leymus secalinus ) is a perennial grass of the Poaceae family, widely distributed in the Eurasian steppes, North America, Northeast my country, Northwest China, North China, and Southwest China. Leymus has the characteristics of strong comprehensive stress tolerance and high nutritional value. At the same time, with its outstanding rhizome asexual reproduction ability, it can quickly clone and grow, and reproduce rapidly in mobile sand dunes, promoting the formation of fixed or semi-fixed sand, and even forming rare herbaceous sand dunes under certain terrain conditions. In recent years, domestic and foreign scholars have mainly conducted preliminary research on the stress resistance of Leymus from the physiological and ecological levels, but the research on the discovery and application of high-quality genes for stress resistance in Leymus has just started, and the screening and identification of specific stress resistance genes is still relatively small. Therefore, it is of great significance to improve the grassland environment to conduct a systematic study on the stress resistance of Leymus at the molecular level, identify the functions of relevant high-quality genes for stress resistance in Leymus, and then guide the cultivation of new forage germplasm from the molecular level.

[0003] Many studies have found that HSFs (Heat Shock Factor) play an important role in plant regulation of high temperature stress and are also an important component of the signal regulation network in plants' response to abiotic stresses such as cold, drought, strong light, heavy metals, salt, and oxidative stress, indicating that HSFs play a key role in regulating plant stress resistance. HSFA It mainly manifests as positive regulation under abiotic stress and is divided into 9 subclasses based on its structure, including A1, A2, A3, A4, A5, A6, A7, A8, and A9. HSFA The functions of genes related to stress resistance have been extensively studied. HSFA1 It is the core regulatory factor for plants to cope with high temperature stress, and it mainly acts as a switch by binding to heat shock proteins under stress environments. HSFA1 It can also induce HSFA2, HSFA3, HSFA7 Various transcription factors such as α, β and β participate in the regulation under high temperature stress. HSFA1 It participates in the removal of reactive oxygen species induced by stress and alleviates the damage caused to plants under stress. HSFA2, HSFA3 In addition to high temperature stress, it also responds to multiple stresses such as salt, drought, and oxidation. HSFA2, HSFA3 Enhanced heat tolerance in Arabidopsis thaliana, and in tall fescue ( Festuca arundinacea ),lily( Lilium longiflorum ) also showed enhanced heat resistance in the overexpression strains. HSFA4 Participated in the regulation process of salt tolerance, strong light, calcium and other stresses. HSFA5Can be used with HSFA4 Combine to form dimers, thereby inhibiting its activity. HSFA6 In addition to increasing plant heat tolerance through the ABA pathway, it can also regulate salt, osmotic and low temperature stress. HSFA7 It can enhance the salt and drought tolerance of plants. HSFA8 The drought resistance of plants can be affected through the flavonoid pathway. HSFA9 It is expressed in large quantities in plant seeds, regulates high temperature, dehydration, and oxidative stress, and is related to seed development and lifespan. HSFA Genes are directly or indirectly involved in the regulation of various stress resistances of plants, especially high temperature tolerance.

[0004] The current study has preliminarily explained its heat tolerance regulation mechanism. In other grasses, heterologous expression of tall fescue or ryegrass ( Lolium perenne ) HSFA Genetic tests have confirmed HSFA Genes can act as positive regulatory factors of abiotic stress, improve plant heat tolerance, and may interact with ABA response factors. As an excellent forage grass with strong stress resistance, Leymus japonicus is a good research object for studying stress resistance traits and functional genes. At present, the Intergovernmental Panel on Climate Change (IPCC) of the United Nations points out that the trend of global warming exceeding 1.5℃ may be unavoidable, and the global biological and ecological development is not optimistic. Therefore, we chose genes related to multiple stress resistance, especially heat resistance. HSFA Gene, in order to preliminarily explore the HSFA Genetic stress responses and mechanisms. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention provides a gene of Leymus japonicus involved in adverse stress and its application. The gene is involved in the regulation related to the stress resistance of Leymus japonicus, providing a theoretical basis and technical support for forage improvement and genetic breeding, the development and utilization of excellent germplasm resources, and the restoration and protection of grassland ecosystems. At the same time, it also enriches the candidate gene pool for the improvement of excellent forage.

[0006] The technical solution adopted by the present invention is as follows: A gene of Leymus japonicus involved in stress LsHSFA , whose nucleotide sequence is shown in SEQ ID NO.1.

[0007] The above genes of Leymus involved in stress LsHSFA The amino acid sequence of the encoded protein is shown in SEQ ID NO.2.

[0008] In the present invention, the transcriptome of Leymus japonicus is sequenced to construct a transcriptome sequence information database of Leymus japonicus. TaHSFAThe homology of the gene was obtained, and a 597bp open reading frame was obtained, which was identified as a key candidate gene for regulating the high temperature tolerance and stress resistance of Leymus thaliana. Since the technology of transgenic Leymus thaliana is not mature enough, the gene was transformed into Arabidopsis thaliana to construct transgenic Arabidopsis thaliana, and its function was verified, and it was determined to be a positive regulatory factor of abiotic stress of Leymus thaliana, directly or indirectly involved in the regulation of high temperature tolerance, and improved the heat tolerance of the plant.

[0009] A gene containing Leymus involved in stress LsHSFA of the recombinant plasmid.

[0010] According to the preferred embodiment of the present invention, the vector of the recombinant plasmid is pEASY -blunt or pCAMBIA1300-GFP.

[0011] A gene containing Leymus involved in stress LsHSFA Or the recombinant bacteria of the above-mentioned recombinant plasmid.

[0012] According to the present invention, the host of the recombinant bacteria is Escherichia coli. Trans 1-T1 or Escherichia coli DH5α or Agrobacterium tumefaciens GV3101.

[0013] The above genes of Leymus involved in stress LsHSFA Application in promoting the development of the aboveground parts of plants.

[0014] The above genes of Leymus involved in stress LsHSFA Application in regulating plant response to heat stress, salt stress or drought stress environment.

[0015] According to the preferred embodiment of the present invention, the application is to transform the gene of Leymus japonicus involved in adverse stress into LsHSFA Transformed into plants to achieve genes involved in stress LsHSFA Overexpression in plants.

[0016] Preferably according to the present invention, the plant is Arabidopsis thaliana or Leymus japonicus.

[0017] Beneficial effects: 1. The present invention discovered for the first time the genes involved in adverse stress in Leymus sibiricus ( LsHSFA ), the nucleotide sequence of which is shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.2.

[0018] 2. The present invention analyzes and verifies the function of genes involved in adverse stress of Leymus gracilis by cloning and identifying, analyzing gene expression and genetic transformation of genes. The results show that genes involved in adverse stress of Leymus gracilis have an important influence on promoting the aboveground development of Leymus gracilis and improving its resistance to stress such as heat resistance, salt resistance and drought resistance. By utilizing the Leymus gracilis stress resistance gene to positively regulate the abiotic stress function, the ability of Leymus gracilis to cope with various stress adversities can be better improved, so that it can adapt to a more complex and harsh ecological environment, improve degraded and desertified grasslands, and provide potential application prospects for the future breeding of Leymus gracilis varieties and the construction of excellent populations.

[0019] 3. The present invention discloses the application of genes of Leymus oxyphylla involved in adverse stress in promoting plant growth and development or improving plant response to stress environment. LsHSFA The function of the gene to promote the development of the aboveground parts, as well as the resistance to heat, drought and salt stress, will help to achieve the goal of improving the survival rate of Leymus oxyphylla, increasing the yield of Leymus oxyphylla and ultimately improving the quality of Leymus oxyphylla in various habitats under the background of global warming. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 For expansion LsHSFA Gene agarose gel electrophoresis diagram; in the figure, lane M is the standard, lane 1 is the amplified sample of the rhizome apical bud of Leymus japonicus, and lane 2 is the amplified sample of the leaf of Leymus japonicus; Figure 2 The agarose gel electrophoresis diagram after double digestion with SpeI and SmaI; in the figure, lane M is the standard, the left figure is the electrophoresis diagram of double digestion with expression vector pCAMBIA1300-GFP, and lanes 1 and 2 are the digestion samples of expression vector pCAMBIA1300-GFP; the right figure is the plasmid pEASY - LsHSFA Double enzyme digestion electrophoresis diagram, lanes 1 and 2 are plasmid pEASY - LsHSFA Enzyme digestion samples; Figure 3 The recombinant vector pCAMBIA1300- LsHSFA -GFP transformed E. coli DH5α positive colony PCR verification agarose gel electrophoresis diagram; in the figure, lane M is the standard, lanes 1-6 are amplified to obtain the target gene, verified as positive transformants; Figure 4 The recombinant vector pCAMBIA1300- LsHSFA -Agarose gel electrophoresis diagram of positive colony PCR verification of Agrobacterium tumefaciens GV3101 transformed with GFP; in the figure, lane M is the standard, and lanes 1-6 are all amplified to obtain the target gene, which is verified as a positive transformant; Figure 5 This is a diagram of the subcellular localization analysis of LsHSFA protein; Figure 6 for LsHSFA Screening pictures of positive seedlings of T1 generation of transgenic Arabidopsis; Figure 7 For T1 generation LsHSFA Gel electrophoresis detection diagram of PCR amplification verification of transgenic Arabidopsis positive seedlings; in the figure, lane M is the standard, and lanes 1-2 and lanes 4-10 are amplified to obtain the target gene, which is verified as a positive transformant; Figure 8 Different tissues of transgenic Arabidopsis LsHSFA Expression analysis bar graph; Figure 9 for LsHSFA Phenotypic and statistical bar graphs of transgenic Arabidopsis and wild-type Arabidopsis; A is the morphological diagram of Arabidopsis after 7 days of vertical culture, B is the bar graph of the length of the main root of Arabidopsis, C is the bar graph of the number of lateral roots of Arabidopsis, and D is the bar graph of overexpression LsHSFA The aboveground phenotype of Arabidopsis thaliana at the seedling stage. F is the bar graph of leaf coverage area of ​​Arabidopsis thaliana at the seedling stage. E is the overexpression LsHSFA Phenotype of the aboveground parts of Arabidopsis during the bolting period, G represents overexpression LsHSFA Histogram of main branch height during the bolting period of Arabidopsis thaliana; Figure 10 for LsHSFA Phenotypes of transgenic Arabidopsis and wild-type Arabidopsis before and after heat treatment; A is the phenotype of different strains at 10 days old under normal culture conditions, and B is the phenotype of different strains after heat stress treatment and continued normal culture for 7 days; Figure 11 Under salt stress LsHSFA Phenotypes and data statistics of transgenic Arabidopsis and wild-type Arabidopsis; A is the root phenotype diagram of Arabidopsis under 50 mM NaCl stress, B is the main root length bar graph of Arabidopsis under salt stress, C is the lateral root number bar graph of Arabidopsis under salt stress, D is the aboveground phenotype diagram of Arabidopsis under salt stress, E is the SOD enzyme activity analysis diagram of Arabidopsis under salt stress, and F is the POD enzyme activity analysis diagram of Arabidopsis under salt stress; Figure 12 Under drought stress LsHSFA Phenotypes and data statistics of transgenic Arabidopsis and wild-type Arabidopsis; A is the root phenotype of Arabidopsis under 50 mM mannitol simulated drought treatment, B is the main root length bar graph of Arabidopsis under drought stress treatment, C is the lateral root number bar graph of Arabidopsis under drought stress treatment, D is the aboveground phenotype of Arabidopsis under drought stress treatment, E is the SOD enzyme activity analysis of Arabidopsis under drought stress treatment, and F is the POD enzyme activity analysis of Arabidopsis under drought stress treatment; Figure 13 Under ABA stress LsHSFAPhenotypes and data statistics of transgenic Arabidopsis and wild-type Arabidopsis; A is the root phenotype of Arabidopsis under 50 μM ABA treatment, B is the bar graph of the main root length of Arabidopsis under ABA stress treatment, C is the bar graph of the number of lateral roots of Arabidopsis under ABA stress treatment, D is the aboveground phenotype of Arabidopsis under ABA stress treatment, E is the SOD enzyme activity analysis diagram of Arabidopsis under ABA stress treatment, and F is the POD enzyme activity analysis diagram of Arabidopsis under ABA stress treatment. DETAILED DESCRIPTION

[0021] The following examples are provided for a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified. The quantitative tests in the following examples were repeated three times, and the results were averaged.

[0022] Example 1 Leymus chinensis LsHSFA Identification of gene sequences The transcriptome sequence information of Leymus chinensis was determined by transcriptome sequencing technology, and a Leymus chinensis transcriptome sequence information database was constructed. Among all the Leymus chinensis transcriptome sequence information, wheat was selected to identify candidate genes resistant to high temperature stress in the Leymus chinensis genome. TaHSFA The gene (NCBI: FJ968107.1) was used as a template to compare and search for similar sequences in the transcriptome sequence of Leymus chinensis. It was found that there was a 597 bp ORF (open reading frame) in the transcriptome sequence of Leymus chinensis, which was similar to TaHSFA The gene homology is 96.82% and is named LsHSFA , whose nucleotide sequence is shown in SEQ ID NO.1.

[0023] LsHSFA The protein encoded by the gene contains 198 amino acids, and its amino acid sequence is shown in SEQ ID NO.2. Its molecular mass is 23493.44Da, and its isoelectric point PI value is 4.93. LsHSFA protein is a hydrophilic protein, and its protein transmembrane domain prediction result is: the entire peptide chain of LsHSFA protein is located outside the cell membrane, and there is no transmembrane domain.

[0024] Example 2 Leymus chinensis LsHSFA Construction of gene recombination vector Total RNA was extracted from Leymus japonicus and converted into cDNA. The corresponding primers were designed for gene amplification. After agarose gel electrophoresis, the target band was recovered and compared with pEASY -blunt vector ligation, transformed into E. coli Trans1-T1 competent cells, sequence and analyze the sequence, pick the positive clones and shake the bacteria for plasmid extraction, add restriction sites and perform double restriction digestion with the vector pCAMBIA1300-GFP, and connect pCAMBIA1300-GFP with T4 ligase LsHSFA The target gene fragment is then transferred into Escherichia coli DH5α competent cells, the positive clones are picked again for plasmid extraction, and finally transferred into Agrobacterium GV3101 to preserve the positive bacteria. The specific operation steps are as follows.

[0025] 1. Total RNA Extraction The rhizome apical buds and leaves of Leymus japonicus were used as materials and extracted according to the operating procedures of the Plant Total RNA Extraction Kit (Nanjing Novogene). The reagents and consumables used were RNase-free. The RNA concentrations of the rhizome apical buds and leaves were detected by the ultra-micro spectrophotometer Nano-300, which were 683.6 ng / μL and 829.1 ng / μL, respectively, and the A260 / A280 was around 2.0, which was of good quality and could meet the requirements of subsequent reverse transcription experiments.

[0026] 2. Obtaining cDNA According to the instructions of the reverse transcription kit (Novozyme), the RNA extracted from the apical buds and leaves of the rhizomes of Leymus japonicus were reverse transcribed to synthesize cDNA, and the cDNA products were used for target gene amplification.

[0027] 3. LsHSFA Gene cloning (1) Based on LsHSFA The full-length sequence of the gene and the restriction enzyme sites of the expression vector used were designed to include the specific primers of the entire ORF (excluding the stop codon), and the restriction enzyme sites were added to the 5' and 3' ends of the primers. The primer sequences were: LsHSFA-F-SmaI: 5'-CCCGGGATGGAGAACGAGTTCCAGGATG-3' (SEQ ID NO. 3), LsHSFA-R-SpeI: 5'-ACTAGTACGAGCTTCGACGGTTTGGTTC-3' (SEQ ID NO. 4); (2) Using the first strand of cDNA obtained above for PCR LsHSFA The specific primers were used for amplification with 2×PhantaMax high-fidelity enzyme (Novozyme). The amplification reaction conditions were 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, and 72°C extension for 5 min after 35 cycles; (3) Detection by 1% agarose gel electrophoresis, such as Figure 1As shown, the amplified fragments were all consistent with the size of the target gene. The target gene fragments were recovered by cutting the gel according to the instructions of the gel recovery kit (Tiangen Biochemical).

[0028] 4. Connect pEASY -blunt vector and E. coli Trans Transformation of 1-T1 competent cells Mix 4 μL of gel recovery product with 1 μL of cloning vector pEASY -blunt vector was gently mixed and reacted at 25℃ for 30min. All ligation products were transferred to 50µL E. coli melted on ice in a clean bench. Trans In 1-T1 competent cells, gently flick the bottom of the tube to mix slowly, place on ice for 30 minutes, heat shock in 42℃ water bath for 1 minute, and quickly place on ice for 2 minutes; add 900µL of LB liquid culture medium in the clean bench, shake and culture at 37℃ and 200rpm for 1 hour; centrifuge at 5000rpm for 1 minute, pour out most of the supernatant in the clean bench, retain 150µL of supernatant, gently aspirate and resuspend the bacteria with a gun, and then spread all of it on LB solid culture medium containing 100 μg / mL kanamycin. After sealing the culture dish with sealing film, turn it upside down in a 37℃ incubator and culture for 14-16 hours.

[0029] After the colonies grow on the plate, use a sterile disposable needle to pick up a single white colony with neat edges and put it into 5 mL of LB liquid medium containing 100 μg / mL kanamycin, shake the bacteria at 37°C and 200 rpm for 12 hours, and use the above-mentioned LsHSFA-F-SmaI and LsHSFA-R-SpeI as primers for PCR amplification verification. The PCR reaction program is: 95°C pre-denaturation for 3 minutes; 95°C denaturation for 15 seconds, 58°C annealing for 15 seconds, 72°C extension at 1 kb / min, and 72°C extension for 5 minutes after 35 cycles; store at 4°C; after 1% agarose gel electrophoresis to detect positive clones, send them to the sequencing company for detection, and compare the sequencing results with LsHSFA The gene sequences were compared, and the colonies with correct sequencing were used to extract the recombinant plasmid using a plasmid extraction kit (Beijing Kangwei) pEASY - LsHSFA .

[0030] 5. Leymus LsHSFA Construction of gene overexpression vector The recombinant plasmid was digested by double enzyme digestion. pEASY - LsHSFA The expression vector pCAMBIA1300-GFP was double-digested with SpeI and SmaI in a PCR instrument at 37°C for 2 h, and the digested products were detected by electrophoresis on 1% agarose gel. Figure 2 As shown, judging by the strip size LsHSFAThe gene and pCAMBIA1300-GFP vector have been correctly cut. After the target band is recovered from the gel, the vector after enzyme cutting and the target gene are mixed at a mass concentration ratio of 3:1, T4 DNA ligase is added, and the reaction is carried out at 25℃ for 2h to obtain the ligation product. The ligation product is transferred into Escherichia coli DH5α competent cells to obtain the recombinant vector pCAMBIA1300-GFP- LsHSFA . Pick a single colony for PCR verification of positive bacteria, and the electrophoresis results are as follows Figure 3 As shown, the positive single colony consistent with the target gene was cultured and sequenced. The sequencing results were consistent with LsHSFA The gene sequence comparison results were completely consistent, indicating that pCAMBIA1300-GFP- LsHSFA The overexpression vector was constructed successfully. The plasmid was extracted from the positive transformant and transferred into Agrobacterium GV3101 competent cells by freeze-thaw method. Single colonies were picked for colony PCR detection. The target bands were consistent, such as Figure 4 As shown, it proves that the transformation into Agrobacterium was successful, and Agrobacterium-positive transformants were obtained. The bacteria were shaken and the bacterial liquid was preserved for later use.

[0031] Example 3 Leymus chinensis LsHSFA Verification of gene function Subcellular localization was used to identify the specific location of LsHSFA protein in the cell. After Arabidopsis thaliana was cultured to an appropriate age, it was transformed by Agrobacterium-mediated method, the T3 representative phenotype was observed, and statistical analysis was performed to analyze its function. The specific operation steps are as follows.

[0032] 1. Subcellular localization of LsHSFA First, the subcellular localization of LsHSFA protein was predicted using the online prediction software https: / / wolfpsort.hgc.jp. The results showed that LsHSFA protein may be localized in the nucleus (nucl) or cytoplasm (cyto). Subcellular localization was verified by injecting tobacco epidermis. The Agrobacterium positive transformant constructed in Example 2 was activated by shaking the bacteria until the OD 600 After the OD value is 1.2-1.5, resuspend the Agrobacterium cells with tobacco suspension to 600 The infection solution was 0.9-1.0, and after standing at room temperature for 3 hours, the infection solution was injected into the lower epidermal cells of the tobacco leaves from the back with a disposable syringe, and the injection range was marked. The tobacco epidermal cells were transiently transformed, and after 72 hours of culture, the lower epidermal cells of the tobacco leaves at the marked position were torn off, and the expression and localization of LsHSFA protein in the tobacco epidermal cells were observed using a laser confocal microscope. The results are shown in Figure 5 As shown, green fluorescent protein GFP was expressed throughout the cells and LsHSFA protein was localized in the cytoplasm.

[0033] The above tobacco suspension (100 mM) was prepared as follows: 0.213 g of MES (2-morpholineethanesulfonic acid) and 0.09521 g of MgCl2 were weighed on a precision balance and dissolved in 100 mL of sterile ddH2O, pH was adjusted to 5.6-5.7, 0.5 g of glucose and 100 μL of AS solution (100 mM) were added, and the mixture was prepared immediately before use; Acetosyringone (AS) solution (100 mM): Weigh 392.4 mg of acetosyringone powder and dissolve it in 12 mL of 95% ethanol. Add 8 mL of sterile ddH2O and mix thoroughly. Sterilize the AS solution with a 0.22 mm filter membrane in a clean bench and dispense into sterilized EP tubes and store at -20°C.

[0034] 2. Obtained by Agrobacterium-mediated genetic transformation LsHSFA Transgenic Arabidopsis Place Arabidopsis seeds in a 1.5 mL centrifuge tube, add 75% ethanol to the centrifuge tube in a clean bench, and shake and wash for 1 min; after the seeds sink to the bottom of the tube, remove the ethanol, add 10% sodium hypochlorite and shake and wash for 10 min; discard the sodium hypochlorite, and then wash with sterile water 3-5 times to thoroughly wash the sodium hypochlorite; sow the seeds on the MS culture medium with a sterilized pipette tip, wrap with sealing film, and place at 4℃ away from light for 48 h; the culture dish after light protection is placed at 22℃, 16 h light / 8 h dark, and light intensity of 40 μmoL•m -2 •s -1 After culturing in the culture room for 2 weeks, the well-growing wild-type Arabidopsis thaliana were transplanted into nutrient soil and maintained regularly until they bolted and produced flower buds.

[0035] Select Arabidopsis thaliana with good growth, cut off the fruit pods and flowers that have completed self-pollination, and water them thoroughly the day before infection; resuspend the Agrobacterium cells in 1 / 2MS liquid medium to a concentration of OD 600 The value is about 0.8-1, and 0.01% Silwet77 is added to obtain the Arabidopsis infection solution. The Arabidopsis inflorescence is completely immersed in the infection solution, gently shaken and kept for 30 seconds, then wrapped with plastic wrap to maintain humidity, cultured in the dark for 24 hours, and then transferred to normal growth conditions for 7-9 days before secondary infection; wait for the seeds to mature and harvest the T0 generation LsHSFA Transgenic Arabidopsis seeds.

[0036] 3. Screening and preliminary examination of transformed plants The harvested T0 generation seeds were dried in an oven at 38°C, disinfected with 10% sodium hypochlorite and washed, and then sown in MS medium containing 100 mg / L hygromycin for T1 generation. LsHSFA Screening of transgenic Arabidopsis thaliana, the screening results are as follows Figure 6As shown, Arabidopsis thaliana with normal rooting on the culture medium was transplanted into nutrient soil. After growing for one week, Arabidopsis thaliana DNA was extracted using the CTAB method. The extracted DNA was used as a template. LsHSFA The specific primers LsHSFA-F-XbaI and LsHSFA-R-SpeI were used for PCR verification. The electrophoresis results were as follows: Figure 7 The sample amplified the target fragment band, indicating that the T1 generation was successfully obtained. LsHSFA Transgenic Arabidopsis thaliana. LsHSFA Transgenic Arabidopsis thaliana was further cultivated and screened by genetic segregation ratio until the T3 generation was homozygous positive. LsHSFA Transgenic Arabidopsis thaliana.

[0037] 4. LsHSFA Gene tissue expression analysis For analysis LsHSFA The expression pattern of the gene in various tissues of the plant was screened and the homozygous positive T3 generation was screened. LsHSFA Transgenic Arabidopsis thaliana was tested by qRT-PCR and analyzed LsHSFA The expression of genes in roots, stems, leaves and flowers was different. Using the cDNA obtained for qPCR as a template, quantitative primers were designed and measured. LsHSFA The expression level of the gene was measured with Arabidopsis actin as the internal reference gene and the expression level in the root as the control.

[0038] The primer sequences used for qPCR are: LsHSFA-F: 5'-ACGCTATTGTCTTGAGGGC-3' (SEQ ID NO.5), LsHSFA-R: 5'-GCATTGCCTTGATGTAGTTG-3' (SEQ ID NO. 6); After preparing the reaction system according to the requirements of the fluorescence quantitative PCR kit, the pre-denaturation was carried out at 95℃ for 10 min; the denaturation was carried out at 95℃ for 15s; the annealing was carried out at 60℃ for 1 min; the cycle number was 40, and the melting curve analysis was carried out according to the recommended program of the ABI 7500 fluorescence quantitative PCR instrument. Three biological replicates were performed for each sample to analyze the expression of genes in different tissues. The results are shown in Figure 8 As shown: LsHSFA The gene was expressed in all tissues, with the highest expression in leaves, followed by roots, flowers, and stems.

[0039] 5. LsHSFA Effects of ectopic gene expression on root development in Arabidopsis Wild-type Arabidopsis was used as the control group and LsHSFA Transgenic Arabidopsis thaliana was used as the treatment group, and wild-type Arabidopsis thaliana and LsHSFAThe seeds of transgenic Arabidopsis were sown in culture dishes containing MS medium. After vernalization at 4°C for 3 days, they were transferred to an environment with a temperature of 22°C and 16 h of sunshine / 8 h of darkness for 7 days. Then the seedlings were transferred to a square (13×13 cm) MS medium and cultured vertically for 7 days. The seedlings were then moved to a 13 cm×13 cm square plate and cultured vertically under light for 10 days. Both the control group and the treatment group had three biological replicates. Image J software was used to measure the root length, Excel 2019 was used for preliminary data statistics and bar graph drawing, and stst was used for one-way analysis of variance and significant difference analysis (p<0.05). The results are shown in Figure 9 As shown in AC, through the phenotypic observation of the main root and lateral root of each strain, LsHSFA Transgenic Arabidopsis thaliana ( 35S:LsHSFA ) showed no significant difference in taproot length or lateral root number compared with wild-type Arabidopsis thaliana (Col), indicating that LsHSFA The gene had no significant effect on root morphology under normal culture conditions.

[0040] 6. LsHSFA Effects of ectopic gene expression on shoot development in Arabidopsis Wild-type Arabidopsis was used as the control group and LsHSFA Transgenic Arabidopsis thaliana was used as the treatment group. Wild-type Arabidopsis thaliana cultured in MS medium for 7 days and LsHSFA Transgenic Arabidopsis seedlings were transferred to nutrient soil and cultured normally. Starting from the first day of transfer, photos were taken every day and the aboveground phenotypes were observed. There were three biological replicates for both the control group and the treatment group. Image J software was used to measure the leaf coverage area and main branch height, and the data statistics and analysis were the same as step 5. The results are shown in Figure 9 As shown in DG, when the number of culture days is the same, LsHSFA The transgenic Arabidopsis leaves grow bolting points earlier than the wild type. By measuring the leaf coverage area, the leaf coverage area of ​​the transgenic strain is about 2.5 times that of the wild type, and it reaches maturity faster than the wild type Arabidopsis in terms of morphology. When the normal culture is maintained until about the fifth week, the transgenic strains at the bolting stage LsHSFA The transgenic lines bolted faster than the wild type, with bolting heights about twice that of the wild type, and produced pods earlier than the wild type. Although the wild type Arabidopsis eventually reached the same height as the transgenic lines, this suggests that LsHSFA Although the gene cannot make Arabidopsis plants larger, it can accelerate their growth, indicating that LsHSFA The gene is related to growth and development regulation.

[0041] 7. Heterologous Overexpression LsHSFA Effects on stress resistance of Arabidopsis Since the transgenic technology of Leymus japonicus is not yet mature, the selected LsHSFA The stress resistance experiments of transgenic Arabidopsis were carried out. Through the stress treatment experiments in culture dishes and nutrient soil and the detection of stress resistance enzyme activity, the effects of heterologous expression of the gene in Arabidopsis on stress resistance were explored, and then the LsHSFA Stress resistance-related functions of genes.

[0042] In the heat stress experiment, wild type and overexpressing LsHSFA After sterilization, seeds of different strains of genes were evenly sown on the same MS culture dish. After being treated at 4℃ in the dark for two days, the culture dish was placed in an environment of 16 h light / 8 h dark and 22℃ for about 10 days. Then, it was placed in a plant incubator and treated at 37℃ for 30 min and 42℃ for 1 h. Figure 10 A) was placed in a normal culture room, and the recovery of Arabidopsis thaliana treated with heat stress was observed every day, and the recovery of Arabidopsis thaliana was recorded and photographed. Figure 10 As shown in B, wild-type Arabidopsis (Col-0) and LsHSFA Transgenic Arabidopsis thaliana ( LsHSFA-L1, LsHSFA-L2, LsHSFA-L3 ), after seven days of heat treatment recovery, wild-type Arabidopsis plants died, and the surviving plants also showed growth retardation. The leaves of the transgenic Arabidopsis strains were healthier, showing a light green color, with only a few leaves slightly dark red on the back. The recovery was good, and the plants grew normally. This shows that overexpression in Arabidopsis LsHSFA Genes can improve heat tolerance in plants. LsHSFA The gene was a heat stress response gene, which was consistent with the predicted result.

[0043] In the salt stress experiment, wild type and LsHSFA After sterilization, transgenic Arabidopsis seeds were sown on MS culture dishes at 4°C in the dark for 2 days, and then the MS culture dishes were placed in a light environment of 16 h / darkness of 8 h and 22°C for 7 days to germinate. After germination, the wild-type and transgenic seedlings were transferred to square (13 cm × 13 cm) MS culture dishes containing 50 mM NaCl, and the culture dishes were placed vertically for 14 days. Photographs were taken to record the growth status of the Arabidopsis roots under the above treatments. At the same time, the germinated wild-type and transgenic seedlings were transferred to MS culture dishes containing 50 mM NaCl. LsHSFATransgenic Arabidopsis was planted in nutrient soil, with five plants transplanted into each square pot in a regular arrangement. The plants were cultured at 23°C and under conditions of 14 h light / 10 h darkness. They were cultured normally until they were four weeks old and then subjected to salt stress treatment: a gradient treatment with different concentrations of NaCl solution was used. The treatment method was to water the Arabidopsis with NaCl solution until water slightly seeped out of the pot. On the first day, 50 mM NaCl was used, 100 mM NaCl was used on the fourth day, and 200 mM NaCl was used on the seventh day. The aboveground leaves were cut the next day, quickly frozen in liquid nitrogen, and stored at -80°C. The SOD and POD enzyme activities were detected according to the instructions of the SOD kit (Suzhou Keming) and the POD kit (Jiangsu Yisejiu). The control and treatment were all biologically replicated three times. The results are shown in Figure 2. Figure 11 As shown, LsHSFA Transgenic Arabidopsis thaliana ( 35S:LsHSFA ) Compared with wild-type Arabidopsis thaliana (Col), the number of lateral roots of the transgenic lines increased significantly, indicating that the growth of lateral roots may be related to the positive regulation of this gene. In addition, the root richness and aboveground growth of the transgenic lines were better than those of the wild type, indicating that this gene improved resistance to salt stress. In addition, the POD enzyme activity of the transgenic lines was significantly higher than that of the wild type and the transgenic lines not subjected to salt stress, indicating that Leymus japonicus LsHSFA The gene may respond to salt stress by regulating POD enzyme.

[0044] In the drought stress experiment, wild type and LsHSFA After sterilization, transgenic Arabidopsis seeds were sown on MS culture dishes at 4°C in the dark for 2 days, and then the MS culture dishes were placed in a light / dark environment of 16 h / 8 h, 22°C for 7 days to germinate. After germination, the wild-type and transgenic seedlings were transferred to square (13 cm × 13 cm) MS culture dishes containing 50 mM mannitol, and the culture dishes were placed vertically for 14 days. Photographs were taken to record the growth status of the Arabidopsis roots under the above treatments. At the same time, the germinated wild-type and transgenic seedlings were transferred to MS culture dishes containing 50 mM mannitol. LsHSFA Transgenic Arabidopsis thaliana was planted in nutrient soil, with five plants transplanted into each square pot in a regular arrangement. The plants were cultured at 23°C with 14 h of light and 10 h of darkness. The plants were cultured normally until they were four weeks old and then subjected to drought stress treatment: watering was stopped, the plants were naturally dry for 7 days, and the aboveground leaves were cut the next day, frozen in liquid nitrogen, and stored at -80°C. The test was performed according to the instructions of the SOD kit (Suzhou Keming) and the POD kit (Jiangsu Yisejiu). The control and treatment were repeated three times. The results are shown in Table 1. Figure 12 As shown, LsHSFA Transgenic Arabidopsis thaliana ( 35S:LsHSFA ) compared with wild-type Arabidopsis thaliana (Col), LsHSFA Transgenic plants showed more lateral roots, indicating LsHSFAThe gene can respond to drought stress and promote lateral root growth in the process of stress resistance, thus facing the drought environment. In addition, wild-type Arabidopsis seedlings died under drought treatment, but transgenic Arabidopsis did not die, and induced faster bolting after stress, which further illustrates that LsHSFA Genes can respond to drought stress and affect the growth and development of plants, improving the drought resistance of plants. At the same time, we found that there was no significant difference in the SOD and POD enzyme activities between the untreated wild type and the overexpression strains, but under drought treatment, the SOD enzyme activity of the transgenic strains increased more significantly than that of the wild type. These experimental results show that LsHSFA The gene may resist the damage caused by drought stress by increasing the activity of SOD enzyme and improve the drought resistance of Arabidopsis.

[0045] In the ABA (abscisic acid) stress experiment, wild-type and transgenic Arabidopsis seeds were sterilized and sown on MS culture dishes at 4°C in the dark for 2 days. The MS culture dishes were then placed in an environment of 16 h light / 8 h dark and 22°C for 7 days to allow them to germinate. The germinated wild-type and transgenic seedlings were transferred to square (13 cm × 13 cm) MS culture dishes containing 50 μM ABA, and the culture dishes were placed vertically for 14 days. Photographs were taken to record the growth status of the Arabidopsis roots under the above treatments. At the same time, the germinated wild-type and transgenic Arabidopsis were placed in a 13 cm × 13 cm square MS culture dish containing 50 μM ABA. LsHSFA Transgenic Arabidopsis was planted in nutrient soil, with five plants transplanted into each square pot in a regular arrangement. The plants were cultured at 23°C with 14 h of light and 10 h of darkness. The plants were cultured normally until they were four weeks old and then subjected to ABA stress treatment: a gradient of ABA solutions of different concentrations was used. The treatment method was to water the Arabidopsis with ABA solution until water slightly seeped out of the pot. On the first day, 50 μM ABA solution was used, on the fourth day, 75 μM ABA solution was used, and on the seventh day, 100 μM ABA solution was used. The next day, the aboveground leaves were cut, immediately frozen in liquid nitrogen, and stored at -80°C. The test was performed according to the instructions of the SOD kit (Suzhou Keming) and the POD kit (Jiangsu Yisejiu). The control and treatment were biologically replicated three times. The results are shown in the figure below. Figure 13 As shown, LsHSFA Transgenic Arabidopsis thaliana ( 35S:LsHSFA ) had no significant difference in taproot length and lateral root number compared with wild-type Arabidopsis thaliana (Col), indicating LsHSFA The gene could not enhance the plant's resistance to ABA stress, and the enzyme activity change trend was the same as that of the wild type, with no significant difference, indicating that the gene may not be involved in the ABA response pathway.

[0046] In summary, LsHSFA It improves the plant's heat resistance, drought resistance and salt resistance, but has no obvious resistance to ABA stress.LsHSFA The growth of lateral roots in transgenic lines under salt stress may be a way for the gene to respond to salt stress and to be positively regulated. It may also participate in the tolerance to salt stress by regulating POD enzyme instead of SOD enzyme. LsHSFA The transgenic lines of the gene may mainly respond to drought stress by increasing the activity of SOD enzymes related to stress resistance, thereby enhancing the drought resistance of plants; in addition, LsHSFA The gene cannot enhance plant resistance to ABA stress and may not be involved in the response to ABA signals.

Claims

1. A gene in Leymus japonicus involved in stress LqCy , characterized in that, The gene LqCy The nucleotide sequence is shown in SEQ ID NO.

1.

2. The gene of Leymus japonicus involved in adverse stress according to claim 1 LqCy The encoded protein is characterized in that The amino acid sequence of the protein is shown in SEQ ID NO.

2.

3. A gene comprising the Leymus japonicus gene involved in adverse stress as claimed in claim 1 LqCy of the recombinant plasmid.

4. The recombinant plasmid according to claim 3, characterized in that The vector of the recombinant plasmid is pEASY -blunt or pCAMBIA1300-GFP.

5. A gene comprising the Leymus japonicus gene involved in adverse stress as claimed in claim 1 LqCy Or the recombinant bacteria of the recombinant plasmid according to claim 3.

6. The recombinant bacterium according to claim 5, characterized in that The host of the recombinant bacteria is Escherichia coli Trans 1-T1 or Escherichia coli DH5α or Agrobacterium tumefaciens GV3101.

7. The gene of Leymus japonicus involved in adverse stress according to claim 1 LqCy Application in promoting the development of the aboveground parts of plants.

8. The gene of Leymus japonicus involved in adverse stress according to claim 1 LqCy Application in regulating plant response to heat stress, salt stress or drought stress environment.

9. The use according to any one of claim 7 or claim 8, characterized in that: The application is to transform the gene of Leymus japonicus involved in adverse stress LqCy Transformed into plants to achieve genes involved in stress LqCy Overexpression in plants.

10. The use according to any one of claim 7 or claim 8, characterized in that: The plant is Arabidopsis thaliana or Leymus japonicus.

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