Fibraurea mairei dreb gene and application thereof in improving cold resistance of plants

Abstract

This invention belongs to the field of agricultural biotechnology, specifically relating to a type of sisal. DREB Genes and their application in improving plant cold resistance. This invention provides sisal. DREB The gene expression level increased significantly after low-temperature stress, indicating a close relationship between its expression and the response of sisal to low-temperature stress. The study found that transgenic methods can significantly improve the expression level of sisal. DREB Genes were overexpressed, along with sisal DREB Increased gene expression levels led to improved cold resistance in transgenic Arabidopsis thaliana. Overexpression of sisal in plants also improved cold resistance. DREB The discovery that genes can enhance plant cold resistance can be used to breed new varieties or seedlings of cold-resistant plants, providing a theoretical basis and genetic resources for genetic breeding, which is of great significance to agricultural production.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biotechnology, specifically relating to a sisal DREB gene and its application in improving plant cold resistance. Background Technology

[0002] DREB The dehydration-responsive element binding gene family plays a crucial role in plant growth regulator signaling pathways in response to abiotic stress. The proteins they encode can specifically bind to the DRE / CRT (dehydration response element / C-repeat) and G / ACCGAC cis-acting elements in the promoter regions of abiotic stress response genes. Currently, these genes have been reported in over 60 plant species. DREB Whole-genome analysis of gene families, with the number of published plant genomes increasing year by year, has made the analysis of gene families more complex. DREB The evolutionary mechanisms of gene families at the genome level have become possible. With the increasing amount of biological information each year, the exploration of... DREB Understanding the mechanisms by which gene families regulate plant responses to abiotic stress is of great significance for elucidating their biological functions.

[0003] Conducted in Arabidopsis thaliana DREB Gene function identification work began relatively early, and the biological functions of many genes have been elucidated, mainly involving water stress and heat stress, and participating in the regulation of leaf senescence. In recent years, with the deepening of crop molecular biology research, studies have also been conducted on other crops. DREB Research on gene regulation related to abiotic stress. For example, OsDREB1C It participates in regulating nitrogen nutrient absorption and photosynthesis in rice. SlDREBA4 Involved in regulating the tomato's response to heat stress, GmDREB2 It participates in regulating drought stress responses. In addition, Populus euphratica... PeDREB2a It can improve the drought and salt tolerance of transgenic Arabidopsis thaliana, eucalyptus EgrDREB3 It can enhance the salt tolerance of transgenic Arabidopsis thaliana.

[0004] Sisal ( Agave sisalana Perr. ex Engelm. Sisal is a hard-leaved fiber crop widely cultivated in tropical and subtropical regions. Its uses are broad, primarily in fisheries, shipping, aerospace, mining, transportation, oil fields, and textiles. Sisal is relatively tolerant of high temperatures but sensitive to low-temperature damage, easily affected by late spring frosts, which can cause large-scale yield reductions in severe cases. Currently, research on the molecular biology of sisal is relatively weak, and there are few reports on cold-resistance genes in sisal. Summary of the Invention

[0005] The purpose of this invention is to provide a sisal DREBGenes and their application in improving plant cold resistance can be used to enhance plant cold resistance and cultivate cold-resistant plants.

[0006] This invention provides sisal DREB Gene or sisal DREB The application of gene-encoded proteins in improving plant cold resistance, specifically sisal. DREB The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein is shown in SEQ ID NO.2.

[0007] Preferably, the plant includes monocotyledonous plants or dicotyledonous plants.

[0008] Preferably, the plant includes one or more of sisal and Arabidopsis thaliana.

[0009] Preferably, by overexpressing sisal DREB Gene or sisal DREB Gene-encoded proteins enhance a plant's cold resistance.

[0010] The present invention also provides a formulation for improving the cold resistance of plants, wherein the active ingredient of the formulation includes the aforementioned sisal. DREB Recombinant gene expression vectors or recombinant Agrobacterium.

[0011] The present invention also provides a method for cultivating cold-resistant plants, the method being (1) or (2): (1) By improving the sisal content of the target plant DREB The expression level of genes was adjusted to obtain plants with stronger cold resistance than the target plant. (2) By enhancing the sisal in the target plant DREB By increasing the activity of gene-encoded proteins, plants with stronger cold resistance than the target plant can be obtained.

[0012] Beneficial effects of the present invention: The present invention provides a sisal plant related to cold resistance. DREB Genes and Sisal DREB The protein encoded by the gene, the sisal DREB The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the sisal... DREB The amino acid sequence of the gene-encoded protein is shown in SEQ ID NO.2. This invention has revealed that sisal... DREB Genes and Sisal DREB The protein encoded by the gene can enhance the cold resistance of plants and is used to cultivate cold-resistant plants. The technical solution provided by this invention can be applied to the genetic breeding of cold-resistant varieties of sisal, providing a theoretical basis and genetic resources for cultivating new cold-resistant plant varieties or seedlings. Attached Figure Description

[0013] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0014] Figure 1 The sisal provided by the present invention DREB Gene agarose gel electrophoresis verification results; bands 1, 2, and 3 are the electrophoresis results of PCR products, and band M is the marker; Figure 2 The sisal provided by the present invention DREB Gene expression levels in sisal after exposure to low temperature stress, where * indicates p <0.05, ** indicates p <0.01; Figure 3 A map of the pEGAD-DREB expression vector; Figure 4 Wild-type Arabidopsis thaliana (WT) and sisal provided for this invention DREB Growth of the gene after overexpression in Arabidopsis thaliana under low temperature treatment and control (normal conditions); OE1, OE2, and OE3 are different overexpression lines; Figure 5 Wild-type Arabidopsis thaliana (WT) and sisal provided for this invention DREB Statistical results of malondialdehyde and three antioxidant enzyme activities after gene overexpression in Arabidopsis thaliana under low temperature treatment and control (normal conditions), respectively. OE1, OE2, and OE3 represent different overexpression lines; * indicates... p <0.05, ** indicates p <0.01. Detailed Implementation

[0015] To facilitate understanding of the present invention, a more comprehensive description will be provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention. Unless otherwise specified, the reagents and methods used in the present invention are conventional.

[0016] Example 1: Sisal DREB Gene amplification The inventor provides a sisal DREB The gene, whose nucleotide sequence was obtained by sequencing and analyzing the transcriptome database of sisal.

[0017] Potted sisal seedlings were used as experimental materials and subjected to a 6℃ low-temperature stress treatment. Sisal leaves were collected at 0h, 6h, 12h, 18h and 24h after stress. Total RNA was extracted from the leaves using the column-based RNA extraction kit TaKaRa MiniBEST Universal RNAExtraction Kit (TaKaRa, Japan). The RNA samples from the five stages were mixed and then reverse transcribed using a commercial reverse transcription kit to obtain cDNA.

[0018] according to DREB Specific primers were designed based on the gene nucleotide sequence, and PCR amplification was performed. The specific primer sequences are as follows: Forward primer: 5'-ATGGAAACAACAGCAGCAGC-3' (SEQ ID NO.4); Reverse primer: 5'-TCAAATAGAGTAACTCCACAGCGAC-3' (SEQ ID NO.5).

[0019] The PCR amplification reaction system (20 μL) is as follows: 1 μL (10 pmol) of forward primer, 1 μL (10 pmol) of reverse primer, 10 μL of EasyTaq® PCR SuperMix, 1 μL of sisal cDNA (template) and 7 μL of ddH2O.

[0020] The PCR amplification reaction program was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 15 s, 60℃ annealing for 15 s, 72℃ extension for 15 s, for 30 cycles, and 72℃ final extension for 2 min.

[0021] Sanger sequencing was performed on the PCR amplification products to obtain sisal. DREB The gene sequence, its nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.2. The PCR amplification products were subjected to agarose gel electrophoresis, and the agarose gel electrophoresis results are shown below. Figure 1 As shown, after gelation, the sisal is purified to obtain sisal. DREB Gene sequence.

[0022] SEQ ID NO.1: 5'-ATGGAAACAACAGCAGCAGCAGGAACCAACTCCTCCTCCTCCTCCTTCCCTCGCTCCTCGGCGTCAGACGAGGAGGCCTACGCCACGGTGTCGTCCCTGCCCCCGAAGCGCCGTGCCGGGCGCACCAAGTTCCGCGAGACGCGGCACCCCGTGTACAAGGGCGTCCGGCGCCGCAACTCCGACCGCTGGGTCTGCGAGGTCCGCGAGCCCAACAAGAAGTCCCGAATCTGGCTCGGCACTTTCCCGACAGCCGAGATGGCCGCCCGGGCCCACGACGTCGCCGCCATTGCCCTCCGCGGCCGCTCCGCCTGCCTCAACTTCGCCGACTCCGCCTGGCTCCTCCCGGTGCCGGACTCCTCCAGCCCCAGGGACATTCAACGGGCCGCCGCTGCAGCGGCCGAGGCCTTCCGCCCGCCCTCCTCGGAGACTAACGATCAGACTATCGCAGTTGTTCAGACAACGGCGGCCGGCACTGAGACGGGGCGCGTTTCTGATGGCAGTGTGGTGGATGATCAGCCGTTGTTTTTTGAGCATGGGTTGAATTTGGGGATGCAGGGCTACTTCGACATGGCGCAGGGGATGCTGATCGATCCGCCGCCGACGGAGATGATGCCGGATGTCGATGGGGAGAGCGACGGCGAAATGTCGCTGTGGAGTTACTCTATTTGA-3'.

[0023] SEQ ID NO.2: METTAAAGTNSSSSSFPRSSASDEEAYATVSSLPPKRRAGRTKFRETRHPVYKGVRRRNSDRWVCEVREPNKKSRIWLGTFPTAEMAARAHDVAAIALRGRSACLNFADSAWLLPVPDSSSPRDIQRAAAAAAEAFRPPSSETNDQTIAVVQTTAAGTETGRVSDGSVVDDQPLFFEHGLNLGMQGYFDMAQGMLIDPPPTEMMPDVDGESDGEMSLWSYSI*.

[0024] Example 2: Sisal DREBGene expression analysis Using real-time quantitative PCR, sisal was designed DREB Gene-specific quantitative primers were used, and sisal was selected. PP2A Gene (SEQ ID NO.3) was used as an internal reference gene, and specific internal reference primers were designed (forward primer: 5'-CCTCCTCCTCCTTCGGTTTG-3' (SEQ ID NO.6); reverse primer: 5'-GCCATGAATGTCACCGCAGA-3' (SEQ ID NO.7)). Potted sisal seedlings were used as experimental material and subjected to 6℃ low-temperature stress treatment. Sisal leaves were collected at 0h, 6h, 12h, 18h, and 24h after stress. Total RNA was extracted from the leaves using the TaKaRa MiniBEST Universal RNA Extraction Kit (TaKaRa, Japan). cDNA was obtained by reverse transcription using a commercial reverse transcription kit and then analyzed by real-time quantitative PCR. Each cDNA sample was prepared in triplicate. The obtained CT values ​​were used as a 2-1 ΔΔCT The method is used to calculate the sisal DREB The relative expression levels of genes were used to clarify the effects of low-temperature stress on sisal. DREB Gene expression status.

[0025] The specific quantitative primer sequences are as follows: Forward primer: 5'-CAGTTGTTCAGACAACGGCG-3' (SEQ ID NO.8); Reverse primer: 5'-GGATCGATCAGCATCCCCTG-3' (SEQ ID NO.9).

[0026] The real-time quantitative PCR reaction system (TransStart Tip Green qPCR SuperMix kit) is as follows: 0.4 μL forward primer, 0.4 μL reverse primer, 5 μL TransStart Tip Green qPCR SuperMix, 0.5 μL sisal cDNA (template), 0.2 μL Reference Dye, and 3.5 μL ddH2O.

[0027] The reaction program was as follows: pre-denaturation at 94℃ for 30 seconds; denaturation at 94℃ for 5 seconds, annealing at 60℃ for 30 seconds, extension at 60℃ for 30 seconds, for 40 cycles.

[0028] The results showed that: Sisal DREB Gene expression levels increased significantly after low temperature stress, peaked at 12 h of treatment, and then gradually decreased. Figure 2 ).

[0029] The above results indicate that sisal DREB The gene expression level increased significantly after low temperature stress treatment, which is closely related to the response of sisal to low temperature stress. It has potential application value in the cold-resistant breeding of sisal and provides a theoretical basis for the breeding of new sisal varieties.

[0030] SEQ ID NO.3: 5'-ATGGCGATGCAAGGGCAAGGGATCGATCCGGCGCTGCTCGACGACATCATCGGCAGGCTGCTCGAGGTCCGATCGGCGAGGCCTGGGAAGCAAGTCCAGCTGTCGGAGGCGGAGATCCGCCAGCTCTGCGTCGCGTCGAGGGAGATCTTTCTTCAGCAGCCCAATCTCCTCGAGCTCGAGGCGCCGATCAAAATCTGCGGTGACATTCATGGCCAATACAGTGACCTTTTAAGGCTATTCGAATATGGAGGTTTTCCTCCTGCGGCTAATTATTTGTTTTTAGGAGATTATGTGGACCGGGGAAAACAGAGCTTGGAAACAATATGCCTTCTTCTTGCTTATAAGATCAAGTACCCTGAGAACTTTTTTCTTCTGAGAGGAAACCATGAATGTGCTTCTATAAATAGGATATATGGATTTTATGATGAATGTAAGCGTCGGTTTAATGTGAGATTATGGAAGGTCTTCACTGATTGCTTTAACTGCCTGCCTGTAGCTGCTCTTATAGATGACAAAATATTATGCATGCATGGTGGCCTTTCCCCAGATCTGTCAAACCTGGACCAGATTAAAAGCATAACTCGTCCTACTGACGTCCCTGACACTGGTTTACTATGTGACCTGCTCTGGTCTGATCCTGGCACTCAAGTTCAAGGGTGGGGAATGAATGATAGAGGGGTTTCATACACTTTCGGTTCTGATAAGGTGTCTGAATTCTTATCAAAGCATGATCTGGATCTTGTGTGTCGTGCGCATCAGGTTGTGGAAGACGGCTATGAGTTCTTTGCAGACAGACAACTCGTCACTATATTCTCAGCACCAAATTACTGTGGTGAATTTGATAATGCTGGTGCTATGATGAGTGTTGATGAAACCTTAATGTGCTCTTTCCAAATCTTAAAGCCTGCAGAAAAGAAGCCAAAATTTATGATGTCCACAAAAGTATGA-3'.

[0031] Example 3 Sisal DREBApplication of genes in improving plant cold resistance 3.1 Construction of sisal DREB gene expression vector The pEGAD expression vector was linearized by double digestion with restriction endonucleases EcoRI and BamHI. The digestion products were separated by 1% agarose gel electrophoresis, and the linearized pEGAD vector fragments were recovered by gel excision. The sisal obtained in Example 1 was then used... DREB The gene and the linearized pEGAD vector fragment were ligated using T4 DNA ligase to obtain the pEGAD-DREB expression vector. Figure 3 The pEGAD-DREB expression vector was transformed into Agrobacterium GV3101 competent cells via electroporation (15-20 kV / cm, 4.5-5.0 ms). The cells were plated on LB agar containing 100 mg / L gentamicin, 50 mg / L rifampin, and 100 mg / L kanamycin. After incubation at 28°C for 2 days, single colonies were picked for colony PCR identification. Sisal was used for further analysis. DREB Gene-specific primers were used for amplification, and positive clones were screened. The obtained positive clones were inoculated into LB liquid medium containing 100 mg / L gentamicin, 50 mg / L rifampin and 100 mg / L kanamycin, and cultured at 28°C with shaking for 24 h. After that, 1 mL of bacterial culture was taken out and the strain was preserved for Arabidopsis transformation.

[0032] 3.2 Arabidopsis transformation Add 2 mL of bacterial culture to 50 mL of LB liquid medium containing 100 mg / L gentamicin, 50 mg / L rifampin, and 100 mg / L kanamycin, and incubate at 28°C with shaking until the bacterial concentration reaches OD500. 600 The value is 0.8-1.0. Centrifuge the obtained bacterial culture at 4000 rpm for 5 min, discard the supernatant, and resuspend the bacterial cells in infection buffer to OD. 600 The value is 0.6; the infection buffer formulation is as follows: sucrose (5%), Silwet L-77 (0.05%), and acetylsuccinone (AS) 100 μmol / L.

[0033] Select healthy Arabidopsis plants that have bolted for one to two weeks. Remove the siliques and open flowers, leaving the apical meristem and flower buds. Immerse the treated inflorescences in a resuspended bacterial solution for 1 minute, then cover with a black lid and incubate in the dark for 24 hours. Remove the black lid and incubate under light (22℃±2℃, light intensity 100μmol). m -2 s -1 ).

[0034] 3.3 Screening and Identification of Transgenic Arabidopsis Plants One month after the Arabidopsis thaliana transformants were grown, mature seeds were collected. Plump seeds were selected, disinfected with a 2% sodium hypochlorite solution for 15 minutes, and after discarding the sodium hypochlorite solution, washed 3-5 times with sterile water. The seeds were then sown in MS medium containing 50 mg / L kanamycin. The growth conditions were 22℃±2℃, 16h light / 8h dark, and a light intensity of 100 μmol / L. m -2 s -1 After one week of cultivation, normally germinating positive plants were selected and planted in soil for further cultivation and management, and T1 generation seeds were collected. T1 generation seeds were planted, and leaves from the T1 generation plants were taken for transgenic plant PCR testing to confirm the acquisition of transgenic positive plants. The transgenic positive plants were then cultivated and managed, and T2 generation seeds were collected.

[0035] 3.4 Transgenic T2 generation seeds (with three biological replicates: OE-1, OE-2, and OE-3) and wild-type Arabidopsis thaliana seeds (WT) were planted in soil and cultured under the same growth conditions as in step 3.3 until the seedling stage. Two treatment groups were set up: one group continued to grow under normal conditions as a control, and the other group was treated at -5℃ for 6 hours, after which normal culture was resumed. Morphological changes of each line were observed and recorded. The results are shown in […]. Figure 4 Simultaneously, physiological and biochemical indicators were measured from the treated leaves. MDA content was determined using a malondialdehyde (MDA) content assay kit (purchased from Beijing Solarbio Science & Technology Co., Ltd.). The activities of three antioxidant enzymes—peroxidase (POD) activity assay kit (purchased from Beijing Solarbio Science & Technology Co., Ltd.), superoxide dismutase (SOD) activity assay kit (purchased from Beijing Solarbio Science & Technology Co., Ltd.), and catalase (CAT) activity assay kit (purchased from Beijing Solarbio Science & Technology Co., Ltd.)—were measured. The results are shown below. Figure 5 .

[0036] The results showed that sisal DREB No significant freezing damage was observed in Arabidopsis thaliana plants overexpressing the gene after low-temperature treatment. Under low-temperature stress, the MDA content of transgenic Arabidopsis thaliana (OE-1, OE-2, OE-3) was lower than that of wild type, while the activities of three antioxidant enzymes were higher than those of wild type, indicating that sisal... DREB Genes can improve the cold resistance of plants and can be applied to the genetic breeding of cold-resistant plants to cultivate new varieties or seedlings of cold-resistant plants.

[0037] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Overexpression of sisal DREB The application of genes in improving plant cold resistance is characterized by, The sisal DREB The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the plant is Arabidopsis thaliana.

2. A method for cultivating cold-resistant plants, characterized in that, The method involves increasing the content of the sisal as described in claim 1 in the target plant. DREB The expression level of genes was adjusted to obtain plants with stronger cold resistance than the target plant. The plant in question is Arabidopsis thaliana.

Images