Application of transcription factor AsNF-YB3 in regulating drought resistance of oat

Abstract

This invention discloses transcription factors As NF-YB3 Its application in regulating drought resistance in oats falls under the field of biotechnology. (Transcription factors) As NF-YB3 The amino acid sequence is shown in SEQ ID NO.1. As NF-YB3 The CDS sequence of the gene is shown in SEQ ID NO.2, and the gDNA sequence is shown in SEQ ID NO.3. Overexpression As NF-YB3 The gene significantly enhances the drought resistance of oats, while also increasing the activity of CAT and SOD enzymes in oats, slowing down the water loss rate of detached leaves, and improving seedling survival rate under drought stress. AsNF-YB3 forms a heterotrimeric complex with AsNF-YA1 and AsNF-YC5, and its 4bp insertion / deletion mutation in the promoter region regulates oat drought resistance by affecting AsARF1 binding and gene expression. This provides a new gene resource and technical pathway for agricultural stress-resistance breeding.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to transcription factors. AsNF-YB3 Application in regulating drought resistance in oats. Background Technology

[0002] Drought is the most significant disaster factor in agriculture, accounting for over 65% of losses, far exceeding floods, storms, or volcanic activity. Oats ( Avena sativa Oats, as an annual crop with both food and feed value, are not only rich in protein, fat, and vitamin B1, but also an important source of high-quality forage for livestock worldwide, demonstrating significant economic and ecological value. However, oats require significantly more water during their growth period than other cereal crops, and drought stress (especially during critical growth periods) poses a serious threat to their production, thus restricting the development of the oat industry.

[0003] Therefore, in the context of climate change, breeding drought-resistant oat varieties and analyzing drought-resistant genes have become urgent priorities for ensuring global food and pasture security.

[0004] Traditional breeding mainly relies on phenotypic selection, hybridization, backcrossing and other methods to screen superior strains through field identification and screening over many years and generations. This process is time-consuming, inefficient and easily affected by environmental conditions, and has certain limitations in the targeted improvement of complex traits (such as stress resistance).

[0005] In contrast, the development of new breeding technologies such as plant molecular biology, genetics, and transgenics has made it possible to locate, modify, and regulate target genes at the genomic level, shortening the breeding process, improving selection efficiency, and increasing the accuracy of improvement.

[0006] Based on these technologies, drought resistance, stress tolerance, and other traits can be optimized more effectively, which to some extent makes up for the shortcomings of traditional breeding in terms of gene resource utilization and selection efficiency, and provides a new technical path for stress-resistant breeding of crops such as oats.

[0007] As one of the world's most important grains, oats are of great theoretical and applied value for discovering key drought-resistant genes and analyzing their molecular regulatory networks, which is crucial for breeding new drought-resistant oat varieties. Summary of the Invention

[0008] The purpose of this invention is to provide transcription factors. AsNF-YB3 In regulating drought resistance in oats, overexpression of this gene can improve the drought tolerance of transgenic oats. This characteristic of improving the drought resistance of transgenic plants can be applied to crops through transgenic technology to address the increasingly serious environmental problems.

[0009] To achieve the above objectives, the present invention provides transcription factors. AsNF-YB3Application in regulating drought resistance in oats, transcription factors AsNF-YB3 The amino acid sequence is shown in SEQ ID NO.1.

[0010] Preferably, encoding transcription factors AsNF-YB3 genes AsNF-YB3 The CDS sequence is shown in SEQ ID NO.2, encoding transcription factor AsNF-YB3 genes AsNF-YB3 The gDNA sequence is shown in SEQ ID NO.3.

[0011] Preferred, transcription factors AsNF-YB3 or encoding transcription factors AsNF-YB3 genes AsNF-YB3 Increased activity and / or expression levels in oats enhance plant drought resistance.

[0012] Preferably, the improved drought resistance of oats includes at least one of the following (1)-(5): (1) Under drought stress, the survival rate of oat seedlings was higher than that of control oats; (2) Oat yield was higher than control oat yield under drought stress; (3) Under drought stress, the relative water content of oat leaves was higher than that of the control oat; (4) The rate of water loss from detached oat leaves was lower than that of control oats; (5) The activity of CAT and / or SOD in oats was higher than that in control oats.

[0013] On the other hand, the present invention also provides a method for cultivating transgenic oats with improved drought resistance, by introducing a gene capable of expressing drought resistance into recipient oats. AsNF-YB3 Nucleic acid molecules of transcription factors were used to produce transgenic oats, which showed improved drought resistance compared to the recipient oats; transcription factors AsNF-YB3 The amino acid sequence is shown in SEQ ID NO.1; it encodes a transcription factor. AsNF-YB3 genes AsNF-YB3 The CDS sequence is shown in SEQ ID NO.2; encoding transcription factor AsNF-YB3 genes AsNF-YB3 The gDNA sequence is shown in SEQ ID NO.3.

[0014] Preferably, the ability to express [the substance] is introduced into the recipient oats. AsNF-YB3 Transcription factor nucleic acid molecules are introduced into the recipient oats containing... AsNF-YB3 This is achieved through gene recombination expression vectors.

[0015] Preferably, the recombinant expression vector is introduced into the recipient oat by transforming plant cells or tissues through methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electroporation or Agrobacterium-mediated transformation, and the transformed plant tissue is then cultured into plants.

[0016] Therefore, the transcription factor of the present invention AsNF-YB3 Its application in regulating the drought resistance of oats has the following beneficial effects: it can utilize oat-derived nutrients... Avena sativa L.) gene AsNF-YB3 Transgenic plants were obtained by introducing oats. These transgenic plants exhibited higher drought resistance than the non-transgenic recipient plants and increased the relative water content of oat leaves under drought stress, as well as the activities of superoxide dismutase (SOD) and catalase (CAT), thereby enhancing the plant's tolerance to drought stress. This indicates that... AsNF-YB3 The transcription factors they encode have drought-resistant functions and are of great significance in breeding and research to improve plant drought resistance.

[0017] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0018] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This study aims to conduct a genome-wide association study (GWAS) on oat seedling survival rate under drought stress and identify candidate genes; where A represents the GWAS analysis and B represents... AsNF-YB3 The mutation site is located 657 bp upstream of the promoter region; C represents the effect of the presence or absence of a 4 bp deletion on drought-induced water loss. AsNF-YB3 The expression levels were grouped; the D-plot shows the drought survival rate grouped according to the presence or absence of a 4bp deletion; Figure 2 for AsNF-YB3 Expression level analysis under drought conditions; Figure 3 for AsNF-YB3 Expression level detection results of transgenic plants; where A is... AsNF-YB3 PCR genotyping results of transgenic plants; B is... AsNF-YB3 Relative expression levels in WT and two independent overexpression (OE) lines; Figure 4 for AsNF-YB3 Statistics on the condition of OE materials before drought and after rehydration; where A is... AsNF-YB3 Phenotypic comparison of OE materials before drought and after rehydration; B is... AsNF-YB3 Statistical chart of drought survival rate of OE materials; Figure 5 for AsNF-YB3 Statistics on the condition of gene knockdown plants before drought and after rehydration; where A represents the condition before and after drought and after rehydration. AsNF-YB3 Phenotypic comparison of gene knockdown plants; B is... AsNF-YB3 Gene knockdown in plants AsNF-YB3 Statistical graph of expression levels; C is AsNF-YB3 Statistics on drought survival rate of gene knockout plants; Figure 6 for AsNF-YB3 Statistical chart of water loss rate of detached blades made of OE material; Figure 7 The results show the detection results of CAT and SOD activities in wild-type (WT), OE1, and OE5 plants; where A represents the CAT activity detection result and B represents the SOD activity detection result. Figure 8 for NF-YB3 Tissue-specific analysis; Figure 9 Subcellular localization analysis of AsNF-YB3; Figure 10 Results of yeast one-hybrid experiment (Y1H); Figure 11 Results of Electrophoretic Mobility Variation Experiment (EMSA); Figure 12 Results of dual-luciferase reporter gene assay (dual-Luc); Figure 13 The results of the interaction verification between AsNF-YB3, AsNF-YC5, and AsNF-YA1 are shown. Among them, A is the result of the co-immunoprecipitation experiment of AsNF-YA1 and AsNF-YB3; B is the result of the co-immunoprecipitation experiment of AsNF-YA1 and AsNF-YC5; and C is the result of the co-immunoprecipitation experiment of AsNF-YB3 and AsNF-YC5. Indicates nonspecific bands; Figure 14 The results of dual fluorescence complementation experiments of AsNF-YC5 with AsNF-YA1, AsNF-YB3 with AsNF-YC5, and AsNF-YB3 with AsNF-YA1; Figure 15 The results are for the interaction verification between AsNF-YB3, AsNF-YC5, and AsNF-YA1. Detailed Implementation

[0020] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0021] To make the objectives, technical solutions, and advantages of this application clearer, more thorough, and more complete, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and embodiments. The following detailed descriptions are all illustrations of embodiments, intended to provide further detailed explanation of the present invention. Unless otherwise specified, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0022] The instruments, equipment, reagents, and materials used in the examples were all obtained commercially.

[0023] Example 1 Transcriptome data from 211 oat varieties (National Crop Germplasm Bank) under drought conditions were used to perform InDels / SVs-based genome-wide association analysis (GWAS) to screen for genetic loci leading to differences in drought resistance. A significant association peak was detected on chromosome 1A, such as... Figure 1 As shown in Figure A, the gene encoding nuclear factor-Y transcription factor was identified. AsNF-YB3 .

[0024] like Figure 1 As shown in Figure B, there is a 4bp insertion / deletion at -657bp upstream of the promoter of this gene (Borris37 carries this 4bp sequence, while XymC06 lacks this 4bp sequence), which contains auxin-responsive elements (TGA motif, AACGAC, ARF transcription factor binding site) and shows a significant association with drought resistance.

[0025] Based on this insertion / deletion mutation, oat germplasm resources were divided into two haplotype groups: Hap1 (deleted 4bp) and Hap2 (present 4bp). For example... Figure 1 The CD shown carries AsNF-YB3 Hap2 Allele germplasm resources are more abundant than those carrying alleles. AsNF-YB3 Hap1 The alleles exhibited significantly higher gene expression levels and stronger drought stress resistance, suggesting that this variation may affect gene expression and drought resistance.

[0026] Example 2 transcription factors AsNF-YB3 And the acquisition of its encoding gene.

[0027] (1) Transcription factors AsNF-YB3 and its encoding gene clones.

[0028] Drought-resistant oat seed Borris37 was used for cultivation under the following conditions: 18℃, 50% relative humidity, and a photoperiod of 14 hours light / 10 hours dark. When the plants reached the two-leaf stage (14-day-old seedlings), seedling leaves were quick-frozen in liquid nitrogen, ground, and total RNA was extracted using Trizol (Invitrogen) reagent. Prime Script was then used to analyze the RNA. TM Reverse transcription was performed using an RT kit (TaKaRa, Japan) to obtain cDNA. This cDNA was then used as a template for PCR amplification with F1 and R1 primers. The amplified product was subjected to 1% agarose gel electrophoresis to obtain a 525 bp PCR amplification product.

[0029] Sequencing revealed that the PCR product derived from the drought-resistant oat variety Borris37 contained the nucleotides shown in SEQ ID NO.2, and the gene shown in SEQ ID NO.3 was named... AsNF-YB3 The protein encoded by this gene, SEQ ID NO.1, is named AsNF-YB3.

[0030] SEQ ID NO.1: MSDGPTSPPGGGSDDGGGGGGGGFGGVREQDRFLPIANISRIMKKAIPANGKIAKDAKETVQECVSEFISFITSEASDKCQREKRKTINGDDLLWAMATLGFEEYIEPLKVYLQKFREGDSKVTSKSGDGSVKKDALGHGGTTSSSAQGMGQQVAYNQGMVYMQPQYHNGDIPN.

[0031] SEQ ID NO.2: ATGTCGGACGGGCCGACGAGCCCCCCGGGCGGCGGGAGCGACGACGGCGGCGGTGGCGGCGGGGGCGGGTTCGGCGGCGTCAGGGAACAGGACAGGTTCCTGCCCATCGCCAACATCAGCCGCATCATGAAGAAGGCCATCCCGGCCAACGGCAAGATTGCCAAGGACGCCAAGGAGACCGTGCAGGAGTGCGTCTCCGAGTTCATCTCCTTCATCACCAGCGAGGCGAGCGACAAGTGCCAGCGGGAGAAACGCAAGACCATCAACGGCGACGACCTGCTGTGGGCGATGGCGACGCTGGGCTTTGAGGAATACATCGAACCCCTCAAGGTCTATCTGCAGAAGTTCAGAGAGGGTGATAGTAAGGTAACATCAAAGTCTGGCGATGGCTCTGTTAAAAAGGATGCACTTGGTCATGGAGGAACAACTAGCTCAAGTGCGCAAGGGATGGGCCAACAAGTAGCATACAACCAAGGAATGGTTTATATGCAACCTCAGTACCATAATGGGGACATCCCAAACTGA。

[0032] SEQ ID NO.3:

[0033] The primer sequences described above are as follows: F1: AsNF-YB3 -F: 5′-ATGTCGGACGGGCCGACG-3′, SEQ ID NO.4; R1: AsNF-YB3 -R: 5′-GTTTGGGATGTCCCCATTAT-3′, SEQ ID NO. 5.

[0034] (2) Recombinant vector pUBI:AsNF-YB3 Construction: Will AsNF-YB3 Borris37 The coding sequence of the type gene was added to pCAMBIA330-GFP The vector contains the bar gene for screening transgenic positive lines; a GFP fluorescent tag is inserted after the multiple cloning restriction site; and it contains the maize ubiquitin promoter. ubi ) EcoRI Homologous arms of the restriction enzyme sites were amplified by PCR using primers F2 and R2. Then... EcoRI The vector was digested with a single enzyme, and the digestion product was homologously recombinated with the PCR product. This was then transformed into the top 10 strains of *E. coli* to obtain the corresponding... AsNF-YB3 Genotype recombinant vector pCAMBIA3300-AsNF-YB3 Named pUBI:AsNF-YB3 .

[0035] F2: pUBI-AsNF-YB3 -F: 5′-tcgctagaggatccgATGTCGGACGGGCCGACG-3′, SEQ IDNO.6; R2: pUBI-AsNF-YB3 -R2: 5′-gtcgacggagctcgaGTTTGGGATGTCCCCATTAT-3′, SEQ ID NO. 7.

[0036] (3) Obtaining recombinant Agrobacterium tumefaciens: Recombinant plasmid pUBI:AsNF-YB3 Transform Agrobacterium tumefaciens strain GV3101 to obtain a recombinant plasmid. pUBI: AsNF-YB3 Positive strains.

[0037] (4) Genetically modified AsNF-YB3 Obtaining oats: Recombinant Agrobacterium pUBI:AsNF-YB3 Immature embryos infected with Monica germplasm were harvested to obtain T1 generation seeds. Whole-genome DNA was extracted from T1 generation transgenic plants, and PCR was performed to identify transgene positivity using primers F3 and R3. RNA from positive plants was reverse transcribed to obtain cDNA, which was then used to identify the gene in oats. AsACTIN2As an internal reference, primers QF1 and QR1 were used. Specific primers QF2 and QR2 were used for... AsNF-YB3 Gene expression levels were detected, with wild-type Monica as a control. The sequences of the primers are shown in Table 1 below.

[0038] Table 1: AsNF-YB3 qPCR primers

[0039] T1 generation represents the seeds produced by the current generation of transformation and the plants grown from them. T1 generation positive transgenic plants can be used for drought phenotype detection, enzyme activity detection, etc.

[0040] Example 3 (1) AsNF-YB3 Positive regulation of drought resistance in oats: Leaves of oat seedlings cultured in an 18℃ constant temperature incubator (culture conditions: 50% relative humidity, photoperiod of 14 hours light / 10 hours dark) were placed on a clean table for drought treatment. Samples were taken at 0h, 1h, 2h, 3h, 4h, 6h, and 9h, and quickly ground into powder in liquid nitrogen (samples from three plants were mixed). Total RNA was extracted from the leaves using Trizol reagent, reverse transcribed into cDNA, and then quantitatively analyzed by qPCR using QF3 and QR3. Figure 2 As shown, this indicates that AsNF-YB3 is upregulated by drought and positively regulates oat drought resistance.

[0041] QF3: AsNF-YB3-qPCR-F: 5′-GCAGAAGTTCAGAGAGGGTG-3′, SEQ ID NO.14; QR3: AsNF-YB3-qPCR-R: 5′-ACTTGTTGGCCATCCCTTG-3′, SEQ ID NO. 15.

[0042] (2) Take the T1-transfer AsNF-YB3 obtained according to Example 2 (4). Borris37 Oat lines (OE1 and OE5, with wild-type Monica oat as the transformation recipient) were subjected to RT-qPCR detection. Results are as follows: Figure 3 As shown, T1 is a substitute for AsNF-YB3 Oatmeal OE1 and OE5 AsNF-YB3 The expression level was significantly higher than that of wild-type oats, indicating that the T1 generation of transgenic oats... AsNF-YB3 Oat OE1 and OE5 are positive genetically modified oats.

[0043] T1 generation seedlings sown for one week AsNF-YB3Oat lines (OE1 and OE5) and wild-type Monica oat plants were transferred to white boxes containing a 1:1 ratio of nutrient soil to vermiculite. Under normal conditions, after reaching the two-leaf stage, they were subjected to a drought treatment (i.e., watering was stopped) for approximately 12 days. When the degree of leaf wilting in the transgenic plants differed significantly from that of the wild type, they were rehydrated. After 3-5 days of rehydration, the survival rate of each line was recorded (plants with normal leaf color and normal growth were defined as surviving plants, and plants showing scorched leaves and unable to grow normally were defined as dead plants; the survival rate was the percentage of surviving plants in each line out of the total number of plants). The experiment was conducted in triplicate, with at least 10 plants from each line in each replicate. The average value was used for statistical analysis. The results are as follows: Figure 4 As shown, after 12 days of drought treatment followed by rehydration, the T1 generation shifted... AsNF-YB3 The leaves of genetically modified oats were less dry than those of wild-type oats, and their survival rate was significantly higher.

[0044] (3) Oats AsNF-YB3 Obtaining gene-silencing lines: The Tobacco Crisp Virus-mediated Virus-Induced Gene Silenting (TRV-VIGS) system was used. Primers were designed via the SGN-VIGS website to obtain gene silencing lines from... AsNF-YB3 Three specific 200-base-pair fragments were amplified from the cDNA of the gene using primers F4, R4; F5, R5 and F6, R6, and cloned into the pTRV2 vector using NC cloning technology.

[0045] pTRV1 and pTRV2 were then transformed into Agrobacterium tumefaciens (GV3101 strain), and strains carrying pTRV1 and various pTRV2 strains were also transformed. TRV2, V-AsNF-YB3-1, V-AsNF-YB3-2, V-AsNF-YB3-3 Mix in a 1:1 ratio with a culture medium containing 19.62 mg / L acetylsuccinone, 400 mg / L cysteine ​​and 5 mL / L Tween-20.

[0046] Oat seeds germinated for 24 hours were immersed in a vacuum of 20 kPa for 5 minutes, followed by overnight culture at 28°C and 180 rpm with shaking. After co-culture, the seeds were washed with sterile water to remove Agrobacterium-dependent bacteria from the surface and sown in soil. Leaf tissue was collected at the two-leaf stage, and qPCR quantitative analysis was performed using QF4 and QR4 primers to obtain gene-silenced plants.

[0047] The primer sequences used are shown in Table 2 below.

[0048] Table 2: AsNF-YB3-VIGS primers

[0049] Successfully infected V-AsNF-YB3-1, V-AsNF-YB3-2, V-AsNF-YB3-3Gene-silenced lines were transplanted into identically sized boxes containing 50g of soil (nutrient soil: vermiculite = 1:1), and their survival rates were statistically analyzed. The results are as follows: Figure 5 As shown, it can be seen that after 12 days of drought treatment, AsNF-YB3 The leaves of the silent type oats were more withered than those of wild oats, and their survival rate was significantly lower than that of wild oats.

[0050] (4) AsNF-YB3 Statistical analysis of water loss from detached leaves of transgenic materials: Germination for one week AsNF-YB3 Transgenic lines were transplanted into square boxes. When they reached the three-leaf stage, the second true leaf (four leaves per group, divided into 3 groups) was selected for an in vitro leaf water loss experiment. The cut leaves were immediately weighed as the weight at 0h. Thereafter, they were weighed every hour. The weight at 0h minus the weight at each time point equals the water loss weight for that time period. The water loss rate is calculated by dividing the water loss weight by the weight at 0h. Figure 6 As shown, the water loss rate of WT was significantly faster than that of OE1 and OE5. A two-tailed t-test was used to determine statistical significance. Indicates the comparison with WT results. P <0.05, the difference is highly significant. Indicates the comparison with WT results. P <0.01 indicates a highly significant difference.

[0051] therefore, AsNF-YB3 The expression levels of transcription factor-encoding genes were significantly correlated with drought resistance in oat seedlings, increasing [their ability to resist drought]. AsNF-YB3 Gene expression levels can significantly improve oat seedling survival rate, slow down water loss rate, and increase oat drought resistance.

[0052] Example 4 AsNF-YB3 Enzyme activity analysis of transgenic materials.

[0053] Wild-type Monica (WT) and AsNF-YB3 OE1 and OE5 were planted in square boxes containing 50g of soil. When they reached the three-leaf stage, the second true leaf (approximately 0.1g) was taken as the normal treatment group. Simultaneously, leaves from the same location were placed on a clean surface for drought treatment. When the water loss reached approximately 30% (approximately 0.1g), the samples were collected and quickly frozen in liquid nitrogen. Each treatment group contained at least four biological replicates. The activities of reactive oxygen species (ROS) scavenging enzymes, including catalase (CAT) and superoxide dismutase (SOD), were then measured. These endogenous compounds were quantitatively analyzed spectrophotometrically. Results are as follows: Figure 7 As shown, under drought stress conditions, AsNF-YB3 Both CAT and SOD activities showed significant changes in the overexpression lines. P<0.05). The results show that... AsNF-YB3 It improved the ROS scavenging capacity under drought stress.

[0054] Example 5 Tissues from mature oats of normal growth stage, including roots, leaves, flowers, seeds, flag leaves, and roots and leaves from seedlings (samples from three plants combined), were rapidly ground into powder in liquid nitrogen. Total RNA was extracted from the leaves using Trizol reagent, reverse transcribed into cDNA, and then quantitatively analyzed by qPCR using QF5 and QR5 primers. Figure 8 As shown, it was found AsNF-YB3 It is widely expressed in oat root, leaf, flag leaf, flower and seed tissues.

[0055] QF5: AsNF-YB3-qPCR-F: 5′-GCAGAAGTTCAGAGAGGGTG-3′, SEQ ID NO. 24; QR5: AsNF-YB3-qPCR-R: 5′-ACTTGTTGGCCATCCCTTG-3′, SEQ ID NO. 25.

[0056] The recombinant Agrobacterium tumefaciens constructed in Example 2 pUBI:AsNF-YB3 and P19 (Auxiliary strains) Mix at a volume ratio of 1:1, 5000 g Centrifuge for 5 min, discard the supernatant, resuspend the bacterial cells in 1 mL of infection buffer containing 150 μM acetylsyringone, and co-inject into Nicotiana benthamiana epidermal cells. After culturing for 48 h, observe the GFP fluorescence signal at an excitation wavelength of 488 nm. Figure 9 As shown, this indicates that AsNF-YB3 is located in the cell nucleus.

[0057] Example 6 Previous genome-wide association analysis identified a 4bp InDel located at AsNF-YB3 The promoter region of this InDel contains the binding site for ARF. To investigate whether ARF binds in oats and its effects on... AsNF-YB3 The following experiments were conducted to investigate the effects on gene expression.

[0058] (1) Yeast one-hybrid experiment (Y1H) to verify AsARF1 and AsNF-YB3 Borris37 Type-A promoter binding: To evaluate whether AsARF is effective against... AsNF-YB3 Two haplotype variants of the promoter exhibit differential binding characteristics. Sixty-seven potential AsARF proteins were identified in the oat genome, among which drought-induced AsARFs were screened for subsequent validation experiments. AsARF1 specifically binds to... AsNF-YB3 Borris37 .

[0059] AsARF1 The encoding region is inserted into the pGADT7 carrier. EcoRI The site was used to obtain the recombinant plasmid pGADT7- AsARF1 Using the genomic DNA of drought-resistant oat germplasm Borris37 and drought-sensitive oat XymC06 as templates, samples containing the genomic DNA of both species were analyzed. AsNF-YB3 The 100bp sequence of the promoter was cloned into... pAbAi The vector was then transformed into yeast strain Y1H Gold. The yeast library was screened according to the manufacturer's instructions. An empty AD plasmid was used as a negative control; the yeast solution was serially diluted and seeded onto SD-Ura-Leu plates with or without 200 ng / mL AbA. Figure 10 As shown, this indicates that AsARF1 can specifically bind to AsNF- YB3 Borris37 (Including a 4-bp) promoter, but not binding AsNF-YB3 XymC06 (Excluding the 4-bp) promoter.

[0060] (2) Electrophoretic mobility variation experiment (EMSA) to verify AsARF1 and AsNF-YB3 Borris37 Type promoter binding: AsARF1 Encoding area insertion pGEX4T-1 carrier SalI A recombinant glutathione S-transferase, GST-AsARF1, was constructed by fusing a GST tag to a multiple cloning site. This protein was then co-incubated with a biotinylated probe, a competing probe, or a mutant probe using a LightShift chemiluminescent EMSA kit (ThermoFisher) at 25°C for 20 minutes. The binding reaction was terminated by adding 5 times the concentration of native sample loading buffer. Samples were separated by native polyacrylamide gel electrophoresis (native-PAGE). The binding signal was detected using a nucleic acid detection kit (ThermoFisher). Figure 11 As shown, the recombinant (GST)-AsARF1 protein and the protein containing the TGA sequence... AsNF-YB3 Borris37 When the probes interact, they produce a strong band with significant displacement, while... AsNF-YB3 XymC06 The binding of the probe was significantly weakened, indicating that AsARF1 binds to the TGA sequence. AsNF-YB3 Borris37 Probe. The probe DNA sequence is shown in Table 3 below.

[0061] Table 3: Primers for AsNF-YB3 probe

[0062] (3) Dual-Luc assay to verify AsARF1 and AsNF-YB3 Borris37 Type promoter binding: AsARF1 Cloning the open reading frame (ORF) to pCAMBIA330-35S-GFP 35S was constructed in the vector: AsARF1 Effect plasmid. AsNF-YB3 100-base-pair promoter fragment insertion pNC-Green-LUC Vectors were used to construct reporter plasmids, and to construct... AsNF-YB3 Borris37 Promoter-driven Luc reporter genes and AsNF-YB3 XymC06 Promoter-driven Luc reporter genes. 35S: AsARF1 The effect plasmid was co-transformed with two reporter plasmids into Agrobacterium tumefaciens GV3101 ( pSoup Tobacco leaves were infected. After incubation at 25°C for 48 hours, leaf tissue samples were collected, and the activities of firefly luciferase (LUC) and kidney luciferase (REN) were detected using a dual-luciferase reporter system (Promega) combined with a luminescence analyzer system (Promega). Figure 12 As shown, under drought stress conditions, AsARF1 The existence of AsNF-YB3 Borris37 Promoter-driven LUC activity is significantly higher than AsNF-YB3 XymC06 Promoter-driven activity.

[0063] Example 7 (1) Screening of AsNF-YB3 interacting proteins: NF-Y is an evolutionarily conserved heterotrimeric transcription factor (TF) complex in eukaryotes. This transcription factor is composed of three subunits: NF-YA, NF-YB, and NF-YC, and participates in the regulation of plant growth and development and responses to abiotic stresses (including drought stress). Mammalian studies have confirmed that NF-YB and NF-YC form a core heterodimer through histone folding domains, mediating non-specific DNA interactions; while NF-YA achieves sequence-specific recognition by binding to the CCAAT frame, thereby regulating downstream target genes.

[0064] To further elucidate the functional characteristics of AsNF-YB3, a drought-induced protein, AsNF-YC5, which interacts with AsNF-YB3, was identified using a yeast two-hybrid assay. Furthermore, based on transcriptome data, the oat NF-YA family, induced to express under drought stress, was cloned, among which AsNF-YA1 interacts with AsNF-YB3.

[0065] (2) Co-IP verification of the interaction between AsNF-YB3, AsNF-YC5, and AsNF-YA1: To further confirm whether AsNF-YB3 interacts with AsNF-YC5 and AsNF-YA1, AsNF-YB3 protein was fused with a GFP tag, AsNF-YA1 protein with a Myc tag, and AsNF-YC5 protein with both GFP and Myc tags. Tobacco leaves were then co-infected with AsNF-YB-GFP and AsNF-YA1-Myc, AsNF-YC5-GFP and AsNF-YA1-Myc, and AsNF-YB-GFP and AsNF-YC5-Myc. Forty-eight hours after transformation, total protein was extracted using lysis buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40, a 1x protease inhibitor mixture [Roche, catalog number 4693116001], and 10 mM dithiothreitol).

[0066] The test tube was then filled with 12,000... g Centrifuge for 10 minutes, and the supernatant is the total protein. Then, incubate 1 mL of the total protein extract with anti-GFP magnetic beads (MBL, catalog number #D153-11) at 4°C for 2 hours. Wash the magnetic beads 3-5 times with GFP lysis buffer, separate the protein by SDS-PAGE, and perform Western blotting analysis using anti-GFP antibody (Abclone, catalog number #AE012) and anti-Myc antibody (Sigma, catalog number #M4439). Results are as follows... Figure 13 As shown, there are pairwise interactions between AsNF-YB3 and AsNF-YC5 and AsNF-YA1.

[0067] (3) Bimolecular fluorescence complementarity (BiFC) experiment to verify the interaction between AsNF-YB3 and AsNF-YC5 and AsNF-YA1: AsNF-YA1 and AsNF-YB3 Coding region cloned to pSPYNE173-nYFP carrier SalI The recombinant plasmid site is named as follows: AsNF- YA1-nYFP and AsNF-YB3-nYFP ;Will AsNF-YC5 and AsNF-YA1 Coding region cloned to pSPYNE173-cYFP carrier SalI The recombinant plasmid site is named as follows: AsNF-YC5-cYFP and AsNF-YA1-cYFP .

[0068] AsNF-YA1-nYFP was co-expressed with AsNF-YC5-cYFP, AsNF-YB3-nYFP with AsNF-YC5-cYFP, and AsNF-YB3-nYFP with AsNF-YA1-cYFP in Nicotiana benthamiana leaves for 48 hours. GFP fluorescence was observed using a confocal microscope (Carl Zeiss LSM900) at an excitation wavelength of 488 nm. Figure 14 As shown, there are pairwise interactions between AsNF-YB3 and AsNF-YC5 and AsNF-YA1.

[0069] (4) Yeast three-hybrid experiment (Y3H) to verify the interaction between AsNF-YB3, AsNF-YC5 and AsNF-YA1: AsNF-YB3 and AsNF-YC5 The coding sequence was cloned to pBridge carrier AsNF-YA1 Clone to pGADT7 The vector was then transformed into the yeast strain Y2HGold (WEIDI). The transformed yeast cells were inoculated into plates containing SD-Leu-Trp medium and SD-Leu-Trp-His-Ade-Met medium and incubated at 30°C for 3 days. Single colonies were shaken overnight, and OD... 600 Approximately 1.0, adjust each bacterial culture to 10. 0 10 -1 10 -2 Yeast growth was observed after 1-2 days of uniform spotting on two defective culture media. Results were as follows: Figure 15 As shown, AsNF-YA1, AsNF-YB3, and AsNF-YC5 assemble to form a heterotrimeric complex.

[0070] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. Transcription factors AsNF-YB3 Its application in regulating drought resistance in oats is characterized by: transcription factors AsNF-YB3 The amino acid sequence is shown in SEQ ID NO.

1.

2. The transcription factor according to claim 1 AsNF-YB3 Its application in regulating drought resistance in oats is characterized by: Encoding transcription factors AsNF-YB3 genes AsNF-YB3 The CDS sequence is shown in SEQ ID NO.2, encoding transcription factor AsNF-YB3 genes AsNF-YB3 The gDNA sequence is shown in SEQ ID NO.

3.

3. The transcription factor according to claim 2 AsNF-YB3 Its application in regulating drought resistance in oats is characterized by: transcription factors AsNF-YB3 or encoding transcription factors AsNF-YB3 genes AsNF-YB3 Increased activity and / or expression levels in oats enhance drought resistance in plants.

4. The transcription factor according to claim 3 AsNF-YB3 Its application in regulating the drought resistance of oats is characterized by, Improved drought resistance in oats includes at least one of the following (1)-(5): (1) Under drought stress, the survival rate of oat seedlings was higher than that of control oats; (2) Oat yield was higher than control oat yield under drought stress; (3) Under drought stress, the relative water content of oat leaves was higher than that of the control oat; (4) The rate of water loss from detached oat leaves was lower than that of control oats; (5) The activity of CAT and / or SOD in oats was higher than that in control oats.

5. A method for cultivating transgenic oats with improved drought resistance, characterized in that: Introducing the expression of the receptor oat into the oat AsNF-YB3 Nucleic acid molecules of transcription factors were used to obtain transgenic oats, which showed improved drought resistance compared to the recipient oats. transcription factors AsNF-YB3 The amino acid sequence is shown in SEQ ID NO.1; Encoding transcription factors AsNF-YB3 genes AsNF-YB3 The CDS sequence is shown in SEQ ID NO.2; Encoding transcription factors AsNF-YB3 genes AsNF-YB3 The gDNA sequence is shown in SEQ ID NO.

3.

6. The method for cultivating transgenic oats with improved drought resistance according to claim 5, characterized in that: Introducing the expression of the receptor oat into the oat AsNF-YB3 Transcription factor nucleic acid molecules are introduced into the recipient oats containing... AsNF-YB3 This is achieved through gene recombination expression vectors.

7. The method for cultivating transgenic oats with improved drought resistance according to claim 6, characterized in that: The recombinant expression vector is introduced into recipient oats by transforming plant cells or tissues through methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electroporation, or Agrobacterium-mediated transformation, and then the transformed plant tissues are cultured into plants.

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