Application of U2AF65B gene in regulation and control of seed germination, seedling growth and ABA stress

Abstract

The invention discloses application of a U2AF65B gene in regulation and control of seed germination, seedling growth and ABA stress, mutation of U2AF65b is beneficial to promotion of seedling growth, and the extension speed of a U2AF65B mutant at the root and the growth amount of seedlings are remarkably increased; after ABA treatment, the germination rate of a wild type plant is significantly lower than that of a U2AF65B mutant, the U2AF65B mutant has a higher germination rate under ABA treatment, the gene has a regulation effect on the germination ability of the plant under stress conditions, the U2AF65B influences the growth and development of the plant by regulating an ABA signal channel, the ABA content of ABA stress hormone is increased when the stress comes, and the U2AF65B mutant has a higher germination rate than the U2AF65B mutant. The germination ability of the U2AF65B mutant seeds is improved, and the adversity resistance can be improved due to low expression quantity or no expression of the gene; the U2AF65B mutant has a phenotype for promoting the growth of seedlings by increasing the length of roots; according to the phenotype that the germination rate of the mutant can be increased under exogenous ABA or when the stress comes, the screened seeds are cultivated, the seed selection is accurate, the time is saved, and the coping capacity of the seeds to the stress is improved when the seeds face the stress.

Description

Technical Field

[0001] The invention belongs to the technical field of biology, and particularly relates to application of a U2AF65B gene in regulating seed germination and seedling growth as well as ABA stress. Background Art

[0002] Abscisic acid (ABA) plays a very important role in plant gene regulation, especially in coping with adversity and regulating growth and development. For example, abscisic acid plays an important role in seed maturation and dormancy. In the seed maturation stage, abscisic acid promotes endosperm drying and seed maturation; in the dormancy stage, abscisic acid maintains the dormant state of seeds and prevents seeds from germinating under unsuitable conditions. In addition, abscisic acid regulates the growth and development of plants, especially in root growth and lateral bud elongation. When plants are subjected to adversity or stress, abscisic acid can inhibit growth, thereby allocating resources to adapt to environmental changes. In short, abscisic acid can play an important regulatory role in plants through multiple pathways such as regulating gene expression, affecting growth and development, and adversity response, helping plants adapt to and respond to changes in the external environment.

[0003] Precursor mRNA (pre-mRNA) is the original transcript in eukaryotes, and its coding sequence consists of multiple discontinuous exons and introns. The spliceosome is an important organelle responsible for splicing pre-mRNA, that is, connecting exons and removing introns. This process is not only mechanical connection and cutting, but also involves complex regulatory mechanisms. The result of splicing is the generation of mature mRNA, which is one of the key steps in gene expression. Through splicing, cells can generate a variety of different mRNA isoforms, thereby producing different protein isoforms, which is essential for cell function and regulation.

[0004] U2 small nuclear ribonucleoprotein particle (snRNP) auxiliary factor 65kDa (U2AF65) is a splicing factor that promotes the assembly of splicing precursors. The role of U2AF65 in alternative splicing (AS) has been elucidated. However, little is known about the function of the large subunit U2AF65B. Patent CN107119056A discloses the application of a splicing auxiliary factor AtU2AF65b in the regulation of plant flowering period. The application of U2AF65b in the regulation of plant flowering period reveals the application of the splicing auxiliary factor AtU2AF65B derived from Arabidopsis thaliana in the regulation of plant flowering period. By increasing the expression level of this gene, the flowering time of the plant can be effectively delayed; conversely, reducing its expression level helps to promote early flowering of annual plants or shorten the reproductive period of perennial plants. Patent CN116286953A discloses the use of U2AF65b in regulating plant salt stress resistance. The use of U2AF65b in regulating plant salt stress resistance discloses the use of U2AF65B in regulating plant resistance to salt stress. Overexpression of the U2AF65B gene in grapes can significantly enhance the adaptability of grapes to salt stress, which has important application prospects for improving soil salinization problems.

[0005] Arabidopsis thaliana is a model plant widely used in botanical research. So far, no clear conclusion has been drawn on the role of the U2AF65b gene in regulating seed germination and seedling growth and the role of abscisic acid. Summary of the invention

[0006] Technical issues solved:

[0007] This application aims to address the technical problems in the prior art, such as the lack of understanding of the function of the large subunit U2AF65B, and the lack of clear conclusions on the research on the U2AF65b gene in regulating seed germination and seedling growth and the role of abscisic acid. It provides the application of the U2AF65B gene in regulating seed germination and seedling growth and ABA stress.

[0008] Technical solution:

[0009] To achieve the above objectives, this application is implemented through the following technical solutions:

[0010] Application of U2AF65B gene in regulating seed germination, seedling growth and ABA stress.

[0011] Beneficial effects:

[0012] The present application provides the use of the U2AF65B gene in regulating seed germination and seedling growth and ABA stress, which has the following beneficial effects compared with the prior art:

[0013] 1. The existing technology can adjust the flowering period of plants by regulating the expression level of U2AF65b, that is, the application in the regulation of plant flowering period. In view of the effect of U2AF65B on seed germination and seedling growth, the present application can optimize the growth performance of plant seedlings by regulating the expression of U2AF65B, and the screened seeds are more targeted and efficient; the role in this aspect also helps to make more precise interventions in plant growth regulation. The existing technology may pay more attention to other regulatory factors, and U2AF65B provides an additional regulatory point; U2AF65B, as a regulatory factor of seed germination and seedling growth, can be used as a new biomarker for screening and evaluating the stress resistance of plants, which has practical application value in the field of plant research and breeding;

[0014] 2. The existing technology discloses the use of U2AF65B in regulating plant resistance to salt stress. Overexpression of U2AF65B can significantly enhance the adaptability of grapes to salt stress, which has important application prospects for improving soil salinization problems, that is, U2AF65B has a positive regulatory effect; the present application shows that U2AF65B has a negative regulatory effect in ABA, that is, low or no expression of U2AF65B promotes seed germination. ABA is a stress hormone. The results of the present invention are not limited to salt stress, but can also be extended to other stress conditions, such as water stress, temperature stress, etc. Therefore, more effective anti-stress strategies can be developed to help plants better cope with complex environmental challenges. Compared with the study of a single stress, this comprehensive study may provide a more comprehensive solution. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is an amino acid sequence comparison diagram of U2AF65B in grape, rice and Arabidopsis thaliana;

[0016] Figure 2 The Arabidopsis thaliana AtU2AF65b T-DNA insertion mutant of the present application promotes root elongation and leads to an increase in seedling biomass, wherein A is an identification diagram of the mutant by RT-PCR, B is a germination rate diagram of the mutant and the wild-type wt on 1 / 2MS medium at 3, 4, 5 and 7 days of germination, CD is a root length diagram of the mutant and the wild-type wt when grown vertically on 1 / 2MS medium for 7 days, and E is a diagram of the fresh weight of the mutant and the wild-type WT when grown vertically on 1 / 2MS medium for 7 days;

[0017] Figure 3 This application is 1mol L -1Figure 1. The germination of Arabidopsis thaliana AtU2AF65B T-DNA insertion mutant seeds was promoted under ABA conditions, where A is the mutant and wild type wt in the presence of 1 μmol L -1 Phenotypic diagram of germination on 1 / 2MS medium containing ABA for 13 days. B is the phenotype of mutant and wild type wt on 1 μmol L -1 The germination rate graph on ABA MS medium at 13 days of germination;

[0018] Figure 4 This is a diagram of the subcellular localization of the OsU2AF65b gene of the present application in rice. DETAILED DESCRIPTION

[0019] The following specific embodiments further illustrate the present invention. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the contents disclosed in the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope limited by the appended claims of the application.

[0020] Embodiment 1:

[0021] Amino acid sequence alignment of U2AF65B:

[0022] 1Sequence search

[0023] TAI R (https: / / www.arab i dops i s.org / ) was used to search for the Arabidopsis U2AF65b gene (AT1G60900) to obtain the protein sequence of Arabidopsis, and phytozome v 13 (https: / / phytozome.jgi.doe.gov) was used to search for the rice and grape U2AF65b genes (LOC_Os11 g45590.1 and VIT_204s0023g01450.1), and the protein sequences of rice (OsU2AF65B), grape (VvU2AF65B) and Arabidopsis (AtU2AF65B) were obtained, respectively.

[0024] The specific sequence is shown in Table 1:

[0025] Table 1 Protein sequences of U2AF65B in rice, grape and Arabidopsis

[0026]

[0027]

[0028]

[0029] 2 Multiple sequence alignment

[0030] The three sequences were aligned using the sequence alignment software MEGA, and the alignment results were beautified using the gene DOC software.

[0031] 3 Results

[0032] First, we compared the amino acid sequences of grape VvU2AF65B, rice OsU2AF65B, and Arabidopsis AtU2AF65B proteins. We found that most regions were highly conserved, i.e. Figure 1 The background in the middle is black, indicating that the amino acids are the same, especially the three special amino acids at positions 100-140, 180-220, 280-320, 400-440 and 480-520 are the same. This indicates that these genes have maintained a high degree of conservation during evolution and may perform similar functions in these plants. Although most regions are conserved, there are certain differences in some parts of the sequence (such as positions 60-100 and 220-260), that is, gray or white background, and the cap indicates different amino acids. These differences may cause the protein to have specific adaptive functions in different species. For example, the middle part of the sequence (approximately positions 240-260) shows some species-specific differences, which may correspond to the specific regulatory functions of these genes in different species or interactions with other proteins.

[0033] Example 2

[0034] T-DNA insertion mutation of Arabidopsis thaliana AtU2AF65b promotes seed germination and root growth

[0035] 1 Experimental materials:

[0036] Arabidopsis thaliana wild type Col-0 (WT) and AtU2AF65b (AT1 G60900) mutant (SALK_055049C, T-DNA insertion, 65b-2).

[0037] AtU2AF65b (AT1 G60900) mutant seeds were purchased through the AraShare science (https: / / www.arashare.cn) platform (NAS code: N664616).

[0038] 2 Test methods

[0039] 2.1 Preparation of culture medium

[0040] Conventional culture medium (1 / 2MS culture medium): Use MS culture medium with production number M5519-50L, weigh 0.443 g, dissolve in 100 mL of distilled water by heating, and sterilize at 121°C for 15 minutes.

[0041] 2.2 Arabidopsis planting, mutant identification and seed germination data collection Wild-type and mutant seeds were sterilized with 10% Bl each for 25 minutes, shaken every 10 minutes, and finally washed with sterilized ultrapure water for 3 times, 2-5 minutes each time. Then the seeds were evenly planted on conventional culture medium. The culture dish with seeds was placed upside down in a 4℃ refrigerator for 2 days, and then transferred to a light incubator (23℃, 16h / 8h day / night, humidity of 84%-90%) and placed horizontally for culture. From the 3rd to the 7th day, the number of germination of seeds of each material in the culture dish of conventional culture medium was counted, and the root length of the seeds on the 7th day of germination was measured. RNA of wild-type and mutant seedlings was extracted and reversely transcribed into cDNA, and the target band was amplified by RT-PCR using synthetic primers (F: 5'-TGGGTCATGATCTGGATTAGG-3'; R: 5'-TATTGGCCGCAAGTCTATACG-3'). Each experiment was repeated 3 times. SPSS19.0 was used for one-way analysis of variance (ANOVA), and Tukey's test or independent sample t test was performed at the p < 0.05 level. Histograms were drawn using GraphPad Prism 9.0.0.

[0042] 3 Results

[0043] The AtU2AF65b mutant (65b-2) was identified, and the results showed that no transcripts of U2AF65b were detected, such as Figure 2 As shown in A.

[0044] The number of germinations of the mutant (65b-2) and wild-type plants (wt) cultured on MS medium was counted on the 3rd, 4th, 5th and 7th days of germination, and the corresponding germination rates were obtained. The results showed that there was no significant difference between the two, as shown in Figure 2 . Figure 2 As shown in B. Their root lengths were further measured and it was found that the root length of the mutant was significantly longer than that of the wild type, as shown in Figure 2 As shown in C and D. The fresh weight of the plants of the two materials was weighed at the same time, and it was found that the fresh weight of the mutant was significantly higher than that of the wild type, as shown in Figure 2 As shown in E, the mutation of U2AF65b is beneficial to promoting seedling growth, especially root elongation.

[0045] Figure 2 Identification of the A mutant by RT-PCR; Figure 2 Germination rates of the middle B mutant and wild-type wt on 1 / 2MS medium at 3, 4, 5, and 7 days (50 seeds were 1 replicate, with 3 replicates in total); Figure 2 Root length of C and D mutants and wild type wt when grown vertically on 1 / 2MS medium for 7 days; Figure 2Fresh weight of plants of E mutant and wild type WT grown vertically on 1 / 2MS medium for 7 days (20 plants were replicated). Data in B, D, and E are the mean ± standard error of 3 replicates, and different letters indicate significant comparisons by Tukey's test or independent sample t-test (p=0.05).

[0046] Figure 2 The data of B, D, and E are shown in Table 2

[0047] Table 2 Germination rate (%) of wild type (wt) and mutant (65b-2) on 1 / 2MS medium at 3, 4, 5 and 7 days

[0048]

[0049]

[0050] Table 3 Root length and fresh weight of wild type (wt) and mutant (65b-2) grown vertically on 1 / 2MS medium for 7 days

[0051]

[0052] RT-PCR was used to detect whether the gene was expressed, and it was found that no transcription product of U2AF65B was detected, indicating that the material was a mutant. In the seed germination and seedling growth experiments, under normal growth conditions, compared with the wild type, the root elongation speed and seedling growth of the mutant plants increased significantly, while the germination rate did not change significantly.

[0053] Example 3

[0054] T-DNA insertion mutation of Arabidopsis thaliana AtU2AF65b promotes germination of Arabidopsis seeds under ABA stress:

[0055] 1 Experimental materials:

[0056] Arabidopsis thaliana wild type Col-0 (WT) and AtU2AF65b (AT1 G60900) mutant (SALK_055049C, T-DNA insertion, 65b-2).

[0057] Arabidopsis thaliana AtU2AF65b (AT1 G60900) mutant seeds were purchased through the AraShare science (https: / / www.arashare.cn) platform (NAS code: N664616).

[0058] 2 Test methods

[0059] 2.1 Preparation of culture medium

[0060] Preparation of ABA stock solution: Weigh 0.026432 g of ABA and dissolve it in 95% ethanol. Add ultrapure water to make up to 100 mL to make the concentration 1 mmol L -1 The prepared mother solution was stored in a refrigerator at 4°C.

[0061] Conventional culture medium (1 / 2MS culture medium): Use MS culture medium with production number M5519-50L, weigh 0.443 g, dissolve in 100 mL of distilled water by heating, and sterilize at 121°C for 15 minutes.

[0062] Contains 1 μmol L -1 Preparation of ABA medium: Add 100 μL of 1 mmol L -1 ABA stock solution containing 1 μmol L -1 ABA medium.

[0063] 2.2 Planting of materials and statistics and photography of seed germination

[0064] The wild-type and mutant seeds were sterilized with 10% Bl each for 25 minutes, shaken every 10 minutes, and finally washed with sterilized ultrapure water for 3 times, each time for 2 to 5 minutes. The seeds were then evenly placed in a solution containing 1 μmol L -1 Cultured in a conventional culture medium containing ABA. Place the culture dish with the seeds upside down in a 4°C refrigerator for 2 days, then transfer it to a light incubator (23°C, 16h / 8h day / night, humidity 84% to 90%) and culture it horizontally. Count the number of germinations of seeds of each material in the culture medium containing ABA on day 13 and take photos. Repeat 3 times. Perform an independent sample t-test at the p<0.05 level. Use GraphPad Prism 9.0.0 to draw a histogram.

[0065] 3 Results

[0066] After statistical analysis of the germination number, it was found that after adding ABA to the culture medium, the germination rate of the wild type was significantly lower than that of the mutant. Figure 3 As shown in A and B.

[0067] A represents the difference between mutant and wild type in the presence of 1 μmol L -1 Phenotypes of the mutant and wild type wt on 1 / 2MS medium containing ABA at 13 days of germination. -1 Germination rate on ABA-containing MS medium after germination for 13 days. Figure 3 The data in B are the mean ± standard error of 3 replicates, and different letters indicate significant comparisons by t-test of independent samples (p = 0.05).

[0068] Figure 3 The data of B are shown in Table 4

[0069] Table 4 Wild type (wt) and mutant (65b-2) in the presence of 1 μmol L -1 Germination rate on 1 / 2MS medium containing ABA at 13 days

[0070]

[0071] RT-PCR was used to detect whether the gene was expressed, and it was found that the transcription product of U2AF65B could not be detected, indicating that the material was a mutant. After ABA treatment during seed germination, the germination rate of the wild type was significantly affected and the germination rate was reduced, which was significantly different from the mutant plant 65b-2, that is, the mutant showed a higher germination rate phenotype.

[0072] Example 4

[0073] Localization of rice OsU2AF65B in the nucleus and cytoplasm:

[0074] 1. Experimental Materials

[0075] Rice Nipponbare seedlings

[0076] 2. Test methods

[0077] 2.1 Vector construction

[0078] (1) Amplification of the target fragment OsU2AF65B: RNA was extracted from Nipponbare seedlings, and the cDNA synthesized by reverse transcription was used as a template. The specific primers (F: 5′

[0079] -CCTGTTGTTTGGTGTTACTTAAGCTTATGGCGGACGACC

[0080] ACGC-3′ and R:5′

[0081] -TCCTCGCCCTTGCTCACCATGGATCCCTACGCGTCATATTCCGCACTA

[0082] GA-3′) for RT-PCR amplification. The amplification system includes: 2μL cDNA template, 25μL 2×Phanta Mix, 2μL primer F, 2μL primer R, 19μL double distilled water, and a total volume of 50μL. The PCR reaction procedure is 95℃ pre-denaturation for 3min; then 35 cycles as follows: 95℃15s, 57℃15s, 72℃30s / 1kb; finally 72℃5min, and stored at 4℃. After the PCR reaction is completed, use agarose gel electrophoresis to detect. If the amplified band shows a single target band, cut the gel and recover it for later use.

[0083] (2) Vector double enzyme digestion: The enzyme digestion reaction system includes: 8 μL pGREEN-UBI-GFP vector, 1 μL H i nd III, 1 μL BamHI, 5 μL CutSmart buffer, 35 μL double distilled water, and a total volume of 50 μL. Incubate in a PCR instrument for 1 h, detect by agarose gel electrophoresis, and cut out the linearized vector band for later use.

[0084] (3) Gel recovery: Use the Tiangen TIANGEN common agarose gel DNA recovery kit (DP209-03) to recover the amplified target fragment and linearized vector fragment. After recovery, store the target band at -20°C for future use or immediately perform transformation and ligation.

[0085] (4) Connection: Use Vazume's One-step ligation was performed using the One Step Cloning Kit, and the operation was performed according to the instructions provided, as follows: the reaction system was configured according to the instructions, and then the reaction was incubated in a 37°C water bath for 30 min, cooled on ice for 2 min, and then transformed immediately.

[0086] (5) Transformation: This step was performed according to the instructions provided by Tiangen's DH5α competent cells.

[0087] (6) Identification of recombinant vector colonies by PCR: After the recombinant vector is transformed, the colonies of positive transformations are tested by PCR. The specific steps are as follows:

[0088] ① Prepare the reaction system according to the PCR system in the PCR eight-tube series.

[0089] ② Use a sterilized toothpick to pick up multiple single colonies in the clean bench, first streak amplification on LB solid resistance medium, and then place the toothpick in a PCR tube and stir.

[0090] ③ Place the culture plate with colony amplification upside down in a 37℃ incubator for 12 hours. At the same time, place the PCR tube into the PCR instrument according to the above procedure.

[0091] ④Observe the electrophoresis results on a gel UV imager, select the positive colonies corresponding to the target bands, and culture them in 2 mL of LB liquid culture medium containing the corresponding antibiotics at 37°C on a shaker (200 rpm) overnight.

[0092] (7) The plasmid extracted from the correct bacterial solution sent for sequencing is the target cloning vector to obtain the OsU2AF65B-PGREEN II-UBI-GFP fusion protein. The plasmid was extracted according to the instructions provided by the plasmid extraction kit of TIANGEN Company and stored at -20°C for future use.

[0093] 2.2 Cultivation of etiolated rice seedlings

[0094] The rice seeds were soaked in carbendazim and clean water for 1 day each, germinated at 28°C for 1 day, and cultured in the dark until the seedlings grew to about 10-12 cm.

[0095] 2.3 Obtaining rice protoplasts

[0096] (1) Preparation of solution:

[0097] ① Enzyme solution (25 ml final concentration): 1.5% cellulase, 0.4% chromatase, 0.4M mannitol, 20mM KCL, 20mM MES (PH5.7, 2-morpholineethanesulfonic acid), heat in a 55℃ water bath for 10 min to dissolve. Cool on ice to room temperature, add 10mM CaCL2, 0.1% (bovine serum albumin), filter at 0.45μm, store at -20℃, and prepare before use.

[0098] ② Rinsing solution W5: 154mM NaCL, 125mM CaCL2, 5mM KCL, 2mM MES (PH 5.7)

[0099] ③Suspension MMg: 0.4M mannitol, 15mMMgCL2·6H2O, 4mMMES-KOH (PH5.7), 0.45μm filtered, and stored at 4℃.

[0100] (2) Isolation of protoplasts

[0101] Weigh about 0.2g of yellow rice seedlings, cut the rice stems into small pieces and place them in the enzymatic solution, and evacuate for 1h. Perform enzymatic hydrolysis in the dark at 25℃ for 4 hours. Pass the enzymatic mixture through a 60-mesh filter, and centrifuge the filtrate at 150×g for 5min. Collect the protoplast precipitate, rinse and suspend it with W5 solution. Centrifuge at 150g for 5min, remove the supernatant, and suspend the obtained protoplasts with W5 solution again.

[0102] (3) Purification of protoplasts

[0103] Suspend the solution containing protoplasts in a freshly prepared 20% sucrose solution. Centrifuge at 100×g for 5 minutes and collect the protoplasts at the interface between the solution and the sucrose solution. Rinse and suspend with W5 solution. Centrifuge at 200×g for 5 minutes, remove the supernatant, and dissolve the obtained protoplasts in W5 solution. Take the protoplast solution and drop it on a blood cell counting plate for counting, and suspend it with MMg solution to make the protoplast cell concentration reach 1-2*10 6 Pieces / mL.

[0104] 2.4 Conversion

[0105] 2 μg of recombinant plasmid was added to 70 μL of rice protoplast suspension and mixed well. An equal volume of the constructed plasmid mixture was combined with 40% PEG4000 solution at a ratio of 1:5 (volume ratio), incubated at 28°C overnight, and then observed using a laser confocal microscope.

[0106] 3 Results

[0107] The model plant rice was selected to construct the OsU2AF65B-PGREEN II-UBI-GFP fusion protein, in which PGREEN II-UBI-GFP was used as a control and transferred into protoplasts. The experimental results showed that the green fluorescence of the OsU2AF65B-PGREEN II-UBI-GFP fusion protein could be observed in the nucleus and cytoplasm, indicating that OsU2AF65B is a nuclear and cytoplasmic dual-localization protein.

[0108] The embodiments described above are part of the embodiments of the present invention, rather than all of the embodiments. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

Claims

1. Application of U2AF65B gene in regulating seed germination, seedling growth and ABA stress.

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