Application of the oat AsGDSL135 gene in seed vigor regulation

Abstract

This invention discloses the application of the oat AsGDSL135 gene in seed vigor regulation, belonging to the field of seed biotechnology. The nucleotide sequence of the oat AsGDSL135 gene is shown in SEQ ID NO.14, and the amino acid sequence is shown in SEQ ID NO.13. Transforming the oat AsGDSL135 gene into Arabidopsis thaliana or rice allows for overexpression of the AsGDSL135 gene in seeds, thereby improving seed vigor and accelerating the germination rate of aging seeds. Utilizing this gene can help in the screening and breeding of high-vigor plant varieties.

Description

Technical Field

[0001] This invention belongs to the field of seed biotechnology, specifically relating to the application of the oat AsGDSL135 gene in seed vigor regulation. Background Technology

[0002] Seed germination is a crucial initial stage in the plant life cycle, vital for plant growth and development, population reproduction, and agricultural production. During this process, seeds need to rapidly transition from a dormant state to an active metabolic state to provide sufficient energy and resources to support seedling establishment. High-vitality seeds can gain an early advantage in growth, enhance stress resistance, promote robust seedling growth, and transmit genetic advantages, ensuring population reproduction. Therefore, in agricultural production, the selection, production, and application of high-vitality seeds should be given high priority to fully leverage their advantages and contribute to sustainable agricultural development and food security.

[0003] GDSL motif lipases are an important subfamily of lipases. GDSL esterases / lipases have a unique structure, with their active site, a serine residue, located near the N-terminus. The secondary structure of GDSL proteins generally consists of multiple α-helices and β-sheets. The GDSL lipase family possesses a highly flexible active site, which undergoes conformational changes upon substrate binding. This broad substrate binding capability allows lipases to perform multiple functions. Although GDSL lipases play important roles in many biological processes, their role in oat seed germination remains insufficiently understood, particularly regarding whether a direct regulatory relationship exists between them and seed vigor. Summary of the Invention

[0004] The purpose of this invention is to provide a new use for the oat AsGDSL135 gene, specifically, the application of the AsGDSL135 gene in seed vigor regulation.

[0005] The objective of this invention can be achieved through the following technical solutions:

[0006] The oat AsGDSL135 gene in this invention has the nucleotide sequence shown in SEQ ID NO.14 and the amino acid sequence shown in SEQ ID NO.13.

[0007] The application of the oat AsGDSL135 gene in seed vigor regulation in this invention.

[0008] The preferred application is overexpression of the AsGDSL135 gene to improve seed vigor.

[0009] The construction and gene function verification of oat AsGDSL135 gene overexpression material in this invention includes the following steps:

[0010] (1) Obtain the nucleotide and amino acid sequences of the oat AsGDSL135 gene;

[0011] (2) Using oat cDNA as a template, PCR amplification was performed with specific primers, and the PCR product was purified and recovered.

[0012] (3) After secondary PCR amplification, the vector was constructed into the pEGOEP35S-H-GFP vector to obtain the pEGOEP35S-H-GFP-AsGDSL overexpression vector;

[0013] (4) Transform the plasmid containing the target fragment of the AsGDSL135 gene from step (3) and perform colony PCR verification.

[0014] Furthermore, in step (2), the upstream primer sequence for PCR amplification is shown in SEQ ID No. 3 of the sequence listing, and the downstream primer sequence is shown in SEQ ID No. 4 of the sequence listing.

[0015] Furthermore, in step (3), the upstream primer sequence for PCR amplification is shown in SEQ ID No. 1 of the sequence listing, and the downstream primer sequence is shown in SEQ ID No. 2 of the sequence listing.

[0016] The beneficial effects of this invention are:

[0017] 1. This invention constructs an overexpression vector of the AsGDSL135 gene and transfects it into Arabidopsis thaliana and rice, confirming that the gene can positively regulate seed germination.

[0018] 2. This invention provides a foundation for screening and breeding high-vitality rice varieties and high-vitality Arabidopsis varieties, which is of great significance to production. Attached Figure Description

[0019] Figure 1 This is a graph showing the electrophoresis results of the target gene fragment after PCR amplification.

[0020] Figure 2 This is a structural diagram of the overexpression vector;

[0021] Figure 3 This is a graph showing the results of bacterial culture PCR detection.

[0022] Figure 4 Image showing the PCR identification results of homozygous positive rice seedlings;

[0023] Figure 5 Figure showing the relative expression levels in homozygous positive rice seedlings;

[0024] Figure 6 A statistical chart showing the number of Arabidopsis thaliana seeds that germinated within 0 to 9 days;

[0025] Figure 7 Images showing the germination of wild-type and transgenic Arabidopsis seeds;

[0026] Figure 8 Statistical charts showing the germination potential and germination rate of wild-type and transgenic rice seeds. Detailed Implementation

[0027] ① Plant materials: The gene cloning material was 'Monida' oat seeds. The oat seeds were treated by placing them in germination dishes and allowing them to absorb water for 32 hours under 8 hours of light, 16 hours of darkness, and a constant temperature of 20℃. The embryos were then used for total RNA extraction and subsequent gene amplification. The Arabidopsis seeds were 'Columbia' wild-type seeds. The 'Nipponbare' rice seeds were purchased from Wuhan Aidijing Biotechnology Co., Ltd.

[0028] ② Reagents, vectors, and bacterial strains: Total RNA extraction kit was purchased from Beijing Huayueyang Biotechnology Co., Ltd.; EasyScript® One-Step gDNA Removal and cDNA Synthesis SuperMix kit (AE311-02) and Escherichia coli DH5α were purchased from Beijing TransGen Biotechnology Co., Ltd.; Agarose gel DNA recovery kit (DP209-03) was purchased from Beijing Tiangen Biotech Co., Ltd.; Intermediate vector for cloning the target gene, Agrobacterium tumefaciens competent cells GV3101, was purchased from Beijing Qingke Biotechnology Co., Ltd.; M519 MS medium powder was purchased from Phytotechlab; Kanamycin and rifampin were purchased from Sigma-Aldrich; Plant overexpression vector pEGOEP35S-H was purchased from Wuhan Aidijing Biotechnology Co., Ltd.

[0029] ③The primers used in the examples for gene cloning, positive identification of Escherichia coli culture PCR, positive identification of Agrobacterium culture PCR, and identification of positive and gene expression levels of transgenic materials in the later stages are shown in Table 1.

[0030] Table 1

[0031]

[0032] Example 1: Gene Cloning

[0033] ① Amplification: Total RNA was extracted from oat seeds according to the instructions of the total RNA extraction kit. Reverse transcription was performed using the EasyScript® One-Step gDNA Removal and cDNA Synthesis SuperMix kit (AE311-02). Using the reverse transcription product as a template, PCR amplification was performed with specific primers AsGDSL-F / R to obtain the target fragment of the AsGDSL135 gene. The results were detected by agarose gel electrophoresis. Figure 1 As shown in the figure; finally, the target fragment was recovered using an agarose gel DNA recovery kit (DP209-03).

[0034] ② Ligation of the target fragment with the cloning vector:

[0035] The recovered target fragment was amplified by secondary PCR using primers pEG-AsGDSL-F / R to obtain the AsGDSL135 gene fragment for constructing the overexpression vector. This fragment was cloned into the 677bp position of the plant expression vector pEGOEP35S-H-GFP, thus constructing the target fragment overexpression vector (pEGOEP35S-H-GFP-AsGDSL overexpression vector). The enzyme digestion and ligation reaction conditions are shown in Table 2, and the overexpression vector structure is as follows. Figure 2 As shown.

[0036] Table 2

[0037]

[0038] ③ Overexpression vector transformation, bacterial detection, and sequencing: Mix 100 μL of DH5α E. coli competent cells with 2-5 μL of the ligation product from step ② and incubate on ice for 30 min; then quickly place in a 42℃ water bath for 90 s, followed by an ice bath. Add 500 μL of LB liquid medium and culture until the cells return to normal growth. Spread the bacterial culture evenly on Kana-resistant LB solid medium plates; after 30 min, incubate overnight at 37℃. Colony PCR detection was performed using the 35S-F / eGFP-cx primer pair, and the results are as follows: Figure 3 As shown, the band size is approximately 1440 bp; the detected positive plaques were picked and cultured. After plasmid extraction, Sanger sequencing was performed, and the sequences were consistent, indicating successful construction of the overexpression vector.

[0039] Example 2: Genetic transformation of the target gene in Arabidopsis thaliana

[0040] (1) Agrobacterium transformation

[0041] After thawing GV3101 competent Agrobacterium on ice, the overexpression vector was added, and the culture was transformed according to the instructions. The bacterial culture was then evenly spread on plates containing kanamycin (50 mg / L) and rifampin (50 mg / L) and incubated at 28°C for 3 days. After colonies grew, 5 single clones were selected for PCR identification. After confirming the positive clones, they were propagated and stored for subsequent experiments.

[0042] (2) Arabidopsis thaliana cultivation

[0043] After sterilizing with sodium hypochlorite, Arabidopsis seeds were evenly sown on 1 / 2 MS medium and vernalized at 4°C for 3 days. They were then transferred to a light incubator to promote germination. Approximately 10 days after germination, the seeds were transplanted into potting soil and placed in an artificial climate chamber (22°C, 16h light / 8h darkness), with regular watering.

[0044] (3) Infection of Arabidopsis thaliana inflorescences

[0045] Agrobacterium was inoculated onto LB agar plates containing kanamycin (50 mg / L) and rifampin (50 mg / L), and cultured for 1 day. The culture was then transferred to 10 mL of LB liquid medium containing antibiotics and propagated on a shaker at 28°C and 200 rpm. When the OD600 of the bacterial suspension reached 0.6–0.8, the suspension was centrifuged at 5000 rpm for 5 min, and the precipitate was resuspended in 20 mL of MS liquid medium. 1 g of sucrose and 10 μL of Silwet L-120 were added to prepare the infection solution.

[0046] Apply the inoculum evenly to the Arabidopsis thaliana flower buds with a brush, ensuring they are moist. Incubate in the dark at 22°C for 2 days, covering with a plastic bag to maintain humidity. Then resume normal growth, repeat the inoculum treatment after one week, and finally harvest the T0 generation seeds.

[0047] (4) Screening of transgenic positive materials

[0048] After drying the T0 generation seeds, a small amount was sterilized with sodium hypochlorite and evenly sown on 1 / 2 MS solid medium containing hygromycin (50 mg / L). Positive seedlings were selected. The positive seedlings were then cultured under standard conditions to harvest T1 generation seeds. T1 generation seeds were sown at a rate of 60 seeds / plate on 1 / 2 MS solid medium containing hygromycin. The number of seedlings, ungerminated seeds, and abnormal seedlings was counted. Normally growing seedlings were selected for transplanting and continued cultivation to harvest T2 generation seeds. If T2 generation seeds did not exhibit segregation (normal germination), they were considered homozygous transgenic material. Homozygous plants were transplanted into nutrient soil for propagation to obtain T3 generation transgenic seeds.

[0049] (5) Detection of relative expression levels in transgenic homozygous plants

[0050] Ten days after germination of transgenic homozygous Arabidopsis seeds, ten seedlings from each line were selected for total RNA extraction and cDNA synthesis. Using the cDNA as a template, the Arabidopsis EFE1a gene as an internal control gene, and the GDSL-qF / R gene as the target gene primer, qPCR analysis was performed using the SYBR Green I method.

[0051] Example 3: Genetic transformation of the target gene in rice

[0052] (1) Induction and differentiation of callus tissue

[0053] Rice seeds were sterilized using conventional seed treatment methods and cultured at 28℃ under 12h light / 12h dark conditions until seedlings reached the appropriate transformation stage. Approximately 1.5–2.0 cm of appropriately grown rice seedlings were cut and placed in MS medium (containing 1.0 mg / L 2,4-D) for callus induction. The medium was changed every 7–10 days until the callus enlarged and became white or milky white granular.

[0054] (3) Agrobacterium transformation

[0055] This step also uses Agrobacterium-mediated genetic transformation, using GV3101 carrying the target gene plasmid for transformation. After culturing to OD600=0.6~0.8, 50μM acetylsalicylic acid induction solution is added and incubated for 4h to enhance transformation ability.

[0056] Under aseptic conditions, callus tissue was suspended in Agrobacterium solution and allowed to stand for 30 min to promote full contact. Subsequently, the callus tissue was transferred to MS medium containing 50 mg / L dapsone and 1.0 mg / L 2,4-D and cultured for 1–2 days for recovery.

[0057] (4) Screening and regeneration

[0058] The transformed callus was transferred to a selection medium (MS medium containing 50 mg / L hygromycin B) to screen for resistant callus. After 2–3 weeks, callus with resistance markers grew. This was then transplanted into a growth substrate containing a low concentration of plant hormone (0.5 mg / L BAP) to induce rooting and complete the regeneration process.

[0059] (5) Plant transplantation

[0060] When the transformed plants reach the appropriate stage of development, they are removed from the culture medium and transplanted into pots containing nutrient soil. Suitable humidity and temperature are maintained to promote their growth. Finally, they are transplanted into a field environment for growth observation.

[0061] (6) Plant resistance testing

[0062] Using hygromycin gene-specific primers, conventional PCR was used to amplify and detect whether rice seedlings contained the hygromycin gene.

[0063] (7) Detection of relative expression levels in transgenic plants

[0064] DNA was extracted from the leaves of resistant rice seedlings. PCR amplification of AsGDSL135 overexpression was performed using 35S-F and eGFP-CX primers. Seven homozygous positive seedlings were obtained. The PCR identification results of the positive seedlings are as follows: Figure 4 As shown (Note: M: DNAladder AL2000; +: AsGDSL plasmid positive control; -: wild rice DNA negative control; other bands are positive seedling samples). RNA was extracted from homozygous positive seedlings and reversed to cDNA. Using the OsUBI gene as an internal control and GDSL-qF / R as primers, qPCR analysis was performed to verify AsGDSL135 expression. The results are shown below. Figure 5 As shown.

[0065] Example 4: Vigor detection of transgenic Arabidopsis thaliana seeds and transgenic rice seeds

[0066] ① The AsGDSL135 gene was overexpressed in Arabidopsis thaliana, and homozygous seeds were subjected to controlled deterioration treatment after harvest. Expression level determination revealed that the OE-49 overexpressing Arabidopsis thaliana plant had the highest AsGDSL135 gene expression level, and this line was used for subsequent experiments. Seed vigor detection results for AsGDSL135 overexpressing Arabidopsis thaliana are shown below. Figures 6-7 As shown in the figure, after 7 days of cultivation at 22℃, the germination rate of non-aged Arabidopsis seeds using the transgenic AsGDSL135 gene reached almost 100%, indicating that these seeds had high viability and were well-developed, making them suitable for subsequent aging experiments. After 5 days of controlled deterioration treatment, wild-type (WT) and transgenic seeds showed different germination patterns. On day 9 of germination, the germination rate of aged seeds tended to stabilize, with WT germination rate at only 50%, while OE-49 germination rate was 64%, significantly higher than WT (P<0.05). Furthermore, in the early stage of germination of aged Arabidopsis seeds (day 3), the germination rate of OE-49 was also significantly higher than WT (P<0.05), indicating that the AsGDSL135 gene can accelerate the germination rate of aged seeds.

[0067] ② The relative expression levels of the AsGDSL135 gene in homozygous positive rice seedlings showed that OE36 and OE43 plants had the highest expression levels. OE36 and OE43 lines were selected as the materials for subsequent experiments. Rice seeds were aged for 9 days, and the germination percentage was measured on days 5 and 14 of germination. Germination potential and germination rate were calculated, and the results are as follows: Figure 8As shown in the figure, for unaged seeds, the germination potential of WT was 72%, OE36 was 78%, and OE43 was 72%, with no significant difference among the three (P>0.05). However, aging significantly (P<0.05) reduced the germination potential of seeds, with WT germinating at only 27%, while the germination potential of transgenic rice (overexpressing AsGDSL135) aged seeds significantly (P<0.05) increased, with OE36 germinating at 37% and OE43 at 49%. Furthermore, overexpression of the AsGDSL135 gene also significantly (P<0.05) increased the germination rate of aged seeds. After 9 days of aging, the germination rate of WT was only 50%, while the germination rates of OE36 and OE43 were 68% and 69%, respectively. These results indicate that overexpression of the AsGDSL135 gene can significantly enhance the vigor of rice seeds.

[0068] While specific embodiments of the present invention have been described in detail, they should not be construed as limiting the scope of protection of the present invention. Various modifications and variations that can be made by those skilled in the art without inventive effort within the scope described in the claims are still within the scope of protection of the present invention.

Claims

1. An application of the oat AsGDSL135 gene in enhancing seed vigor, wherein the nucleotide sequence of the oat AsGDSL135 gene is shown in SEQ ID NO.14 and the amino acid sequence is shown in SEQ ID NO.13; the seed is Arabidopsis thaliana or rice seed.

2. The application according to claim 1, characterized in that: Seed vigor was enhanced by overexpressing the oat AsGDSL135 gene in seeds.

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