Application of oat AsSAD8 gene in seed vigor regulation

Abstract

The invention discloses application of an oat AsSAD8 gene in seed activity regulation and control, and belongs to the technical field of seed biology. The nucleotide sequence of the oat AsSAD8 gene is as shown in SEQ ID NO. 1, and the amino acid sequence of the oat AsSAD8 gene is as shown in SEQ ID NO. 2. The over-expression of the AsSAD8 gene significantly reduces the germination rate of the aged seeds of arabidopsis thaliana and rice, indicates the potential negative effect of the gene in the seed activity, and reveals that the expression of the AsSAD8 gene is inhibited, the seed aging is delayed, the seed life is prolonged, and the seed preservation quality is improved.

Description

Technical Field

[0001] This invention belongs to the field of seed biotechnology, specifically relating to the application of the oat AsSAD8 gene in seed vigor regulation. Background Technology

[0002] Agricultural production, as the cornerstone of human survival and development, highly depends on high-vitamin seeds. High-vitamin seeds are not only a key factor in ensuring high-yield and high-quality crops, but also a core element for agriculture to cope with complex and ever-changing environments and achieve sustainable development. However, during the storage period after harvesting, seeds inevitably undergo a series of physiological and biochemical metabolic changes. These changes act like hidden "killers," seriously threatening seed viability.

[0003] With the continuous development of molecular biology techniques, regulating seed vigor at the gene level has become a research hotspot. By identifying and studying genes related to seed vigor and their expression regulation mechanisms during seed development, maturation, and storage, it is hoped that new pathways and methods can be provided to improve seed preservation quality. Currently, although some genes related to seed vigor have been discovered, the regulation of seed vigor is a complex biological process involving the interaction of multiple genes and signaling pathways. Many unknown genes and mechanisms still require further exploration and research. Summary of the Invention

[0004] The purpose of this invention is to provide a new use for the oat AsSAD8 gene, specifically, the application of the AsSAD8 gene in seed vigor regulation.

[0005] The application of the oat AsSAD8 gene in seed vigor regulation in this invention: reducing seed vigor by overexpressing the oat AsSAD8 gene in seeds.

[0006] The objective of this invention can be achieved through the following technical solutions: (1) Obtain the nucleotide and amino acid sequences of the oat AsSAD8 gene; (2) Using oat cDNA as a template, PCR amplification was performed with specific primers, and the PCR product was purified and recovered. (3) The recovered PCR product was used to construct the PEGOEP35S-H-GFP vector to obtain the overexpression vector; (4) Transform the plasmid containing the target fragment of the AsSAD8 gene from step (3) and perform colony PCR verification.

[0007] (5) Screening of Arabidopsis thaliana and rice AsSAD8 gene overexpression materials and identification of seed vigor phenotype.

[0008] Furthermore, the oat AsSAD8 gene in this invention has the nucleotide sequence shown in SEQ ID NO.1 and the amino acid sequence shown in SEQ ID NO.2.

[0009] Furthermore, in step (2), oat cDNA is used as a template to clone the gene sequence by PCR. The upstream primer sequence of the PCR is shown in SEQ ID NO.5 of the sequence listing, and the downstream primer sequence is shown in SEQ ID NO.6 of the sequence listing.

[0010] Further, in step (3), the primer sequence of the gene with homologous recombination adapter is first cloned by PCR using the CDS fragment of the AsSAD8 gene as a template. The upstream primer sequence of the PCR 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. Then, the target fragment with the AsSAD8 gene is constructed into PEGOEP35S-H-GFP to obtain the overexpression vector.

[0011] Furthermore, the upstream primer sequence used in step (5) to screen positive overexpression materials of rice is shown in SEQ ID NO.7, and the downstream primer sequence is shown in SEQ ID NO.8.

[0012] Furthermore, in step (5), the PCR detection primer sequences are as follows: the upstream primer sequence is shown in SEQ ID NO.9 of the sequence listing, and the downstream primer sequence is shown in SEQ ID NO.10 of the sequence listing. The beneficial effects of this invention are: 1. This study reveals that the AsSAD8 gene can reduce seed vigor by inhibiting its expression, thereby delaying seed senescence, extending seed lifespan, and improving seed preservation quality. Attached Figure Description

[0013] Figure 1 This is a graph showing the electrophoresis results of the target gene fragment after PCR amplification. Figure 2 This is a graph showing the results of bacterial culture PCR detection. Figure 3 This is a statistical chart showing the number of Arabidopsis thaliana seeds germinating. Figure 4 Images showing the germination of wild-type and transgenic Arabidopsis seeds; Figure 5 Image showing the PCR identification results of homozygous positive rice seedlings; Figure 6 Figure showing the relative expression levels in homozygous positive rice seedlings; Figure 7 Statistical charts showing the germination potential and germination rate of wild-type and transgenic rice seeds. Detailed Implementation

[0014] 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.

[0015] ② Reagents, vectors, and 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.

[0016] ③The primer sequences used in the examples are shown in Table 1.

[0017] Table 1

[0018] Example 1: Gene Cloning ① Using 'Monida' oat cDNA as a template, the target fragment of the AsSAD8 gene was amplified by PCR using specific primers AsSAD-F and AsSAD-R (e.g., Figure 1 The CDS length of the AsSAD8 gene shown is 1140 bp, and the size of the bright band is as expected. The recovered AsSAD8 gene product was then ligated into the PEGOEP35S-H-GFP vector. The primers used in the overexpression vector construction were pEG-AsSAD-F / R. The overexpression vector was then transformed into competent E. coli cells, and PCR amplification was performed on the target bacterial culture. (The detection results are shown below.) Figure 2 As shown, the sample was verified by first-generation DNA sequencing, and all sequencing results were consistent with the original sequence.

[0019] Example 2: Vigor detection of transgenic Arabidopsis thaliana seeds and transgenic rice seeds ① AsSAD8 was overexpressed in Arabidopsis thaliana. Ten days after germination of homozygous transgenic Arabidopsis seeds, ten seedlings from each line were selected for total RNA extraction and cDNA synthesis. Using cDNA as a template and the Arabidopsis EFE1a gene as an internal control gene, qPCR analysis was performed using SAD-qF / R primers. OE-3 showed the highest overexpression level among all lines. Harvested homozygous seeds were subjected to controlled deterioration treatment to detect seed viability. The viability test results are shown below. Figures 3-4 As shown in the figure, after culturing unaged Arabidopsis seeds at 22℃ for 2 days, the germination rate was almost 100%, and the seeds all exhibited high viability, indicating that all seeds developed well in the early stages and could be used for subsequent aging-related experiments. Germination tests were conducted on WT and OE-3 seeds aged for 5 days. The results showed that the germination rate of aged Arabidopsis seeds tended to stabilize after day 9. At this point, the germination rate of WT seeds was only 50%, and the germination rate of OE-3 seeds was 42%, significantly lower than that of WT seeds (P<0.05). The results indicate that overexpression of AsSAD8 reduces seed viability.

[0020] ② A genetic transformation vector for overexpressing AsSAD8 in rice was constructed and an overexpression line was successfully generated. DNA was then extracted from the leaves of resistant rice seedlings, and PCR amplification of the overexpressed AsGDSL135 was performed using 35S-F and eGFP-CX primers. A total of 6 homozygous positive rice seedlings were obtained (PCR identification results are shown below). Figure 5 (As shown in the image), then RNA from positive seedlings was extracted and reversed into cDNA. Using the OsUBI gene as an internal control and SAD-qF / R as primers, qPCR analysis was performed. The results are shown in the image. Figure 6 As shown, OE27 and OE30 showed the highest expression levels, and these two lines were selected as the materials for subsequent experiments. Germination tests were conducted on transgenic rice seeds after aging for 9 days. Germination potential and germination rate were calculated on days 5 and 14 of germination. The results are as follows: Figure 7 As shown, aging reduces seed germination potential and germination rate, but there was no significant difference in germination potential between WT and the OE27 and OE30 lines overexpressing AsSAD8 (P>0.05). In the germination rate determination 14 days after germination, the germination rate of aged WT was 50%, while in aged OE27 and OE30, the germination rate decreased significantly (P<0.05), to only 42%. These results indicate that overexpression of AsSAD8 reduces seed vigor.

[0021] 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 AsSAD8 gene in seed vigor regulation, wherein the nucleotide sequence of the oat AsSAD8 gene is shown in SEQ ID NO.1 and the amino acid sequence is shown in SEQ ID NO.

2.

2. The application according to claim 1, characterized in that: Seed viability was reduced by overexpressing the oat AsSAD8 gene in the seeds.

3. The application according to claim 2, characterized in that: The seeds are Arabidopsis thaliana or rice.

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