Application of AmCS gene in improvement of anti-aging capability of seeds and anti-aging plant breeding

Abstract

The invention belongs to the technical field of plant genetic engineering, and particularly relates to application of an AmCS gene in improvement of seed aging resistance and anti-aging plant breeding. The invention provides application of an AmCS gene in improvement of anti-aging capability of plant seeds and / or anti-aging plant breeding. The sequence of the AmCS gene is shown as SEQ ID NO: 3. Experiments prove that overexpression of the allium mongolicum regel AmCS gene in heterologous plants can improve the anti-aging ability of plant seeds, and the invention provides a new way for anti-aging plant breeding.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically involving AmCS Application of genes in enhancing seed anti-aging ability and in breeding anti-aging plants. Background Technology

[0002] Sand onion is a key species in desert steppe ecosystems, possessing important ecological characteristics such as drought and barrenness tolerance, and contributing to desert ecological restoration. Its unique flavor and nutritional value have also made it a distinctive resource in desert steppes with good economic potential. However, due to the lack of genomic information and the absence of a genetic transformation system, research on sand onion gene function has long been limited, making it difficult to directly elucidate its stress-resistance biological mechanisms.

[0003] Seed senescence is a core issue affecting agricultural production efficiency and the long-term safe preservation of germplasm resources. Essentially, it is a series of oxidative damage processes triggered by the accumulation of reactive oxygen species (ROS), including increased levels of malondialdehyde (MDA), a lipid peroxidation product, loss of cell membrane integrity, and inhibition of germination-related signaling pathways, ultimately leading to a significant decline in seed viability. Currently, the unique anti-senescence gene resources derived from desert plants (such as sand onion) have not been fully explored. As a representative species adapted to extreme environments, sand onion may contain anti-senescence gene resources in its genome.

[0004] Therefore, screening a *Allium tuberosum* gene resource from the gene bank that can effectively enhance seed anti-aging ability and can be effectively expressed in heterologous plants has become an urgent problem for those skilled in the art. Summary of the Invention

[0005] To address the lack of effective gene resources for enhancing seed anti-aging capabilities in existing technologies, this invention provides... AmCS The application of genes in enhancing the anti-aging ability of plant seeds includes the following technical solutions: This invention provides AmCS The application of genes in improving the anti-aging ability of plant seeds and / or in breeding anti-aging plants, the aforementioned AmCS The gene sequence is shown in SEQ ID NO:3.

[0006] Preferably, the anti-aging ability includes: reducing oxidative damage to seeds caused by aging and / or increasing the germination rate of aged seeds.

[0007] Preferably, the plant includes Arabidopsis thaliana.

[0008] This invention also provides an overexpression AmCS Gene-based biological materials, the AmCSThe gene sequence is shown in SEQ ID NO:3.

[0009] Preferably, the biomaterial includes amplification of the... AmCS Primer pairs of genes, containing the above AmCS overexpression recombinant vectors of the gene and / or containing the above AmCS Recombinant microorganisms that overexpress genes.

[0010] Preferably, the primer pair includes primer pairs as shown in SEQ ID NO:1 and SEQ ID NO:2.

[0011] Preferably, the overexpression recombinant vector includes a backbone vector, wherein the backbone vector includes pCAMBIA1300-35S.

[0012] Preferably, the overexpressed recombinant microorganism includes a basic microorganism, which includes Agrobacterium.

[0013] The present invention also provides the application of the biomaterials described in any of the preceding claims in improving the anti-aging ability of plant seeds and / or in the breeding of anti-aging plants.

[0014] This invention also provides a method for improving the anti-aging ability of plant seeds, comprising the following steps: Overexpression in plants AmCS Genes are cultivated until they form seeds, which are then harvested to obtain seeds with enhanced anti-aging properties. The AmCS The gene sequence is shown in SEQ ID NO:3.

[0015] The beneficial effects of this invention are as follows: This invention provides AmCS The application of genes in regulating the anti-aging ability of plant seeds and / or in the breeding of anti-aging plants, the aforementioned AmCS The gene sequence is shown in SEQ ID NO:3. This invention experimentally verified the efficacy of *Allium tuberosum*. AmCS Overexpression of genes in heterologous plants can enhance the anti-aging ability of plant seeds, providing a new approach for plant breeding.

[0016] This invention will... AmCS The gene was constructed in the pCAMBIA1300-35S vector and stably overexpressed in Arabidopsis thaliana using Agrobacterium-mediated genetic transformation. Experimental results showed that in the transgenic lines… AmCS The transcriptional level of the gene was significantly increased, the seed germination rate was significantly higher than that of the wild type, the seedlings grew well, and the malondialdehyde (MDA) content and relative conductivity were significantly lower than those of the wild type. After artificial accelerated aging treatment, the transgenic seeds still maintained high germination viability.

[0017] This invention not only successfully established a functional verification system for the *Allium chinense* gene in *Arabidopsis thaliana* for the first time, providing a reliable technical platform for the functional analysis of *Allium chinense* stress-resistance genes, but also provided new candidate genes with clear functions and valuable genetic materials that can be directly used for subsequent research in plant anti-aging breeding, demonstrating good application potential and market prospects. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0019] Figure 1 sand onion AmCS Electrophoresis results of PCR amplification of gene clones; Where M represents the molecular weight standard of DNA; C represents... AmCS Gene PCR products; Figure 2 The results of PCR testing were obtained by selecting single colonies after transforming DH5α with the recombinant plasmid pCAMBIA1300-AmCS and plating them on a plate. Where M is the DNA molecular weight standard; lanes 1-4 are all positive colony amplification products; Figure 3 sand onion AmCS PCR screening of transgenic Arabidopsis thaliana lines; Wherein, - represents the negative control; + represents the positive control, with an expected band of 385 bp; M represents the DNA molecular weight standard; OE1~OE19 are 19 overexpression lines respectively; Figure 4 sand onion AmCS Analysis of gene expression levels in transgenic Arabidopsis thaliana; Figure 5 Phenotypic characteristics of transgenic Arabidopsis seeds and seedling growth; Where A represents the germination rate statistics; B represents a schematic diagram of seedling growth phenotypes. Figure 6 Analysis of the anti-aging ability of transgenic Arabidopsis seeds; In this table, A represents the result of malondialdehyde (MDA) content determination; B represents the result of relative conductivity determination. Detailed Implementation

[0020] This invention provides AmCS The application of genes in improving the anti-aging ability of plant seeds and / or in breeding anti-aging plants, the aforementioned AmCS The gene sequence is shown in SEQ ID NO:3.

[0021] In one embodiment, the anti-aging ability includes: reducing oxidative damage to seeds caused by aging and / or increasing the germination rate of aged seeds. In one embodiment, reducing oxidative damage to seeds caused by aging includes: reducing malondialdehyde (MDA) content in the plant and / or reducing the plant's electrical conductivity. In one embodiment, the anti-aging plant breeding includes: increasing the germination rate of aged plant seeds, reducing MDA content in the plant, and / or reducing the plant's electrical conductivity to improve the plant's anti-aging ability. In one embodiment, the plant includes Arabidopsis thaliana. In one embodiment, the method of improving the anti-aging ability of plant seeds or the method of anti-aging plant breeding includes: overexpressing the [specific ingredient] in the plant. AmCS Genes can be used to enhance the anti-aging ability of plant seeds or to obtain anti-aging plants.

[0022] This invention also provides overexpression AmCS Gene-based biological materials, the AmCS The gene sequence is shown in SEQ ID NO:3.

[0023] As one implementation, the biomaterial includes amplification of the... AmCS Primer pairs of genes, containing the above AmCS overexpression recombinant vectors of the gene and / or containing the above AmCS Recombinant microorganisms that overexpress genes. As one embodiment, the primer pair includes primer pairs as shown in SEQ ID NO:1 and SEQ ID NO:2. As one embodiment, the overexpression recombinant vector includes a backbone vector. As one embodiment, the backbone vector includes a plant transgenic binary vector. As one embodiment, the plant transgenic binary vector includes pCAMBIA1300-35S. As one embodiment, the restriction enzyme site on pCAMBIA1300-35S is... EcoR I and Hind III. In one embodiment, the overexpressed recombinant microorganism includes a basic microorganism. In one embodiment, the basic microorganism includes Agrobacterium. In one embodiment, the Agrobacterium includes Agrobacterium tumefaciens GV3101.

[0024] The present invention also provides the application of the biomaterials described above in improving the anti-aging ability of plant seeds and / or in the breeding of anti-aging plants.

[0025] This invention also provides a method for enhancing the anti-aging ability of plant seeds, comprising the following steps: Overexpression of the above in plants AmCS Genes are cultivated until they germinate, and then harvested to obtain seeds with enhanced anti-aging capabilities.

[0026] As one implementation method, the overexpression of the [specific substance] in plantsAmCS The gene sequencing steps include: using restriction endonucleases EcoR I and Hind III pairs AmCS The gene and pCAMBIA1300-35S vector were double-digested with enzymes, and the digestion products were ligated with T4 DNA ligase to obtain an overexpression recombinant vector. The recombinant expression plasmid was transformed into competent cells of Agrobacterium tumefaciens GV3101 to obtain overexpressing recombinant microorganisms. Plants in the initial flowering stage were transfected with the overexpressing recombinant microorganisms using the inflorescence immersion method to obtain the overexpressing recombinant microorganisms. AmCS Genetically modified plants.

[0027] To further illustrate the present invention, the following description, in conjunction with the accompanying drawings and embodiments, explains the invention. AmCS The application of genes in enhancing seed anti-aging ability and in the breeding of anti-aging plants is described in detail, but it should not be construed as limiting the scope of protection of this invention.

[0028] Unless otherwise specified, the reagents and instruments used in this invention are all conventional reagents and instruments in the art and can be purchased. Unless otherwise specified, the experimental methods used in this invention are all conventional operations in the art.

[0029] Example 1 AmCS Gene effect verification experiment 1. AmCS Gene fragment cloning After soaking and germinating the seeds, total RNA was extracted from the seeds according to the instructions of the plant total RNA extraction kit (TaKaRa, Code No. 9767). Then, the total RNA from the seeds was reverse transcribed into first-strand cDNA using a cDNA synthesis kit (TaKaRa, Code No. 6210A).

[0030] Using the obtained cDNA as a template, specific primers AmCS-F / R were designed, and the primer sequences are shown in Table 1. PCR amplification was performed using the primers and KOD-FX high-fidelity DNA polymerase. The products were detected by 1.0% agarose gel electrophoresis. The target band was excised and purified using a gel imaging system. The agarose gel electrophoresis results are shown below. Figure 1 As shown. According to Figure 1 The agarose gel electrophoresis results shown indicate that the size of the target band of the amplified product is consistent with the expected target fragment (1461 bp).

[0031] Table 1 Cloning primer sequences

[0032] In Table 1, Acta is the protective base, gggtctcG or gggtctcT is the restriction site, and CACC or CGCC is the sticky end.

[0033] The target fragment obtained by PCR amplification using the primers shown in Table 1 is 1461 bp in length, and the nucleotide sequence of the target fragment is shown in SEQ ID NO:3.

[0034] AmCS

[0035] 2. Carrier Construction Use restriction endonucleases EcoR I and Hind III. The target fragment shown in SEQ ID NO:3 and the modified plant expression vector pCAMBIA1300-35S were double-digested to obtain the digested products.

[0036] The modified plant expression vector pCAMBIA1300-35S was purchased from Wuhan Aidijing Biotechnology Co., Ltd.

[0037] The enzyme digestion products described above were ligated with T4 DNA ligase to obtain the recombinant expression plasmid pCAMBIA1300-35S::AmCS.

[0038] 3. Plasmid transformation The recombinant expression plasmid pCAMBIA1300-35S::AmCS was transformed into DH5α competent cells to obtain transformants. The transformants were then plated on LB agar containing 50 mg / L kanamycin and incubated upside down at 37°C for 16 h. Single colonies were then picked. The single colonies growing on the plates are likely clones containing the pCAMBIA1300-35S::AmCS recombinant plasmid.

[0039] Using selected single colonies as templates, PCR identification of single colonies was performed using universal vector primers 35S-F and eGFP-cx. The results are as follows: Figure 2 As shown, if the PCR amplification product shows a specific band at the expected size position (approximately 1500 bp) when detected by agarose gel electrophoresis, it indicates that the colony may contain a recombinant plasmid of the expected size, which can be used as a positive clone candidate.

[0040] Depend on Figure 2 As can be seen, plasmids were further extracted from colonies displaying the expected bands, and Sanger sequencing was performed using primers eGFP-cx. Sequence alignment confirmed that it matched the one shown in SEQ ID NO:3. AmCS The gene sequences were completely identical, indicating that the recombinant expression vector pCAMBIA1300-35S::AmCS was successfully constructed.

[0041] Table 2. Colony PCR Primer Sequences

[0042] 4. Preparation of dyeing solution and inflorescence dyeing The successfully constructed recombinant plasmid pCAMBIA1300-35S::AmCS was introduced into competent cells of Agrobacterium tumefaciens GV3101 by electroporation. The transformed cells were screened on LB solid medium containing 50 mg / L rifampin and 50 mg / L kanamycin to finally obtain the Agrobacterium tumefaciens engineered strain containing the target gene.

[0043] The Agrobacterium engineered strain containing the target gene was resuspended in 1 / 2 MS liquid medium, and then the surfactant Silwet L-77 was added. The volume percentage of Silwet L-77 in the suspension was 0.02%. The concentration of the engineered bacterial suspension was adjusted to OD0.05. 600 The value was 0.8, and the engineered bacteria suspension was obtained and immediately used for inflorescence inoculation.

[0044] Wild-type Arabidopsis thaliana (Columbia-0 ecotype) plants in the bolting and flowering stage (early flowering period) were selected. After removing the formed siliques, the inflorescences were immersed in the OD. 600 Immerse the bacteria in an engineered bacterial suspension with a value of 0.8 for 40 seconds, gently agitating during the process to ensure full contact. After removal, gently aspirate or blot dry with sterile filter paper to remove excess bacterial solution. Incubate in the dark at 80%–90% humidity for 2 days, then transfer to normal light and temperature conditions for cultivation until harvesting T0 generation seeds.

[0045] The normal light and temperature conditions are as follows: 100~150 μmol m -2 s -1 The photoperiod is 16 hours of light / 8 hours of darkness. The temperature is 22±2℃ (light period) / 18±2℃ (dark period). The relative humidity is approximately 50-60%.

[0046] 5. Screening of positive plants After sterilizing the T0 generation seeds described in step 4, they were sown in a selection medium containing 50 mg / L hygromycin and cultured under light with a light condition of 16 h light / 8 h darkness, and a concentration of 100~120 μmol / L. -2 s -1 The temperature was kept constant at 22±2℃ until the seeds germinated, yielding Arabidopsis thaliana T1 generation plants. Positive seedlings were then selected from the Arabidopsis thaliana T1 generation plants.

[0047] T1 generation Arabidopsis plants were screened. Fresh young leaves were collected from T1 plants when they had grown 6-8 true leaves but had not yet bolted, serving as the positive seedling leaf group. Wild-type Arabidopsis served as the negative control. Genomic DNA was extracted from the positive seedling leaves using the CTAB method. Using the genomic DNA from the positive seedling leaves as a template, PCR verification was performed using the specific primers hpt-F / R for the hygromycin phosphotransferase gene (hpt gene) to detect the presence of the hpt gene in Arabidopsis. The results are as follows: Figure 3 As shown, specific bands of the same size as those in the positive control could be detected in the transformed plants. Among the 20 Arabidopsis thaliana plants that were co-transformed, PCR detection confirmed that 19 of them were positive plants, with a positive transformation rate of 95%.

[0048] Table 3 Primers for hpt gene detection

[0049] 6. Expression level analysis Wild-type Arabidopsis thaliana (WT) and Arabidopsis thaliana positive seedlings (OE) from step 5 were used as samples. Expression levels were analyzed using the SYBR Green method on a real-time quantitative PCR instrument. Actin As an internal reference gene, it was detected using specific primers AmCSq-F / R and Actin-F / R. AmCS The relative expression levels of genes, the results are as follows: Figure 4 As shown, it can be seen that in transgenic lines AmCS The relative expression level of the gene's mRNA was significantly higher than that of the wild type, confirming that the gene has been successfully heterologously overexpressed in Arabidopsis thaliana.

[0050] Table 4 RT-qPCR Primer Sequences

[0051] 7. Artificial aging treatment of seeds The T1 generation positive seedlings were continuously self-pollinated until the seeds of the T3 generation homozygous Arabidopsis thaliana were harvested. The seeds of wild-type Arabidopsis thaliana and the T3 generation homozygous Arabidopsis thaliana were then subjected to aging treatment to obtain aged wild-type Arabidopsis thaliana seeds and overexpression-treated seeds. AmCS Genetic aging in Arabidopsis seeds.

[0052] Before aging, the seeds need to be equilibrated at 20℃ and 85% humidity for 3 days. Then, the seeds are placed in empty petri dishes, and the empty petri dishes containing the seeds are placed in a desiccator containing saturated KCl solution (relative humidity 85%). The Arabidopsis seeds are then treated at 42℃ for 5 days to obtain aged Arabidopsis seeds.

[0053] 8. Seed germination experiment Using wild-type Arabidopsis thaliana seeds (WT), overexpression AmCS Arabidopsis thaliana seeds (OE), wild-type aged Arabidopsis thaliana seeds (WT+ACK), and overexpression of the gene AmCS Aged Arabidopsis thaliana seeds (OE+ACK) were used as samples. 300 seeds were prepared for each group, and the 300 seeds were divided into three equal portions as three replicates for seed germination experiments. The experimental steps are as follows: After surface sterilization, the seeds were sown in MS medium and subjected to 16 h light / 8 h dark conditions at 100–120 μmol / L. -2 s -1 Germination was carried out at a constant temperature of 22±2℃ for 10 days, and the germination rate was counted. The results are as follows: Figure 5 As shown in Figure A, under normal conditions without aging treatment, the germination rate of OE seeds was 92.67%, significantly higher than the 79.33% germination rate of WT seeds. After artificial accelerated aging treatment, the transgenic lines still maintained high viability; the germination rate of OE+ACK group seeds reached 79.33%, while the germination rate of WT+ACK group seeds significantly decreased to 64.67%. These results indicate that overexpression... AmCS The gene can significantly improve the germination ability and anti-aging ability of Arabidopsis seeds.

[0054] Germinated Arabidopsis thaliana plants were exposed to light intensities of 100–150 μmol / m². -2 s -1 The photoperiod was 16 h light / 8 h dark. The plants were cultured at 22±2℃ (light period) / 18±2℃ (dark period) and relative humidity of approximately 50-60% for 3-4 weeks. The appearance of different groups of Arabidopsis thaliana was then recorded. The results are as follows: Figure 5 As shown in Figure B, the growth of transgenic seedlings is significantly better than that of wild-type Arabidopsis thaliana under the same treatment.

[0055] 9. Measurement of anti-aging physiological indicators The malondialdehyde (MDA) content in Arabidopsis seeds of the WT, OE, WT+ACK, and OE+ACK groups was determined using the thiobarbituric acid method. The results are as follows: Figure 6 As shown in Figure A. The relative conductivity of Arabidopsis seeds from different groups was measured using a conductivity meter. The specific conductivity measurement method followed standard plant physiology methods. The results are shown in Figure A. Figure 6 As shown in B. From Figure 6 As shown in Figures A and B, in terms of physiological indicators, under the same treatment, the malondialdehyde (MDA) content and relative conductivity of the transgenic lines were significantly lower than those of the wild type, indicating that... AmCS Genes effectively enhance seed vigor and stress resistance by mitigating cell membrane lipid peroxidation damage and maintaining membrane structural integrity.

[0056] In summary, this invention successfully constructed a sand onion. AmCS Gene overexpression vectors were developed, and transgenic Arabidopsis lines were obtained via Agrobacterium-mediated inflorescence staining. A series of functional analyses confirmed heterologous overexpression. AmCS The gene significantly improves the germination rate and anti-aging ability of Arabidopsis seeds. This study not only elucidates... AmCS The biological functions of genes provide experimental evidence and also provide important germplasm materials and technical basis for using genetic engineering technology to improve the stress resistance of plant seeds.

[0057] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments without creative effort, as shown in these embodiments, and these embodiments all fall within the protection scope of the present invention.

Claims

1. AmCS The application of genes in improving the anti-aging ability of plant seeds and / or in breeding anti-aging plants, the aforementioned AmCS The gene sequence is shown in SEQ ID NO:

3.

2. The application as described in claim 1, characterized in that, The anti-aging ability includes: reducing oxidative damage to seeds caused by aging and / or increasing the germination rate of aged seeds.

3. The application as described in claim 1, characterized in that, The plants mentioned include Arabidopsis thaliana.

4. Overexpression AmCS Gene-based biomaterials, characterized in that, The AmCS The gene sequence is shown in SEQ ID NO:

3.

5. The biomaterial as described in claim 4, characterized in that, The biomaterial includes the amplification of the... AmCS Primer pairs of genes, containing the above AmCS overexpression recombinant vectors of the gene and / or containing the above AmCS Recombinant microorganisms that overexpress genes.

6. The biomaterial as described in claim 5, characterized in that, The primer pairs include those shown in SEQ ID NO:1 and SEQ ID NO:

2.

7. The biomaterial as described in claim 5, characterized in that, The overexpression recombinant vector includes a backbone vector, which includes pCAMBIA1300-35S.

8. The biomaterial as described in claim 5, characterized in that, The overexpressed recombinant microorganisms include basic microorganisms, including Agrobacterium.

9. The application of the biomaterial according to any one of claims 4 to 8 in improving the anti-aging ability of plant seeds and / or in the breeding of anti-aging plants.

10. A method for improving the anti-aging ability of plant seeds, characterized in that the steps include... include: Overexpression in plants AmCS Genes are cultivated until they form seeds, which are then harvested to obtain seeds with enhanced anti-aging properties. The AmCS The gene sequence is shown in SEQ ID NO:3.

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