Application of gene SGAM1 in mitochondrial activity regulation to enhance salt tolerance and drought tolerance of plants

Abstract

The invention discloses application of a gene SGAM1 in mitochondrial activity regulation to enhance salt tolerance and drought tolerance of plants. The partial reduction of the expression of the gene SGAM1 can significantly improve the salt tolerance and drought tolerance of the plant, so that the salt-tolerant and drought-tolerant transgenic plant can be cultivated.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering, and particularly relates to the application of gene SGAM1 in regulating mitochondrial activity to enhance plant salt tolerance and drought tolerance. Background Art

[0002] Under the severe background of extreme global climate change, the drought and salinization problems faced by cultivated land have become key abiotic stress factors that need to be addressed urgently, significantly threatening the production safety of many economic and food crops. When plants are subjected to salt stress, they will undergo a series of physiological and metabolic changes, including but not limited to slow growth of stems and roots, weakened photosynthesis, and redistribution of respiratory metabolism. These changes are directly related to the energy production, distribution and utilization efficiency of plants. In particular, mitochondria, as the "energy factory" in plant cells, have a decisive role in the plant's resistance to stress when in salt stress.

[0003] Existing studies have shown that under drought and salt stress, the functional adjustment of plant mitochondria involves complex physiological processes and involves the synergistic effects of multiple mechanisms. First, mitochondria respond to the negative effects of stress through their unique adaptation mechanisms, such as the regulation of the electron transport chain (ETC), the control of reactive oxygen species (ROS) levels, and the change of tricarboxylic acid cycle (TCA) activity. These adaptations not only help maintain the energy metabolism balance of cells, but also promote the expression of stress-related genes and activate the defense mechanism of plants. In addition, mitochondria also play an important role in signal transmission under salt stress, namely the so-called "retrograde signal", which transmits stress signals to the cell nucleus and initiates the transcription and expression of a series of stress response genes, thereby enhancing the stress resistance of plants. At the same time, mitochondria also support key processes such as ion transport, biosynthesis of compatible solutes, and detoxification of ROS in cells under stress by adjusting the supply of ATP and reductants. These functions are essential for stabilizing cell functions and alleviating toxicity.

[0004] However, there is still insufficient research on how to directly enhance plant stress resistance by regulating mitochondrial activity, especially in the practical operation level, which lacks systematic methods and application cases. Therefore, developing a method that can effectively regulate mitochondrial activity and improve the stress resistance of plants under salt stress is of great significance for sustainable agricultural development and ecological environmental protection. Summary of the invention

[0005] The purpose of the present invention is to provide an application of a gene SGAM1 in regulating mitochondrial activity to enhance plant salt tolerance and drought tolerance, so as to solve the problem of soil salinization.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] Application of gene SGAM1 in regulation of mitochondrial activity to enhance salt and drought tolerance in plants.

[0008] The characteristics of the gene SGAM1 are as follows:

[0009] (1) A DNA molecule with a genomic sequence as shown in SEQ ID NO.1;

[0010] (2) a DNA molecule whose CDS sequence is shown in SEQ ID NO. 2;

[0011] (3) A protein encoded by a DNA sequence defined in (1) or (2) under stringent conditions, wherein the amino acid sequence is shown in SEQ ID NO.3.

[0012] Preferably, the expression level of SGAM1 is regulated by precisely editing specific sites of the SGAM1 gene.

[0013] Preferably, the gene SGAM1 is used to cultivate salt-tolerant and drought-tolerant transgenic plants.

[0014] Preferably, the plant is a vegetable or a horticultural crop.

[0015] Preferably, the plant is one of rapeseed, Chinese cabbage, cucumber or pepper.

[0016] Beneficial effects: The present invention provides an application of the gene SGAM1 in regulating mitochondrial activity to enhance plant salt tolerance and drought tolerance. Partial reduction of the expression of the gene SGAM1 can significantly improve plant salt tolerance and drought tolerance, thereby cultivating salt-tolerant and drought-tolerant transgenic plants. The expression level of the gene SGAM1 can be applied to new targets for plant genetic improvement. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Determination of editing sites and gene expression levels of wild-type WT and SGAM1 mutants;

[0018] Figure 2 The phenotypes of wild type WT and SGAM1 mutants under 120 mM salt stress;

[0019] Figure 3 The phenotypes of wild type WT and mutant sgam1-1 under drought stress;

[0020] Figure 4 Sequence alignment of SGAM1 homologous genes in important economic crop species. DETAILED DESCRIPTION

[0021] The present invention is further explained below in conjunction with the examples. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent companies unless otherwise specified.

[0022] Example 1: Editing and expression analysis of different sites of the SGAM1 gene

[0023] In previous studies, we screened and obtained the SGAM1 mutant sgam1-1 from the mutant library, and the T-DNA insertion site showed that there was a T-DNA insertion in its 3'-UTR region. At the same time, we ordered a second mutant sgam1-2 from the TAIR stock, and the T-DNA insertion of this mutant occurred in the 5'-UTR region. In addition, we also introduced a base insertion into the wild-type (WT) background through CRISPR-Cas9 gene editing technology, resulting in a stop codon in the first exon ( Figure 1 A).

[0024] Five seedlings were selected from the wild type (WT) and three mutants of the SGAM1 gene, and the seedling tissues were ground into light green powder using liquid nitrogen. Subsequently, RNA was extracted using the TRIzol method, and centrifuged and washed according to the experimental steps to remove DNA, protein and other impurities, and finally pure RNA was obtained. The extracted RNA was quality tested to ensure that its A260 / A280 ratio was between 1.8 and 2.1 to confirm that the RNA was suitable for subsequent experiments. A reverse transcription reaction system was prepared to reverse transcribe the extracted RNA into cDNA. Subsequently, the expression level of SGAM1 was detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction (qRT-PCR). The primers used spanned the first intron and were named Primer 1 and Primer 2, corresponding to F1 and R1 in the figure. Arabidopsis ATCIN2 was used as the internal reference gene, and the primers for ATCIN2 were Primer 3 and Primer 4. . The specific sequences of PCR primers are as follows:

[0025] Primer 1: 5'-CCCAGCTCTCAAGCTATCTG-3';

[0026] Primer 2: 5'-GTGTACGAATCCGGCATCAC-3';

[0027] Primer 3: 5'-GCCATCCAAGCTGTTCTCTC-3';

[0028] Primer 4: 5'-GCTCCGTAGTCAACAGCAACAA-3';

[0029] The ΔΔCt method was used to calculate the relative changes in the expression of the SGAM1 gene in the wild type (WT) and each SGAM1 mutant. The data were normalized with the internal reference gene ATCIN2 to compare the differences in the expression levels of SGAM1 between different samples. Figure 1 As shown in B.

[0030] Example 2: Analysis of salt tolerance of different SGAM1 mutants

[0031] Place an appropriate amount of seeds in a 1.5mL centrifuge tube, and first use 75% ethanol containing 0.05% Tween-20 for surface sterilization for 20 minutes. After removing the supernatant, shake with 95% ethanol for 3 minutes, and then spread the seeds on sterile filter paper. This process must be carried out in a sterile clean bench and blown dry. Next, we sowed the seeds of each strain in a pre-prepared 1 / 2MS medium containing 0.8% Agar, and placed the culture dish at 4°C for two days in the dark. Subsequently, the culture dish was transferred to a light incubator for vertical culture. The culture conditions were 22°C, the light cycle was 16h light / 8h dark, and the relative humidity was controlled at 40%-60%. After 2 days of vertical growth, the seedlings were moved to a 1 / 2MS medium containing 120mMNaCl and continued to grow vertically for 5 days. Then, the phenotype was observed and photographed. The results are as follows Figure 2 A, the scale bar is 1 cm. Subsequently, the taproot length of the seedlings was measured using Image J software, and the relative values ​​of the taproot length of the sgam1-1, sgam1-2, and sgam1-3 mutant lines relative to the wild type (WT) were calculated, where the average root length of the wild type seedlings was set to 1. The results are shown in Figure 2 B.

[0032] Example 3: Analysis of drought tolerance of WT and SGAM1 gene mutant plants

[0033] WT and sgam1-1 seedlings germinated for 8 days in the culture medium were transferred to soil and cultured under normal conditions for 20 days. Watering was stopped for 14 days to simulate drought conditions. The phenotypes were observed and photographed during this period. Watering was then resumed, and the recovery ability of the wild type (WT) and sgam1-1 mutant lines was observed again one day after rehydration. Figure 3 As shown, after 14 days of drought treatment, the sgam1-1 mutant showed stronger drought stress tolerance than the wild type WT, and after 1 day of rewatering, the sgam1-1 mutant showed faster recovery ability than the wild type WT.

[0034] Example 4: Multiple sequence alignment of SGAM1 in Arabidopsis thaliana in different species

[0035] By using ClustalW to perform multiple sequence alignments of homologous protein sequences of SGAM1 in different species, we found that SGAM1 has high sequence conservation in multiple important horticultural crops, including rapeseed (Cruciferae), Chinese cabbage (Cruciferae), cucumber (Cucurbitaceae) and pepper (Solanaceae). Specifically, the sequence identity of SGAM1 with rapeseed reached 86%, the identity with Chinese cabbage was also 86%, and the identity with cucumber and pepper was 66% and 67%, respectively. This result not only shows the importance of SGAM1 in these crops, but also provides new genetic resources and theoretical support for the use of homologous genes in vegetables to improve salt tolerance and drought resistance.

[0036] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of gene SGAM1 in regulating mitochondrial activity to enhance plant salt and drought tolerance.

2. The use according to claim 1, characterized in that: The characteristics of the gene SGAM1 are as follows: (1) A DNA molecule with a genomic sequence as shown in SEQ ID NO.1; (2) a DNA molecule whose CDS sequence is shown in SEQ ID NO. 2; (3) A protein encoded by a DNA sequence defined under stringent conditions as described in (1) or (2), wherein the amino acid sequence is shown in SEQ ID NO.

3.

3. The use according to claim 1, characterized in that: The expression level of SGAM1 can be regulated by precisely editing the specific sites of the SGAM1 gene.

4. The use according to claim 1, characterized in that: The gene SGAM1 is used to cultivate salt-tolerant and drought-tolerant transgenic plants.

5. The use according to claim 1 or 4, characterized in that: The plants are vegetables or horticultural crops.

6. The use according to claim 5, characterized in that: The plant is one of rapeseed, Chinese cabbage, cucumber or pepper.

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