Breeding improvement method for improving drought resistance of alfalfa based on SLAC1 gene

Abstract

The invention belongs to the field of plant molecular biology, and particularly relates to a variety improvement method for improving drought resistance of alfalfa based on an SLAC1 gene. The gene segment can be used for regulating stomatal movement of alfalfa and enhancing drought resistance of alfalfa. According to the invention, the alfalfa MtSLAC1 gene is cloned, and an overexpression vector of the alfalfa MtSLAC1 gene is constructed, so that the gene is successfully transformed into alfalfa or other plants, and overexpression of the MtSLAC1 gene is realized. A series of functional verification experiments including a drought stress experiment, an in-vitro leaf water loss experiment, stomatal phenotype analysis, Cl <-> outflow detection by a non-invasive micro-measurement technology and the like prove that the MtSLAC1 gene plays an important role in drought stress response, and can reduce water loss and remarkably improve the drought resistance of plants by regulating stomatal closing and Cl <-> outflow. The method has wide application potential, can be used in the fields of agriculture, ecological restoration, plant molecular biology research and the like, and particularly has important economic and social values in the planting industry in arid regions.

Description

Technical Field

[0001] The present invention belongs to the field of research on plant drought resistance mechanisms, and particularly relates to a drought resistance method and application based on the regulation of stomatal closure by the alfalfa MtSLAC1 gene. This method regulates the expression of the MtSLAC1 gene in alfalfa guard cells to regulate the opening and closing of stomata, thereby improving the drought resistance of plants under drought stress. Background Art

[0002] The stomata on plant leaves are important gateways for gas exchange between plants and the external environment, directly affecting plant growth and survival. Under drought stress conditions, plants regulate the opening and closing of stomata to reduce water loss, thereby enhancing drought resistance. The movement of stomata is regulated by the turgor pressure of guard cells, and the regulation of the turgor pressure of guard cells depends on the activities of a series of ion channels and transporters.

[0003] In recent years, with the intensification of climate change, drought events have occurred frequently, especially in the northwestern region of China, posing a serious threat to the development of agriculture and animal husbandry. Alfalfa, as an excellent forage plant, is widely planted in the northwestern region of China. However, due to the lack of water resources in this region, drought stress poses a huge threat to the planting area and yield of alfalfa. Therefore, studying the drought resistance mechanism of alfalfa, especially its stomatal regulation mechanism, has important theoretical significance and practical application value.

[0004] In the process of plant drought resistance response, abscisic acid (ABA) is an important signaling molecule that can reduce water loss by regulating the closure of stomata. The ABA signal activates ion channels and transporters in guard cells, inducing the efflux of anions (such as Cl - and NO3 - ), thereby reducing the turgor pressure of guard cells and driving the closure of stomata. Among them, SLAC1 (Slow Anion Channel-Associated 1) is an important anion channel protein, which has been proven to be a key factor in the ABA-mediated stomatal closure process in Arabidopsis and other plants.

[0005] However, there is currently no report on the function of alfalfa MtSLAC1 in drought resistance response. In this study, through the cloning, expression analysis and function verification of the alfalfa MtSLAC1 gene, its role in drought stress response was clarified, and a gene fragment involved in alfalfa drought resistance was constructed, providing a theoretical basis and technical support for cultivating alfalfa varieties with strong drought resistance. Summary of the Invention

[0006] The present invention belongs to the field of plant molecular biology, and specifically relates to a method for constructing a gene fragment involved in alfalfa drought resistance. This gene fragment can be used to regulate the stomatal movement of alfalfa and enhance its drought resistance.

[0007] The object of the present invention is to construct a vector based on SLAC1 that can participate in the drought resistance of alfalfa, specifically including the following steps:

[0008] Clone the alfalfa MtSLAC1 gene: Through molecular biology techniques, the MtSLAC1 gene was cloned from alfalfa (Medicago truncatula), and its sequence was analyzed.

[0009] Construct an expression vector: Insert the MtSLAC1 gene into a plant expression vector for expression in host plants.

[0010] Plant transformation: Transform the recombinant expression vector into alfalfa or other plants to obtain transgenic plants overexpressing MtSLAC1.

[0011] I. Functional verification of MtSLAC1

[0012] (1) Selection of target gene and obtaining of mutants

[0013] Selection of target gene: By consulting the literature, we learned that the SLAC1 gene is particularly important for plants. SLAC1 is a slow anion channel located on the plasma membrane of guard cells, which can efflux anions under drought stress, thereby closing stomata to maintain the water content of plant leaves. In previous studies, it was known that the SLAC1 channel protein plays a key role in the closure of plant stomata. The homologous gene 4g101880 of the SLAC1 ion channel protein in Medicago truncatula was used as the research object by querying the website MtrunR108_HiC Protal. The nucleotide sequence encoding the Medicago truncatula SLAC1 ion channel protein is shown in SEQ ID NO.1.

[0014] Obtaining of mutant seeds: We ordered a retrotransposon-inserted SLAC1 loss-of-function mutant from the Medicago truncatula mutant seed bank.

[0015] (2) Phenotypic analysis of wild-type and mutant plants

[0016] Vernalize wild-type Medicago truncatula wild-type and mutant seeds: Wash with concentrated sulfuric acid for 8 minutes, wash 6 times with ultrapure water, place them in a petri dish lined with moist filter paper, incubate in the dark at 4°C for 3 days, and then transfer them to nutrient soil after the cotyledons open under light for 2 days.

[0017] Drought experiment: To study the function of alfalfa MtSLAC1 under drought stress, in this project, the growth states of WT and mtslac1-2 mutants were observed under well-watered and drought conditions. After the wild type and mutants were grown in a 24°C incubator with sufficient water and nutrients for 2 weeks, watering was stopped for drought treatment. After 10 - 14 days of drought treatment, wild-type leaves were cut and quickly frozen in liquid nitrogen and stored at -80°C. After 2 weeks, photos were taken and the fresh weight of the plants was measured. The results are as Figure 1 shown. Under well-watered conditions, there were no significant differences in the growth phenotypes of WT and mtslac1-2 plants ( Figure 1 A). In contrast, under drought conditions, the growth state of the mtslac1-2 mutant was significantly weaker than that of WT plants ( Figure 1 B), and its above-ground fresh weight was also significantly lower than that of WT plants ( Figure 1 C).

[0018] Real-time quantitative PCR (qPCR) detection of SLAC1 expression: RNA was extracted from the cut alfalfa leaves using an RNA extraction kit, reverse-transcribed into cDNA, and qPCR primers were designed. Medicago truncatula R108 cDNA was used as a template. A qPCR instrument was used to detect the expression of SLAC1 under drought stress. The expression of SLAC1 was significantly induced by drought stress ( Figure 1 D). The above experiments show that MtSLAC1 plays an important role in the process of alfalfa responding to drought stress.

[0019] (3) Phenotypic analysis of wild-type and mutant detached leaves

[0020] Detached leaf water loss experiment: To study whether MtSLAC1 participates in the process of drought stress response by inhibiting leaf water loss, the phenotypes and water loss rates of detached leaves of WT plants and mtslac1-1 and mtslac1-2 mutants were analyzed. Ten leaves of wild-type and mutant plants that were about 4 weeks old and grown with sufficient water and nutrients were cut and placed on white paper at room temperature. The fresh weight was measured every 10 minutes. Compared with WT, the leaves of mtslac1-1 and mtslac1-2 mutants showed a phenotype of accelerated water loss. After 30 minutes of detachment, the mutant leaves lost water severely and the leaves withered and shrank, while the leaves of WT plants basically remained unchanged ( Figure 2 A). The statistical results of the water loss rate showed that the water loss rates of the leaves of mtslac1-1 and mtslac1-2 mutants were significantly higher than those of WT plants ( Figure 2 B). The above results show that MtSLAC1 plays a key role in maintaining leaf water.

[0021] (4) Phenotypic analysis of wild-type and mutant stomata

[0022] ABA treatment of leaves to observe stomatal experiments: Under drought stress, plants promote stomatal closure through the ABA signal, thereby achieving the purpose of reducing water loss. To explore whether SLAC1 plays a role in stomatal closure, we treated the leaves of 4-week-old WT plants and mtslac1-1 and mtslac1-2 mutants with 10 μM ABA and observed and counted the stomatal aperture. It can be seen that when no exogenous ABA was applied, the stomatal apertures of mtslac1-1 and mtslac1-2 mutants were already significantly larger than those of WT plants( Figure 3 A). When ABA was present, the stomata of WT plants could be closed within 2 hours( Figure 3 A). In contrast, mtslac1-1 and mtslac1-2 mutants hardly responded to ABA at all, and their stomata remained in the nearly circular open state without ABA treatment( Figure 3 A). ABA could significantly reduce the stomatal aperture of WT plants, and there was no significant difference in the stomatal aperture of mtslac1-1 and mtslac1-2 mutants before and after ABA treatment, that is, they were in a continuously extremely open state( Figure 3 B). The above results indicate that MtSLAC1 is essential for ABA-induced stomatal closure.

[0023] (5) Detection of Cl- efflux in wild-type and mutants by non-invasive micro-test technology

[0024] Non-invasive micro-test experiment: Under drought stress, plants activate the ABA signal, promote the efflux of anions in leaf guard cells, and then close the stomata to reduce water loss. We tore off the lower epidermis of the leaves of 4-week-old wild-type and mutant plants and placed them in a 10 μM ABA treatment solution, treated them under high light for 2 hours, and used non-invasive micro-test technology (Non-invasive Micro-test Technology, NMT) to detect the transmembrane flow of Cl- in leaf stomatal guard cells( Figure 4 A). When no ABA was present, there was no obvious Cl- efflux in the guard cells of WT and mtslac1-1 mutants( Figure 4 B). Two minutes after adding ABA, the guard cells of WT plants showed a significant Cl- efflux, and reached a peak at 7 minutes, and then gradually decreased. Even after 20 minutes, the Cl- efflux was still significantly higher than that under the condition without ABA( Figure 4 B). In contrast, the guard cells of mtslac1-1 mutants hardly showed any Cl- efflux, only showing a weak efflux during 8-11 minutes after adding ABA, but then returning to the basal state( Figure 4B). Statistical results of the average Cl- flow rate before and after adding ABA in this study showed that ABA could strongly induce the Cl- efflux of WT stomatal guard cells, but not that of mtslac1-1 mutant guard cells. Figure 4 C). The above results indicate that ABA-induced Cl- efflux in stomatal guard cells is dependent on the channel protein MtSLAC1.

[0025] The above experiments have proved that MtSLAC1 is crucial for alfalfa to cope with drought stress.

[0026] II. Construction of overexpression vector

[0027] (1) Cloning of target gene

[0028] Obtaining the entry vector: On the MtrunR108_HiC Protal website, use the AtSLAC1 DNA sequence for alignment to find MtSLAC1 and design specific primers for amplifying MtSLAC1. Using wild-type alfalfa R108 cDNA as a template, the MtSLAC1 CDS was amplified.

[0029] Put MtSLAC1 into the pDONR vector through BP reaction to form an entry vector. Send it to a biological company for sequencing, and the correctly sequenced ones are stored at -20 °C for subsequent construction of expression vectors.

[0030] (2) Construction of expression vector

[0031] Construction of overexpression vector: Construct MtSLAC1 into the pEarleyGate202 vector through LR reaction and transform it into Agrobacterium tumefaciens EHA105.

[0032] (3) Genetic transformation of alfalfa

[0033] Take 4-week-old alfalfa leaves without damage or lesions as explants, and use Agrobacterium-mediated (EHA105) genetic transformation to transform pEG202-MtSLAC1 into alfalfa leaves to form callus. After growing into alfalfa plants, verify whether the transformation is successful through genotype identification, and then obtain homozygous mutants through hybridization.

[0034] III. Application and promotion

[0035] The MtSLAC1 expression vector constructed in this invention can be used for gene improvement of alfalfa or other plants to improve their drought resistance. This has important economic and social value for agricultural production, especially for planting in arid regions.

[0036] The present invention constructs a gene fragment involved in the drought resistance of alfalfa and its construction method, especially enhancing the drought resistance of plants by regulating the expression of the MtSLAC1 gene. This technology has broad application potential in the fields of agriculture, ecological restoration, and plant molecular biology research. The specific applications and promotion are as follows:

[0037] (1) Agricultural applications

[0038] By cloning and constructing the MtSLAC1 gene, the present invention can overexpress this gene in alfalfa, thereby significantly improving the drought resistance of plants. This technology can be directly applied to the variety improvement of alfalfa to cultivate alfalfa varieties with stronger drought resistance, which is particularly suitable for planting in arid regions of northwestern China. By using transgenic technology to introduce the MtSLAC1 gene into other crops (such as wheat, corn, etc.), the survival ability and yield stability of these crops under drought stress conditions can also be significantly improved.

[0039] (2) Ecological restoration

[0040] In arid and semi-arid regions, vegetation restoration and ecological restoration are important ecological engineering tasks. In the present invention, the MtSLAC1 gene can significantly improve the drought resistance of plants, so it can be used to restore degraded ecosystems in these regions. By introducing this gene into drought-tolerant plants, the vegetation restoration can be accelerated and the ecological environment can be improved.

[0041] (3) Plant molecular biology research

[0042] The research methods and technical means of the present invention (such as gene cloning, expression vector construction, plant transformation, etc.) provide new research tools and ideas for the field of plant molecular biology. Researchers can use these technologies to further explore key genes and their functions in the drought resistance mechanisms of other plants, providing theoretical support for plant drought resistance research.

[0043] (4) Promotion strategies

[0044] Technical verification and demonstration: Further verify the drought resistance effect of the MtSLAC1 gene under laboratory and field conditions, and demonstrate its application value in actual planting through demonstration projects.

[0045] Technology transfer: License this technology to seed companies, agricultural science and technology enterprises or research institutions to promote its application in actual production.

[0046] Training and promotion: Popularize the application methods and advantages of this technology to farmers, agricultural technicians and scientific researchers through training sessions, technical exchange meetings, etc.

[0047] (5) Market potential

[0048] With the intensification of global climate change, the impact of drought stress on agricultural production has become increasingly significant. The technology of the present invention can significantly improve the drought resistance of plants, especially in the planting industry in arid regions, where there is a huge market demand. By promoting this technology, the drought resistance of crops can be significantly enhanced, thereby increasing agricultural yields and food security.

[0049] (6) Policy Support

[0050] The government can support the promotion and application of the present invention through policies such as agricultural subsidies and scientific research funding, especially providing financial and technical support for large-scale planting projects in arid regions. In addition, relevant departments can formulate corresponding technical standards and promotion plans to ensure the safety and effectiveness of this technology.

[0051] (7) Ecological Assessment

[0052] Before large-scale promotion of this technology, it is necessary to comprehensively evaluate the ecological safety of genetically modified plants to ensure that they will not have a negative impact on the local ecosystem. At the same time, through the implementation of ecological restoration projects, the application effect of this technology in the actual ecological environment can be further verified. Description of the Drawings

[0053] Figure 1 is the drought phenotype and statistical chart of the SALC1 mutant;

[0054] Figure 2 is the water loss of detached leaves of the SALC1 mutant and the statistical chart;

[0055] Figure 3 is the stomatal phenotype and statistical chart of stomatal aperture of the SALC1 mutant after ABA treatment;

[0056] Figure 4 is the in-situ live statistics of Cl- efflux of the SLAC1 mutant and WT by non-invasive micro-test technology and the statistical chart.

Claims

1. A method for improving alfalfa drought resistance based on the SLAC1 gene, characterized in that: The gene fragment is derived from alfalfa, and the gene fragment encodes a protein involved in drought resistance regulation, especially the channel protein MtSLAC1, which can mediate Cl - and / or NO3 - outflow, thereby regulating the closure of stomata to reduce water loss.

2. The construction method according to claim 1, characterized in that: The gene fragment is further constructed into an expression vector to express the MtSLAC1 protein in host cells.

3. The construction method according to claim 1, characterized in that: This gene fragment is used to improve plant drought resistance, especially by transforming plant cells to enhance their stomatal closure ability and water retention capacity.

4. The construction method according to claim 1, characterized in that: The DNA was cloned from Medicago truncatula, and the protein sequence similarity between it and Medicago sativa is more than 99%, and it has the same function in Medicago sativa.

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