Application of kiwifruit acflz27 gene in improving plant tolerance to salt and osmotic stress

Abstract

This invention discloses kiwifruit AcFLZ27 The application of genes in improving plant tolerance to salt and osmotic stress, the AcFLZ27 The nucleotide sequence of the gene is shown in SEQ ID NO.1. Overexpression of the gene... AcFLZ27 Following gene expression, the plant's tolerance to salt and osmotic stress is enhanced, significantly reducing damage to seedlings caused by salt and osmotic stress and significantly increasing seedling growth rate. Under saline irrigation conditions, the survival rate of overexpressing plants is significantly improved, playing an important role in breeding new salt- and drought-resistant kiwifruit varieties. This has a substantial impact on my country's agriculture, forestry, and horticulture industries, and has broad application prospects and research value.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to kiwifruit. AcFLZ27 Application of genes in improving plant tolerance to salt and osmotic stress. Background Technology

[0002] kiwi( Actinidia spp. Originally from humid, acidic environments, kiwifruit has relatively weak overall resistance to adverse conditions, especially drought and salt stress. In areas with seasonal drought or uneven rainfall, it easily leads to insufficient water absorption by roots, leaf wilting, and decreased photosynthetic efficiency, causing flower and fruit drop, smaller fruits, and reduced quality. In severe cases, it can even cause plant death, significantly reducing orchard yield and economic benefits. Kiwifruit thrives in soils with a pH of 5.5–6.5. In saline-alkali soils (pH > 7.5, salt content > 0.3%), it is prone to ion poisoning, osmotic imbalance, and oxidative damage, leading to hindered root growth, yellowing and scorched leaf edges, tree decline, and even the death of the entire plant, severely limiting available land resources and industrial expansion. Current technologies for addressing drought and salt stress in production have significant shortcomings. 1. Lack of stress-resistant germplasm and varieties: Existing main cultivated varieties (such as "Hongyang") generally have weak salt and drought resistance, and there is a lack of commercially viable varieties with high stress resistance that can be directly promoted; 2. Traditional breeding has a long cycle and low efficiency: Kiwifruit is a perennial plant with a complex genetic background. Conventional hybridization breeding to select salt-tolerant and drought-resistant varieties takes more than 10 years, and it is difficult to balance resistance with quality and yield. 3. High cultivation costs and poor sustainability: Irrigation and soil improvement require large investments and consume a lot of water, making it difficult to sustain in arid and water-scarce areas in the long term; 4. Unclear molecular mechanisms of stress resistance and limited available gene resources: The functional verification and breeding application of identified salt-tolerant and drought-resistant genes are still insufficient, making it difficult to support efficient molecular design breeding.

[0003] Therefore, identifying key genes for salt and drought tolerance in kiwifruit, elucidating their molecular regulatory mechanisms, and developing efficient genetic engineering or molecular marker-assisted breeding technologies to create new germplasm that combines high quality, high yield, and strong salt and drought tolerance are of significant theoretical and practical value for improving kiwifruit's stress resistance, expanding its cultivation area, reducing production costs, and ensuring stable, efficient, and sustainable development of the industry. This invention addresses these industry pain points and technological bottlenecks by providing a controllable, efficient, and industrially scalable salt and drought tolerant technology solution. Summary of the Invention

[0004] The purpose of this invention is to provide a kiwifruit AcFLZ27 Application of genes in improving plant tolerance to salt and osmotic stress.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows: kiwiAcFLZ27 Genes, the ones mentioned AcFLZ27 The nucleotide sequence of the gene is shown in SEQ ID NO.1 (the... AcFLZ27 The gene has the sequence number Ach00g330121.1 in the Hongyang kiwifruit gene database, and its amino acid sequence is shown in SEQ ID NO.2.

[0006] During the resistance screening process, this invention discovered that kiwifruit... AcFLZ27 This invention aims to improve the tolerance of plants to salt and osmotic stress, thus providing the aforementioned kiwifruit. AcFLZ27 Application of genes in improving plant tolerance to salt and osmotic stress.

[0007] The functions of the genes protected by this invention include not only those described above. AcFLZ27 The gene, including the function of homologous genes with high homology (such as above 80%; more preferably above 90%; more preferably above 95%; more preferably above 98%) in improving plant tolerance to salt and osmotic stress.

[0008] contain AcFLZ27 The gene expression vector pK2GW7 also enhances the plant's salt tolerance and osmotic stress resistance. The constructed plant expression vector can be directly used for Agrobacterium-mediated genetic transformation to create new stress-resistant woody plant varieties, which can be used for plant improvement.

[0009] To improve the desirable traits of plants, this invention also discloses a method for improving plant stress tolerance, wherein the aforementioned... AtFLZ27 Genes were introduced into the target plant to obtain transgenic plants, which showed higher salt tolerance and osmotic stress resistance than the target plant.

[0010] Specifically, AtFLZ27 Specifically, the gene can be introduced into the target plant via the overexpression vector. In this method, the overexpression vector can be transformed into plant cells or tissues using conventional biological methods such as Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electroporation, and Agrobacterium-mediated transformation, and the transformed plant tissues can be cultured into plants.

[0011] The present invention also discloses a plant breeding method, wherein the method is as follows (1) or (2): (1) By increasing the activity of AcFLZ27 protein in the target plant, plants with stronger tolerance to salt stress and osmotic stress than the target plant were obtained; (2) By increasing the content of the target plant AcFLZ27 Gene expression was used to obtain plants with stronger tolerance to salt and osmotic stress than the target plant; Used to promote the growth of target plantsAcFLZ27 The gene expression method is selected from at least one of the following: (1) Introducing a substance containing [a substance] into the target plant cells AcFLZ27 Recombinant expression vectors encoding genes, to obtain AcFLZ27 Overexpressing plants; (2) Through site-specific genome editing, in the target plant endogenous AcFLZ27 A strong promoter is inserted upstream of the transcription start site of the gene; (3) Through site-specific genome editing, in the target plant endogenous AcFLZ27 Transcription enhancer elements are inserted upstream and downstream of the gene.

[0012] The preferred embodiment of the present invention is embodiment (1), that is, achieving it through genetic modification. AcFLZ27 Gene overexpression.

[0013] Preferably, the target plant is Arabidopsis thaliana or kiwifruit.

[0014] In this invention, there are no particular limitations on the plants suitable for gene transformation, as long as they are suitable for gene transformation operations, such as various crops, flowering plants, or forestry plants. The plants may be (but are not limited to): dicotyledons, monocotyledons, or gymnosperms.

[0015] As one implementation method, the "plant" mentioned includes, but is not limited to, Arabidopsis thaliana and kiwifruit, especially Actinidia rubra julibrissin, and any gene that has the gene or is homologous to it is applicable.

[0016] The term "plant" as used in this invention includes the whole plant, its parent and offspring plants, and different parts of the plant, including seeds, fruits, buds, stems, leaves, roots (including tubers), flowers, tissues, and organs, all of which contain our target gene or nucleic acid. The term "plant" also includes plant cells, suspension cultures, callus tissue, embryos, meristematic regions, gametophytes, sporophytes, pollen, and microspores; similarly, each of these objects contains the target gene / nucleic acid.

[0017] This invention includes any plant cell, or any plant obtained or obtainable by the methods described herein, as well as all plant parts and their propagules. This patent also includes transformed cells, tissues, organs, or whole plants obtained by any of the foregoing methods. The only requirement is that the offspring exhibit the same genotype or phenotypic characteristics, and that offspring obtained using the methods of this patent have identical characteristics.

[0018] This invention also extends to the harvestable parts of the plants as described above, but is not limited to seeds, leaves, fruits, flowers, stems, roots, rhizomes, tubers, and bulbs. It further relates to other derivatives of the plant after harvest, such as dried granules or powders, oils, fats and fatty acids, starches, or proteins. This invention also relates to foods or food additives obtained from the relevant plants.

[0019] Advantages of this invention: This invention isolates the coding from Hongyang kiwifruit. AcFLZ27 The complete cDNA of the gene was constructed into a plant expression vector, and then genetic transformation was completed using Agrobacterium-mediated transformation to obtain transgenic plants. Stress experiments showed that overexpression of the AcFLZ27 gene effectively alleviated damage to seedlings caused by salt and osmotic stress, and significantly improved the growth rate and survival rate of seedlings under stress conditions.

[0020] This study is the first to clone and functionally identify the salt and osmotic stress tolerance characteristics of the kiwifruit AcFLZ27 gene, filling a gap in the application of this gene in plant stress resistance research and breeding. This gene has a clear function, strong stress resistance specificity, and excellent stress tolerance, effectively compensating for the shortcomings of traditional stress resistance breeding, such as long cycles and low breeding efficiency.

[0021] From an application perspective, AcFLZ27 This gene can be used to create new stress-resistant materials for woody plants, and also to improve the stress resistance traits of various economic crops. It is suitable for a wide range of cultivation scenarios, including kiwifruit cultivation and the planting of agricultural and forestry crops. The accompanying Agrobacterium-mediated transformation technology system is mature and can be directly implemented on a large scale in breeding practices. The stress-resistant new varieties bred based on this gene can be adapted to planting in saline-alkali land, arid land, and other marginal lands. This can not only expand the suitable planting areas for crops, reduce yield losses due to stress, and improve the economic benefits of the agricultural, forestry, and horticultural industries, but also reduce the cost of artificial improvement of saline-alkali and arid lands, and help to make efficient use of idle marginal lands. It has a substantial driving effect on the high-quality development of my country's agriculture, forestry, and horticulture industries, and has extremely high scientific research value and industrial application prospects. Attached Figure Description

[0022] Figure 1 This is a chart comparing the growth status of wild-type (WT) and overexpression lines after salt stress treatment, as well as statistical data on plant height and fresh weight growth status.

[0023] Figure 2 This data represents the measurements of proline and antioxidant enzyme activity in wild-type (WT) and overexpression lines after salt treatment.

[0024] Figure 3 This is a chart comparing the growth status of wild-type (WT) and overexpression lines after osmotic stress treatment, as well as statistical data on root length and fresh weight growth status. Detailed Implementation

[0025] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer with the description. However, unless otherwise specified, the specific experimental methods involved in the following embodiments are conventional methods or implemented according to the conditions recommended in the manufacturer's instructions.

[0026] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. Unless otherwise specified, the experimental methods in the following embodiments are all conventional methods. Unless otherwise specified, the reagents and materials used can be purchased commercially.

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be used in this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0028] Example 1 AcFLZ27 Gene acquisition and transgenic vector construction AcFLZ27 The full-length cDNA of (Ach00g330121.1) was cloned from the *Actinidia chinensis* cv. Hongyang sequence in the kiwifruit database. Its nucleotide sequence is shown in SEQ ID NO.1, which consists of 669 bases encoding 222 amino acids, and its sequence is shown in SEQ ID NO.2. Total RNA was extracted from *Actinidia chinensis* seedlings using an RNA extraction and isolation kit, and then amplified using a One-Step RT-PCR kit, followed by PCR amplification to clone the target gene. The primers used are as follows: Forward primer: GGGGACAAGTTTGTACAAAAAAGCAGGCTACATGGCGGATTTGAAGGGAAAC (SEQ IDNO.3); Reverse primer: GGGGACCACTTTGTACAAGAAAGCTGGGTCTGCTGCAGCCACCCCAGC (SEQ IDNO.4); Subsequently, the Gateway cloning method was used to... AcFLZ27The gene was ligated into the pK2GW7 vector. The constructed vector was then transformed into Agrobacterium EHA105 via a liquid nitrogen freeze-thaw method. Positive clones were screened using LB agar plates containing 50 mg / L rifampicin and 50 mg / L spectinomycin. Positive clones were inoculated into LB liquid medium (containing antibiotics: 50 mg / L rifampicin and 50 mg / L spectinomycin) and cultured on a shaker at 28°C and 220 rpm until OD600 = 0.3-0.5. Subsequently, the clones were infected into Arabidopsis flowers. Normal positive transgenic seedlings were obtained by screening with 50 mg / L kanamycin. The results were verified by semi-quantitative RT-PCR, finally yielding homozygous transgenic cells containing the overexpressing gene. AcFLZ27 Genetically modified plants.

[0029] Example 2 AcFLZ27 Gene function verification Seeds of sterilized wild-type WT and overexpression lines (OE4, OE5, and OE9) were sown on 1 / 2 MS medium and cultured at a constant temperature of 23℃ (16 / 8 h light / dark) for 5–7 days. Healthy seedlings with uniform growth were selected and transplanted into 10 × 10 cm pots (nutrient soil: vermiculite = 2:1). These pots were then placed in an incubator with a photoperiod of 16 h light, 8 h dark, and a light intensity of 100 μmol·m⁻¹. - ²·s - ¹, The cultivation temperature was set to 23℃. After the seedlings had grown for 2-3 weeks, both groups of plants were evenly irrigated with an equal amount of 200 mM NaCl solution, once every 3 days. After 6 days of treatment, the plant height and fresh weight were observed and recorded, and samples were taken. At the same time, rosette leaves were collected for the determination and analysis of physiological indicators (proline, antioxidant enzymes).

[0030] See Figure 1 The experimental results showed that under salt stress treatment, the growth differences between WT and overexpression lines gradually became apparent. On day 11 of treatment, the salt stress effect continued to accumulate, and the difference between WT and overexpression lines reached its maximum. WT plants exhibited severe lodging and wilting, with most leaves turning yellow, white, or even necrotic, and the plants were on the verge of death. In contrast, the overexpression lines maintained a relatively intact plant structure and better growth. Among them, the OE9 line performed the best, with only a few older leaves showing yellowing, indicating... AcFLZ27 Overexpression significantly improved the tolerance of Arabidopsis thaliana to long-term salt stress. Under normal growth conditions, there was no significant difference in plant height between the WT and overexpressing lines. After salt stress treatment, the plant height of all lines was inhibited, but the decrease in WT was significantly greater than that of the overexpressing lines. Among them, the OE9 line showed the best plant height maintenance ability, followed by OE4 and OE5, indicating that... AcFLZGene overexpression effectively alleviated the inhibitory effect of salt stress on plant growth. Under normal growth conditions, there was no significant difference in fresh weight between WT and OE lines, and their biomass accumulation levels were consistent. Under salt stress treatment, salt stress caused a decrease in fresh weight in all lines, but the degree of decrease differed significantly between the two. The wild-type showed a sharp drop in fresh weight and severe biomass loss, indicating that salt stress severely damaged the cellular structure and metabolic function of WT, leading to cell dehydration, inhibited biomass synthesis, and inability to maintain normal nutrient accumulation, further demonstrating its salt-sensitive characteristics. Although the fresh weight of the overexpressing lines (OE5, OE4, OE9) decreased, it was significantly higher than that of the WT at the same time point.

[0031] See Figure 2 Under normal growth conditions, the proline content of all lines remained at a low basal level. After salt stress treatment, the proline content of all lines increased significantly, but the increase in the overexpression lines was significantly higher than that in the WT line, with the OE9 line showing the highest increase, approximately 2-3 times that of the WT line. The results indicate that... AcFLZ27 Gene overexpression can promote the accumulation of proline under salt stress and initiate a stronger osmotic protection response.

[0032] Superoxide dismutase (SOD) is the rate-limiting enzyme in the first step of the ROS scavenging cascade. Its main function is to disproportionate superoxide anions generated under salt stress into hydrogen peroxide, reducing the direct damage of superoxide anions to cells and laying the foundation for subsequent H2O2 scavenging. Under salt stress treatment, salt stress induced an increase in SOD activity in all lines, but the SOD activity of overexpressing lines was significantly higher than that of WT lines. Among them, the OE9 line had the highest activity, followed by OE4 and OE5, indicating that... AcFLZ Gene overexpression can significantly activate SOD activity and enhance its ability to disproportionate superoxide anions.

[0033] Under salt stress, the POD activity of the overexpressing lines was significantly higher than that of the WT lines, with some lines reaching highly significant levels. The OE9 line showed the highest POD activity, effectively decomposing H2O2 produced by SOD dismutation. The OE4 and OE5 lines showed the next highest activity, synergistically interacting with SOD to construct a second line of defense for ROS scavenging, preventing secondary oxidative damage caused by H2O2 accumulation, and further enhancing the plant's antioxidant defense capabilities.

[0034] Under salt stress treatment, the CAT activity of the OE strain was significantly higher than that of the WT strain, with the OE9 strain exhibiting the best activity. It can efficiently remove H2O2 from mitochondria and peroxisomes, complementing POD and covering the H2O2 removal needs of the entire cell. This further improves the ROS removal cascade, ensuring that intracellular ROS is rapidly and thoroughly removed, and maintaining redox homeostasis. AcFLZ27 Overexpression of Arabidopsis thaliana genes significantly activates the activity of three antioxidant enzymes, SOD, POD, and CAT, constructing an SOD-POD-CAT antioxidant enzyme scavenging cascade to achieve efficient scavenging of ROS under salt stress.

[0035] (2) Seeds of wild-type Arabidopsis thaliana WT and overexpression lines (OE4, OE5 and OE9) were disinfected. The seeds were vernalized at 4℃ for 48 h, cultured conventionally for 3 days, and then transferred to 1 / 2 MS solid medium containing 300 mM mannitol and 400 mM mannitol, respectively. They were placed vertically in an incubator and cultured at 23℃ (light / dark 16 / 8 h) for 7 days. During the culture period, the plant growth status was observed regularly, and the root length and fresh weight of the seedlings were measured and recorded.

[0036] See Figure 3 Under normal growth conditions, there was no significant difference in root length between WT and the overexpression lines OE4, OE5, and OE9, all remaining within the 4.5-5.0 cm range, confirming that the root development of the overexpression lines was consistent with the wild type under normal conditions. There was no significant difference in fresh weight between WT and the overexpression lines, both remaining stable within the 3.8-4.0 mg range. On 1 / 2 MS medium supplemented with 300 mM Mannitol, the root length of WT decreased sharply to approximately 2.2 cm, with an inhibition rate exceeding 50%; although the root length of the overexpression lines decreased somewhat (approximately 3.8-4.0 cm), it was significantly higher than that of WT, and the decrease was much smaller than that of the wild type, reflecting the superior tolerance of the root system to moderate stress. The fresh weight of WT decreased to approximately 1.5 mg, a reduction exceeding 60%; although the fresh weight of the overexpression lines decreased (approximately 2.5-2.7 mg), it was significantly higher than that of WT, reflecting stronger stability in biomass accumulation under stress. On 1 / 2 MS medium supplemented with 400 mM Mannitol, the root length of the WT line further shrank to approximately 1.8 cm, almost losing its elongation function. Although the root length of the overexpression line decreased to approximately 3.0 cm, it was still significantly higher than that of the WT line, indicating that the overexpression line could still maintain basic root growth under severe stress. The fresh weight of the WT line decreased to approximately 0.8 mg, and its biomass was almost depleted. The fresh weight of the overexpression line was maintained at approximately 1.5-1.6 mg, significantly higher than that of the WT line, further verifying its ability to maintain growth under severe osmotic stress.

[0037] The embodiments described above are merely preferred embodiments of the present invention and are only used to explain the present invention. They are not intended to limit the scope of the present invention. For those skilled in the art, other implementation methods can be easily made by substitution or modification based on the technical content disclosed in this specification. Therefore, all changes and improvements made on the principle of the present invention should be included within the scope of the patent application of the present invention.

Claims

1. Kiwi fruit AcFLZ27 The application of genes in improving plant tolerance to salt and osmotic stress is characterized by, The AcFLZ27 The nucleotide sequence of the gene is shown in SEQ ID NO.

1. Overexpression of the gene... AcFLZ27 After gene modification, the plant's tolerance to salt and osmotic stress increased; the plant in question is Arabidopsis thaliana.

2. The application according to claim 1, characterized in that, By constructing an overexpression vector and introducing it into Arabidopsis thaliana, transgenic Arabidopsis thaliana overexpressing the vector was obtained. The transgenic Arabidopsis thaliana showed higher tolerance to salt stress and osmotic stress than the wild type.

3. The application according to claim 1, characterized in that, Starting with wild-type Arabidopsis thaliana, the constructed overexpression vector was transformed into Agrobacterium EHA105 using the liquid nitrogen freeze-thaw method, and then the expression vector was transferred into Agrobacterium-mediated flower dipping method. AcFLZ27 Genes were transferred into Arabidopsis thaliana and overexpressed through resistance selection. AcFLZ27 Transgenic Arabidopsis thaliana.

4. A plant breeding method, characterized in that, The method is as follows (1) or (2): (1) By increasing the activity of AcFLZ27 protein in the target plant, plants with stronger tolerance to salt stress and osmotic stress than the target plant were obtained; (2) By increasing the content of the target plant AcFLZ27 Gene expression was used to obtain plants with stronger tolerance to salt and osmotic stress than the target plant; The AcFLZ27 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence of the AcFLZ27 protein is shown in SEQ ID NO.2; the target plant is Arabidopsis thaliana.

5. The method according to claim 4, characterized in that, Promote the growth of target plants AcFLZ27 The gene is expressed in the form of overexpression. AcFLZ27 Gene.

6. A method for improving the tolerance of plants to salt stress and osmotic stress, characterized in that, The method is to... AcFLZ27 Genes were introduced into a target plant to obtain transgenic plants. These transgenic plants exhibited higher tolerance to salt and osmotic stress than the target plant. The target plant was Arabidopsis thaliana. AcFLZ27 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

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