Highland barley polyamine transporter gene HvPUT5 and application thereof

Abstract

The invention discloses a polyamine transporter encoding gene HvPUT5 derived from highland barley and application of the polyamine transporter encoding gene HvPUT5 in improvement of cell osmotic stress tolerance, and relates to the technical field of plant stress resistance molecular breeding and functional gene mining. The HvPUT5 is positioned in cytoplasm, and the over-expression of the gene in a yeast system can obviously improve the survival rate of the yeast under the condition of high osmotic pressure, which indicates that the gene has a positive effect on enhancing the osmotic stress tolerance. The HvPUT5 has a good application prospect, can be used as a candidate gene resource and a molecular marker for drought-resistant breeding, and is applied to genetic improvement and molecular breeding research of crop stress resistance characters.

Description

Technical Field

[0001] The present invention relates to the fields of plant molecular biology and genetic engineering, and relates to a polyamine transporter gene derived from hulless barley ( Hulless barley, Hordeum vulgare L.) and its applications. var. nudum Background Art

[0002] Polyamines (PAs) are a class of low-molecular-weight aliphatic cationic compounds that are widely present in plants and mainly include putrescine (Put), spermidine (Spd), and spermine (Spm). They play a crucial role in plant stress resistance, especially against drought and other abiotic stresses (Shao et al., 2022). Polyamines play an active regulatory role in plants' response to drought stress by stabilizing cell membrane structures, scavenging reactive oxygen species (ROS), and regulating the expression of stress-related genes. Exogenous application of polyamines can enhance the drought resistance of plants, manifested as increased water use efficiency, antioxidant activity, and osmotic adjustment ability (Milon et al., 2020). Under drought stress conditions, the polyamine content in plants usually increases. PAs regulate the reactive oxygen species homeostasis, enhance the antioxidant system, and stabilize cell membranes (Alqurashi, 2023). Polyamines also affect gene expression, cell signal transduction, and metabolic processes, thus promoting plant growth and development under stress conditions (Roy et al., 2024). In addition, some microbial preparations that can regulate polyamine metabolism can also further enhance the drought resistance of plants by increasing the endogenous polyamine content and inducing the expression of stress-related genes. In maize, drought stress can induce the accumulation of putrescine, and the conversion of putrescine to spermidine and spermine is more significant in drought-resistant varieties. This conversion process helps to enhance the antioxidant enzyme activity and improve the osmotic adjustment ability, playing a key role in improving the drought resistance of maize (Chen et al., 2023). Polyamine transporters are mainly divided into two categories: PUT (Polyamine Uptake Transporters) and BAT (Bidirectional Amino acid Transporters). In barley, PUTs and BATs represent two types of transporters with different structural and functional characteristics. Among them, PUTs mainly mediate the uptake of exogenous polyamines, while BATs may be involved in the redistribution of polyamines between cells. These proteins help to maintain polyamine homeostasis by regulating the distribution and transport of polyamines within cells, thus enhancing the abiotic stress tolerance of plants at multiple levels such as signal transduction, osmotic adjustment, and antioxidant defense.

[0003] ​Polyamine transporters play an important regulatory role in plant abiotic stress responses. In sweet orange, polyamine transporter genes (CsPUTs) show differential expression under drought stress conditions, and some genes such as CsPUT1 and CsPUT4 are significantly up-regulated in leaves (Alhag et al., 2021). This indicates that polyamine transporters may play a positive role in plants' response to drought stress by regulating the distribution and homeostasis of polyamines. In tomato, the promoter regions of the SlPUT gene family contain cis-acting elements related to drought (such as MBS elements), low temperature, and hormones (such as ABA, MeJA) responses, suggesting that it may be involved in the regulatory responses of plants to various abiotic stresses. Transcriptome data further show that multiple SlPUT genes (slPUT3 and slPUT5) are significantly up-regulated under drought stress conditions, suggesting that they may play a positive role in the drought resistance mechanism of tomato by regulating the transport and homeostasis of polyamines (Zhong et al., 2023). OsPUT1, as a rice polyamine transporter, overexpression of OsPUT1 can enhance the drought resistance of rice, play a positive role in plant response to drought stress by regulating the homeostasis of intracellular polyamines (such as putrescine and spermidine), slowing down water loss, and increasing the survival rate of plants under drought conditions (Mulangi et al., 2012).

[0004] Highland barley is an important agro-ecological crop in the Qinghai-Tibet Plateau and other high-altitude regions. As a major crop unique to the plateau, highland barley has attracted much attention due to its excellent stress tolerance and unique nutritional components. During the long-term evolution process, highland barley has formed a high degree of adaptability to extreme environments, showing good tolerance to high altitude, drought, strong wind, cold, and nutrient-poor environments, and having characteristics such as a short growth cycle. With the intensification of global warming, drought stress has become the main abiotic factor limiting the yield of highland barley. However, the drought resistance response mechanism of highland barley has not been fully analyzed at the molecular level.

[0005] Therefore, it is necessary to develop a method for molecular breeding of plant stress resistance. Summary of the Invention

[0006] The present invention belongs to the technical fields of molecular breeding of plant stress resistance and functional gene mining, and specifically relates to a polyamine transporter-encoding gene (HvPUT5) derived from highland barley and its application in enhancing the tolerance of cells to osmotic stress.

[0007] In the first aspect of the present invention, there is provided a polyamine transporter-encoding gene derived from highland barley HvPUT5 , and its nucleotide sequence is as shown in SEQ ID NO.1.

[0008] In the second aspect of the present invention, a hulless barley polyamine transporter HvPUT5 is provided. The amino acid sequence of the protein is shown in SEQ ID NO.2. The protein encoded by the gene has a multi-transmembrane structure and belongs to the major facilitator superfamily.

[0009] In the third aspect of the present invention, a recombinant expression vector is provided, and the recombinant expression vector can express the hulless barley polyamine transporter HvPUT5.

[0010] Furthermore, the recombinant expression vector includes at least one of an Escherichia coli expression vector, a yeast expression vector, a Bacillus subtilis expression vector, a Lactobacillus expression vector, a Streptomyces expression vector, a filamentous fungus expression vector, a plant expression vector, an insect expression vector, or a mammalian cell expression vector.

[0011] In the fourth aspect of the present invention, a recombinant bacterium or an engineered host cell line of the recombinant expression vector is provided.

[0012] Furthermore, the host cell includes one of an Escherichia coli host cell, a yeast host cell, a Bacillus subtilis host cell, a Lactobacillus host cell, an actinomycete host cell, a filamentous fungus host cell, and an insect cell.

[0013] In the fifth aspect of the present invention, applications of the gene, the hulless barley polyamine transporter HvPUT5, the recombinant expression vector, the recombinant bacterium or the engineered host cell line in improving the cellular osmotic stress tolerance of plants are provided.

[0014] In the sixth aspect of the present invention, a method for improving the cellular osmotic stress tolerance of plants is provided. The method includes: introducing the HvPUT5 gene or the expression vector into a target plant, and applying stress conditions to improve the cellular osmotic stress tolerance of the plant.

[0015] Furthermore, the stress conditions include: Osmotic stress: drought conditions simulated by mannitol, NaCl, or PEG, and the stress treatment concentration is ≥0.8 M mannitol, ≥150 mM NaCl, or ≥15% PEG6000.

[0016] Furthermore, the plant includes one of hulless barley, corn, wheat, and tomato.

[0017] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages: 1. The present invention provides a polyamine transporter encoding gene derived from hulless barley HvPUT5And its applications. By integrating the physiological phenotypes and transcriptome data of hulless barley seedlings under drought stress, the weighted gene co-expression network analysis (WGCNA) was used to lock the key module MEsalmon2, and the core gene HvPUT5 (SEQ ID NO.1) with the highest topological connectivity was screened out from it. This gene encodes an MFS superfamily transporter containing 12 transmembrane domains (SEQ ID NO.2), and its subcellular localization is in the cytoplasm. Yeast heterologous expression experiments showed that overexpression of HvPUT5 could increase the survival rate of engineered bacteria under 1.5 M mannitol stress by 42 - 56% (p < 0.01), confirming its biological function of significantly enhancing the cell osmotic adjustment ability.

[0018] 2. The HvPUT5 gene plays a positive regulatory function in the response to osmotic stress, has good application prospects, is expected to be used as a candidate gene resource for improving drought tolerance, and can also be developed into an auxiliary screening marker in molecular breeding, providing a theoretical basis and technical support for stress-resistant molecular breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is the correlation analysis between gene expression modules and physiological traits in the drought stress response of hulless barley based on WGCNA. Among them, A: The hierarchical clustering diagram of gene modules obtained by WGCNA clustering analysis; B: The correlation matrix between gene modules and physiological parameters; among them, enzyme activity-related parameters are labeled with "e", photosynthetic rate-related parameters are labeled with "GasEx", chlorophyll fluorescence-related parameters are labeled with "FLR", plant physiology-related parameters are labeled with "phy", and reactive oxygen species (ROS)-related detection parameters are labeled with "ROS". C: The co-expression network analysis diagram of genes within the MEsalmon2 gene module, where the color depth of each gene node represents its connectivity within the module, and the darker the color, the higher the connectivity.

[0020] Figure 2 It is the identification of the drought stress gene HvPUT5 in hulless barley. Among them, A: Gene cloning and annotation. The bar graph represents the length of the amino acid sequence, and the number is the number of amino acids. The IPR code represents the domain position and name annotated in the InterPro database; B: The confocal microscopy observation results of HvPUT5 fused with YFP in tobacco leaf epidermal cells. The images include fluorescence images (YFP), bright field images (BF), and merged images (merged).

[0021] Figure 3Growth status of yeast transformed with empty vector pYES2 or pYES2-HvPUT5 under control conditions (left) and 1.5 M mannitol stress conditions (right). Detailed implementation manners

[0022] The present invention will be specifically described below in combination with the detailed implementation manners and examples, and the advantages and various effects of the present invention will be presented more clearly therefrom. Those skilled in the art should understand that these detailed implementation manners and examples are used to illustrate the present invention, rather than to limit the present invention.

[0023] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as having the meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as the general understanding of those skilled in the art to which the present invention pertains. In case of contradiction, this specification shall prevail.

[0024] Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment, etc. used in the present invention can be obtained through market purchase or can be obtained by existing methods.

[0025] To solve the technical problems of the present invention, the general idea of the present invention is as follows: First, by performing a weighted gene co-expression network analysis (WGCNA) on the physiological parameters and transcriptome data of multiple hulless barley varieties under drought stress conditions at the seedling stage, a co-expression module MEsalmon2 closely related to drought response was identified, and a core gene with a significantly higher connectivity was screened out in this module. This gene was functionally annotated as encoding a polyamine uptake transporter and named HvPUT5.

[0026] Further, through protein domain analysis, it was confirmed that the HvPUT5 protein has typical transmembrane transport structural characteristics and belongs to the major facilitator superfamily (MFS). Subcellular localization experiments showed that this protein was specifically localized in the cytoplasm, indicating that it may mediate polyamine transport functions in the intracellular environment.

[0027] To verify the function of this gene, the present invention overexpressed HvPUT5 in the yeast strain INVSc1 and performed growth and survival analysis under 1.5 M mannitol stress conditions. The results showed that the overexpression of HvPUT5 significantly improved the survival rate of yeast cells under high osmotic pressure conditions, indicating its positive role in enhancing the tolerance of cells to osmotic stress.

[0028] The present application will be described in detail below in combination with examples and experimental data.

[0029] Example 1 HvPUT5 Discovery of the gene Construction of the WGCNA co-expression network and mining of candidate genes Multiple hulless barley varieties (YC83, YC85, YC88, ZY97, ZY1100, and ZY1252) were selected, and drought treatment was set at the seedling stage. Systematic determinations were made on relevant physiological and biochemical indexes, including antioxidant enzyme activities (such as catalase CAT, peroxidase POD, superoxide dismutase SOD), photosynthetic rate-related parameters, chlorophyll fluorescence parameters, plant height, fresh weight, dry weight, chlorophyll content, and reactive oxygen species (ROS) level, etc. At the same time, transcriptome data of the corresponding samples were obtained. The WGCNA package in R language was used to construct a co-expression network. Through module-trait association analysis, the module Mesalmon2 highly correlated with plant height, fresh weight, dry weight, and chlorophyll content under drought stress was screened out, and a core gene with a significantly higher connectivity was identified therein, annotated as encoding a polyamine transporter (HvPUT5). The nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2.

[0030] Example 2. Analysis of the protein domain of HvPUT5 and subcellular localization After the target gene was cloned in the present invention, the protein sequence thereof was annotated for domains by using the InterPro database to identify the position and type of functional domains. The amino acid lengths of each protein and their domain distributions were shown through bar charts, and the IPR code was used to indicate the specific position and name of the domain to assist in analyzing its potential function. The CDS sequence of HvPUT5 was cloned into the restriction enzyme sites of NruI and AvrII of the expression vector pEARLEYGATE 101 with a YFP tag (for the vector, see the literature: Earley, Keith, Jeremy R. Haag, Olga Pontes, Kristen Opper, Tom Juehne, Keming Song, and Craig S. Pikaard (2006). Gateway-compatible vectors for plant functional genomics and proteomics. The Plant J. 45:616-629.) to construct a fusion expression vector. The vector was introduced into the epidermal cells of tobacco leaves through Agrobacterium-mediated transient transformation, and a laser confocal microscope was used to observe the subcellular localization of the protein.

[0031] The results of the protein structure analysis of HvPUT5 are as Figure 2 shown in Figure 2 A. The protein contains 12 transmembrane α-helices ( 152 , 189 , 207 ), and the substrate-binding domain is located in the 3rd - 5th helices (Glu

[0032] The results of protein subcellular localization are as Figure 2 shown in B. The YFP fluorescence signal is evenly distributed in the cytoplasm, indicating that HvPUT5 is localized in the cytoplasm.

[0033] Example 3. Functional verification of HvPUT5 in yeast The CDS of HvPUT5 (the sequence is shown in SEQ ID NO.1) was cloned into the EcoRI and SacI restriction sites of the pYES2 yeast overexpression vector (purchased from Shanghai Weidi Biotechnology Co., Ltd., product number YC1050S), transformed into the yeast strain INVSc1 (purchased from Shanghai Weidi Biotechnology Co., Ltd., product number YC1050S), and the osmotic stress environment was simulated by treatment with mannitol (concentration 1.5 M).

[0034] The results are as Figure 3 shown. Under non-stress conditions, there was no significant difference in the survival rate between the target gene transformation group and the empty vector control group of yeast. After treatment with 1.5 M mannitol for 48 hours, the transformed yeast strains expressing HvPUT5 showed a significantly higher survival rate than the control group, indicating that HvPUT5 helps to improve the tolerance of yeast to osmotic stress.

[0035] The results show that the survival rate of the yeast strain overexpressing HvPUT5 under osmotic stress conditions is significantly higher than that of the empty vector control group, indicating that HvPUT5 can enhance the tolerance of cells to osmotic stress.

[0036] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent substitution methods and are all included in the protection scope of the present invention.

[0037] Finally, it should also be noted that the term "comprises", "comprising" or any other variation thereof is intended to cover a non-exclusive inclusion, such that a process, method, article or device comprising a series of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article or device.

[0038] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications once they learn the basic creative concept. Therefore, the appended claims are intended to be construed to include the preferred embodiments as well as all changes and modifications falling within the scope of the present invention.

[0039] Obviously, those skilled in the art can make various modifications and variations to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these modifications and variations.

Claims

1. A gene encoding a polyamine transporter from highland barley HvPUT5 , characterized in that, Its nucleotide sequence is shown as SEQ ID NO.

1.

2. A highland barley polyamine transporter HvPUT5, characterized in that The amino acid sequence of the protein is shown in SEQ ID NO.

2. The protein encoded by the gene has a multi-transmembrane structure and belongs to the main transport protein superfamily.

3. A recombinant expression vector, characterized in that: The recombinant expression vector is capable of expressing the highland barley polyamine transporter HvPUT5 described in claim 2.

4. The recombinant expression vector according to claim 3, characterized in that The recombinant expression vector includes at least one of an Escherichia coli expression vector, a yeast expression vector, a Bacillus subtilis expression vector, a lactic acid bacteria expression vector, a Streptomyces expression vector, a filamentous fungus expression vector, a plant expression vector, an insect expression vector, or a mammalian cell expression vector.

5. A recombinant bacterium or engineered host cell line comprising the recombinant expression vector according to any one of claims 3 to 4.

6. The recombinant bacteria or engineered host cell line according to claim 5, characterized in that: The host cell includes one of an Escherichia coli host cell, a yeast host cell, a Bacillus subtilis host cell, a lactic acid bacteria host cell, an actinomycete host cell, a filamentous fungus host cell, and an insect cell.

7. Use of the gene described in claim 1, the highland barley polyamine transporter HvPUT5 described in claim 2, the recombinant expression vector described in any one of claims 3-4, and the recombinant bacteria or engineered host cell line described in any one of claims 5-6 in improving the cell osmotic stress tolerance of plants.

8. A method for improving the cell osmotic stress tolerance of a plant, characterized in that: The method comprises: introducing the HvPUT5 gene of claim 1 or the expression vector of claims 3-4 into a target plant, and applying stress conditions to improve the cell osmotic stress tolerance of the plant.

9. The method according to claim 8, characterized in that The stress adopts osmotic stress: drought conditions simulated by mannitol, NaCl or PEG, and the treatment concentration of the osmotic stress is selected from one of the following: ≥0.8M mannitol, ≥150mM NaCl or ≥15% PEG6000.

10. The method according to claim 8, characterized in that The plant includes one of highland barley, corn, wheat and tomato.

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