Application of FVE gene in regulating high temperature tolerance of plants

Abstract

The application discloses application of an FVE gene in regulating high-temperature resistance of plants and belongs to the technical field of plant molecular biology.The application discloses application of a gene FVE related to chromatin remodeling and its coded protein in response of Arabidopsis to environmental high temperature.It is found in the application that the FVE deletion mutant fve-4 is not sensitive to environmental high temperature, and the mutant phenotype can be complemented by constructing a transgenic plant line, which indicates that the FVE gene is related to regulation of response of plants to high temperature.It is found by whole gene transcriptome sequencing (RNA-seq) and fluorescence real-time quantitative (qPCR) analysis that the fve-4 is not sensitive to environmental high temperature due to serious disorder of an auxin signal pathway in the body of the fve-4.The application provides a new factor capable of promoting high-temperature resistance of plants, which provides an important basis for response of plants to global climate warming.

Description

Technical Field

[0001] This invention relates to the field of plant molecular biology, specifically to the application of an FVE gene in regulating plant heat tolerance. Background Technology

[0002] Environmental temperature affects plant growth and development, as well as crop yield and fruit quality. As sessile organisms, plants cannot escape environmental stress or natural disasters; therefore, the much-discussed global warming seriously harms crop growth and yield.

[0003] Although plants cannot escape adversity, they adapt to their environment by adjusting their morphological changes. Studies have shown that plants can distinguish temperature differences of 1°C, and changes in environmental temperature affect plant morphogenesis. For example, in Arabidopsis thaliana, under high-temperature conditions below heat stress, the rosette leaves develop longer petioles, and the leaves elongate upwards, increasing the angle between the leaves and the ground. Photo-grown Arabidopsis seedlings also exhibit hypocotyl elongation under high temperatures. This series of morphological changes in plants to adapt to high temperatures is called "thermal morphogenesis." Thermal morphogenesis mainly involves reducing the plant's contact with hot soil, increasing the surface area exposed to air, increasing transpiration rates, and lowering leaf surface temperature. Therefore, studying the thermal morphogenesis process is essential to improving plant heat tolerance. However, current technology has not reported that the FVE gene can regulate plant heat tolerance. Summary of the Invention

[0004] To address the aforementioned shortcomings of existing technologies, the purpose of this invention is to provide an application of the FVE gene in regulating plant heat tolerance.

[0005] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: providing an application of the FVE gene in regulating plant heat tolerance; wherein, the nucleotide sequence of the FVE gene is shown in SEQ ID NO:1.

[0006] Furthermore, the plant is Arabidopsis thaliana.

[0007] Furthermore, the FVE gene was knocked out, making the plant unresponsive to high temperatures.

[0008] Furthermore, genetic complementation of plants with the FVE gene knocked out was used to promote the plant's response to high temperatures.

[0009] An agent for regulating plant heat tolerance, comprising the aforementioned FVE gene or the protein encoded by that gene.

[0010] A method for regulating plant heat tolerance includes the following steps:

[0011] Regulating plant responses to high temperatures by knocking out or reintroducing the FVE gene.

[0012] The present invention has the following beneficial effects:

[0013] This invention discloses the application of the gene FVE, associated with chromatin remodeling, and its encoded protein in the response of Arabidopsis thaliana to high temperatures. The invention found that the FVE deletion mutant fve-4 is insensitive to high temperatures, and transgenic plant lines were constructed to restore the mutant phenotype, indicating that the FVE gene is involved in regulating plant responses to high temperatures. Analysis using whole-genome transcriptome sequencing (RNA-seq) and quantitative real-time PCR revealed that the insensitivity of fve-4 to high temperatures stems from severe dysregulation of its auxin signaling pathway. This invention provides a novel factor that can promote plant heat tolerance, offering important evidence for plant responses to global warming. Attached Figure Description

[0014] Figure 1 This is a schematic diagram of the mutation sites of the fve-4 mutant.

[0015] Figure 2 Phenotypic diagrams for Col (control group), fve-4, fve-4; pFVE::HA-FVE; fve-4; P35S::HA-FVE.

[0016] Figure 3 This is a diagram showing differential gene expression analysis across the entire transcriptome.

[0017] Figure 4 To verify the expression map of auxin gene using real-time quantitative PCR.

[0018] Figure 5 This is a diagram of gene ontology function analysis. Detailed Implementation

[0019] The examples given below are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, conditions in the examples are performed under standard conditions or as recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0020] The FVE gene (FLOWERING LOCUS VE) belongs to the MSI1 (Multicopy Suppressor of IRA1) family, also known as MS1-4. This gene was isolated from Arabidopsis thaliana, with a full-length cDNA of 1524 bp, and its CDS sequence is shown in SEQ ID NO:1. The protein encoded by this gene is a structural protein of approximately 55.7 kDa, consisting of 507 amino acids, and its amino acid sequence is shown in SEQ ID NO:2. The FVE protein contains a conserved WD-40repeat domain, which is conserved in multiple species and its function is related to chromatin remodeling.

[0021]

[0022] MESDEAAAVSPQATTPSGGGTGASGPKKRGRKPKTKEDSQTPSSQQQSDVKMKESGKKTQQSPSVDEKYSQWKGLVPILYDWLANHNLVWPSLSCRWGPQLEQATYKNRQRLYLSEQTDGSVPNTLVI ANCEVVKPRVAAAEHISQFNEEARSPFVKKYKTIIHPGEVNRIRELPQNSKIVATHTDSPDVLIWDVETQPNRHAVLGAANSRPDLILTGHQDNAEFALAMCPTEPFVLSGGKDKSVVLWSIQDHITT IGTDSKSGSIIKQTGEGTDKNESPTVGPRGVYHGHEDTVEDVAFSPTSAQEFCSVGDDSCLILWDARTGTNPVTKVEKAHDADLHCVDWNPHDDNLILTGSADNTVRLFDRRKLTANGVGSPIYKFE GHKAAVLCVQWSPDKSSVFGSSAEDGLLNIWDYDRVSKKSDRAAKSPAGLFFQHAGHRDKVVDFHWNASDPWTIVSVSDDCETTGGGGTLQIWRMSDLIYRPEEEVVAELEKFKSHVMTCASKP(SEQ ID NO:2).

[0023] Example 1:

[0024] I. Plant Materials

[0025] Col wild-type, fve-4 is mutated in the Col background (see schematic diagram of the mutation site). Figure 1 fve-4; pFVE::HA-FVE and fve-4; P35S::HA-FVE are transgenic lines of Arabidopsis thaliana by transferring the pGWB513-pFVE-gFVE and pGWB513-p35s-gFVE vectors into Arabidopsis thaliana using the flower immersion transgenic method, and the desired transgenic lines are selected by antibiotic screening.

[0026] II. Phenotypic Observation

[0027] After sterilization, the seeds of the above-mentioned plant materials were placed at 4℃ for 2-3 days, germinated and grew at 21℃ under short-day conditions for 4 days, and then transferred to 27℃ or maintained at 21℃ for 4 days to continue growing before taking pictures. The hypocotyls of the plants were measured and observed using ImageJ.

[0028] This invention uses the elongation of the hypocotyl in Arabidopsis thaliana under high environmental temperatures as an indicator to determine whether Arabidopsis is sensitive to high temperatures. This invention utilizes an Arabidopsis thaliana mutant, fve-4, which lacks the FVE gene and was found to be insensitive to high temperatures. Genetic replacement was performed on this mutant material using transgenic technology, with the FVE self-promoter and the constitutive promoter 35S (pFVE::HA-FVE; p35S::HA-FVE) used for replacement. It was found that the transgenic plants could restore the high-temperature insensitivity phenotype of fve-4. This demonstrates that the FVE gene plays a crucial role in the Arabidopsis response to high environmental temperatures (see details...). Figure 2 ).

[0029] III. RNA Extraction, RNA Sequencing, and RT-qPCR

[0030] RNA extraction: Seedlings cultured at 21°C for 7 days were transferred to 27°C or maintained at 21°C and treated at time point ZT1. Seedlings were collected at time point ZT13. Total RNA was extracted using the Eastep Super Total RNA Extraction Kit (Promega, LS1040). Three independent biological replicates were set up for each line and treatment condition.

[0031] RNA sequencing: Sequencing libraries were constructed using the NEBNext Ultra RNA Library Preparation Kit (NEB, 7530L) according to the manufacturer's instructions. The libraries were sequenced on a NovaSeq 6000 platform, generating 150bp paired-end reads. Adapter excision and low-quality read filtering were performed using fastp (version 0.20.1). Reads were aligned to the Arabidopsis genome (TAIR10) using Hisat2 (version 2.1.10), gene read counts were calculated using HTseq (version 0.11.2), and TPM (Transcripts Per Million) values ​​were generated using R. Differential gene expression analysis was performed using DESeq2 (version 1.26.0). Genes with a change in expression level exceeding two-fold and a corrected P-value < 0.05 were identified as differentially expressed genes. Gene ontology (GO) enrichment analysis was performed using DAVID (https: / / david.ncifcrf.gov / ).

[0032] RT-qPCR: Reverse transcription was performed using the HiScript III First-Strand cDNA Synthesis Kit (Vazyme, R312-02). Quantitative PCR was performed using ChamQ Universal SYBR qPCR premix (Vazyme, Q711-02) on an Applied Biosystems QuantStudio 6Flex or Bio-Rad CFX384 real-time quantitative PCR instrument. PP2A was used as an internal control gene for normalization. The primer sequences for real-time quantitative PCR are as follows:

[0033] YUC8

[0034] F: 5'-GACTGCTCGGTTCGATGAGAC-3' (SEQ ID NO. 3);

[0035] R: 5'-TGAATCACCTCACCGGAAAAC-3' (SEQ ID NO.4);

[0036] IAA29

[0037] F: 5'-ATCCTCTGGAATCCGAGTCTTC-3' (SEQ ID NO.5);

[0038] R: 5'-TACTGGTGGCCATCCAACAAC-3' (SEQ ID NO. 6);

[0039] SAUR20

[0040] F: 5'-CCTTGTCATGAAGATACGTTCATC-3' (SEQ ID NO.7);

[0041] R: 5'-CACAAAAATGGCATCCATTCTCTAAAC-3' (SEQ ID NO. 8);

[0042] SAUR22

[0043] F: 5'-GACAAATAGAGAATTATAAATGGCTCTG-3' (SEQ ID NO. 9);

[0044] R: 5'-ATGAATTAAGTCTATATCTAACTCGGAAA-3' (SEQ ID NO. 10);

[0045] PP2A

[0046] F: 5'-TCGGATGACGATTCTTCGTGCAG-3' (SEQ ID NO. 11);

[0047] R: 5'-CTTGGTCGACTATCGGAATGAGAG-3' (SEQ ID NO. 12).

[0048] For the specific results mentioned above, please see Figures 3-5 ,Depend on Figures 3-5 It can be known that:

[0049] This invention analyzes the differences in gene expression profiles between fve-4 and wild-type Col using whole-genome transcriptome analysis. It reveals that the downregulated genes include several auxin synthesis and cell elongation-related genes, such as PRE1, SAUR20, and YUC8. This invention utilizes real-time quantitative PCR to verify the expression of some auxin synthesis genes in this mutant under high environmental temperatures. The results are consistent with the transcriptome analysis: in the control group, the expression of these genes significantly increases under high environmental temperatures. However, compared to the control group, the expression levels of these genes in the mutant are significantly decreased under high environmental temperatures.

[0050] This invention uses gene ontology to classify and analyze genes upregulated and downregulated in the whole transcriptome of fve-4 and Col under high environmental temperatures. It was found that downregulated fve-4 genes were enriched in the auxin signaling pathway and in response to red and far-red light signaling pathways. Upregulated genes were enriched in glycoside production and stress response signaling pathways.

[0051] The above results all indicate that the FVE gene can affect Arabidopsis thaliana auxin signaling, thereby influencing Arabidopsis thaliana's ability to respond to high temperatures by promoting the elongation of hypocotyl cells.

[0052] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of the FVE gene in regulating plant heat tolerance; among which, The nucleotide sequence of the FVE gene is shown in SEQ ID NO:1; the regulation is achieved by knocking out the FVE gene to inhibit hypocotyl elongation in plants under high temperature conditions, so as to achieve plant non-response to high temperature; the plant is Arabidopsis thaliana; the high temperature is 27°C.

2. The application according to claim 1, characterized in that, Genetic complementation of plants with the FVE gene knocked out can enhance their response to high temperatures.

3. A method for regulating plant heat tolerance, comprising the following steps: The plant's response to high temperature is regulated by knocking out or reintroducing the FVE gene as described in claim 1; the regulation is achieved by knocking out the FVE gene to inhibit hypocotyl elongation under high temperature conditions, so as to achieve non-response to high temperature; the plant is Arabidopsis thaliana; the high temperature is 27°C; the plant's response to high temperature is promoted by genetically reintroducing the FVE gene knocked out.

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