Arabidopsis long non-coding RNA ARTA and its application in plant drought tolerance

Abstract

This invention discloses a long non-coding RNA (lncRNA) ARTA from Arabidopsis thaliana and its application in plant drought tolerance. The nucleotide sequence of the lncRNA ARTA is shown in SEQ ID NO.1. Using a purchased lncRNA ARTA T-DNA insertion mutant as material, this invention found that the loss of function of lncRNA ARTA makes plants insensitive to ABA. Subsequently, an overexpression vector was constructed by cloning the full-length ARTA, and ARTA-overexpressing transgenic materials in a mutant background were obtained using the Agrobacterium-mediated flower immersion method. In soil drought phenotypic experiments, plants overexpressing ARTA showed stronger drought tolerance, while the ARTA loss-of-function mutant was sensitive to drought. In the determination of water loss rate in detached leaves, the lncRNA ARTA loss-of-function mutant showed a faster water loss rate, further validating the results regarding soil drought. This indicates that lncRNA ARTA plays a positive role in plant drought adaptation, that is, lncRNA ARTA can enhance plant tolerance to drought.

Description

Technical Field

[0001] This invention relates to molecular biology techniques and the field of genetics, specifically to the identification of an Arabidopsis long non-coding RNA ARTA and its application in plant drought stress. Background Technology

[0002] Long non-coding RNAs (lncRNAs) are a class of RNAs, longer than 200 nt, widely found in eukaryotes and lacking protein-coding capabilities. In recent years, with the development of sequencing technology and bioinformatics, a large number of lncRNAs have been identified in plants. Studies of their biological functions have revealed their involvement in regulating multiple biological processes within plants at various levels, such as transcription, post-transcriptional processes, translation, and epigenetic modifications. In Arabidopsis, long non-coding RNA MAS is cold-induced and can bind to WDR5a, a core protein component of the COMPASS-like complex, recruiting the complex to the MAF4 gene locus and activating MAF4 expression to inhibit premature flowering. Arabidopsis long non-coding RNAs COOLAIR and COLDAIR participate in plant flowering regulation by affecting histone modification at the FLC locus. Potato long non-coding RNA StFLORE1 regulates plant drought tolerance by regulating StFLORE1 expression. Arabidopsis long non-coding RNA ASCO can competitively bind to NSRs with mRNA, interfering with alternative splicing of NSR proteins in downstream auxin-responsive genes, thereby affecting lateral root growth.

[0003] Abiotic stress is one of the main factors affecting plant growth, development, and geographical distribution, and the ABA signaling pathway is one of the important pathways for plant responses to abiotic stress. Previous studies have shown that long non-coding RNAs (LNCs) participate in regulating plant responses to abiotic stress. In Arabidopsis thaliana, the LNC DRIR participates in the plant's response to drought and salt stress; overexpression of DRIR enhances the plant's drought and salt tolerance. In Arabidopsis thaliana, overexpression of the cotton LNC RNA lncRNA973 enhances the plant's salt tolerance, while overexpression of the cotton LNC RNA lncRNA354 leads to dwarfing, reduced root dry weight, and decreased salt tolerance. In alfalfa, high-throughput sequencing has revealed a large number of lncRNAs responding to osmotic and salt stresses. Furthermore, some lncRNAs responding to low temperature and drought stresses have been identified in the whole alfalfa genome. Although in recent years, a large number of lncRNAs involved in various abiotic stresses have been identified in plants using a combination of sequencing technology and bioinformatics, their mechanisms of action remain unclear and require further investigation. Therefore, we delve into long non-coding RNAs associated with plant responses to ABA and drought, and study their roles in plant ABA response and drought stress response, thereby providing a scientific basis for further improving the regulatory mechanism of plant drought stress response and the breeding of new drought-resistant crop varieties. Summary of the Invention

[0004] In view of the above-mentioned prior art, the purpose of this invention is to provide a long non-coding RNA ARTA related to plant ABA response, explore its molecular mechanism in plant drought tolerance, and thus provide the application of this long non-coding RNA in improving plant drought tolerance.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] In a first aspect, the present invention provides a long non-coding RNA associated with plant ABA response, which is lncRNAARTA, having the nucleotide sequence shown in SEQ ID NO.1.

[0007] In a second aspect, the present invention provides a T-DNA insertion mutant of the Arabidopsis long non-coding RNA ARTA described above.

[0008] In a third aspect, the present invention provides a plant overexpression vector containing the above-mentioned Arabidopsis long non-coding RNA ARTA and a recombinant bacterium.

[0009] In a fourth aspect, the present invention provides the application of the above-mentioned Arabidopsis long non-coding RNA ARTA plant expression vector or recombinant bacteria in improving plant drought resistance.

[0010] A fifth aspect of the present invention provides the application of the above-mentioned Arabidopsis long non-coding RNA ARTA in improving plant drought tolerance, comprising the following steps:

[0011] (1) Construct a plant overexpression vector containing the Arabidopsis long non-coding RNA ARTA shown in SEQ ID NO.1;

[0012] (2) The plant overexpression vector from step (1) was transformed into the mutant, which increased the expression level of long non-coding RNA ARTA in Arabidopsis thaliana, thereby enhancing the plant's drought resistance.

[0013] Compared with the prior art, the beneficial effects of the present invention are:

[0014] This invention marks the first identification of a long non-coding RNA (ARTA) in Arabidopsis thaliana that participates in the plant ABA response and whose overexpression enhances drought tolerance. This ARTA can be used as a genetic resource in agriculture, generating practical application value through the breeding of drought-resistant transgenic plants. Attached Figure Description

[0015] Figure 1 The image shows an agarose gel electrophoresis result of PCR amplification of lncRNA ARTA, where the lncRNA ARTA fragment is 1247 bp in length.

[0016] Figure 2 For the colony PCR results of the overexpression vector pCAMBIA1300-ARTA, the DNA marker used was: Plus II;

[0017] Figure 3 To detect the expression level of lncRNA ARTA in mutant and overexpressing transgenic plants by qRT-PCR;

[0018] Figure 4 The ABA phenotypes of lncRNA ARTA mutants and overexpressing transgenic plants on plates are shown in Figure A, which is a phenotypic diagram of the plants, and Figure B is a statistical analysis of the elongation of the aboveground parts and roots.

[0019] Figure 5 The drought phenotypes of lncRNA ARTA mutants and overexpressing transgenic plants in soil are shown. A is a diagram of plant drought phenotypes, and B is a statistical representation of plant survival rates after drought rehydration.

[0020] Figure 6 The results show the changes in water loss rate of detached leaves from lncRNA ARTA mutants and overexpressing transgenic plants. Detailed Implementation

[0021] The following embodiments of the present invention are illustrative, and the detailed steps are intended to provide further explanation of this application. Unless otherwise specified, all techniques used herein are conventional techniques, and the scientific terms used are common terms in the technical field to which this application pertains.

[0022] As described in the background section, lncRNAs are closely related to plant responses to abiotic stresses, but lncRNAs associated with drought stress responses and their mechanisms of action are rarely reported. Therefore, the purpose of this invention is to provide a long non-coding RNA, ARTA, that participates in the plant ABA response and whose overexpression can improve plant drought tolerance.

[0023] This invention utilizes high-throughput sequencing technology (RNA-Seq) to screen for a long non-coding RNA, ARTA, in Arabidopsis thaliana that is associated with the plant ABA response. Further analysis determined the sequence length of this long non-coding RNA to be 1247 bp, and its nucleotide sequence is shown in SEQ ID NO.1.

[0024] This invention also cloned the lncRNA ARTA transcript sequence, ligated lncRNA ARTA into the expression vector pCAMBIA1300 via BamHI and EcoRI restriction sites, and identified by colony PCR and sequencing. The successfully constructed overexpression vector plasmid was then transformed into Agrobacterium GV3101. Subsequently, the mutants were infected using the Agrobacterium-mediated flower immersion method to obtain the corresponding stably overexpressing lncRNA ARTA transgenic plants. Phenotypic identification experiments revealed that the lncRNA ARTA mutants were insensitive to ABA but sensitive to drought, while overexpression of lncRNA ARTA caused the plants to exhibit the opposite phenotype, namely sensitivity to ABA and enhanced drought tolerance.

[0025] In conclusion, using plant overexpression vectors to increase the expression level of long non-coding RNA ARTA can improve the drought resistance of plants.

[0026] To make the technology used in this application clearer, the technical solution of this application will be described in detail below with reference to specific embodiments. The experimental materials used in the embodiments of this invention are all conventional experimental materials in the art and can be purchased through commercial channels. Unless otherwise specified, the experimental methods and conditions are performed according to conventional procedures.

[0027] Example 1: Cloning of lncRNA ARTA

[0028] 1. Materials and Reagents

[0029] 1.1 Materials

[0030] Arabidopsis thaliana Columbia ecotype (Colobia-0, Col-0)

[0031] 1.2 Reagents

[0032] M519 and M524 culture media were purchased from PhytoTechnology Laboratories; PrimeSTAR Max DNA Polymerase (2x) was purchased from Takara; chloroform, anhydrous ethanol, and isopropanol were purchased from Sinopharm; DEPC-Treated Water and TRIzol Reagent were purchased from Ambion; Sodium chloride, MES monohydrate, and agar powder were purchased from BBI; TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix and pEASY-BluntZero Cloning Kit were purchased from Transgen; Gel Extraction Kit was purchased from Omega; 2x Es Master Mix (Dye) was purchased from CWBIO; and Tryptone and YEAST EXTRACT were purchased from OXOID.

[0033] 2. Experimental Methods

[0034] 2.1 Transcriptome sequencing of lncRNAs

[0035] Total RNA was extracted from ABA-treated and untreated 2-week-old wild-type Arabidopsis thaliana using the Trizol method. Strand-specific RNA-seq libraries were prepared and deep sequenced at the Shanghai Center for Plant Stress Biology (Shanghai, China). These libraries were constructed according to the manufacturer's instructions using TruSeq Stranded mRNA (Illumina, San Diego, CA, USA). The quality of the RNA-seq libraries was assessed using an Advanced Analytical (IA, USA) fragment analyzer, and the resulting libraries were sequenced on an Illumina HiSeq 2500 instrument, producing 100 or 125 nucleotide end reads. Based on the characteristics of long non-coding RNAs, further filtering and analysis were performed using bioinformatics to screen for long non-coding RNAs associated with the ABA response, including lncRNA ARTA.

[0036] 2.2 Detection of lncRNA ARTA

[0037] 2.2.1 Total RNA Extraction

[0038] Two-week-old wild-type Arabidopsis seedlings were used as subjects. Total RNA extraction was performed according to the TriZol instruction manual. The specific operation was as follows: (1) Take about 0.1g of fresh plant material into a 1.5mL RNA-free centrifuge tube, freeze it in liquid nitrogen, break it up with a homogenizer and place it in liquid nitrogen; (2) Add 1mL of TriZol, shake vigorously on a shaker to mix well, and let it stand for 5min; (3) Add 200μL of chloroform, shake vigorously on a shaker to mix well, and let it stand for 5min; (4) Place it in a pre-cooled 4℃ centrifuge and centrifuge at 12000rpm. Centrifuge for 15 min; (5) Take 450 μL of supernatant and add an equal volume of isopropanol, shake to mix, and let stand for 15 min; (6) Place in a pre-cooled 4℃ centrifuge and centrifuge at 12000 rpm for 15 min; (7) Discard the supernatant, add 75% ethanol prepared with DEPC water to wash the precipitate twice; (8) Discard the 75% ethanol, and centrifuge briefly once at 4℃, and remove the residual ethanol with an RNA-free pipette tip; (9) Open the centrifuge tube cap, let stand for 5 min, and then add 35 μL of DEPC water to dissolve. The quality of RNA was detected by agarose gel electrophoresis. The OD260, OD280 and OD230 of the extracted total RNA were measured by NanoPhotometer, the RNA concentration was recorded, and stored at -80℃ for later use.

[0039] 2.2.2 Reverse transcription reaction

[0040] Using the extracted total RNA (3 μg) as a template, the reaction system was prepared according to the TransScript One-Step cDNASynthesis SuperMix instructions, and the mixture was incubated at 42℃ for 30 min and 85℃ for 5 sec to obtain cDNA for later use.

[0041] 2.2.3 Primer design and amplification

[0042] Based on the nucleotide sequence of lncRNA ARTA, primers for lncRNA ARTA overexpression were designed using Primer 5.0 software. Full-length amplification was performed using the high-fidelity enzyme PrimeSTAR Max DNA Polymerase (2x). The primers are as follows:

[0043]

[0044] The reaction system and reaction procedure are as follows:

[0045] reagents Volume added (μL) cDNA 3 PrimeSTAR Max DNA Polymerase(2x) 25 10μM Forward Primer 1.5 10μM Reverse Primer 1.5 <![CDATA[RNA-free H2O]]> Add to a total volume of 50 μL

[0046] Step 1: 98℃ for 3 minutes

[0047] Step 2: 98℃ for 10 seconds

[0048] Step 3: 55℃ for 15 seconds (adjust according to the annealing temperature of each primer)

[0049] Step 4: 72℃ for 1 min (adjust according to the length of the amplified fragment, 1kb / 1min)

[0050] Repeat steps 2-4 35 times.

[0051] Step 5: 72℃ for 10 minutes

[0052] Step 6: Keep warm at 12℃

[0053] After the PCR setup program is complete, take samples and run them on a 1% agarose gel, then use ChemiDoc. TM Observe gel images using the MP-Bio-Rad gel imaging system ( Figure 1 Cut off the corresponding size bands from the gel, use the Omega Gel Extraction Kit to recover the PCR product from the gel, and finally add 30 μL of sterile ddH2O to elute. After obtaining the sample, use a spectrophotometer to detect the concentration. Store at 4℃ or -20℃ for later use.

[0054] 2.2.4 Identification of nucleotide sequences

[0055] According to the pEASY-Blunt Zero Cloning Kit instructions, 1 μL of the recovered product and 1 μL of the pEASY-Blunt Zero Cloning vector were added, gently mixed, and reacted at 25°C for 5 min. After incubating on ice for 1 min, the mixture was directly transformed into E. coli DH5α competent cells. The plates were incubated overnight at 37°C. Single clones were picked and cultured for 10-12 h for colony PCR detection. The plasmids of positive single clones were extracted and sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing and identification, which confirmed the existence of the lncRNA ARTA in Arabidopsis thaliana.

[0056] Example 2: Overexpression of lncRNA ARTA

[0057] 1. Materials and Reagents

[0058] 1.1 Materials

[0059] The background is the Arabidopsis Col-0 lncRNA ARTA mutant (CS834193, arta-2).

[0060] 1.2 Reagents

[0061] Silwet L-77 was purchased from Sigma, and the ClonExpress II One Step Cloning Kit and plasmid extraction kit were purchased from Novizan Biotechnology Co., Ltd.

[0062] 2. Experimental Methods

[0063] 2.1 Construction of lncRNA ARTA overexpression vector

[0064] The plant overexpression vector pCAMBIA1300 was linearized with BamHI and EcoRI, and the correctly sequenced lncRNA ARTA sequence fragment was ligated into the vector using ClonExpress II recombinase. The reaction was carried out at 37°C for 30 min, followed by transformation into *E. coli* DH5α. An appropriate amount of bacterial culture was plated onto LB agar plates containing kanamycin and incubated overnight at 37°C. Single colonies were picked and streaked for 10-12 h, followed by colony PCR detection. Figure 2 Positive single clones were selected for culture by shaking, and plasmids of the corresponding single clones were extracted and sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. After comparison with the lncRNA ARTA sequence, the vector construction was confirmed to be successful. Plasmid extraction was performed according to the plasmid extraction kit instructions.

[0065] 2.2 Transformation of Agrobacterium GV3103 with lncRNA ARTA overexpression vector

[0066] The successfully constructed pCAMBIA1300-ARTA plasmid was transformed into Agrobacterium GV3101. Approximately 1 μg of plasmid was added to every 50 μL of competent cells, and the mixture was gently stirred by tapping the bottom of the tube. The tube was then incubated sequentially on ice for 10 min, in liquid nitrogen for 1 min, in a 37°C water bath for 5 min, and in an ice bath for 5 min. Subsequently, 800 μL of antibiotic-free LB broth was added, and the tube was incubated at 28°C with shaking for 2–3 hours. 100 μL of the bacterial culture was spread onto an LB agar plate containing kanamycin and rifampin. The plate was inverted and incubated at 28°C for 48 hours. Single colonies were then picked for colony PCR identification.

[0067] 2.3 Agrobacterium infection of Arabidopsis plants

[0068] The above-mentioned pCAMBIA1300-ARTA Agrobacterium was streaked on LB (containing kanamycin and rifampicin) solid medium and cultured until cells grew. A small amount of cells was then picked and placed in 5 mL of LB (containing kanamycin and rifampicin) liquid medium and cultured overnight at 28°C with shaking (until the medium became turbid). After preservation, all cells were transferred to 50 mL of fresh LB (containing kanamycin and rifampicin) liquid medium and cultured at 28°C with shaking until turbidity (OD600 = 1.0). The cells were collected by centrifugation at 4000 rpm for 15 min and then resuspended in transformation solution (1 / 2 MS (M524), 50 g / L sucrose, pH adjusted to approximately 5.8, and 200 μL of Silwet L-77). The suspended cells were directly immersed in the aboveground parts of Arabidopsis thaliana plants in full bloom for about 1 min, then completely wrapped with plastic wrap to retain moisture, and placed back in the incubation room in the dark for 24 hours. After removing the plastic wrap, the plants were placed under normal light conditions for further culture.

[0069] Example 3: Phenotypic identification of lncRNA ARTA mutants and overexpressing plants

[0070] 1. Materials and Reagents

[0071] 1.1 Materials

[0072] Arabidopsis wild-type Col-0, arta-2, and ARTA overexpressing plants

[0073] 1.2 Reagents

[0074] Abscisic acid (ABA) was purchased from Sigma, and TB Green Advantage qPCR premixes were purchased from Takara.

[0075] 2. Experimental Methods

[0076] 2.1. Primer Design

[0077] Based on the nucleotide sequence of lncRNA ARTA, qPCR primers were designed using Primer 5.0 software. The product length was approximately 138 bp. UBQ3 was used as an internal control gene. The primer sequences are as follows:

[0078]

[0079] 2.2.Quantitative real time PCR

[0080] RNA was extracted from Col-0, arta-2, and lncRNA ARTA overexpressing plants, and reverse transcribed (using the same method as in Example 1). Using cDNA diluted 20-fold as a template, real-time quantitative PCR was performed on a BioRad CFX96 quantitative PCR instrument using the TB Green Advantage qPCR premixes kit to detect the expression level of lncRNA ARTA. Arabidopsis thaliana UBQ3 was used as an internal control gene to verify the effectiveness of the mutants and overexpression. The following reaction system was prepared on ice:

[0081]

[0082]

[0083] Quantitative real-time PCR was performed using a two-step method. The amplification program consisted of 95℃ for 30 s pre-denaturation, followed by 45 cycles of PCR at 95℃ for 15 s and 55℃ for 30 s. Melting curve analysis was then performed to obtain the Ct value. UBQ3 gene expression level was used as a standard internal control, and the relative level of lncRNA ARTA expression in each sample was measured using 2^-ΔΔCt. Figure 3 Each sample was replicated three times, and each biological replicate was performed three times technically.

[0084] 2.3 Identification of ABA sensitivity phenotypes on flat plates

[0085] (1) Prepare 1 / 2 MS medium (1L):

[0086]

[0087] After sterilizing at 121℃ for 21 minutes, pour the contents into a 10×10cm square dish and let it air dry.

[0088] (2) Seed washing: Select an appropriate amount of Col-0, arta-2, lncRNA ARTA overexpression seeds, first wash with 75% alcohol for 30s to 1min, absorb the alcohol, add 1mL of water to wash once, then wash with sodium hypochlorite: water = 1:8 for 10 minutes, and then wash with sterilized ultrapure water 6 to 7 times.

[0089] (3) Seeding: Place the washed seeds onto the solidified 1 / 2 MS medium;

[0090] (4) Low temperature treatment: Wrap the plate with seeds in newspaper and put it in a chromatography cabinet at 4℃ for 3 days.

[0091] (5) Growth and development: Take out the plate from (4) and place it in a PERCIVAL incubator (22℃, 16h light / 8h dark). After the seeds germinate, culture for 4 days.

[0092] (6) Stress treatment: Four-day-old seedlings of almost the same size were transferred to 1 / 2 MS medium containing ABA and 1 / 2 MS medium without ABA respectively and then placed in a PERCIVAL incubator for vertical culture for 6 to 8 days.

[0093] (7) Results Analysis: Photos were taken, and the root length (Image J) and above-ground fresh weight (weighing) of each seedling were recorded. Figure 4 ).

[0094] 2.4 Soil drought stress

[0095] Seedlings (non-bolting) that grew normally for 3 weeks in soil (vermiculite: nutrient soil = 3:1) under constant temperature and long day (16h light / 8h darkness) at 22℃ were dried for about 2 weeks and then rehydrated for 3 days. Phenotypic characteristics were observed. Wild-type Col-0 was used as the control. Figure 5 ).

[0096] 2.5 Detection of plant water loss rate

[0097] Three-week-old seedlings (non-bolting) grown in normal soil under constant temperature and long-day conditions (16h light / 8h darkness) at 22℃ were selected. Five to six rosette leaves of equal size were selected as one replicate, for a total of three replicates. The weight of the cut rosette leaves was measured at different time points, and the data were then analyzed. Figure 6 ).

[0098] 2.6. Experimental Results

[0099] According to qRT-PCR results, lncRNA ARTA was almost not expressed in arta-2, but its expression level in overexpressing plants was significantly higher than that in wild-type plants (P < 0.05, two-sample independent t-test). Figure 3 Furthermore, in phenotypic experiments, arta-2 was insensitive to ABA but sensitive to drought stress, and its detached leaf water loss rate was higher than that of the wild type. In contrast, lncRNA ARTA overexpressing plants exhibited the opposite phenotype: they were sensitive to ABA but showed strong tolerance to drought stress, and their detached leaf water loss rate was comparable to that of the wild type. Figure 4-6 These results further demonstrate that lncRNA ARTA is not only involved in the plant response to ABA, but also emphasize that overexpression of lncRNA ARTA can enhance the drought resistance of plants.

[0100] The above description merely illustrates preferred embodiments of the present invention and is quite specific and detailed, but it should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications, improvements, and substitutions within the spirit and principles of this application, and these all fall within the protection scope of the present invention.

Claims

1. A long non-coding RNA from Arabidopsis thaliana ARAT Its characteristics are, Its nucleotide sequence is shown in SEQ ID NO.

1.

2. Containing the Arabidopsis long non-coding RNA as described in claim 1 ARTA Plant expression vectors.

3. Recombinant bacteria containing the plant expression vector of claim 2.

4. The Arabidopsis long non-coding RNA of claim 1 ARTA The application of the plant expression vector of claim 2 or the recombinant bacteria of claim 3 in improving the drought resistance of Arabidopsis thaliana.

5. Arabidopsis long non-coding RNA ARTA Its application in improving the drought resistance of Arabidopsis thaliana is characterized by, Includes the following steps: (1) Constructing a long non-coding RNA of Arabidopsis thaliana containing the RNA shown in SEQ ID NO.1 ARTA Plant overexpression vectors; (2) The plant overexpression vector from step (1) was transformed into the mutant, which increased the long non-coding RNA in Arabidopsis thaliana. ARTA The expression level of Arabidopsis thaliana can be increased to enhance its drought resistance.

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