A method for improving stress tolerance of plants based on pre-rRNA and use of pre-rRNA

Abstract

The application provides a method for improving stress tolerance of plants based on pre-rRNA and application of pre-rRNA, and the method comprises the following steps: using a stress-induced expression promoter to regulate transcription of any continuous fragment on pre-rRNA, so that transcription is not started under normal growth conditions, transcription is started to generate risiRNA when plants are subjected to abiotic stress, and stress tolerance of the plants is improved; and the nucleotide sequence of the pre-rRNA is shown as SEQ ID NO. 1. The risiRNA is a stress tolerance factor, and can help plants to cope with environmental stress of abiotic stress. The stress-induced expression promoter regulates transcription of any continuous fragment on pre-rRNA to generate a risiRNA precursor, and the stress-induced expression promoter regulates transcription of the risiRNA precursor to generate risiRNA when plants are subjected to abiotic stress. The method can improve stress tolerance of plants under stress conditions by activating gene expression to generate risiRNA under stress conditions, and meanwhile, the problem of growth inhibition caused by expression under non-stress conditions can be avoided.

Description

Technical Field

[0001] This invention relates to the technical field of genetic engineering, and in particular to a method for improving plant stress tolerance based on pre-rRNA and the application of pre-rRNA. Background Technology

[0002] Globally, drought is one of the most critical factors leading to reduced plant yields. Due to the widespread uneven distribution of water resources and the dramatic increase in modern water demand, crops worldwide face an even more severe drought threat, necessitating the discovery of key factors regulating plant drought tolerance and the creation of drought-resistant germplasm through genetic engineering. Currently, drought-resistant genes in plants have been reported. However, while overexpressing drought-resistant genes using traditional constitutive promoters improves drought tolerance, the genes are also highly expressed under normal growth conditions, easily leading to slower plant growth and reduced yields. This significantly limits the practical application of using drought-resistant genes to improve germplasm. Therefore, discovering more drought-resistant factors and developing appropriate application methods is crucial. Furthermore, in field production, drought stress often occurs alongside high light stress. High light damages chloroplasts, causing a large accumulation of reactive oxygen species in cells, reducing photosynthetic efficiency, and ultimately leading to reduced crop yields. Similarly, in genetic engineering breeding, balancing plant high light tolerance with yield is also very important. Summary of the Invention

[0003] The purpose of this invention is to provide a method for improving plant stress tolerance based on pre-rRNA and the application of pre-rRNA.

[0004] The first aspect of this invention provides a method for improving plant stress tolerance based on pre-rRNA, characterized in that it includes: The stress-induced expression promoter is used to regulate the transcription of risiRNA precursors to produce risiRNA precursors, so that the transcription is not initiated in plants under normal growth conditions, but is initiated to generate risiRNA when plants are subjected to non-biological stress, thereby improving the stress tolerance of plants. The risiRNA precursor is any continuous fragment on the pre-rRNA, and the nucleotide sequence number of the pre-rRNA is shown in SEQ ID NO.1.

[0005] Furthermore, the length of any of the continuous segments ranges from 200 to 400 base pairs.

[0006] Furthermore, the stress-induced expression promoter is proAtRD29A, and its nucleotide sequence number is shown in SEQ ID NO.2.

[0007] Furthermore, the use of stress-induced expression promoters to regulate the transcription of risiRNA precursors includes: Construct a risiRNA expression vector based on the stress-induced expression promoter proAtRD29A; The expression vector was transformed into plants to obtain proAtRD29A:risiRNA transgenic plants; The transgenic plants do not produce risiRNA when not subjected to abiotic stress, but initiate transcription to produce risiRNA when subjected to abiotic stress, thereby improving the plant's stress tolerance.

[0008] Furthermore, the construction of the risiRNA expression vector based on the stress-induced expression promoter proAtRD29A includes: The proAtRD29A containing homologous arms, the reverse sequence of the pre-rRNA transcript fragment, and the intron-pre-rRNA transcript fragment were obtained by PCR amplification. Linearized vector fragments were prepared by double digestion of the pRS415 vector with HindIII and SacI restriction endonucleases. The above fragments were gelled and recovered, then transferred into BY4741 yeast competent cells for recombinant reaction; After the recombinant vector was sequenced without errors, the recombinant fragment was excised by double digestion with HindIII and SacI restriction endonucleases. The expression vector was obtained by ligating the pGWB640 vector, which had been linearized by double digestion with HindIII and SacI restriction endonucleases, using T4 DNA ligase and transforming it into E. coli.

[0009] Furthermore, the transgenic plant is Arabidopsis thaliana.

[0010] Furthermore, the transformation of the expression vector into the plant includes: The expression vector was transformed into Arabidopsis thaliana using the Agrobacterium-mediated flower-dipping method to obtain proAtRD29A:risiRNA transgenic plants.

[0011] A second aspect of the present invention provides an application of pre-rRNA, wherein any continuous fragment on the pre-rRNA is used to transcribe a risiRNA precursor into a plant when the plant is subjected to abiotic stress, and the risiRNA precursor is digested to generate risiRNA to improve the stress tolerance of the plant. The nucleotide sequence number of the pre-rRNA is shown in SEQ ID NO.1.

[0012] Furthermore, any of the aforementioned continuous segments are used to improve the drought resistance of plants.

[0013] Furthermore, any of the aforementioned continuous segments are used to improve the plant's tolerance to high light intensity.

[0014] The beneficial effects of this plan are as follows: This application provides a method for enhancing plant stress tolerance based on pre-rRNA and an application of pre-rRNA. risiRNA is an abiotic tolerance factor that helps plants cope with abiotic stress. Any continuous fragment of the pre-rRNA is transgenic into the plant along with a stress-inducible expression promoter. When the plant is subjected to abiotic stress, the transcribed risiRNA precursor is processed by endonuclease within the plant to generate the mature risiRNA, thereby enhancing the plant's stress tolerance. This method, through the use of a stress-inducible expression promoter, activates gene expression to generate risiRNA only under stress conditions, thus improving plant tolerance under stress conditions and avoiding growth inhibition caused by expression under non-stress conditions. Attached Figure Description

[0015] Figure 1 Map of the pGWB640-proAtRD29A-risiRNA plant expression vector; Figure 2 A schematic diagram illustrating the construction of the expression carrier; Figure 3 Northern blot was used to detect the abundance of risiRNA in plants after drought stress treatment (U6 was used as an internal control). Figure 4 The phenotype of proAtRD29A:risiRNA transgenic plants in drought tolerance tests; Figure 5 A comparison of Fv / Fm values ​​in proAtRD29A:risiRNA transgenic plants during high light tolerance testing; Figure 6 The phenotype of the proAtRD29A:risiRNA transgenic plant in the high light tolerance test. Detailed Implementation

[0016] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0017] Small interfering RNAs (siRNAs) are a class of 21-24 base-length non-coding small RNA molecules derived from double-stranded RNA transcripts. They are widely found in eukaryotes and can bind to Argonaute (AGO) proteins to form RNA-induced silencing complexes (RISCs), thereby silencing the expression of downstream target genes at the transcriptional or post-transcriptional levels. siRNAs derived from ribosomal RNA (rRNA) are called rRNA-derived small interfering RNAs (risiRNAs). The production of risiRNA by expressing specific rRNA precursor (pre-rRNA) sequences in plants can be achieved using RNAi expression vectors through transcription of a stem-loop transcript. The stem-loop structure consists of an intron sequence in the central "loop" region and two inversely complementary sequences in the two "stem" regions at either end. The double-stranded region of the "stem" portion of the stem-loop transcript is cleaved by the Dicer-like (DCL) endonuclease in vivo, ultimately forming the mature siRNA. Therefore, risiRNA can be generated by replacing the specific sequence into the "stem" regions at both ends of the RNAi stem-loop transcript.

[0018] Constitutive promoter-driven gene overexpression is often accompanied by pleiotropic effects. Plants overexpressing drought-resistant genes driven by the conventional cauliflower mosaic virus 35S promoter (proCaMV35S) often exhibit improved drought resistance but also phenotypes of stunted growth and reduced fertility. proAtRD29A is a promoter derived from the Arabidopsis thaliana Response to Dehydration 29A (AtRD29A) gene. This promoter is a stress-specific promoter, initiating gene transcription only when the plant is under stress, and not under normal growth conditions.

[0019] However, the application of the Arabidopsis stress-induced expression promoter proAtRD29A to novel non-biological tolerance factors such as risiRNA in improving plant drought resistance is still relatively rare.

[0020] This embodiment discloses a ribosomal RNA precursor (pre-rRNA), whose nucleotide sequence number is shown in SEQ ID NO.1.

[0021] Any continuous fragment of 200-400 base pairs on the pre-rRNA can be used for inducible expression under abiotic stress to improve plant stress tolerance. Specifically, examples of continuous fragments are shown below: ①The risiRNA-ETS fragment, whose nucleotide sequence number is shown in SEQ ID NO.4; ②The risiRNA-18S fragment, whose nucleotide sequence number is shown in SEQ ID NO.5; ③The risiRNA-4875 fragment, whose nucleotide sequence number is shown in SEQ ID NO.6; ④The risiRNA-7957 fragment, whose nucleotide sequence number is shown in SEQ ID NO.7.

[0022] Among them, the risiRNA-ETS fragment, risiRNA-18S fragment, risiRNA-4875 fragment, and risiRNA-7957 fragment can all be transcribed under the regulation of stress-induced expression promoters. After transcription, risiRNA hairpin structure precursors (stem-loop structures) are produced, which are then processed by endonucleases in the plant to produce mature risiRNA, thereby improving the plant's stress tolerance. They can enhance the plant's tolerance to drought, high salinity, high temperature, low temperature, and strong light.

[0023] Abiotic stress refers to pressures caused by environmental factors that adversely affect plant growth and development, such as drought, high salinity, high temperature, low temperature, and strong light. These stress conditions activate stress responses within plants, affecting their normal physiological functions.

[0024] Inducible expression refers to the phenomenon where gene expression levels are significantly increased under specific stimuli (such as non-biological stress). Unlike constitutive expression, inducible expression ensures that genes are transcribed and translated in large quantities only when needed, thereby avoiding unnecessary metabolic burdens or negative effects on plants under normal growth conditions.

[0025] Stress tolerance refers to a plant's ability to maintain normal growth, development, and reproduction when subjected to abiotic stresses. Improving a plant's stress tolerance helps it survive adverse environments and maintain yield.

[0026] Furthermore, this embodiment uses the risiRNA-7957 fragment as an example for illustration.

[0027] Specifically, this embodiment discloses a method for the inducible expression of risiRNA-7957. The method uses the stress-induced expression promoter proAtRD29A to regulate the transcription of the risiRNA-7957 precursor fragment, enabling the risiRNA to be generated when the plant is subjected to abiotic stress, but not under normal plant growth conditions. The nucleotide sequence number of the stress-induced expression promoter proAtRD29A is shown in SEQ ID NO.2. Based on the stress-specific response of the proAtRD29A promoter and the regulatory function of risiRNA on the plant's tolerance to drought and high light, this invention provides an application of risiRNA in improving plant tolerance to stressful environments.

[0028] This embodiment also discloses an expression vector containing a hairpin transcript of the risiRNA-7957 RNAi precursor driven by the proAtRD29A promoter, denoted as pGWB640-proAtRD29A-risiRNA-7957, whose nucleotide sequence number is shown in SEQ ID NO.3. See also Figure 1 .

[0029] Based on the principle of siRNA generation, the sequence “7957” (SEQ ID NO. 7), a fragment at the junction of the 25S and 3' ETS regions of the Arabidopsis pre-rRNA transcript that can generate risiRNA, was expressed using an RNAi expression vector to produce risiRNA. The RNAi stem-loop structure of this expression vector is transcribed by the stress-specific promoter proAtRD29A (SEQ ID NO. 2), and risiRNA is expressed only under stress conditions. Transforming the constructed RNAi expression vector into Arabidopsis yields proAtRD29A:risiRNA-7957 transgenic plants.

[0030] Furthermore, the proAtRD29A:risiRNA-7957 transgenic plant disclosed in this embodiment contains the aforementioned expression vector. This plant can activate a tolerance protection mechanism during periods of non-biological stress, while maintaining a normal physiological state during non-stress periods. The transgenic plant is Arabidopsis thaliana.

[0031] This invention constructs Arabidopsis plants that express risiRNA-7957 driven by the proAtRD29A promoter, enabling risiRNA to accumulate and be expressed only when the plant is under stress. This improves the plant's tolerance to drought and high light intensity without affecting its growth, development, and fertility under normal growth conditions, providing an effective solution for creating plant germplasm that is both drought-tolerant and light-tolerant.

[0032] This embodiment discloses a method for improving plant stress tolerance based on pre-rRNA, including: S1. Obtain the individual fragments used for recombination, see [link to documentation]. Figure 2 , S11. PCR amplification yielded proAtRD29A (containing a 5' HindIII restriction endonuclease site), the reverse sequence of the risiRNA-7957 transcript fragment, and the intron-risiRNA-7957 transcript fragment (containing a 3' SacI restriction endonuclease site). Specifically, the AtRD9A fragment with a 5' adapter was obtained by PCR amplification using primers PRS415-HindIII-AtRD29A-F (nucleotide sequence number shown in SEQ ID NO. 8) and AtRD29A-R (nucleotide sequence number shown in SEQ ID NO. 12); the reverse sequence of the 7957' transcript fragment with a 5' adapter was obtained by PCR amplification using primers AtRD29A-7957'-F (nucleotide sequence number shown in SEQ ID NO. 9) and 7957'-R (nucleotide sequence number shown in SEQ ID NO. 13); and the intron-7957 transcript fragment with 5' and 3' adapters was obtained by PCR amplification using primers 7957'-intron-F (nucleotide sequence number shown in SEQ ID NO. 10) and PRS415-SacI-7957-R (nucleotide sequence number shown in SEQ ID NO. 11).

[0033] S12. The pRS415 vector was prepared by double digestion with HindIII and SacI restriction endonucleases to prepare a linearized large fragment of the vector, which was then recovered. The double digestion reaction system is shown in Table 1.

[0034] Table 1. Double enzyme digestion reaction system

[0035] S2. The fragments are transferred into yeast cells to carry out the recombination reaction; S21. Pretreatment of Carrier DNA: After melting the Carrier DNA, place it in a 95°C metal bath for 5 minutes, then quickly place it in an ice bath for at least 3 minutes.

[0036] S22. Take 100 μL of BY4741 yeast competent cells, thaw them on ice, and pre-cool each DNA fragment. The reaction system for recombining each fragment in yeast competent cells is shown in Table 2.

[0037] Table 2 Yeast Recombination Reaction System

[0038] S23. After centrifuging at 5000 rpm for 1 min at room temperature, discard the supernatant, resuspend in 400 μL of sterile water, centrifuge again at 5000 rpm for 30 s at room temperature, discard the supernatant, add 100 μL of sterile water to resuspend and spread on yeast nutrient deficiency medium PM2201 SD / -Leu, and incubate at 28℃ for 48 h-96 h.

[0039] S3, positive clones of yeast colonies detected by PCR; S31. Pick 8 or more single colonies and place them in 30 μL of 20 mM NaOH solution to rupture the cell walls. The cell wall rupture procedure is shown in Table 3.

[0040] Table 3 Yeast cell wall breaking program

[0041] S32. After the cell wall is broken, take 1 μL of the cell wall breaking liquid as a bacterial culture, use appropriate primers to perform bacterial PCR on each fragment involved in recombination, and select single clones with the correct bands to be sent to the biotechnology company for sequencing.

[0042] S4, Transformation of Escherichia coli; After the recombinant vector was sequenced without errors, the recombinant fragment was excised by double digestion with HindIII and SacI restriction endonucleases; it was then ligated into the pGWB640 vector, which had been linearized by double digestion with HindIII and SacI restriction endonucleases, using T4 DNA ligase, and transformed into E. coli to obtain the expression vector.

[0043] Specifically, positive clones verified by sequencing can be used to extract plasmids. These plasmids are recombinant vectors containing the inverse sequence of the proAtRD29A7957' transcript fragment and the intron-7957 transcript fragment recombined on pRS415. To enable expression of this fragment in plants, the recombinant vector is double-digested with HindIII and SacI, and then ligated to the pGWB640 expression vector using T4 DNA ligase, resulting in the vector named pGWB640-proAtRD29A-7957. Details of the T4 ligation reaction system are shown in Table 4. The vector is then transformed into *E. coli*, and positive clones are screened by colony PCR and sequenced again. Specific operational steps can be found in the previously described methods. The identification of positive clones is detailed in [Table 4]. Figure 3 .

[0044] The endonuclease catalog number is: HindIII-HF® Cat # NEB R3104; SacI-HF NEB Cat # R3156.

[0045] Table 4. T4 Connection Reaction System

[0046] S5, Agrobacterium-transformation Positive clones verified by sequencing can be used to extract plasmids. The specific steps for transforming Agrobacterium with this vector can be found in the previously described methods. For information on identifying positive clones, please refer to [link to documentation]. Figure 3 .

[0047] S6. Agrobacterium-mediated flower-dipping genetic transformation in Arabidopsis thaliana. S61. Preparations before conversion Prepare 5-week-old Arabidopsis thaliana plants that are in good growth condition and have many open inflorescences. Water them with an appropriate amount of Flower Power Nutrient Fertilizer solution the day before conversion to fully open the stomata. Before conversion, cut off the siliques that have already grown on the Arabidopsis thaliana plants to improve the positive rate of seed harvesting after infection.

[0048] S62, Agrobacterium tumefaciens (expanded culture) The Agrobacterium tumefaciens culture medium containing the constructed plant expression vector, as described above, was inoculated into LB broth containing both Spec and Rif antibiotics for expansion. 100 μL of the culture medium was inoculated into 5 mL of the dual-antibiotic LB broth and cultured overnight at 28°C and 220 rpm. The next day, all of the expanded culture was added to 150 mL of the dual-antibiotic LB broth and cultured overnight at 28°C and 220 rpm until the OD600 of the expanded culture reached 1.2.

[0049] S63, Agrobacterium-mediated transformation of Arabidopsis thaliana. (1) After the cultured bacterial solution is centrifuged at 6000 rpm for 5 min to enrich the bacterial cells, the supernatant culture medium is discarded, and the bacterial cells are resuspended in 150 mL of infection solution. The infection solution formula is shown in Table 5. Table 5. Formula for Agrobacterium-mediated transformation of Arabidopsis thaliana infection solution

[0050] (2) Immerse the prepared Arabidopsis thaliana in the infection solution for 80 seconds, ensuring the inflorescence is completely submerged. Gently shake off any remaining infection solution from the infected Arabidopsis thaliana, place it on a tray, cover with a light-blocking cloth, and incubate in the dark at 23°C for 24 hours. The next day, place it in a normal growth environment in the culture room and lightly spray the infected Arabidopsis thaliana with water to quickly restore its vitality. Water it again after another day. A second infection can be performed after one week to improve the positive transformation rate. After the Arabidopsis thaliana matures, collect the seeds, dry them, and they can be used for subculturing or selection.

[0051] Drought resistance test: (1) The above transgenic plant proAtRD29A:risiRNA-7957 was screened for antibiotics to obtain T3 homozygous lines (#6-14 and #15-15). proAtRD29A:risiRNA-7957 was then cross-linked with Arabidopsis wild-type (Col-0) and Arabidopsis mutant. rdr6 and dcl234 (The risiRNA pathway in both strains was blocked, serving as a negative control.) The plants were cultured simultaneously under normal watering conditions and drought conditions with water cut-off. The abundance of risiRNA in the plants was detected by Northern blot. The results showed that under normal watering conditions, no risiRNA accumulated in the proAtRD29A:risiRNA-7957 plants, consistent with Col-0. However, under drought conditions with water cut-off for 5 days, compared with Col-0, the proAtRD29A:risiRNA-7957 plants expressed and accumulated risiRNA to varying degrees (Figure 3).

[0052] (2) Further, proAtRD29A:risiRNA-7957 was combined with Col-0, rdr6 and dcl234 Simultaneously, the drought resistance of the plants was tested under water shortage and drought treatment. The experimental results showed that the drought resistance of proAtRD29A:risiRNA-7957 plants was significantly higher than that of Col-0 (Figure 4A).

[0053] (3) Combine proAtRD29A:risiRNA-7957 with Col-0, rdr6 and dcl234 Under normal growth conditions, the growth, development and fertility of the plants were observed simultaneously. The results showed that the proAtRD29A:risiRNA-7957 plants did not show any growth retardation compared with Col-0 (Figure 4B); at the same time, no significant difference was found in the flowering time and number of proAtRD29A:risiRNA-7957 plants and Col-0 (Figure 4C).

[0054] in, Figure 4 Area A in the diagram represents the survival rate of plants after drought stress treatment and rehydration treatment. Area B represents the development of plants after 3 weeks of normal cultivation. Area C represents the bolting status of plants after 5 weeks of normal cultivation. Scale bar = 5 cm.

[0055] High gloss resistance test: High light stress treatment conditions: Four-week-old Arabidopsis transgenic plants proAtRD29A:risiRNA-7957 and Arabidopsis wild-type (Col-0) were subjected to high light stress at 600 μmol·m⁻¹. -2 ·s -1 Cultured under light intensity for 5 days, observe phenotype, and measure Fv / Fm value using a modulated chlorophyll fluorometer (scale bar = 2 cm).

[0056] Results of high light treatment: After high light treatment, the Fv / Fm values ​​of the two transgenic lines of proAtRD29A:risiRNA-7957 (#6-14 and #15-15) were significantly higher than those of the wild-type plant (Col-0), indicating that proAtRD29A:risiRNA-7957 is more tolerant to high light stress. See also Figure 5 and Figure 6 .

[0057] The stress-induced risiRNA transgenic Arabidopsis line proAtRD29A:risiRNA-7957 constructed using this method has the following advantages: 1. Transgenic plants do not express risiRNA-7957 under normal growth conditions, but rapidly express large amounts of risiRNA-7957 under stress induction. 2. Compared to the wild type, the transgenic plants are more drought-resistant.

[0058] 3. Compared to the wild type, the transgenic plants are more tolerant of high light.

[0059] 4. Compared with the wild type, no slow growth or reduced fertility was observed in the transgenic plants.

[0060] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they 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 and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A method for improving plant stress tolerance based on pre-rRNA, characterized in that, include: By using stress-induced expression promoters to regulate the transcription of any continuous segment on pre-rRNA, the plant does not initiate transcription under normal growth conditions, but initiates transcription to generate risiRNA when the plant is subjected to non-biological stress, thereby improving the plant's stress tolerance. The nucleotide sequence number of the pre-rRNA is shown in SEQ ID NO.

1.

2. The method for improving plant stress tolerance based on pre-rRNA according to claim 1, characterized in that, The length of any of the continuous segments ranges from 200 to 400 base pairs.

3. The method for improving plant stress tolerance based on pre-rRNA according to claim 1, characterized in that, The stress-induced expression promoter is proAtRD29A, and its nucleotide sequence number is shown in SEQ ID NO.

2.

4. The method for improving plant stress tolerance based on pre-rRNA according to claim 1, characterized in that, The use of stress-induced promoter expression to regulate the transcription of risiRNA precursors includes: Construct a risiRNA expression vector based on the stress-induced expression promoter proAtRD29A; The expression vector was transformed into plants to obtain proAtRD29A:risiRNA transgenic plants; The transgenic plants do not produce risiRNA when not subjected to abiotic stress, but initiate transcription to produce risiRNA when subjected to abiotic stress, thereby improving the plant's stress tolerance.

5. The method for improving plant stress tolerance based on pre-rRNA according to claim 4, characterized in that, The construction of the risiRNA expression vector based on the stress-induced expression promoter proAtRD29A includes: The proAtRD29A containing homologous arms, the reverse sequence of the pre-rRNA transcript fragment, and the intron-pre-rRNA transcript fragment were obtained by PCR amplification. Linearized vector fragments were prepared by double digestion of the pRS415 vector with HindIII and SacI restriction endonucleases. The above fragments were gelled and recovered, then transferred into BY4741 yeast competent cells for recombinant reaction; After the recombinant vector was sequenced without errors, the recombinant fragment was excised by double digestion with HindIII and SacI restriction endonucleases. The expression vector was obtained by ligating the pGWB640 vector, which had been linearized by double digestion with HindIII and SacI restriction endonucleases, using T4 DNA ligase and transforming it into E. coli.

6. The method for improving plant stress tolerance based on pre-rRNA according to claim 4, characterized in that, The transgenic plant is Arabidopsis thaliana.

7. The method for improving plant stress tolerance based on pre-rRNA according to claim 6, characterized in that, The process of transforming the expression vector into a plant includes: The expression vector was transformed into Arabidopsis thaliana using the Agrobacterium-mediated flower-dipping method to obtain proAtRD29A:risiRNA transgenic plants.

8. An application of a pre-rRNA, characterized in that, Any continuous fragment on the pre-rRNA is used to transcribe risiRNA into risiRNA when the plant is subjected to abiotic stress, thereby improving the plant's stress tolerance; The nucleotide sequence number of the pre-rRNA is shown in SEQ ID NO.

1.

9. The application of the pre-rRNA according to claim 8, characterized in that, Any of the aforementioned continuous segments is used to improve the drought resistance of plants.

10. The application of the pre-rRNA according to claim 8, characterized in that, Any of the aforementioned continuous segments are used to improve the plant's tolerance to high light intensity.

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