KR-20260062961-A - Modified RNA demethylase and nucleic acid encoding the same, uses, plant cultivation methods, and products

Abstract

A modified RNA demethylase, a nucleic acid encoding the modified RNA demethylase, its use, a method for plant cultivation, and a product are provided. Compared to the original RNA demethylase derived from algae or invertebrates, the modified RNA demethylase is obtained by adding a specific peptide fragment sequence to the N-terminus or C-terminus of the original RNA demethylase. The modified RNA demethylase or the nucleic acid encoding the modified RNA demethylase is introduced into an early plant to obtain a target plant with optimized traits compared to the early plant.

Inventors

• 리 지
• 리 웨이펑

Assignees

• 에피플랜트 컴퍼니 리미티드

Dates

Publication Date

20260507

Application Date

20240628

Priority Date

20230831

Claims (20)

1. A modified RNA demethylase obtained by adding at least one peptide sequence to the N-terminus and/or C-terminus compared to an original RNA demethylase derived from an algae or invertebrate.
2. In claim 1, the peptide sequence has the following amino acid sequence characteristics: a) containing at least three amino acids selected from the group consisting of H, K and R; and b) containing at least one amino acid selected from the group consisting of PY, and H, K, and R A modified RNA demethylase having any of the following.
3. A modified RNA demethylase according to claim 1, wherein, when the peptide sequence has the amino acid sequence characteristics of a), at least two amino acids selected from H, K, or R are consecutive or contain V or I.
4. A modified RNA demethylase according to claim 1, wherein the peptide sequence has any of the following amino acid sequence characteristics: (K/R)(K/R)X 1-2 (K/R), (K/R)X 1-2 (K/R)(K/R), (K/R)(K/R)X 10-12 (K/R) 2-5 , (R/K/H)X 2-5 PY, or K(V/I)XKX 1-2 (K/H/R), where X is any amino acid.
5. The modified RNA demethylase according to claim 1, wherein the peptide sequence comprises at least one of RPRK, RKVIK, RNKKKK, PKKKRKV, KRPAATKKAGQAKKKK, TLLLRETMNNLGVSDHAVLSRKTPQPY, and KRTPTAEERERGAKKLRLLEELEDTWLPYL.
6. In claim 1, the modified RNA demethylase comprises at least one of its homologue or variant/conjugate having equivalent function.
7. In claim 1, the original RNA demethylase is a modified RNA demethylase that promotes the demethylation of N6-methyladenine in RNA.
8. In claim 1, the modified RNA demethylase is the same as that shown in sequence list sequence numbers 13 to 48.
9. In claim 1, the invertebrate comprises a modified RNA demethylase containing at least one of acanthaster plankie or sarcogloss.
10. A modified RNA demethylase according to claim 1, wherein the algae include at least one of blue algae, red algae, cryptoflagellates, dinoflagellates, yellow algae, yellow-green algae, diatoms, brown algae, Euglena, green algae, and rotifers.
11. A nucleic acid encoding the modified RNA demethylase as described in claim 1.
12. In paragraph 11, the above nucleic acid is DNA, a nucleic acid.
13. Use for plant introduction of the modified RNA demethylase described in claim 1 or the nucleic acid described in claim 11.
14. In paragraph 13, a use comprising either an increase in yield or an increase in biomass.
15. In paragraph 13, the above plants include at least one of rapeseed plants, tomato, lettuce, sugar beet, rice, corn, soybean plants, potato, wheat, millet, sugarcane, sorghum, cassava, tobacco, alfalfa, rubber grass, cotton, flax, sunflower, camelina, knotweed, cannabis, and poplar.
16. A method for cultivating plants, comprising the steps of introducing the modified RNA demethylase described in claim 1 or the nucleic acid described in claim 11 into the initial plant, and obtaining a target plant having optimized traits by comparison with the initial plant.
17. A method for cultivating plants according to claim 16, wherein the above trait optimization includes at least one of increased yield and increased biomass.
18. A method of plant cultivation according to claim 16, wherein the modified RNA demethylase or the nucleic acid according to claim 1 is introduced in excess to the initial plant.
19. A method of plant cultivation according to claim 16, wherein the initial plant is selected from at least one of rapeseed plants, tomato, lettuce, sugar beet, rice, corn, soybean plants, potato, wheat, millet, sugarcane, sorghum, cassava, tobacco, alfalfa, rubber grass, cotton, flax, sunflower, camelina, knotweed, cannabis, and poplar.
20. A method for cultivating plants according to claim 16, wherein the part of the plant undergoing the introduction treatment comprises at least one of a plant organ, a plant tissue, or a plant cell.

Description

Modified RNA demethylase and nucleic acid encoding the same, uses, plant cultivation methods, and products The present invention relates to the field of genetic engineering and plant cultivation, in particular to modified RNA demethylase, nucleic acid encoding modified RNA demethylase, uses, methods for plant cultivation, and products. Artificial intervention methods for plant cultivation typically encompass various approaches, such as genetic engineering, cell engineering, and microbial engineering. To obtain new plant varieties, the aforementioned methods are generally used. New plant varieties introduced herein include whole plants, plant organs (e.g., flowers, fruits, seeds, roots, stems, leaves, etc.), plant tissues, plant cells, and other parts that may be derived from plants. Newly obtained plant varieties are generally expected to exhibit more optimized traits, such as optimized growth, increased yield, optimized biomass, optimized structure, or optimized cell division. Among the aforementioned optimized traits, increased yield can be achieved through various means, such as increasing the size or weight of individual seeds/thousand-seeds, increasing the number of root tillers, and other factors that may influence plant yield, particularly increasing the desirable yield of the parts obtained from the plant. The aforementioned expectations are often achieved through specific modification methods, such as altering plant DNA or histones, to achieve the purpose of changing plant traits. Even prior to the present invention, research on optimizing plant traits through demethylation of the 6mA region of plant RNA has been conducted. Figure 1 is a schematic diagram of the demethylation process of N6-methyladenine. Figure 2 is a graph comparing plant tubers obtained from the experimental group and the blank group using potatoes as an example. Figure 3 is a comparison image of the entire plant obtained from the experimental group and the blank group, using rice as an example. Specific implementation method To enable those skilled in the art to better understand the technical solution of the present invention, a more detailed description of the present invention is provided below, along with specific embodiments. Sequence modification of the original RNA demethylase derived from birds or invertebrates. Original RNA demethylases obtained from various algae or invertebrates are subjected to gene sequencing, screening, and amplification to obtain corresponding sequence samples. The sequences of original RNA demethylases from various algal sources are shown in sequence list numbers 1–12. Each sequence is recombined into the p1307-FLAG vector via direct PCR to obtain p1307-RNA demethylase-FLAG. Subsequently, the RNA demethylase-FLAG fragment is amplified and recombined into the pGEX-His vector to obtain pGEX-RNA demethylase-FLAG-His, which can be used for subsequent in vitro experiments and peptide sequence introduction. The plasmid pGEX-RNA demethylase-FLAG-His is circularized and recombined using primers containing the peptide sequence, after which the resulting modified gene fragment is reintroduced into the p1307-FLAG vector. This results in the modified RNA demethylase of the present invention, the specific sequence of which is as shown in sequence list numbers 13 to 48. The obtained modified RNA demethylase may introduce a peptide sequence to at least one end of the original protein, either the N-terminus or the C-terminus. For example, introducing a peptide sequence to the N-terminus involves inserting an NLS sequence between the first amino acid M and the second amino acid of the original protein. Similarly, introducing a peptide sequence to the C-terminus involves adding an NLS sequence after the last amino acid of the original protein. Embodiment 1: Method for obtaining a target plant through Agrobacterium infection of callus tissue 1. Induce callus tissue using plant seeds as test material. 1) Sterilization: Take mature plant seeds, remove the hulls manually, and select plump, clean seeds free of sterile spots. Place the seeds in a 100ml sterile beaker and pour in 70% alcohol to disinfect for 2 minutes; discard the alcohol, add a 20% sodium hypochlorite (NaClO) solution, and soak for 30 minutes; discard the sodium hypochlorite solution, rinse the seeds 4 to 5 times with sterile distilled water, and finally soak in sterile distilled water for 30 minutes. 2) Induction culture (aseptic work required): After sterilizing the seeds, place them on sterile filter paper to absorb surface moisture, then transfer 12 to 14 seeds per dish into NB medium containing 2,4-D 2.0 mg/L (pH 5.8). To ensure the induction rate, it is best to keep the germination direction of the seeds parallel to or slightly downward relative to the medium during inoculation; upward or vertically downward orientations should be avoided. After the procedure, cover the culture dishes with sealing film and incubate in a 30°C light incubator with a