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CN-121975803-A - Enhancement of TaDREB3 gene expression using uORF sequence-based gene editing technique

CN121975803ACN 121975803 ACN121975803 ACN 121975803ACN-121975803-A

Abstract

The invention discloses a gene editing technology based on uORF sequence to improve TaDREB3 gene expression. The invention belongs to the technical field of biology, and particularly relates to a method for improving TaDREB3 gene expression by utilizing a gene editing technology based on uORF sequences. The present invention provides a DNA molecule of uORF wherein a) the nucleotide sequence is that of SEQ ID No.1, b) a DNA fragment having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more identity with the nucleotide sequence defined in a) and having the same function, c) a DNA fragment hybridizing with the nucleotide sequence defined in a) or b) under stringent conditions and having the same function. The inhibition of uORF to downstream main ORF translation is verified in protoplast by using dual luciferase report system, gene expression can be enhanced accurately in a moderate range, and a new technical path is provided for crop trait improvement.

Inventors

  • CHEN MING
  • MA YOUZHI
  • ZHAO YAN
  • TANG WENSI
  • ZHOU YONGBIN
  • WANG CHUNXIAO
  • CHEN JUAN
  • XU ZHAOSHI

Assignees

  • 中国农业科学院作物科学研究所

Dates

Publication Date
20260505
Application Date
20251229

Claims (10)

  1. 1. The DNA molecule of a), b) or c) below: a) A DNA molecule with a nucleotide sequence of SEQ ID No. 1; b) A DNA fragment having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more identity with the nucleotide sequence defined in a) and having the same function; c) DNA fragments which hybridize under stringent conditions with the nucleotide sequences defined under a) or b) and which have the same function.
  2. 2. An expression cassette or recombinant vector comprising the DNA molecule of claim 1.
  3. 3. A recombinant microorganism comprising the DNA molecule of claim 1.
  4. 4. A transgenic cell line comprising the DNA molecule of claim 1.
  5. 5. Use of the DNA molecule of claim 1 as a regulatory sequence.
  6. 6. Use of a DNA molecule according to claim 1 for regulating the level of translation initiation of a gene of interest in a plant.
  7. 7. The method of claim 6, wherein said modulating the translation of the gene of interest is inhibiting the expression of the main open reading frame (mORF) of the gene of interest.
  8. 8. Use of a substance which knocks out a DNA molecule according to claim 1 for enhancing the level of translation of a gene of interest, or for increasing the expression of a gene of interest, or for enhancing the activity of a gene of interest.
  9. 9. Use of a DNA molecule according to claim 1 or a recombinant vector or expression cassette according to claim 2, a recombinant microorganism according to claim 3 or a transgenic cell line according to claim 4 for regulating the expression of a gene of interest.
  10. 10. A method of modulating the level of translation or the activity of expression of a gene of interest comprising inhibiting, reducing or silencing the function of the DNA molecule of claim 1 such that the level of translation or the activity of expression of the gene of interest is increased.

Description

Enhancement of TaDREB3 gene expression using uORF sequence-based gene editing technique Technical Field The invention belongs to the technical field of biology, and particularly relates to a method for improving TaDREB3 gene expression by utilizing a gene editing technology based on uORF sequences. Background Wheat is used as staple food for 35% -40% of population worldwide, has high nutritive value, is rich in starch, protein, vitamins and minerals, and its specific gluten protein is suitable for making various wheaten foods. However, drought and salt stress have severely threatened wheat production as climate warming and soil salinization have increased. To address this challenge, the use of transgenic and genetic editing techniques to promote stress resistance in wheat has become an important direction. The multi-gene editing technology can synchronously improve a plurality of agronomic traits and obviously accelerate the breeding process. For example, in rice, researchers successfully obtain new germplasm with high yield and cold resistance by simultaneously editing and controlling genes of spike length, grain length and cold resistance through a CRISPR/Cas9 system, and researches are also carried out by knocking out three genes so as to realize multi-character aggregation of disease resistance, temperature-sensitive sterility and the like. Three-gene multi-site synchronous editing is realized in wheat, so that a foundation is laid for a multi-gene editing system, but the polymerization of the offspring genetic stability and excellent properties in commercial varieties is still to be studied deeply. The efficient and universal wheat polygene editing technology is established, and the analysis of polyploid crop breeding and complex character regulation network is forced to be driven. Upstream open reading frame (uoorf) is an important cis-element regulating gene expression, often affecting the expression of the main open reading frame (mORF) by inhibiting translation initiation or regulating mRNA stability. Plants can also attenuate the inhibitory effect of the uORF by self-regulation or environmental response. In recent years, the development of bioinformatics and translation histology has promoted systematic identification and research of plant uofs, while CRISPR-based uofs editing technology is showing broad application prospects in crop molecular breeding. Disclosure of Invention The main problem to be solved by the invention is how to regulate the translation level of the target gene, thereby influencing the expression level. In order to solve the above problems, the present invention provides a DNA molecule of the following a), b) or c): a) A DNA molecule with a nucleotide sequence of SEQ ID No. 1; b) A DNA fragment having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more identity with the nucleotide sequence defined in a) and having the same function; c) DNA fragments which hybridize under stringent conditions with the nucleotide sequences defined under a) or b) and which have the same function. The above 75% or more identity may be 80%, 85%, 90%, 95% or more identity. The stringent conditions may be hybridization with a solution of 6 XSSC, 0.5% SDS at 65℃and then washing the membrane once with 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS. The invention also provides expression cassettes or recombinant vectors containing the DNA molecules described hereinbefore. The vectors described herein are well known to those of skill in the art and include, but are not limited to, plasmids, phages (e.g., lambda phage or M13 filamentous phage, etc.), cosmids (i.e., cosmids), ti plasmids, or viral vectors. In the present invention, the vector may be an expression vector. The expression vector may be any suitable recombinant expression vector. Suitable vectors include vectors designed for propagation and amplification or for expression or both, such as plasmids and viruses. For example, the vector may be selected from the pUC series (FERMENTAS LIFE SCIENCES, glen Burnie, md.), the pBluescript series (Stratagene, laJolla, calif.), the pET series (Novagen, madison, wis.), the pGEX series (PHARMACIA BIOTECH, uppsala, sweden) and the pEX series (Clontech, palo Alto, calif.). Phage vectors such as λGT10, λGT11, λ ZapII (Stratagene), λEMBL4, and λNM1149 can also be used. Wherein, the recombinant vector can be a recombinant expression vector or a recombinant cloning vector. In a specific embodiment, the recombinant vector may be pGreen II 0800-LUC-uORF-TaDREB3A、pGreen II 0800-LUC-uorf-TaDREB3A、pGreen II 0800-LUC-uORF-TaDREB3B、pGreen II 0800-LUC-uorf-TaDREB3B、pGreen II 0800-LUC-uORF-TaDREB3D、pGreen II 0800-LUC-uorf-TaDREB3D. The structure of the expression vector pGreen II 0800-LUC-uORF-TaDREB3A is described as follows, namely, a DNA fragment with the sequence of SEQ ID No.2 is inserted between BamH I enzyme cutting sites of the original vector pGreen II 0800-LUC, and other sequences of the vector pGreen II 0