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CN-121975810-A - Oil palm U6 promoter and application thereof

CN121975810ACN 121975810 ACN121975810 ACN 121975810ACN-121975810-A

Abstract

The invention discloses an oil palm U6 promoter gene and application thereof, and belongs to the technical field of biology. The nucleic acid sequence of the promoter gene is shown as SEQ ID NO.1, and the invention clones in the oil palm genome for the first time to obtain the oil palm RNA polymerase III type promoter gene, namely the oil palm endogenous U6 promoter gene EgU6, which has high transcription activity and can drive the expression of downstream fluorescent protein mNeonGreen. The invention can provide candidate oil palm endogenous U6 promoter genes for establishing a high-efficiency oil palm gene editing technical system based on a CRISPR/cas9 system.

Inventors

  • ZHANG DAPENG
  • SHI PENG
  • WANG YONG
  • LI ZHIYING
  • YIN MINHUI
  • YU QUN
  • He Xiangman

Assignees

  • 中国热带农业科学院三亚研究院
  • 中国热带农业科学院椰子研究所

Dates

Publication Date
20260505
Application Date
20260409

Claims (9)

  1. 1. The oil palm U6 promoter gene is characterized in that the nucleotide sequence of the oil palm U6 promoter gene is shown in SEQ ID NO. 1.
  2. 2. A biological material comprising the oil palm U6 promoter gene of claim 1, wherein the biological material comprises a recombinant vector, an expression cassette, or a recombinant bacterium.
  3. 3. The biological material according to claim 2, wherein the recombinant vector is obtained by ligating the oil palm U6 promoter gene according to claim 1 with mNeonGreen gene and plant expression vector.
  4. 4. A biomaterial according to claim 3 wherein the plant expression vector is a pUN1301 vector.
  5. 5. The biomaterial according to claim 2, wherein the recombinant bacterium is escherichia coli containing the oil palm U6 promoter gene.
  6. 6. The use of the oil palm U6 promoter gene of claim 1 or the biomaterial of any one of claims 2-5, wherein the use is to drive the transformation of mNeonGreen genes in oil palm protoplasts.
  7. 7. A method for cloning the U6 promoter gene of oil palm, comprising the steps of: (1) The specific primers EgU-F and EgU-R are designed by taking the genomic DNA of the oil palm as a template, wherein the nucleotide sequence of EgU-F is shown as SEQ ID NO.2, and the nucleotide sequence of EgU-6-R is shown as SEQ ID NO. 3; (2) PCR amplification using KOD enzyme; (3) Recovering the amplified product to obtain EgU fragment containing 300bp oil palm U6 promoter gene for seamless cloning, and the nucleotide sequence of the oil palm U6 promoter gene is shown as SEQ ID NO. 1.
  8. 8. A method of constructing a transient transformation vector for oil palm protoplasts, comprising the steps of: (a) Preparing EgU fragment containing 300bp oil palm U6 promoter gene for seamless cloning, wherein the nucleotide sequence of the oil palm U6 promoter gene is shown as SEQ ID NO. 1; (b) Designing specific primers EgU-Neon F and EgU-Neon R, and carrying out PCR amplification by using KOD enzyme with a plasmid containing mNeonGreen genes as a template to obtain a mNeonGreen fragment; The nucleotide sequence of EgU-Neon F is shown as SEQ ID NO.4, and the nucleotide sequence of EgU-Neon R is shown as SEQ ID NO. 5; (c) Cutting pUN1301 vector by enzyme, and recovering vector skeleton fragment; (d) And (3) connecting the EgU fragment, the mNeonGreen fragment and the vector skeleton fragment by adopting a seamless cloning method to obtain an expression vector pEgU-mNeonGreen containing the oil palm EgU promoter gene driven fluorescent protein.
  9. 9. The method of claim 8, wherein the cleavage is a HindIII and BamHI double cleavage.

Description

Oil palm U6 promoter and application thereof Technical Field The invention belongs to the technical field of biology, and particularly relates to an oil palm U6 promoter and application thereof. Background Oil palm (school name: elaeis guineensiss jacq.) is an important tropical economic palm crop, and at present, the breeding of new varieties of oil palm in China is still mainly based on traditional crossbreeding, and the problems of low efficiency and long period exist, so that the progress of high-yield and high-quality breeding of oil palm is seriously hindered. With the development of biotechnology, the application of molecular breeding techniques represented by gene editing techniques to various crops has accelerated the cultivation of new varieties, but the related research of oil palm has yet to be developed. The genome editing technology is an important tool for researching gene functions, can accurately modify genes at specific sites of receptor cell chromosomes, can efficiently generate functional inactivation mutants of specific genes, and can provide high-quality genetic materials for biofunctional genome research. The use of gene editing techniques is highly dependent on transcriptional regulatory elements capable of precisely regulating gene expression. The expression of small molecule RNAs (such as CRISPR/Cas gene edited sgRNAs), the expression of nucleases such as CAS9 and the expression of reporter genes such as fluorescent protein genes are all required to be driven by promoter genes. U6 RNA is a non-coding RNA involved in pre-mRNA splicing, and the U6 promoter gene is a class III RNA polymerase promoter gene, which is a key element driving transcription of U6 RNA genes, and has found great use in CRISPR/Cas9 systems of many species. Although CRISPR/Cas9 genome editing techniques have been widely used in a number of species at present, gene editing techniques for oil palm have not yet been reported. This is mainly due to the fact that although U6 promoter genes have been reported in a large number in many species, exogenous U6 promoter genes are not generally suitable. It can be seen that the lack of a suitable U6 promoter gene has become the limiting factor for current oil palm CRISPR/Cas9 gene editing systems. Therefore, screening U6 promoter genes with functional activity in oil palm has positive significance for the development of oil palm genetic breeding technology. Disclosure of Invention In view of the defects in the prior art, the invention provides an oil palm U6 promoter gene and application thereof. The technical scheme of the invention mainly comprises the following contents: The nucleotide sequence of the oil palm U6 promoter gene is shown in SEQ ID NO. 1. The oil palm U6 promoter gene (EgU) belongs to the RNA polymerase III type promoter gene of the oil palm U6 snRNA gene and is derived from oil palm (Elaeis guineensiss Jacq.) Further, the invention provides a biological material containing the oil palm U6 promoter gene, wherein the biological material comprises a recombinant vector, an expression cassette or recombinant bacteria. The recombinant vector is obtained by connecting the oil palm U6 promoter gene with mNeonGreen gene and plant expression vector. Further, the plant expression vector is preferably a pUN1301 vector. Further, the recombinant bacterium is preferably Escherichia coli containing the oil palm U6 promoter gene. Furthermore, the invention relates to the application of the oil palm U6 promoter gene or the biological material in driving fluorescent protein genes in plant protoplast transformation. The plant comprises a monocot or dicot plant, and in particular, in embodiments of the invention, the plant is oil palm. The fluorescent protein gene is preferably mNeonGreen genes. Further, the present invention provides a method for cloning the oil palm U6 promoter gene, comprising the steps of: (1) The specific primers EgU-F and EgU-R are designed by taking the genomic DNA of the oil palm as a template, wherein the nucleotide sequence of EgU-F is shown as SEQ ID NO.2, and the nucleotide sequence of EgU-6-R is shown as SEQ ID NO. 3; (2) PCR amplification using KOD enzyme; (3) Recovering the amplified product to obtain EgU fragment containing 300bp oil palm U6 promoter gene for seamless cloning, and the nucleotide sequence of the oil palm U6 promoter gene is shown as SEQ ID NO. 1. Specifically, in the examples of the present invention, the PCR amplification reaction procedure of step (2) was 95℃pre-denaturation for 6min,95℃denaturation for 15s,57℃annealing for 15s,72℃extension for 5s,35 cycles, and 72℃final extension for 7min. Further, the invention provides a method for constructing the oil palm protoplast transient transformation vector, which comprises the following steps: (a) Preparing EgU fragment containing 300bp oil palm U6 promoter gene for seamless cloning, wherein the nucleotide sequence of the oil palm U6 promoter gene is shown as SEQ ID NO. 1; (b) Des