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CN-122012504-A - Begonia U6 promoter BsU6-3 and application thereof

CN122012504ACN 122012504 ACN122012504 ACN 122012504ACN-122012504-A

Abstract

The application belongs to the technical field of cultivation of new ornamental plant varieties, and particularly relates to a begonia U6 promoter BsU-3 and application thereof. The nucleotide sequence of the U6 promoter BsU-3 is shown as SEQ ID No.1, and the promoter is mainly used for constructing a begonia CRISPR/Cas9 gene editing system or driving LUC fluorescent protein expression. In the process of gene editing, the endogenous U6 promoter of the receptor plant or a related species is used, so that the method has very important technical significance for improving the transcription efficiency of sgRNA and improving the gene editing efficiency. In the application, the inventor clones series U6 promoters in begonia genome, and takes LUC reporter gene as an example, and performs preliminary comparison analysis on transcriptional activity of the promoters. Based on the results, a good technical foundation can be laid for the subsequent high-efficiency gene editing system construction and further the cultivation of new begonia varieties.

Inventors

  • ZHANG KAIMING
  • ZHAO MENGRU
  • FU CONGCONG
  • YANG YANG
  • LI FEI
  • LIU WEICHAO
  • JIANG WENQIAN
  • LI QINGHUI

Assignees

  • 河南农业大学

Dates

Publication Date
20260512
Application Date
20260309

Claims (5)

  1. 1. The begonia U6 promoter BsU6-3 is characterized in that the nucleotide sequence of the U6 promoter BsU6-3 is shown in SEQ ID No. 1.
  2. 2. Use of the begonia U6 promoter BsU6-3 as claimed in claim 1 in plants for constructing begonia CRISPR/Cas9 gene editing systems or for driving LUC fluorescent protein expression.
  3. A primer pair for PCR amplification of the begonia U6 promoter BsU-3 according to claim 1, which is specifically designed as follows: the forward primer is 5'-ATCAGTCAACTCGACCCGAT-3' of the primer, Reverse primer 5'-GGACCATTTCTCGATTTGTGCG-3'.
  4. 4. The truncated sequence BsU-3.4P of the Begonia U promoter BsU-3 is characterized in that the nucleotide sequence of the truncated sequence BsU6-3.4P is shown as SEQ ID No. 2.
  5. 5. Use of the truncated sequence BsU-3.4P of the begonia U promoter BsU-3 as claimed in claim 4 in plants for constructing a begonia CRISPR/Cas9 gene editing system or for driving LUC fluorescent protein expression 2.

Description

Begonia U6 promoter BsU6-3 and application thereof Technical Field The application belongs to the technical field of cultivation of new ornamental plant varieties, and particularly relates to a begonia U6 promoter BsU-3 and application thereof. Background In plant breeding, biotechnology means such as gene editing and the like are a common new variety cultivation method. The CRISPR/Cas 9-based gene editing system is one of the common technical means in plant breeding due to the technical advantages of high efficiency, accuracy, simplicity and convenience in operation and the like. Begonia plants are widely used in gardens at present due to the characteristics of rich and various leaf shapes and leaf colors and the like. However, the conventional breeding mode of Begonia is still relied on in the existing breeding mode of Begonia. One of the main technical obstacles that hamper the application of CRISPR/Cas9 and other gene editing techniques in Begonia is that related gene editing systems have not been constructed effectively. During gene editing based on CRISPR/Cas9 system, the sgrnas (single guide RNAs) driven to be transcribed by RNA polymerase III are responsible for recognition of the target sequence, so their expression level directly affects editing efficiency. The RNA polymerase III is started by the U6 promoter, so that the deep research of the U6 promoter is an important precondition for constructing the begonia gene editing system technology. Disclosure of Invention The application aims to provide a series of begonia U6 promoters, thereby laying a certain technical foundation for the construction of begonia gene editing system and the further cultivation of new begonia varieties. The technical scheme adopted by the application is detailed as follows. The nucleotide sequence of the begonia U6 promoter BsU-3 is shown as SEQ ID No. 1. The begonia U6 promoter BsU-3 is applied to plants, and is used for constructing a begonia CRISPR/Cas9 gene editing system or driving LUC fluorescent protein expression. The truncated sequence BsU-3.4P of the Begonia U promoter BsU-3 has a nucleotide sequence shown in SEQ ID No. 2. The truncated sequence BsU-3.4P of the begonia U promoter BsU-3 is applied to plants, and is used for constructing a begonia CRISPR/Cas9 gene editing system or driving LUC fluorescent protein expression. The CRISPR/Cas9 gene editing system comprises two parts, namely Cas9 nuclease and sgRNA responsible for recognizing target sequences. Wherein the sgRNA is responsible for transcription by RNA polymerase III, which is driven by the U6 promoter. The existing research shows that the U6 promoter has species specificity, so that the use of the endogenous U6 promoter of the receptor plant or the related species in the gene editing process has very important technical significance for improving the transcription efficiency of sgRNA and improving the gene editing efficiency. In the application, the inventor takes begonia quadricarina as an example, clones a series of U6 promoters in begonia quadricarina genome, and takes a LUC reporter gene as an example, and performs preliminary comparison analysis on transcriptional activity of the promoters. Based on the results, a good technical foundation can be laid for the subsequent high-efficiency gene editing system construction and further the cultivation of new begonia varieties. Drawings FIG. 1 shows the result of the post-PCR amplification electrophoresis detection of 6U 6 promoters in Begonia, and the result of the post-recombinant plasmid electrophoresis detection, wherein FIG. A shows the result of the post-PCR amplification electrophoresis detection of the promoters, FIG. A shows the result of the post-PCR amplification electrophoresis detection of 2 kb DNA Marker, FIG. 1 shows BsU6-1, FIG. 2 shows BsU6-2, FIG. 3 shows BsU-3, FIG. 4 shows BsU-4, FIG. 5 shows BsU-5, FIG. 6 shows BsU-6, FIG. B shows the result of the post-PCR electrophoresis detection of recombinant plasmids, FIG. 2 shows kb DNA Marker, FIG. 1 shows BsU-1 shows LUC, FIG. 2 shows BsU-2 shows LUC, FIG. 3 shows BsU-3 shows LUC, FIG. 4 shows BsU-4 shows LUC, FIG. 5 shows BsU-5 shows LUC, and FIG. 6 shows BsU-6 shows LUC. FIG. 2 is a schematic diagram of recombinant plasmid expression vector structure; FIG. 3 shows the results of transient expression detection of recombinant plasmid expression vectors in Nicotiana benthamiana leaves, wherein A shows the fluorescent signal results of experimental groups, a shows BsU-1:: LUC groups, B shows BsU-2::: LUC groups, c shows BsU-6-3:: LUC groups, d shows BsU-4::: LUC groups, e shows BsU-5:: LUC groups, f shows BsU-6:: LUC groups, and B shows no-load control groups; FIG. 4 shows the result of truncation of the U6-3 promoter, FIG. A shows the schematic representation of truncation of the 5' end of the U6-3 promoter, FIG. B shows the result of electrophoresis of the truncated product of PCR amplification, with band M of 2 kb DNA Marker, band 1 of BsU