Search

CN-118995713-B - Promoter for improving dicotyledon gene editing efficiency and application thereof

CN118995713BCN 118995713 BCN118995713 BCN 118995713BCN-118995713-B

Abstract

The invention relates to a promoter pAtEF alpha for improving dicotyledonous plant gene editing efficiency and application thereof, and belongs to the field of plant genetic engineering. The pAtEF < 1 > alpha promoter of the invention can be used for the expression of adenine base editing proteins, cytosine base editing proteins, cas nucleases and gRNA. The invention also provides an expression cassette group containing pAtEF1 alpha promoter and a recombinant vector containing the expression cassette group. The pAtEF alpha promoter of the invention can be widely applied to the fields of gene editing, base editing and the like of dicotyledonous plants, and has wide application in genetic improvement of dicotyledonous crops such as tomatoes and the like and biosynthesis of high-value metabolites.

Inventors

  • LI JIANFENG
  • XIONG XIANGYU
  • LIN XIAOXIA
  • YAO YUXIN
  • ZHU LUYUAN
  • BAO YING

Assignees

  • 中山大学

Dates

Publication Date
20260508
Application Date
20240914

Claims (9)

  1. 1. A promoter for improving the gene editing efficiency of dicotyledonous plants is characterized in that the promoter is pAtEF1 alpha, and the nucleotide sequence of the promoter pAtEF alpha is shown as SEQ ID NO. 1.
  2. 2. An expression cassette combination for improving gene editing efficiency of dicotyledonous plants, comprising a first expression cassette that is an adenine base editing protein expression cassette, wherein the first expression cassette comprises a pAtEF a promoter sequence of claim 1, and a second expression cassette that is a guide RNA expression cassette.
  3. 3. The expression cassette combination of claim 2, wherein the pAtEF a promoter nucleotide sequence in the first expression cassette is operably linked to the polynucleotide coding sequence of the adenine base editing protein expression cassette, the E9t terminator, the p2 x 35S, hygR element in sequence; the adenine base editing protein expression frame is formed by fusing adenosine deaminase and a nCas-D10A nucleotide sequence from a 5 'end to a 3' end; the adenosine deaminase is TadA e, tadA8s or any one of TadA e and TadA8s carrying a V106W mutation.
  4. 4. The combination of expression cassettes of claim 2, wherein the second expression cassette is obtained by any one of the following ligation methods: a) The nucleotide sequence of promoter pAtEF a of claim 1 operably linked to a polynucleotide coding sequence of a tRNA-mediated guide RNA expression system, or a polynucleotide coding sequence of a dinucleotide-mediated guide RNA expression system, or a polynucleotide coding sequence of a Cys 4-mediated guide RNA expression system, or, B) The nucleotide sequence of the AtU6-26 promoter or AtU6-1 promoter is operably linked to the polynucleotide coding sequence of the guide RNA.
  5. 5. An expression cassette combination for cytosine base editing in a dicotyledonous plant, comprising a first expression cassette that is a cytosine base editing protein expression cassette and a second expression cassette that is a guide RNA expression cassette, wherein at least one of the two expression cassettes comprises the promoter pAtEF a of claim 1; the cytosine base editing protein expression cassette is operably linked in sequence by nucleotides of: The promoter pAtEF a, cytosine base editing protein expression cassette, E9t terminator, p2 x 35S, and HygR element of claim 1; The cytosine base editing protein expression frame consists of nucleotide sequences of cytidine deaminase, nCas-D10A and uracil glycosidase inhibitor protein UGI; The cytidine deaminase is AID10.
  6. 6. An expression cassette combination for dicotyledonous plant gene editing, wherein the set of expression cassettes comprises a first expression cassette that is a Cas nuclease expression cassette and a second expression cassette that is a guide RNA expression cassette, wherein at least one of the two expression cassettes comprises the promoter pAtEF a of claim 1; The Cas nuclease is SpCas9, saCas9 or ScCas.
  7. 7. A recombinant vector comprising the combination of expression cassettes of any one of claims 2-6.
  8. 8. The use of the recombinant vector according to claim 7 in dicotyledonous plant gene editing, wherein the dicotyledonous plant is arabidopsis thaliana or lycopersicon esculentum.
  9. 9. A method for targeted editing of a plant genome comprising the step of introducing the recombinant vector of claim 7 into a plant.

Description

Promoter for improving dicotyledon gene editing efficiency and application thereof Technical Field The invention relates to the technical field of plant genetic engineering, in particular to a promoter for improving dicotyledonous plant gene editing efficiency and application thereof. Background Single Nucleotide Polymorphisms (SNPs) are important factors for plants to evolve in natural environments, and alleles generated by SNP variation confer numerous important agricultural traits. The gene editing technology developed based on the CRISPR/Cas system can play a role of efficiently cutting the target DNA by only relying on single Cas nuclease and guide RNA, is an important tool for researching gene functions and improving crop inheritance, but has the problem of lower accuracy. The base editing technology developed by the CRISPR/Cas gene editing technology can efficiently and accurately carry out base-oriented mutation on the genome under the condition of not generating double-strand break, and is a sharp tool for realizing accurate introduction of SNP loci on the genome. The base editing technology mainly comprises two types of base editing proteins, namely adenine base editing protein (ABE), and the base editing protein is usually composed of nCas-D10A nickase and adenosine deaminase obtained by artificial directed evolution, so that A to G mutation can be realized. Another class is the cytosine base editing proteins (CBEs), which are generally composed of nCas-D10A nickases and cytidine deaminase, which can effect C to T mutation. In the dicotyledonous plant field, the editing efficiency of single base editing proteins, especially ABE, is greatly affected by promoter selection. Kang et al found that using the common p35S or pYao promoter to drive expression of ABE7.10 resulted in little efficient editing in transgenic Arabidopsis, while replacing the pAtRPS A promoter induced up to 85% of A > G editing at the same target (KANG B C,YUN J Y,KIM S T,et al.Precision genome engineering through adenine base editing in plants[J].Nat Plants,2018,4(7):427-431). The promoters used for dicot ABE are varied, and driving ABE using conventional constitutive p35S promoters or pAtUbi promoters tends to result in a large number of chimeric lines and it is difficult to obtain plants containing stable heritable mutations. The meristem specific promoter pAtRPS A can be matched with ABE to effectively generate stable plants which can be subjected to genetic mutation, but the report of the same kind of promoters is not yet seen at home. Thus, there is a need for an autonomously controllable promoter for efficient gene editing in dicotyledonous plants. Disclosure of Invention The invention aims to overcome the defects of the prior art and provide a promoter for improving the gene editing efficiency of dicotyledonous plants and application thereof, the promoter can be used for driving the expression of adenine base editing protein, cytosine base editing protein, cas nuclease and guide RNA, and the promoter can be widely applied to the fields of gene editing, base editing and the like of dicotyledonous plants. In order to achieve the above purpose, the technical scheme adopted by the invention is as follows: In a first aspect, the present invention provides a promoter for improving the efficiency of gene editing in dicotyledonous plants, said promoter being designated pAtEF 1a, said promoter pAtEF a being any one of the following: a) The nucleotide sequence of the promoter pAtEF < 1 > alpha is shown as SEQ ID NO. 1; b) A polynucleotide molecule having 75% or more identity to the nucleotide sequence defined in a) and having pAtEF.alpha.promoter function, or, C) A polynucleotide molecule which hybridizes to the nucleotide sequence defined in a) or b) and which has the function of a pAtEF a promoter. In some embodiments, the promoter has 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO. 1. In a second aspect, the invention provides a set of expression cassettes for adenine base editing in dicotyledonous plants, said set of expression cassettes comprising a first expression cassette being an adenine base editing protein expression cassette and a second expression cassette being a guide RNA expression cassette. In some embodiments, the first expression cassette is operably linked to the polynucleotide coding sequence of the adenine base editing protein by the nucleotide sequence of promoter pAtEF a. In some specific embodiments, the polynucleotide coding sequence of the adenine base editing protein is formed by fusing a nucleotide sequence of an adenosine deaminase with a nucleotide sequence of nCas-D10A from a 5 'end to a 3' end, wherein the nucleotide sequence of the adenosine deaminase is shown as SEQ ID NO:3 from 5,703 th to 6,254 th of the 5 'end, and the nucleotide sequence of the nCas-D10A is shown as SEQ ID NO:3 from 6,3