CN-122003504-A - Luminous related gene derived from photinia serrulata

CN122003504ACN 122003504 ACN122003504 ACN 122003504ACN-122003504-A

Abstract

The present invention provides a novel luminescence-related gene (nucleic acid, expression vector) capable of providing a higher luminescence ability to organisms (particularly plants) than known luminescence-related genes. There is provided a nucleic acid comprising at least one nucleotide sequence selected from the group consisting of a nucleotide sequence encoding a protein comprising the amino acid sequence shown in SEQ ID NO. 1 or an amino acid sequence having 90% or more sequence identity to the sequence shown in SEQ ID NO. 1, a nucleotide sequence encoding a protein comprising the amino acid sequence shown in SEQ ID NO. 2 or 3 or an amino acid sequence having 90% or more sequence identity to the sequence shown in SEQ ID NO. 2 or 3, a nucleotide sequence encoding a protein comprising the amino acid sequence shown in SEQ ID NO. 4 or 5 or an amino acid sequence having 90% or more sequence identity to the sequence shown in SEQ ID NO. 4 or 5, and a nucleotide sequence encoding a protein comprising the amino acid sequence shown in SEQ ID NO. 6 or an amino acid sequence having 90% or more sequence identity to the sequence shown in SEQ ID NO. 6.

Inventors

NAGAI TAKEHARU
HASEBE KOJI
ITANI HIROYUKI

Assignees

国立大学法人大阪大学

Dates

Publication Date: 20260508
Application Date: 20240617
Priority Date: 20230621

Claims (11)

1. A nucleic acid comprising a nucleotide sequence encoding a protein having an oxyfluorescein and a light-emitting function, which nucleic acid comprises an amino acid sequence represented by SEQ ID NO. 7 or an amino acid sequence derived from the amino acid sequence represented by SEQ ID NO. 7, which is obtained by substituting amino acids 22 and 241 with leucine (L) and aspartic acid (D), respectively, and which has 91% or more of sequence identity compared to the full length of the sequence represented by SEQ ID NO. 7, an amino acid sequence represented by SEQ ID NO. 8, or an amino acid sequence derived from the amino acid sequence represented by SEQ ID NO. 8, which is obtained by substituting amino acids 6 and 82 with alanine (A) and valine (V), which has 91% or more of sequence identity compared to the full length of the sequence represented by SEQ ID NO. 8, or an amino acid sequence derived from the amino acid sequence represented by SEQ ID NO. 9, which is obtained by substituting amino acids 206 and 206 of glutamine (Q) with isoleucine (I) and amino acid sequence represented by SEQ ID NO. 9, which has 91% or more of sequence identity compared to the full length of the sequence represented by SEQ ID NO. 8, Wherein the functions of the oxyfluorescein and the luminescence of the protein are superior to those of the protein comprising the amino acid sequence shown in SEQ ID NO. 1.
2. An expression vector comprising the nucleic acid of claim 1 integrated therein.
3. The expression vector of claim 2, which is a plant expression vector.
4. A non-human transgenic organism comprising the nucleic acid of claim 1.
5. The non-human transgenic organism of claim 4 which is a plant cell.
6. The non-human transgenic organism of claim 4 which is a callus.
7. The non-human transgenic organism of claim 4 which is a plant.
8. A method for producing a non-human transgenic organism comprising transforming a plant cell, a callus or a plant body with the expression vector of claim 3.
9. An expression cassette comprising the nucleic acid of claim 1.
10. An expression cassette comprising the expression cassette of claim 9 arranged in a plasmid in tandem with at least one expression cassette selected from the group consisting of: An expression cassette comprising a nucleic acid comprising a nucleotide sequence encoding a protein comprising the amino acid sequence shown in SEQ ID NO. 2 or 3, or an amino acid sequence having 90% or more sequence identity to the sequence shown in SEQ ID NO. 2 or 3 and having the function of catalyzing the reaction producing Hizibetin hispidin from caffeic acid and malonyl-CoA (CoA); An expression cassette comprising a nucleic acid comprising a nucleotide sequence encoding a protein comprising the amino acid sequence shown in SEQ ID NO. 4 or 5 or an amino acid sequence having 90% or more sequence identity to the sequence shown in SEQ ID NO. 4 or 5 and having a function of adding a hydroxyl group to the carbon at position 3 of hispidin, and An expression cassette comprising a nucleic acid comprising a nucleotide sequence encoding a protein comprising the amino acid sequence set forth in SEQ ID NO. 6 or an amino acid sequence having 90% or more sequence identity to the sequence set forth in SEQ ID NO. 6 and having the function of producing caffeic acid from caffeoyl pyruvic acid.
11. An expression cassette assembly comprising an expression cassette according to claim 9, and at least one expression cassette selected from the group consisting of: An expression cassette comprising a nucleic acid comprising a nucleotide sequence encoding a protein comprising the amino acid sequence shown in SEQ ID NO. 2 or 3, or an amino acid sequence having 90% or more sequence identity to the sequence shown in SEQ ID NO. 2 or 3 and having the function of catalyzing the reaction of caffeic acid and malonyl-CoA to hispidin; An expression cassette comprising a nucleic acid comprising a nucleotide sequence encoding a protein comprising the amino acid sequence shown in SEQ ID NO. 4 or 5 or an amino acid sequence having 90% or more sequence identity to the sequence shown in SEQ ID NO. 4 or 5 and having a function of adding a hydroxyl group to the carbon at position 3 of hispidin, and An expression cassette comprising a nucleic acid comprising a nucleotide sequence encoding a protein comprising the amino acid sequence set forth in SEQ ID NO. 6 or an amino acid sequence having 90% or more sequence identity to the sequence set forth in SEQ ID NO. 6 and having the function of producing caffeic acid from caffeoyl pyruvic acid.

Description

Luminous related gene derived from photinia serrulata Technical Field The present invention relates to nucleic acids encoding luminescence-related genes derived from Pleurotus cornucopiae (Mycena luxcoeli or Mycena lux-coeli), vectors comprising the nucleic acids, transgenic organisms comprising the nucleic acids and methods for producing the transgenic organisms. Background Some organisms, such as luminescent bacteria and fireflies, are capable of producing and emitting light, i.e., some organisms are bioluminescent. Bioluminescence is known to be caused by the function of luciferase or photoprotein. The term "luciferase" is a general term of an enzyme that oxidizes a light emitting substance to emit light, and a light emitting substance oxidized by a luciferase is generally referred to as "luciferin". However, luciferases and luciferins are different depending on the kind of organism entirely, and the mechanisms of luminescence reactions are also different from each other. An example of a known photoprotein is aequorin from aequorin crystal (Aequorea coerulescens). Aequorin is known to form complexes with green fluorescent proteins and to emit light according to its nearby calcium concentration. In bioluminescent organisms, the mechanism by which the mushrooms emit light has long been unknown. In 2015, a luciferin structure of a luminous mushroom, that is, a Nambioid mushroom (Neonothopanus nambi), was found (non-patent document 1), and then a luciferin structure and a luminous mechanism were found (non-patent document 2 and patent document 1). In addition, it was found that the introduction of the luminescence-related gene of the umbilicus of Nameko into plants to catalyze the luminescence reaction in plants using caffeic acid inherent to the plants as a precursor of the luminescent substance does not significantly affect the survival of the plants. Therefore, self-luminous plants were constructed (non-patent document 3). With respect to the introduction of luminescence-related genes into plants, for example, genes of luminescent bacteria have been introduced into plants. However, it was found that the use of Nam produced more intense luminescence than the navel-like mushroom gene compared to the use of the gene of the luminescent bacteria. Citation document Patent literature Patent document 1 JP 2020-505028A Non-patent literature Non-patent document 1, K.V. Purtov, et al, angew. Chem. Int. Ed., vol.50, pp.8124-8128, 2015 Non-patent document 2: a.a. Kotlobay et al, proc.Natl. Acad. Sci, U.S. A., vol.115, pp. 12728-12732, 2018 Non-patent literature 3:T, mitiouchkina, et al Nature Biotechnology, vol.38, pp. 944-946, 2020 Summary of The Invention Technical problem The luminous plants can be used as indoor decorations due to their appearance. Further, the self-luminous plant does not require electric power or the like, and thus its application as an environment-friendly illumination can be expected. However, currently, the luminous intensity of luminous plants cannot be said to be sufficient to replace lighting devices. Therefore, development of a luminescent plant that emits more intense light is desired. It is an object of the present invention to provide a novel luminescence-related gene (e.g., nucleic acid, expression vector) that can provide stronger luminescence to organisms (particularly plants) than known luminescence-related genes. It is a further object of the present invention to provide a transgenic organism comprising such a luminescence-related gene introduced therein and a method for producing such a transgenic organism. Solution to the problem The present invention provides the following. [1] A nucleic acid comprising a nucleotide sequence encoding a protein having an oxyfluorescein and a light-emitting function, the nucleic acid comprising an amino acid sequence represented by SEQ ID NO. 7 or an amino acid sequence derived from the amino acid sequence represented by SEQ ID NO. 7, which is obtained by substituting amino acids 22 and 241 with leucine (L) and aspartic acid (D), respectively, and having 91% or more of sequence identity compared to the whole length of the sequence represented by SEQ ID NO. 7, an amino acid sequence represented by SEQ ID NO. 8 or an amino acid sequence derived from the amino acid sequence represented by SEQ ID NO. 8, which is obtained by substituting amino acids 6 and 82 with alanine (A) and valine (V), which has 91% or more of sequence identity compared to the whole length of the sequence represented by SEQ ID NO. 8, or an amino acid sequence derived from the amino acid sequence represented by SEQ ID NO. 9, which is obtained by substituting amino acids 206 and 206 with isoleucine (Q) and having more of sequence identity compared to the whole length of the amino acid sequence represented by SEQ ID NO. 9, which is obtained by oxidizing the amino acid sequence represented by SEQ ID NO. 9, and having a function superior to that of the protein represented by amino aci