Amino acid mutants and their application in enhancing bioluminescence intensity

Abstract

This invention relates to amino acid mutants of TAL, H3H, LUZ, PKS, AROG, or P450, comprising mutations selected from one or more of the following sites: TAL W645 TAL D571 TAL E163 TAL P3 TAL S108 TAL Y245 TAL L162 TAL A498 TAL V186 TAL I634 H3H F139 H3H P200 H3H E379 H3H F4 H3H V142 H3H N6 H3H S7 H3H A197 ;LUZ V29 LUZ C26 LUZ F230 LUZ E181 LUZ V28 LUZ F148 LUZ L67 PKS C79 PKS C121 PKS F266 PKS L193 PKS L321 PKS L382 PKS L405 PKS R330 PKS S366 PKS S402 PKS V196 PKS Y327 AROG N5 AROG K11 AROG K41 AROG E174 AROG A319 AROG K322 AROG G350 Or P450 A11 P450 H18 P450 L31 P450 K50 P450 H94 P450 P95 P450 S97 P450 Y380 This application solves the problems of low luminescence brightness and poor caffeic acid utilization efficiency in self-luminescent plants that rely on fungal FBP systems.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to amino acid mutants and their application in enhancing bioluminescence intensity. Background Technology

[0002] Bioluminescent genes require no external excitation light, have low background signal interference, high detection sensitivity, and excellent biocompatibility, making them a core tool in life science research, clinical medical diagnosis, and environmental monitoring.

[0003] In 2018, the fungal bioluminescence pathway (FBP) was fully elucidated, providing a new paradigm for the development of self-luminescent plants. This pathway uses caffeic acid, an endogenous metabolite in plants, as a starting substrate. It involves a cascade of four core enzymes—HispS, H3H, LUZ, and CPH—to form a metabolic cycle, ultimately emitting fluorescence. This process requires no additional exogenous substrate, and the metabolites have minimal adverse effects on the plant host, naturally adapting to the plant's physiological metabolic system. Leveraging this unique advantage, the FBP system has been successfully heterologously expressed in various plants, including tobacco, Arabidopsis thaliana, and rose, constructing sustainably luminescent engineered plants and becoming a core technological pathway for the current research and development of self-luminescent plants.

[0004] However, existing technologies still have many key defects, resulting in insufficient luminous brightness, which seriously restricts their further application. Summary of the Invention

[0005] To address the technical problems existing in the prior art, this invention proposes amino acid mutants of TAL, H3H, LUZ, PKS, AROG, or P450, which include mutations selected from one or more of the following sites: TAL W645 TAL D571 TAL E163 TAL P3 TAL S108 TAL Y245 TAL L162 TAL A498 TAL V186 TAL I634 H3H F139 H3H P200 H3H E379 H3H F4 H3H V142 H3H N6 H3H S7 H3H A197 ;LUZ V29 LUZ C26 LUZF230 LUZ E181 LUZ V28 LUZ F148 LUZ L67 PKS C79 PKS C121 PKS F266 PKS L193 PKS L321 PKS L382 PKS L405 PKS R330 PKS S366 PKS S402 PKS V196 PKS Y327 AROG N5 AROG K11 AROG K41 AROG E174 AROG A319 AROG K322 AROG G350 Or P450 A11 P450 H18 P450 L31 P450 K50 P450 H94 P450 P95 P450 S97 P450 Y380 .

[0006] The amino acid mutants described above, wherein one or more sites are mutated to the following amino acids: TAL W645V TAL W645A TAL W645L TAL W645T TAL W645I TAL W645S TAL W645N TAL W645F TAL W645G TAL D571A TAL D571V TAL D571L TAL D571G TAL E163A TAL E163S TAL E163A TAL P3A TAL S108P TAL Y245K TAL L162Q TAL A498H TAL V186P TAL I634PH3H F139N H3H F139G H3H F139H H3H F139S H3H F139E H3H F139Q H3H F139A H3H F139K H3H F139R H3H P200R H3H P200K H3H P200Q H3H P200N H3H E379M H3H E379L H3H E379N H3H E379A H3H E379S H3H F4P H3H F4A H3H V142P H3H N6K H3H S7K H3H S7R H3H A197R H3H A197K LUZ V29F LUZ V29W LUZ C26W LUZ C26Y LUZ F230N LUZ E181A LUZ E181Q LUZ E181H LUZ V28F LUZ V28W LUZ F148E LUZ L67P ;PKS C79N 、PKS C121S 、PKS F266N 、PKS L193S 、PKS L321A 、PKS L321K 、PKS L321N 、PKS L321Q 、PKS L321R 、PKS L321S 、PKS L382A 、PKS L382E 、PKS L382K 、PKS L382P 、PKS L382Q 、PKS L382S 、PKS L405G 、PKS L405P 、PKS R330P 、PKS S366P 、PKS S402GPKS V196A PKS Y327A PKS Y327G PKS Y327H PKS Y327K PKS Y327N PKS Y327Q PKS Y327R PKS Y327S PKS Y327T AROG N5T AROG K11Y AROG K11S AROG K41T AROG K41L AROG E174Q AROG A319V AROG A319I AROG K322Q AROG G350T AROG G350S AROG G350M Or P450 A11I P450 A11V P450 H18I P450 H18V P450 L31N P450 K50D P450 K50E P450 K50N P450 H94E P450 P95V P450 P95Q P450 P95A P450 P95E P450 P95I P450 P95T P450 P95S P450 S97A P450 Y380N .

[0007] The amino acid mutants described above include simultaneous mutations selected from the following mutation sites: TAL W645 / D571 TAL W645 / S108 TAL D571 / S108 TAL W645 / D571 / S108 H3H F139 / P200 H3H F139 / E379 H3H P200 / E379 H3H P200N / E379M H3H F139 / P200 / E379 ;LUZ V29 / E181 LUZ V29 / V28 LUZ E181 / V28 LUZ V29 / E181 / V28PKS L382 / L405 PKS L382 / Y327 PKS L405 / Y327 PKS L382 / L405 / Y327 AROG K11 / E174 AROG K11 / K322 AROG K11 / G350 AROG E174 / K322 AROG E174Q / G350M AROG K322Q / G350 AROG K11 / E174 / K322 AROG K11 / E174 / G350 AROG E174 / K322 / G350 Or P450 K50 / P95 .

[0008] The amino acid mutants described above, wherein multiple mutation sites are simultaneously mutated to the following sites: TAL W645V / D571L TAL W645V / S108P TAL W645L / D571L TAL W645L / S108P TAL D571L / S108P TAL W645V / D571L / S108P TAL W645L / D571L / S108P H3H F139H / P200Q H3H F139H / P200N H3H F139H / E379M H3H F139H / E379L H3H P200Q / E379M H3H P200Q / E379L H3H P200N / E379M H3H P200Q / E379L H3H F139H / P200Q / E379M H3H F139H / P200Q / E379L H3H F139H / P200N / E379M H3H F139H / P200N / E379L ;LUZ V29F / E181A LUZ V29F / V28W LUZ V29W / E181A LUZ V29W / V28W LUZ E181A / V28W LUZ V29F / E181A / V28W LUZ V29W / E181A / V28W PKS L382K / L405G PKS L382K / Y327N PKS L382K / Y327Q PKS L382K / Y327T PKS L405G / Y327N PKS L405G / Y327Q PKS L405G / Y327T PKS L382K / L405G / Y327N PKS L382K / L405G / Y327Q PKS L382K / L405G / Y327T AROG K11Y / E174Q AROGK11Y / K322Q AROG K11Y / G350M AROG E174Q / K322Q AROG E174Q / G350M AROG K322Q / G350M AROG K11Y / E174Q / K322Q AROG K11Y / E174Q / G350M AROG E174Q / K322Q / G350M Or P450 K50E / P95Q P450 K50E / P95A P450 K50E / P95E P450 K50E / P95I P450 K50E / P95T P450 K50E / P95S .

[0009] The amino acid mutants described above, wherein the amino acid sequences of TAL, H3H, LUZ, PKS, AROG, and P450 constitute a natural protein or a protein excluding the mutations described in claim 1.

[0010] The amino acid mutants described above, wherein the amino acid sequence of TAL is shown in SEQ ID NO.1, the amino acid sequence of H3H is shown in SEQ ID NO.31, the amino acid sequence of LUZ is shown in SEQ ID NO.70, the amino acid sequence of PKS is shown in SEQ ID NO.90, the amino acid sequence of AROG is shown in SEQ ID NO.132, and the amino acid sequence of P450 is shown in SEQ ID NO.155.

[0011] The coding gene of any of the amino acid mutants described above, wherein the coding gene sequence of TAL is shown in SEQ ID NO. 181, the coding gene sequence of H3H is shown in SEQ ID NO. 182, the coding gene sequence of LUZ is shown in SEQ ID NO. 183, the coding gene sequence of PKS is shown in SEQ ID NO. 184, the coding gene sequence of AROG is shown in SEQ ID NO. 185, and the coding gene sequence of P450 is shown in SEQ ID NO. 186.

[0012] Recombinant vectors, expression cassettes, or recombinant strains that encode genes as described above.

[0013] Application of any of the amino acid mutants described above, or combinations thereof, in improving bioluminescence intensity or increasing the amount of 3-arylacrylic acid synthesized in biological cells; preferably, the 3-arylacrylic acid is selected from the group consisting of: caffeic acid, cinnamic acid, p-coumaric acid, coumaric acid, umbelliferic acid, sinapic acid, and ferulic acid.

[0014] As described above, the combination of mutants is selected from the group consisting of: TAL W645 / H3H P200 TAL W645 / LUZ V29 TAL W645 / PKS S366 TAL W645 / AROG K11 TAL W645 / P450 P95 H3H P200 / LUZ V29 H3H P200 / PKS S366 H3H P200 / AROG K11 H3H P200 / P450 P95 LUZ V29 / PKS S366 LUZ V29 / AROG K11 LUZ V29 / P450 P95 PKS S366 / AROG K11 PKS S366 / P450 P95 AROG K11 / P450 P95 TAL W645 / H3H P200 / LUZ V29 TAL W645 / H3H P200 / PKS S366 TAL W645 / H3H P200 / AROG K11 TAL W645 / H3H P200 / P450 P95 TAL W645 / LUZ V29 / PKS S366 TAL W645 / LUZ V29 / AROG K11 TAL W645 / LUZ V29 / P450 P95 TAL W645 / PKS S366 / AROG K11 TAL W645 / PKS S366 / P450P95 ,TAL W645 / AROG K11 / P450 P95 、H3H P200 / LIGHT V29 / PKS S366 、H3H P200 / LIGHT V29 / FOOD K11 、H3H P200 / LIGHT V29 / P450 P95 、H3H P200 / PKS S366 / FOOD K11 、H3H P200 / PKS S366 / P450 P95 、H3H P200 / AROG K11 / P450 P95 、LIGHT V29 / PKS S366 / RUDE K11 ,LIGHT V29 / PKS S366 / P450 P95 ,LIGHT V29 / AROG K11 / P450 P95 、PKS S366 / AROG K11 / P450 P95 ;TAL W645 / H3H P200 / LIGHT V29 / PKS S366 ,TAL W645 / H3H P200 / LIGHT V29 / AROG K11 ,TAL W645 / H3H P200 / LIGHT V29 / P450 P95 ,TAL W645 / H3H P200 / PKS S366 / AROG K11 ,TAL W645 / H3H P200 / PKS S366 / P450 P95 ,TAL W645 / H3H P200 / AROG K11 / P450P95 ,TAL W645 / LIGHT V29 / PKS S366 / AROG K11 ,TAL W645 / LIGHT V29 / PKS S366 / P450 P95 ,TAL W645 / LIGHT V29 / AROG K11 / P450 P95 ,TAL W645 / PKS S366 / AROG K11 / P450 P95 、H3H P200 / LIGHT V29 / PKS S366 / AROG K11 / 、H3H P200 / LIGHT V29 / PKS S366 / P450 P95 、H3H P200 / LIGHT V29 / AROG K11 / P450 P95 、H3H P200 / PKS S366 / AROG K11 / P450 P95 ,LIGHT V29 / PKS S366 / AROG K11 / P450 P95 ;TAL W645 / H3H P200 / LIGHT V29 / PKS S366 / AROG K11 ,TAL W645 / H3H P200 / LIGHT V29 / PKS S366 / P450 P95 ,TAL W645 / H3H P200 / LIGHT V29 / AROG K11 / P450 P95 ,TAL W645 / H3H P200 / PKS S366 / AROG K11 / P450 P95 ,TALW645 / LUZ V29 / PKS S366 / AROG K11 / P450 P95 H3H P200 / LUZ V29 / PKS S366 / AROG K11 / P450 P95 ; or TAL W645 / H3H P200 / LUZ V29 / PKS S366 / AROG K11 / P450 P95 .

[0015] As described above, the combination of mutants is selected from the group consisting of: TAL W645 / D571 / S108 / H3H F139 / P200 / E379 TAL W645 / D571 / S108 / LUZ V29 / E181 / V28 TAL W645 / D571 / S108 / PKS L382 / L405 / Y327 TAL W645 / D571 / S108 / AROG K11 / E174 / G350 TAL W645 / D571 / S108 / P450 K50 / P95 H3H F139 / P200 / E379 / LUZ V29 / E181 / V28 H3H F139 / P200 / E379 / PKS L382 / L405 / Y327 H3H F139 / P200 / E379 / AROG K11 / E174 / G350 H3H F139 / P200 / E379 / P450 K50 / P95 LUZ V29 / E181 / V28 / PKS L382 / L405 / Y327 LUZ V29 / E181 / V28 / AROG K11 / E174 / G350 / 、LUZ V29 / E181 / V28 / P450 K50 / P95 PKS L382 / L405 / Y327 / AROG K11 / E174 / G350 PKS L382 / L405 / Y327 / P450 K50 / P95 AROG K11 / E174 / G350 / P450 K50 / P95 TAL W645 / D571 / S108 / H3H F139 / P200 / E379 / LUZ V29 / E181 / V28 TAL W645 / D571 / S108 / H3H F139 / P200 / E379 / PKS L382 / L405 / Y327 TALW645 / D571 / S108 / H3H F139 / P200 / E379 / AROG K11 / E174 / G350 ,TAL W645 / D571 / S108 / H3H F139 / P200 / E379 / P450 K50 / P95 ,TAL W645 / D571 / S108 / LIGHT V29 / E181 / V28 / PKS L382 / L405 / Y327 ,TAL W645 / D571 / S108 / LIGHT V29 / E181 / V28 / AROG K11 / E174 / G350 ,TAL W645 / D571 / S108 / LIGHT V29 / E181 / V28 / P450 K50 / P95 ,TAL W645 / D571 / S108 / PKS L382 / L405 / Y327 / RUDE K11 / E174 / G350 ,TAL W645 / D571 / S108 / PKS L382 / L405 / Y327 / P450 K50 / P95 ,TAL W645 / D571 / S108 / RUDE K11 / E174 / G350 / P450 K50 / P95 、H3H F139 / P200 / E379 / LIGHT V29 / E181 / V28 / PKS L382 / L405 / Y327 、H3H F139 / P200 / E379 / LIGHT V29 / E181 / V28 / AROG K11 / E174 / G350 、H3H F139 / P200 / E379 / LIGHT V29 / E181 / V28 / P450 K50 / P95 、H3H F139 / P200 / E379 / PKS L382 / L405 / Y327 / AROG K11 / E174 / G350 、H3H F139 / P200 / E379 / PKS L382 / L405 / Y327 / P450 K50 / P95 、H3H F139 / P200 / E379 / AROG K11 / E174 / G350 / P450 K50 / P95 ,LIGHT V29 / E181 / V28 / PKS L382 / L405 / Y327 / AROG K11 / E174 / G350 ,LIGHT V29 / E181 / V28 / PKS L382 / L405 / Y327 / P450 K50 / P95 、PKS L382 / L405 / Y327 / AROG K11 / E174 / G350 / P450 K50 / P95 ;TAL W645 / D571 / S108 / H3H F139 / P200 / E379 / LIGHT V29 / E181 / V28 / PKSL382 / L405 / Y327 ,TAL W645 / D571 / S108 / H3H F139 / P200 / E379 / LIGHT V29 / E181 / V28 / AROG K11 / E174 / G350 ,TAL W645 / D571 / S108 / H3H F139 / P200 / E379 / LIGHT V29 / E181 / V28 / P450 K50 / P95 ,TAL W645 / D571 / S108 / H3H F139 / P200 / E379 / PKS L382 / L405 / Y327 / AROG K11 / E174 / G350 ,TAL W645 / D571 / S108 / H3H F139 / P200 / E379 / PKS L382 / L405 / Y327 / P450 K50 / P95 ,TAL W645 / D571 / S108 / H3H F139 / P200 / E379 / AROG K11 / E174 / G350 / P450 K50 / P95 ,TAL W645 / D571 / S108 / LIGHT V29 / E181 / V28 / PKS L382 / L405 / Y327 / AROG K11 / E174 / G350 ,TAL W645 / D571 / S108 / LIGHT V29 / E181 / V28 / PKS L382 / L405 / Y327 / P450 K50 / P95 ,TAL W645 / D571 / S108 / LIGHT V29 / E181 / V28 / AROG K11 / E174 / G350 / P450 K50 / P95 ,TAL W645 / D571 / S108 / PKS L382 / L405 / Y327 / RUDE K11 / E174 / G350 / P450 K50 / P95 、H3H F139 / P200 / E379 / LIGHT V29 / E181 / V28 / PKS L382 / L405 / Y327 / AROG K11 / E174 / G350 、H3H F139 / P200 / E379 / LIGHT V29 / E181 / V28 / PKS L382 / L405 / Y327 / P450 K50 / P95 、H3H F139 / P200 / E379 / LIGHT V29 / E181 / V28 / RUDE K11 / E174 / G350 / P450 K50 / P95 、H3H F139 / P200 / E379 / PKS L382 / L405 / Y327 / AROG K11 / E174 / G350 / P450 K50 / P95 LUZ V29 / E181 / V28 / PKS L382 / L405 / Y327 / AROG K11 / E174 / G350 / P450 K50 / P95 TAL W645 / D571 / S108 / H3H F139 / P200 / E379 / LUZ V29 / E181 / V28 / PKS L382 / L405 / Y327 / AROG K11 / E174 / G350 TAL W645 / D571 / S108 / H3H F139 / P200 / E379 / LUZ V29 / E181 / V28 / PKS L382 / L405 / Y327 / P450 K50 / P95 TAL W645 / D571 / S108 / H3H F139 / P200 / E379 / LUZ V29 / E181 / V28 / AROG K11 / E174 / G350 / P450 K50 / P95 TAL W645 / D571 / S108 / H3H F139 / P200 / E379 / PKS L382 / L405 / Y327 / AROG K11 / E174 / G350 / P450 K50 / P95 TAL W645 / D571 / S108 / LUZ V29 / E181 / V28 / PKS L382 / L405 / Y327 / AROG K11 / E174 / G350 / P450 K50 / P95 H3H F139 / P200 / E379 / LUZ V29 / E181 / V28 / PKS L382 / L405 / Y327 / AROG K11 / E174 / G350 / P450 K50 / P95 ; or TAL W645 / D571 / S108 / H3H F139 / P200 / E379 / LUZ V29 / E181 / V28 / PKS L382 / L405 / Y327 / AROG K11 / E174 / G350 / P450 K50 / P95 .

[0016] A method for increasing bioluminescence intensity or increasing the amount of 3-arylacrylic acid synthesized in biological cells includes: expressing LUZ, SKP, H3H, CPH proteins or any of the amino acid mutants described above or a combination of said mutants in plants; preferably, the 3-arylacrylic acid is selected from the group consisting of caffeic acid, cinnamic acid, p-coumaric acid, coumaric acid, umbelliferic acid, sinapic acid and ferulic acid.

[0017] A method for increasing bioluminescence intensity or increasing the amount of 3-arylacrylic acid synthesized in biological cells includes: expressing LUZ, SKP, H3H, CPH, TAL, P450 proteins or amino acid mutants as described above or combinations thereof in plants; preferably, the 3-arylacrylic acid is selected from the group consisting of caffeic acid, cinnamic acid, p-coumaric acid, coumaric acid, umbelliferic acid, sinapic acid, and ferulic acid.

[0018] A method for increasing bioluminescence intensity or increasing the amount of 3-arylacrylic acid synthesized in biological cells includes: expressing LUZ, SKP, H3H, CPH, TAL, P450, AROG proteins or amino acid mutants as described above or combinations thereof in plants; preferably, the 3-arylacrylic acid is selected from the group consisting of caffeic acid, cinnamic acid, p-coumaric acid, coumaric acid, umbelliferic acid, sinapic acid, and ferulic acid.

[0019] This application solves the problems of low luminescence brightness and poor caffeic acid utilization efficiency in self-luminescent plants that rely on fungal FBP systems. Attached Figure Description

[0020] The preferred embodiments of the present invention will now be described in further detail with reference to the accompanying drawings, wherein: Figure 1 This describes the effect of a site-directed single mutation of TAL on the luminescence intensity of plants according to an embodiment of the present invention. Figure 2 This is a photograph of plant luminescence after a site-directed single mutation of TAL according to an embodiment of the present invention; Figure 3 This describes the effect of a site-directed single mutation of TAL on the caffeic acid content in cells according to an embodiment of the present invention. Figure 4 This describes the effect of TAL site-directed multiple mutations on plant luminescence intensity according to an embodiment of the present invention; Figure 5 These are bioluminescent images of plants after site-directed multiple mutations of TAL according to an embodiment of the present invention; Figure 6 This describes the effect of TAL site-directed multiple mutations on cellular caffeic acid content according to an embodiment of the present invention. Figure 7 This describes the effect of a site-directed single mutation of H3H on the luminescence intensity of plants according to an embodiment of the present invention. Figure 8 This is a bioluminescent photograph of a plant after a site-directed single mutation of H3H according to an embodiment of the present invention; Figure 9 This describes the effect of a site-directed single mutation of H3H on the caffeic acid content of cells according to an embodiment of the present invention. Figure 10This describes the effect of H3H site-directed multiple mutations on plant luminescence intensity according to an embodiment of the present invention; Figure 11 These are bioluminescent images of plants after H3H site-directed multiple mutations according to an embodiment of the present invention; Figure 12 This describes the effect of H3H site-directed multiple mutations on cellular caffeic acid content according to an embodiment of the present invention. Figure 13 This describes the effect of a site-directed single mutation of LUZ on the luminescence intensity of plants according to an embodiment of the present invention. Figure 14 This is a photograph of plant luminescence after a site-directed single mutation of LUZ according to an embodiment of the present invention; Figure 15 This describes the effect of a site-directed single mutation of LUZ on cellular caffeic acid content according to an embodiment of the present invention. Figure 16 This describes the effect of LUZ site-directed multiple mutations on plant luminescence intensity according to an embodiment of the present invention; Figure 17 These are bioluminescent images of plants after LUZ site-directed multiple mutations according to an embodiment of the present invention; Figure 18 This describes the effect of LUZ site-directed multiple mutations on cellular caffeic acid content according to an embodiment of the present invention. Figure 19 This describes the effect of a PKS site-directed single mutation on plant luminescence intensity according to an embodiment of the present invention. Figure 20 These are bioluminescent images of plants after PKS site-directed single mutation according to an embodiment of the present invention; Figure 21 This describes the effect of a PKS site-directed single mutation on cellular caffeic acid content according to an embodiment of the present invention. Figure 22 This describes the effect of PKS site-directed multiple mutations on plant luminescence intensity according to an embodiment of the present invention. Figure 23 These are bioluminescent images of plants after PKS site-directed multiple mutations according to an embodiment of the present invention; Figure 24 This describes the effect of PKS site-directed multiple mutations on cellular caffeic acid content according to an embodiment of the present invention. Figure 25 This describes the effect of a site-directed single mutation of AROG on the luminescence intensity of plants according to an embodiment of the present invention. Figure 26 This is a photograph of a plant exhibiting bioluminescence after a site-directed single mutation of AROG according to an embodiment of the present invention; Figure 27 This describes the effect of a site-directed single mutation of AROG on cellular caffeic acid content according to an embodiment of the present invention. Figure 28 This describes the effect of AROG site-directed multiple mutations on plant luminescence intensity according to an embodiment of the present invention. Figure 29 These are bioluminescent images of plants after AROG site-directed multiple mutations according to an embodiment of the present invention; Figure 30 This describes the effect of AROG site-directed multiple mutations on cellular caffeic acid content according to an embodiment of the present invention. Figure 31 This describes the effect of a P450 site-directed single mutation on plant luminescence intensity according to an embodiment of the present invention. Figure 32 This is a photograph of a plant exhibiting bioluminescence after a site-directed single mutation of P450 according to an embodiment of the present invention; Figure 33 This describes the effect of a P450 site-directed single mutation on cellular caffeic acid content according to an embodiment of the present invention. Figure 34 This is an embodiment of the invention regarding the effect of P450 site-directed multiple mutations on plant luminescence intensity; Figure 35 This is a photograph of a plant with P450 site-directed multiple mutations according to an embodiment of the present invention; Figure 36 This describes the effect of P450 site-directed multiple mutations on cellular caffeic acid content according to an embodiment of the present invention. Figure 37 This describes the effect of a combination of two site-directed single mutations on plant luminescence intensity according to an embodiment of the present invention. Figure 38 This is a photograph of a plant bioluminescence based on a dual-gene site-directed single mutation combination according to an embodiment of the present invention. Figure 39 This describes the effect of a combination of two site-directed single mutations on cellular caffeic acid content according to an embodiment of the present invention. Figure 40 This describes the effect of a three-gene site-directed single mutation combination on plant luminescence intensity according to an embodiment of the present invention. Figure 41 This is a photograph of a plant luminescence based on a three-gene site-directed single mutation combination according to an embodiment of the present invention; Figure 42 This describes the effect of a combination of three site-directed single mutations on cellular caffeic acid content according to an embodiment of the present invention. Figure 43 This describes the effect of a four-gene site-directed single mutation combination on plant luminescence intensity according to an embodiment of the present invention. Figure 44 This is a photograph of a plant luminescence based on a four-gene site-directed single mutation combination according to an embodiment of the present invention; Figure 45 This describes the effect of a combination of four site-directed single mutations on cellular caffeic acid content according to an embodiment of the present invention. Figure 46 This describes the effect of a combination of five or six gene site-directed single mutations on plant luminescence intensity according to an embodiment of the present invention. Figure 47 This is a photograph of a plant bioluminescence based on a combination of five or six gene site-directed single mutations according to an embodiment of the present invention. Figure 48 This invention relates to the effect of a combination of five or six gene site-directed single mutations on cellular caffeic acid content according to an embodiment of the present invention. Figure 49 This describes the effect of a multi-gene site-directed multiple mutation combination on plant luminescence intensity according to an embodiment of the present invention; Figure 50 These are bioluminescent photographs of plants with multiple gene site-directed mutation combinations according to an embodiment of the present invention; and Figure 51 This invention relates to the effect of a combination of multiple gene site-directed mutations on the caffeic acid content in cells, according to an embodiment of the present invention. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] In the following detailed description, reference can be made to the accompanying drawings, which form part of this application and illustrate specific embodiments of the present application. In the drawings, similar reference numerals describe substantially similar components in different figures. Specific embodiments of the present application are described in sufficient detail below to enable those skilled in the art to implement the technical solutions of the present application. It should be understood that other embodiments or modifications to the embodiments of the present application may also be utilized.

[0023] This application found that the wild-type TAL, H3H, LUZ, PKS, AROG, and P450 luminescent genes exhibit low intrinsic luminescence intensity, resulting in weak detection signals under low expression levels. Simultaneously, the caffeic acid-mediated luminescence reaction exhibits insufficient catalytic efficiency, failing to meet the detection requirements of high-sensitivity, low-abundance samples. Existing mutational studies are fragmented, lacking systematic site screening and functional validation for the aforementioned five types of luminescent genes, and failing to fully explore the synergistic potential of key sites in enhancing luminescence intensity, caffeic acid binding efficiency, and catalytic activity. Research on single-gene multi-site combination mutations and cross-gene synergistic mutations is still lacking, making it impossible to achieve exponential increases in luminescence intensity through inter-site synergistic effects. Furthermore, the absence of a supporting in vitro / in vivo luminescence imaging validation system hinders the practical application of high-performance luminescence tools.

[0024] Therefore, this application provides high-brightness mutants (single-point, single-gene intra-gene combination, and cross-gene combination) of six major luminescent genes, namely TAL, H3H, nnLUZ, PKS, AROG, and P450, to solve the problems of low brightness and poor caffeic acid utilization efficiency of existing luminescent genes; and further establishes a triple verification system with caffeic acid content, luminescence brightness, and luminescence images as the core to comprehensively verify the technical effects of the mutants and provide reliable experimental basis for their industrial application.

[0025] This invention uses caffeic acid as a specific luminescent substrate and employs a technical route of "conserved site analysis - single-point mutation screening - combined mutation optimization - triple experimental verification" to modify six genes: TAL, H3H, nnLUZ, PKS, AROG, and P450. Key mutation sites that can improve caffeic acid binding efficiency and enhance luminescence brightness are screened out. Single-gene and cross-gene combined mutants are designed, and the superior performance of the mutants is confirmed by detecting caffeic acid content (substrate utilization efficiency) and quantifying luminescence brightness (luminescence intensity). All mutants maintain good structural stability and spectral specificity.

[0026] The bioluminescent organisms referred to in this application are organisms that can emit light after inheriting genes related to the fungal FBP system, including but not limited to bacteria, plants, and animals. According to one embodiment of this application, the bioluminescent organism is a bioluminescent plant. In some embodiments, the plants are selected from algae, bryophytes, ferns, gymnosperms, and angiosperms; preferably, the plants are selected from the group consisting of: Nicotiana benthamiana, Arabidopsis thaliana, tobacco, chrysanthemum, marigold, cosmos, zinnia, coreopsis, echinacea, Shasta daisy, silverleaf chrysanthemum, ageratum, marigold, white chrysanthemum, daisy, asters, cineraria, salvia splendens, sage, lavender, rosemary, mint, patchouli, coleus, rose, rose, tulip, hyacinth, daylily, hosta, spider plant, asparagus fern, aloe vera, liriope, purslane, kale, violet, honesty, carnation, carnation, baby's breath, tall snow lily, dwarf snow lily, jasmine, coral bean, ornamental pepper, tobacco flower, Bauhinia, wisteria, lupin, trifoliate orange. Grass, iris, German iris, butterfly flower, iris, anthurium, peace lily, monstera, pothos, peace lily, calla lily, tomato, pepper, eggplant, pumpkin, rapeseed, colored pepper, colored rapeseed, cabbage, broccoli, cauliflower, cotton, osmanthus, jasmine, lilac, forsythia, winter jasmine, forsythia, poinsettia, crown of thorns, croton, jade tree, kalanchoe, sedum, lotus, sedum, cactus, globular cactus, epiphyllum, epiphyllum, Christmas cactus, purple bamboo plum, wandering japonica, amaryllis, cyclamen, gloxinia, oxalis, bellflower, lobelia, verbena, geranium, nasturtium, gazania, marguerite, bleeding heart, foxglove, ranunculus, columbine, blue plumbago, periwinkle, million bells and sweet alyssum.

[0027] The mutant referred to in this application is a gene sequence obtained by altering one or more nucleotides of a gene, thereby changing the amino acid sequence at one or more sites. This mutant can be a nucleotide mutant, including a nucleotide sequence that differs from the original sequence; or it can be an amino acid mutant, including an amino acid sequence that differs from the original sequence. In some embodiments, the original sequence can be a naturally occurring sequence in an organism or a modified sequence. According to one embodiment of this application, regardless of whether the modified sequence includes mutations not mentioned in this application, as long as it includes the site-directed mutations of this application, it falls under the technical solution of this application.

[0028] Example 1: Vector Construction In this application, various vectors were selected and constructed, and site-directed mutagenesis was performed on these vectors. The vectors involved in this application include genes such as LUZ, PKS, H3H, CPH, TAL, AROG, and P450. The amino acid sequence of CPH is shown in SEQ ID NO. 180. This application is not limited to the base vector used to carry the above genes. In some embodiments, each vector is based on the pCAMBIA2300 plasmid and modified accordingly.

[0029] According to one embodiment of this application, two sets of basic vectors are constructed for site-directed mutagenesis modification and plant transformation in this application.

[0030] Vector A includes fungal FBP expression system genes such as LUZ, PKS, H3H, and CPH.

[0031] Vector B includes genes such as TAL, AROG, and P450, which are used to enhance the luminescence intensity of the fungal FBP system.

[0032] Gene synthesis and amplification: Commercially synthesized genes such as LUZ, PKS, H3H, CPH, TAL, P450, and AROG, or their mutants, along with the CaMV 35S promoter and NOS terminator sequences. High-fidelity PCR enzymes were used to amplify each gene and regulatory element fragment separately, ensuring the integrity and specificity of the amplified fragments.

[0033] The CaMV 35S promoter and NOS terminator are commonly used components in the art, and any CaMV 35S promoter and NOS terminator verified in the art can be used in this application, and their sequences have negligible impact on the technical effects of this application. In some embodiments, the promoters preceding the PKS gene, LUZ gene, H3H gene, CPH gene, TAL gene, P450 gene, and AROG gene are all CaMV 35S, but this application is not limited thereto.

[0034] The gene mutants involved include, but are not limited to: TAL W645V TAL W645A TAL W645L TAL W645T TAL W645I TAL W645S TAL W645N TAL W645F TAL W645G TAL D571A TAL D571V TAL D571L TAL D571G TAL E163A, TAL E163S , TAL E163A , TAL P3A , TAL S108P , TAL Y245K , TAL L162Q , TAL A498H , TAL V186P , TAL I634P etc.; H3H F139N , H3H F139G , H3H F139H , H3H F139S , H3H F139E , H3H F139Q , H3H F139A , H3H F139K , H3H F139R , H3H P200R , H3H P200K , H3H P200Q , H3H P200N , H3H E379M , H3H E379L , H3H E379N , H3H E379A , H3H E379S , H3H F4P , H3H F4A , H3H V142P , H3H N6K , H3H S7K , H3H S7R , H3H A197R , H3H A197K etc.;​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​L321S PKS L382A PKS L382E PKS L382K PKS L382P PKS L382Q PKS L382S PKS L405G PKS L405P PKS R330P PKS S366P PKS S402G PKS V196A PKS Y327A PKS Y327G PKS Y327H PKS Y327K PKS Y327N PKS Y327Q PKS Y327R PKS Y327S PKS Y327T wait; AROG N5T AROG K11Y AROG K11S AROG K41T AROG K41L AROG E174Q AROG A319V AROG A319I AROG K322Q AROG G350T AROG G350S AROG G350M wait; P450 A11I P450 A11V P450 H18I P450 H18V P450 L31N P450 K50D P450 K50E P450 K50N P450 H94E P450 P95V P450 P95Q P450 P95A P450 P95E P450 P95I P450 P95T P450 P95S P450 S97A P450 Y380N etc.; and Any combination of the above mutants, including combinations of different mutations of the same gene, combinations of mutants of different genes, etc.

[0035] Vector linearization: The pCAMBIA2300 vector was double-digested with EcoRI and HindIII. The digestion system (50 μL) consisted of 40 μL pCAMBIA2300 vector, 1 μL EcoRI, 1 μL HindIII, 5 μL 10×Cutsmart buffer, and 3 μL ddH2O. After incubation at 37℃ for 10 h, the digestion was terminated by treatment at 80℃ for 20 min. The linearized vector was recovered by agarose gel electrophoresis.

[0036] The gene combination fragments mentioned above were mixed with the linearized vector at a molar ratio of 1:3 to 1:5, and the seamless cloning kit reagents were added. The ligation was completed by incubation according to the instructions.

[0037] Transformation and Validation: The ligation product was transformed into *E. coli* DH5α competent cells, plated on LB agar plates containing 50 mg / L kanamycin, and incubated at 37°C for 12–16 h. Single colonies were picked for colony PCR identification. Plasmids were extracted from positive clones and Sanger sequencing was performed to verify the insertion direction, sequence integrity, and accuracy of enhancer positions for each fragment. Successful sequencing indicated a successfully constructed recombinant construct.

[0038] Example 2 Plant Transformation In this application, the methods for transforming plants with Agrobacterium include transient transformation and stable transformation. The transient transformation method includes: Agrobacterium transformation: Each recombinant construct was transformed into Agrobacterium GV3101 competent cells by freeze-thaw method, plated on LB agar plates containing rifampicin (50 mg / L) and kanamycin (50 mg / L), and incubated upside down at 28°C for 48 h. Single colonies were picked and transferred to LB liquid medium containing the same antibiotics, and cultured at 28°C and 200 rpm for 16-20 h to obtain seed culture.

[0039] Agrobacterium expansion culture: Inoculate the seed culture at a 1:100 volume ratio into fresh LB broth (containing the same antibiotic), and incubate at 28°C with shaking at 200 rpm until OD. 600 =0.8-1.0, centrifuge at 4℃ and 5000 rpm for 10 min to collect bacterial cells.

[0040] Preparation of infection buffer: The bacterial cells were resuspended in infection buffer (MS basal medium 4.43 g, sucrose 100 g, deionized water to a final volume of 1 L, pH 5.6-5.8, sterilized and then acetylsuccinone to a final concentration of 200 μmol / L) and the OD was adjusted. 600=0.6-0.8, incubate in the dark at 28℃ for 2-3 h.

[0041] Plant infection: Select healthy, disease-free leaves from 4-6 week old Nicotiana benthamiana plants, immerse them in the infection solution for 30-60 seconds, and gently shake to ensure that both sides of the leaves are in contact with the infection solution. This application does not limit the infected plant to Nicotiana benthamiana; other plants for which luminescence is desired can also be used in this process.

[0042] Co-culture and screening: After infection, the leaves were blotted dry with sterile filter paper to remove residual liquid on the surface, and then spread evenly in a petri dish with wet filter paper. They were co-cultured at 25°C in the dark for 2-3 days. Subsequently, they were transferred to MS solid medium containing cephalosporin (500 mg / L) and cultured at 25°C under 16 h light / 8 h dark conditions for 3-5 days.

[0043] Plants containing the seven genes or their mutants mentioned in Example 1 were obtained.

[0044] Among them, mutant plants include, but are not limited to: Single-gene mutants: FBP-ΔTAL, FBP-ΔH3H, FBP-ΔLUZ, FBP-ΔPKS, FBP-ΔAROG, FBP-ΔP450, etc.; Double gene mutants: FBP-ΔTAL / H3H, FBP-ΔTAL / LUZ, FBP-ΔTAL / PKS, FBP-ΔTAL / AROG, FBP-ΔH3H / LUZ, FBP-ΔH3H / PKS, FBP-ΔH3H / AROG , FBP-ΔH3H / P450, FBP-ΔLUZ / PKS, FBP-ΔLUZ / AROG, FBP-ΔLUZ / P450, FBP-ΔPKS / AROG, FBP-ΔPKS / P450, FBP-ΔAROG / P450, etc.; Tri-gene mutants: FBP-ΔTAL / H3H / LUZ, FBP-ΔTAL / H3H / PKS, FBP-ΔTAL / H3H / AROG, FBP-ΔTAL / H3H / P450, FBP-ΔTAL / LUZ / PKS, FBP-ΔTAL / LUZ / AROG, FBP-ΔTAL / LUZ / P450, FBP-ΔTAL / PKS / AROG, FBP-ΔTAL / PKS / P450, FBP-ΔTAL / AROG / P450, FBP-ΔH3H / LUZ / PKS, FBP-ΔH3H / LUZ / AROG, FBP-ΔH3H / LUZ / P450, FBP-ΔH3H / PKS / AROG, FBP-ΔH 3H / AROG / P450, FBP-ΔLUZ / PKS / AROG, FBP-ΔLUZ / PKS / P450, FBP-ΔLUZ / AROG / P450, FBP-ΔPKS / AROG / P450, etc.; Four-gene mutants: FBP-ΔTAL / H3H / LUZ / PKS, FBP-ΔTAL / H3H / LUZ / AROG, FBP-ΔTAL / H3H / LUZ / P450, FBP-ΔTAL / H3H / PKS / AROG, FBP-ΔTAL / H3H / PKS / P450, FBP-ΔTAL / H3H / AROG / P450, FBP-ΔTAL / LUZ / PKS / AROG, FBP-ΔTAL / LUZ / PKS / P450, FBP-ΔTAL / LUZ / AROG / P 450, FBP-ΔTAL / PKS / AROG / P450, FBP-ΔH3H / LUZ / PKS / AROG, FBP-ΔH3H / PKS / AROG / P450, FBP-ΔLUZ / PKS / ARDG / P450, etc.; Five gene mutants: FBP-ΔTAL / H3H / LUZ / PKS / AROG, FBP-ΔTAL / H3H / LUZ / PKS / P450, FBP-ΔTAL / H3H / LUZ / AROG / P45 0. FBP-ΔTAL / H3H / PKS / AROG / P450, FBP-ΔTAL / LUZ / PKS / AROG / P450, FBP-ΔH3H / LUZ / PKS / AROG / P450, etc.; Six gene mutants: FBP-ΔTAL / H3H / LUZ / PKS / AROG / P450, etc.

[0045] Example 3: Detection of luminescence intensity The method for detecting luminescence intensity includes culturing tobacco transformed by the gene in Example 2 for 24-48 hours (36 hours in this application), placing the cultured tobacco leaves in a dark environment, and using a LightScout full-spectrum quantum meter (Shanghai Zequan Technology) with the detection port close to the leaf surface to detect the amount of photons accumulated (shooting conditions: Huawei P30 Pro, ISO6400, time-lapse photography 2s, aperture 1.6 mm). Three biological replicates are set up for each group, and the luminescence intensity data are recorded and statistically analyzed.

[0046] Example 4: Detection of Caffeic Acid Content Sample preparation: Tobacco leaves that had been transformed in Example 2 and cultured for 36 hours were immediately frozen and ground in liquid nitrogen. About 1 g of the frozen tissue was placed in a 50 ml centrifuge tube and freeze-dried. The freeze-dried material was stored at -20°C. Each sample group was prepared and analyzed three times.

[0047] Extraction and filtration: Weigh approximately 50 mg of lyophilized powder, treat with 7 ml of 70% methanol aqueous solution in an ultrasonic bath for 30 minutes, centrifuge at 4,000 rpm for 10 minutes, collect the supernatant, and filter with a Phenex GF / PVDF syringe filter (30 mm in diameter, 0.45 μm in pore size).

[0048] LCMS Analysis: Analysis was performed using a Shimadzu 8030 system (HPLC-DAD-ESI-TQ MS). The column was a Discovery C18 column (4.6 × 150 mm, 5 μm). Mobile phase A was 0.3% aqueous acetic acid (v / v), and mobile phase B was acetonitrile. Gradient run: 0–4 min 10–40% B, 4–5 min 40–80% B, 5–10.5 min 100% B isocratic elution, followed by restoration to initial conditions. Column temperature: 40℃, flow rate: 1 ml / min, injection volume: 20 μl.

[0049] The recorded caffeic acid content is a relative content. Tobacco leaves transformed with unmutated vectors A and B were designated as the WT group, with a caffeic acid content of 1. The caffeic acid content of other mutant groups was a multiple of that of the WT group.

[0050] Example 5: Effect of TAL point mutation on luminescence intensity In this embodiment, genes such as LUZ, PKS, H3H, CPH, TAL, P450, and AROG were transformed into tobacco. Different site-directed mutations of TAL were transformed into different tobacco plants or tobacco leaves, including one or more mutations. The vector construction method and plant transformation method are detailed in Examples 1 and 2, and will not be repeated in this embodiment.

[0051] The amino acid sequences of TAL and its site-directed mutations are shown in Table 1: Table 1

[0052] Figures 1-6 The effects of site-directed single or multiple mutations of TAL on plant luminescence intensity and cellular caffeic acid content were shown.

[0053] like Figures 1-3 As shown, specific site mutations in TAL can increase the caffeic acid content in cells, thereby increasing the luminescence intensity of plant leaves. Mutations at positions 645, 571, and 108 significantly increased caffeic acid content. Furthermore, mutations at the same site to different amino acids had varying effects on caffeic acid accumulation and luminescence intensity. For example, mutations at position 645 (tryptophan (W) to leucine (L) or valine (V), at position 571 (aspartic acid (D)) to leucine (L), and at position 108 (serine (S)) to proline (P) significantly increased luminescence intensity compared to mutations at other sites. Therefore, replacing W at position 645 with the hydrophobic, nonpolar amino acid L or V resulted in a more significant increase in luminescence intensity and a greater increase in caffeic acid content compared to other substitutions.

[0054] like Figure 4-6 As shown, after selecting a combination of single mutation sites with significant technical effects, both the luminescence intensity and the accumulation of caffeic acid were improved compared to the single mutant, and the luminescence intensity could be further improved. Different mutations at the same site had a relatively small impact on the luminescence intensity.

[0055] Those skilled in the art should know that the above mutations are only representative mutation sites selected by the applicant, and do not represent all mutation origins and mutation schemes that affect luminescence intensity and caffeic acid content accumulation.

[0056] Example 6: Effect of H3H point mutation on luminescence intensity In this embodiment, genes such as LUZ, PKS, H3H, CPH, TAL, P450, and AROG were transformed into tobacco. Different H3H site-directed mutations, including one or more mutations, were transformed into different tobacco plants or tobacco leaves. The vector construction method and plant transformation method are detailed in Examples 1 and 2, and will not be repeated in this embodiment.

[0057] The amino acid sequences of H3H and its site-directed mutations are shown in Table 2: Table 2

[0058] Figures 7-12The effects of H3H site-directed single or multiple mutations on plant luminescence intensity and cellular caffeic acid content were shown.

[0059] like Figures 7-9 As shown, specific site mutations in H3H can increase the caffeic acid content in cells, thereby increasing the luminescence intensity of plant leaves. Mutations at positions 139, 200, and 379 significantly increased caffeic acid content. Furthermore, mutations at the same site to different amino acids had varying effects on caffeic acid accumulation and luminescence intensity. For example, mutations at position 139 (phenylalanine (F) to histidine (H), position 200 (proline (P)) to aspartic acid (N) or glutamine (Q), and position 379 (glutamate (E)) to methionine (M) or leucine (L) significantly increased luminescence intensity compared to mutations at other sites.

[0060] like Figure 10-12 As shown, after selecting a combination of single mutation sites with significant technical effects, both the luminescence intensity and the accumulation of caffeic acid were improved compared to the single mutant, and the luminescence intensity could be further improved. Different mutations at the same site had a relatively small impact on the luminescence intensity.

[0061] Those skilled in the art should know that the above mutations are only representative mutation sites selected by the applicant, and do not represent all mutation origins and mutation schemes that affect luminescence intensity and caffeic acid content accumulation.

[0062] Example 7: Effect of LUZ point mutation on luminescence intensity In this embodiment, genes such as LUZ, PKS, H3H, CPH, TAL, P450, and AROG were transformed into tobacco. Different site-directed mutations of LUZ were transformed into different tobacco plants or tobacco leaves, including one or more mutations. The vector construction method and plant transformation method are detailed in Examples 1 and 2, and will not be repeated in this embodiment.

[0063] The amino acid sequences of LUZ and its site-directed mutations are shown in Table 3: Table 3

[0064] Figures 13-18 The effects of LUZ site-directed single or multiple mutations on plant luminescence intensity and cellular caffeic acid content were shown.

[0065] like Figures 13-15As shown, specific site mutations in LUZ can increase the caffeic acid content in cells, thereby increasing the luminescence intensity of plant leaves. Mutations at positions 10, 162, and 9 significantly increased caffeic acid content. Furthermore, mutations at the same site to different amino acids had varying effects on caffeic acid accumulation and luminescence intensity. For example, mutations at position 10 (valine (V) to phenylalanine (F) or tryptophan (W), at position 162 (glutamate (E)) to alanine (A), and at position 9 (valine (V) to tryptophan (W)) significantly increased luminescence intensity compared to mutations at other sites.

[0066] like Figure 16-18 As shown, selecting a combination of single mutation sites with significant technical effects resulted in improved luminescence intensity and caffeic acid accumulation compared to the single mutant, with luminescence intensity increasing by 7-8 times. Different mutations at the same site had a relatively small impact on luminescence intensity.

[0067] Those skilled in the art should know that the above mutations are only representative mutation sites selected by the applicant, and do not represent all mutation origins and mutation schemes that affect luminescence intensity and caffeic acid content accumulation.

[0068] Example 8: Effect of PKS point mutation on luminescence intensity In this embodiment, genes such as LUZ, PKS, H3H, CPH, TAL, P450, and AROG were transformed into tobacco. Different site-directed mutations of PKS were transformed into different tobacco plants or tobacco leaves, including one or more mutations. The vector construction method and plant transformation method are detailed in Examples 1 and 2, and will not be repeated in this embodiment.

[0069] The amino acid sequences of PKS and its site-directed mutations are shown in Table 4. Table 4

[0070] Figures 19-24 The effects of PKS site-directed single or multiple mutations on plant luminescence intensity and cellular caffeic acid content were shown.

[0071] like Figures 19-21As shown, specific site mutations in PKS can increase the caffeic acid content in cells, thereby increasing the luminescence intensity of plant leaves. Mutations at positions 306, 329, and 251 significantly increased caffeic acid content. Furthermore, mutations at the same site to different amino acids had varying effects on caffeic acid accumulation and luminescence intensity. For example, mutations at position 306 (leucine (L) to lysine (K), position 329 (leucine (L) to glycine (G), and position 251 (tyrosine (Y) to asparagine (N), glutamine (Q), or threonine (T) significantly increased luminescence intensity compared to mutations at other sites.

[0072] like Figure 22-24 As shown, after selecting a combination of single mutation sites with significant technical effects, both the luminescence intensity and the accumulation of caffeic acid were improved compared to the single mutant, and the luminescence intensity could be further improved. Different mutations at the same site had a relatively small impact on the luminescence intensity.

[0073] Those skilled in the art should know that the above mutations are only representative mutation sites selected by the applicant, and do not represent all mutation origins and mutation schemes that affect luminescence intensity and caffeic acid content accumulation.

[0074] Example 9: Effect of AROG point mutation on luminescence intensity In this embodiment, genes such as LUZ, PKS, H3H, CPH, TAL, P450, and AROG were transformed into tobacco. Different AROG site-directed mutations, including one or more mutations, were transformed into different tobacco plants or tobacco leaves. The vector construction method and plant transformation method are detailed in Examples 1 and 2, and will not be repeated in this embodiment.

[0075] The amino acid sequences of AROG and its site-directed mutations are shown in Table 5. Table 5

[0076] Figures 25-30 The effects of AROG site-directed single or multiple mutations on plant luminescence intensity and cellular caffeic acid content were shown.

[0077] like Figures 25-27As shown, specific site mutations in AROG all increased the caffeic acid content in cells, thereby increasing the luminescence intensity of plant leaves. Mutations at positions 11, 174, 322, and 350 significantly increased caffeic acid content. Furthermore, mutations at the same site to different amino acids had varying effects on caffeic acid accumulation and luminescence intensity. For example, mutations at position 11 (lysine (K) to tyrosine (Y), position 174 (glutamate (E)) to glutamine (Q), position 322 (lysine (K)) to glutamine (Q), and position 350 (glycine (G)) to methionine (M) significantly increased luminescence intensity compared to mutations at other sites.

[0078] like Figures 28-30 As shown, after selecting a combination of single mutation sites with significant technical effects, both the luminescence intensity and the accumulation of caffeic acid were improved compared to the single mutant, and the luminescence intensity could be further improved. Different mutations at the same site had a relatively small impact on the luminescence intensity.

[0079] Those skilled in the art should know that the above mutations are only representative mutation sites selected by the applicant, and do not represent all mutation origins and mutation schemes that affect luminescence intensity and caffeic acid content accumulation.

[0080] Example 10: Effect of P450 point mutation on luminescence intensity In this embodiment, genes such as LUZ, PKS, H3H, CPH, TAL, P450, and AROG were transformed into tobacco. Different site-directed mutations of P450 were transformed into different tobacco plants or tobacco leaves, including one or more mutations. The vector construction method and plant transformation method are detailed in Examples 1 and 2, and will not be repeated in this embodiment.

[0081] The amino acid sequences of P450 and its site-directed mutations are shown in Table 6. Table 6

[0082] Figures 31-36 The effects of site-directed single or multiple mutations of P450 on plant luminescence intensity and cellular caffeic acid content were shown.

[0083] like Figures 31-33As shown, specific site mutations in P450 can increase the caffeic acid content in cells, thereby increasing the luminescence intensity of plant leaves. Mutations at positions 50 and 95 significantly increased caffeic acid content. Furthermore, mutations at the same site to different amino acids had varying effects on caffeic acid accumulation and luminescence intensity. For example, mutations at position 50 (lysine (K) to glutamic acid (E), and at position 95 (proline (P) to glutamine (Q), alanine (A), glutamic acid (E), isoleucine (I), threonine (T), or serine (S) significantly increased luminescence intensity compared to mutations at other sites.

[0084] like Figures 34-36 As shown, after selecting a combination of single mutation sites with significant technical effects, both the luminescence intensity and the accumulation of caffeic acid were improved compared to the single mutant, and the luminescence intensity could be further improved. Different mutations at the same site had a relatively small impact on the luminescence intensity.

[0085] Those skilled in the art should know that the above mutations are only representative mutation sites selected by the applicant, and do not represent all mutation origins and mutation schemes that affect luminescence intensity and caffeic acid content accumulation.

[0086] Example 11 Effect of dual-gene single-mutation combination on luminescence intensity In this embodiment, the genes LUZ, PKS, H3H, CPH, TAL, P450, and AROG, or their mutants, were transformed into tobacco, with each gene including a maximum of one mutation site. In this embodiment, the selected mutant sites were TAL... W645L H3H P200Q LUZ V29W PKS S366P AROG K11Y and P450 P95A The methods for constructing the vector and transforming the plant are detailed in Examples 1 and 2, and will not be repeated in this example.

[0087] The dual-gene mutants are FBP-ΔTAL / H3H, FBP-ΔTAL / LUZ, FBP-ΔTAL / PKS, FBP-ΔTAL / AROG, FBP-ΔH3H / LUZ, FBP-ΔH3H / PKS, FBP-ΔH3H / AROG, FBP-ΔH3H / P450, FBP-ΔLUZ / PKS, FBP-ΔLUZ / AROG, FBP-ΔLUZ / P450, FBP-ΔPKS / AROG, FBP-ΔPKS / P450, and FBP-ΔAROG / P450, etc., and their luminescence intensity, caffeic acid content, etc. are as follows: Figures 37-39 As shown.

[0088] like Figures 37-39By combining the luminescence intensity of the single point mutant of each gene, the luminescence intensity was significantly increased after the double gene mutation, compared with the unmutated leaves.

[0089] Example 12 Effect of Tri-Gene Single Mutation Combination on Luminescence Intensity In this embodiment, the genes LUZ, PKS, H3H, CPH, TAL, P450, and AROG, or their mutants, were transformed into tobacco, with each gene including a maximum of one mutation site. In this embodiment, the selected mutant sites were TAL... W645L H3H P200Q LUZ V29W PKS S366P AROG K11Y and P450 P95A The methods for constructing the vector and transforming the plant are detailed in Examples 1 and 2, and will not be repeated in this example.

[0090] The three gene mutants are FBP-ΔTAL / H3H / LUZ, FBP-ΔTAL / H3H / PKS, FBP-ΔTAL / H3H / AROG, FBP-ΔTAL / H3H / P450, FBP-ΔTAL / LUZ / PKS, FBP-ΔTAL / LUZ / AROG, FBP-ΔTAL / LUZ / P450, FBP-ΔTAL / PKS / AROG, FBP-ΔTAL / PKS / P450, and FBP-ΔTAL / AROG / P450, FBP-ΔH3H / LUZ / PKS, FBP-ΔH3H / LUZ / AROG, FBP-ΔH3H / LUZ / P450, FBP-ΔH3H / PKS / AROG, FBP-ΔH3H / AROG / P450, FBP-ΔLUZ / PKS / AROG, FBP-ΔLUZ / PKS / P450, FBP-ΔLUZ / AROG / P450, FBP-ΔPKS / AROG / P450, etc., their luminescence intensity, caffeic acid content, etc. Figures 40-42 As shown.

[0091] like Figures 40-42 By combining the luminescence intensity of the single point mutant of each gene, the luminescence intensity was significantly improved after the three-gene mutation, which was significantly improved compared with the leaves with two-gene mutation and those without mutation.

[0092] Example 13 Effect of a combination of four single-mutant genes on luminescence intensity In this embodiment, the genes LUZ, PKS, H3H, CPH, TAL, P450, and AROG, or their mutants, were transformed into tobacco, with each gene including a maximum of one mutation site. In this embodiment, the selected mutant sites were TAL... W645LH3H P200Q LUZ V29W PKS S366P AROG K11Y and P450 P95A The methods for constructing the vector and transforming the plant are detailed in Examples 1 and 2, and will not be repeated in this example.

[0093] The four gene mutants are FBP-ΔTAL / H3H / LUZ / PKS, FBP-ΔTAL / H3H / LUZ / AROG, FBP-ΔTAL / H3H / LUZ / P450, FBP-ΔTAL / H3H / PKS / AROG, and FBP-ΔTAL / H3H / PKS / AROG. PKS / P450, FBP-ΔTAL / H3H / AROG / P450, FBP-ΔTAL / LUZ / PKS / AROG, FBP-ΔTAL / LUZ / PKS / P450, FBP-ΔTAL / LUZ / AROG / P450, FBP-ΔTAL / PKS / AROG / P450, FBP-ΔH3H / LUZ / PKS / AROG, FBP-ΔH3H / PKS / AROG / P450, FBP-ΔLUZ / PKS / ARDG / P450, etc., their luminescence intensity, caffeic acid content, etc., are as follows: Figures 43-45 As shown.

[0094] like Figures 43-45 Combining the luminescence intensity of each gene's single-point mutant, the luminescence intensity was significantly increased after the four-gene mutation, significantly higher than that of the unmutated leaves. However, simultaneous mutations of some genes, such as TAL... W645L LUZ V29W PKS S366P and P450 P95A The increase in luminescence intensity was similar to that of the single mutation, without a significant increase.

[0095] Example 14: Effect of single mutation combinations of five or six genes on luminescence intensity In this embodiment, the genes LUZ, PKS, H3H, CPH, TAL, P450, and AROG, or their mutants, were transformed into tobacco, with each gene including a maximum of one mutation site. In this embodiment, the selected mutant sites were TAL... W645L H3H P200Q LUZ V29W PKS S366P AROG K11Y and P450 P95A The methods for constructing the vector and transforming the plant are detailed in Examples 1 and 2, and will not be repeated in this example.

[0096] The five-gene mutants are FBP-ΔTAL / H3H / LUZ / PKS / AROG, FBP-ΔTAL / H3H / LUZ / PKS / P450, FBP-ΔTAL / H3H / LUZ / AROG / P450, FBP-ΔTAL / H3H / PKS / AROG / P450, FBP-ΔTAL / LUZ / PKS / AROG / P450, FBP-ΔH3H / LUZ / PKS / AROG / P450, etc.; the six-gene mutant is FBP-ΔTAL / H3H / LUZ / PKS / AROG / P450, etc. Their luminescence intensity, caffeic acid content, etc., are as follows: Figures 46-48 As shown.

[0097] like Figures 46-48 Based on the luminescence intensity of the single point mutant of each gene, the luminescence intensity was significantly increased after mutation of five or six genes, which was significantly higher than that of the unmutated leaves.

[0098] Example 15: Effect of Multi-gene, Multi-site Mutation Combinations on Luminescence Intensity In this embodiment, genes such as LUZ, PKS, H3H, CPH, TAL, P450, and AROG, or their mutants, were transformed into tobacco, with each mutant gene including at least one mutation site. In this embodiment, the selected mutant sites are as follows: Group A: TAL W645V +TAL D571L +TAL S108P ; Group B: H3H F139H +H3H P200Q +H3H E379L ; Group C: LUZ V29W +LUZ E181A +LUZ V28W ; Group D: PKS L382K +PKS L405G +PKS Y327Q ; Group E: Arog K11Y +Arog E174Q +Arog G350M ; Group F: P450 K50E +P450 P95A .

[0099] The methods for constructing the carrier and transforming the plant are detailed in Examples 1 and 2, and will not be repeated in this example. Luminescence intensity, caffeic acid content, etc., are as follows... Figures 49-51 As shown.

[0100] like Figures 49-51 Based on the luminescence intensity of single-point mutants of each gene, multi-gene, multi-site mutations significantly increased luminescence intensity compared to unmutated leaves. However, some gene mutation combinations, such as A+B+C+E, showed a similar increase in luminescence intensity as single mutations, without a significant improvement.

[0101] The above embodiments are for illustrative purposes only and are not intended to limit the invention. Those skilled in the art can make various changes and modifications without departing from the scope of the invention. Therefore, all equivalent technical solutions should also fall within the scope of the invention.

Claims

1. Amino acid mutants of TAL, H3H, LUZ, PKS, AROG, or P450, including mutations selected from one or more of the following sites: OF W645 、TAL D571 、TAL E163 、TAL P3 、TAL S108 、TAL Y245 、TAL L162 、TAL A498 、TAL V186 、TAL I634 ; H3H F139 H3H P200 H3H E379 H3H F4 H3H V142 H3H N6 H3H S7 H3H A197 ; LIGHT V29 ,LIGHT C26 ,LIGHT F230 ,LIGHT E181 ,LIGHT V28 ,LIGHT F148 ,LIGHT L67 ; PKS C79 、PKS C121 、PKS F266 、PKS L193 、PKS L321 、PKS L382 、PKS L405 、PKS R330 、PKS S366 、PKS S402 、PKS V196 、PKS Y327 ; RUDE N5 、GOOD K11 、GOOD K41 、GOOD E174 、GOOD A319 、GOOD K322 、GOOD G350 ; or P450 A11 、P450 H18 、P450 L31 、P450 K50 、P450 H94 、P450 P95 、P450 S97 、P450 Y380 。 2. The amino acid mutant according to claim 1, wherein the one or more sites are mutated to the following amino acids: OF W645V 、TAL W645A 、TAL W645L 、TAL W645T 、TAL W645I 、TAL W645S 、TAL W645N 、TAL W645F 、TAL W645G 、TAL D571A 、TAL D571V 、TAL D571L 、TAL D571G 、TAL E163A 、TAL E163S 、TAL E163A 、TAL P3A 、TAL S108P 、TAL Y245K 、TAL L162Q 、TAL A498H 、TAL V186P 、TAL I634P ; H3H F139N H3H F139G H3H F139H H3H F139S H3H F139E H3H F139Q H3H F139A H3H F139K H3H F139R H3H P200R H3H P200K H3H P200Q H3H P200N H3H E379M H3H E379L H3H E379N H3H E379A H3H E379S H3H F4P H3H F4A H3H V142P H3H N6K H3H S7K H3H S7R H3H A197R H3H A197K ; LIGHT V29F ,LIGHT V29W ,LIGHT C26W ,LIGHT C26Y ,LIGHT F230N ,LIGHT E181A ,LIGHT E181Q ,LIGHT E181H ,LIGHT V28F ,LIGHT V28W ,LIGHT F148E ,LIGHT L67P ; PKS C79N 、PKS C121S 、PKS F266N 、PKS L193S 、PKS L321A 、PKS L321K 、PKS L321N 、PKS L321Q 、PKS L321R 、PKS L321S 、PKS L382A 、PKS L382E 、PKS L382K 、PKS L382P 、PKS L382Q 、PKS L382S 、PKS L405G 、PKS L405P 、PKS R330P 、PKS S366P 、PKS S402G 、PKS V196A 、PKS Y327A 、PKS Y327G 、PKS Y327H 、PKS Y327K 、PKS Y327N 、PKS Y327Q 、PKS Y327R 、PKS Y327S 、PKS Y327T ; RUDE N5T 、GOOD K11Y 、GOOD K11S 、GOOD K41T 、GOOD K41L 、GOOD E174Q 、GOOD A319V 、GOOD A319I 、GOOD K322Q 、GOOD G350T 、GOOD G350S 、GOOD G350M ; or P450 A11I 、P450 A11V 、P450 H18I 、P450 H18V 、P450 L31N 、P450 K50D 、P450 K50E 、P450 K50N 、P450 H94E 、P450 P95V 、P450 P95Q 、P450 P95A 、P450 P95E 、P450 P95I 、P450 P95T 、P450 P95S 、P450 S97A 、P450 Y380N 。 3. The amino acid mutant according to claim 1, comprising simultaneous mutations selected from a plurality of mutation sites: OF W645 / D571 、TAL W645 / S108 、TAL D571 / S108 、TAL W645 / D571 / S108 ; H3H F139 / P200 H3H F139 / E379 H3H P200 / E379 H3H P200N / E379M H3H F139 / P200 / E379 ; LIGHT V29 / E181 ,LIGHT V29 / V28 ,LIGHT E181 / V28 ,LIGHT V29 / E181 / V28 ; PKS L382 / L405 、PKS L382 / Y327 、PKS L405 / Y327 、PKS L382 / L405 / Y327 ; RUDE K11 / E174 、GOOD K11 / K322 、GOOD K11 / G350 、GOOD E174 / K322 、GOOD E174Q / G350M 、GOOD K322Q / G350 、GOOD K11 / E174 / K322 、GOOD K11 / E174 / G350 、GOOD E174 / K322 / G350 ; or P450 K50 / P95 。 4. The amino acid mutant according to claim 3, wherein, Multiple mutation sites simultaneously mutate into the following sites: OF W645V / D571L 、TAL W645V / S108P 、TAL W645L / D571L 、TAL W645L / S108P 、TAL D571L / S108P 、TAL W645V / D571L / S108P 、TAL W645L / D571L / S108P ; H3H F139H / P200Q H3H F139H / P200N H3H F139H / E379M H3H F139H / E379L H3H P200Q / E379M H3H P200Q / E379L H3H P200N / E379M H3H P200Q / E379L H3H F139H / P200Q / E379M H3H F139H / P200Q / E379L H3H F139H / P200N / E379M H3H F139H / P200N / E379L ; LIGHT V29F / E181A ,LIGHT V29F / V28W ,LIGHT V29W / E181A ,LIGHT V29W / V28W ,LIGHT E181A / V28W ,LIGHT V29F / E181A / V28W ,LIGHT V29W / E181A / V28W ; PKS L382K / L405G 、PKS L382K / Y327N 、PKS L382K / Y327Q 、PKS L382K / Y327T 、PKS L405G / Y327N 、PKS L405G / Y327Q 、PKS L405G / Y327T 、PKS L382K / L405G / Y327N 、PKS L382K / L405G / Y327Q 、PKS L382K / L405G / Y327T ; RUDE K11Y / E174Q 、GOOD K11Y / K322Q 、GOOD K11Y / G350M 、GOOD E174Q / K322Q 、GOOD E174Q / G350M 、GOOD K322Q / G350M 、GOOD K11Y / E174Q / K322Q 、GOOD K11Y / E174Q / G350M 、GOOD E174Q / K322Q / G350M ; or P450 K50E / P95Q 、P450 K50E / P95A 、P450 K50E / P95E 、P450 K50E / P95I 、P450 K50E / P95T 、P450 K50E / P95S 。 5. The amino acid mutant according to claim 1, wherein, The amino acid sequences of TAL, H3H, LUZ, PKS, AROG, and P450 constitute natural proteins or proteins that do not include the mutations described in claim 1.

6. The amino acid mutant according to claim 5, wherein, The amino acid sequence of TAL is shown in SEQ ID NO.1, the amino acid sequence of H3H is shown in SEQ ID NO.31, the amino acid sequence of LUZ is shown in SEQ ID NO.70, the amino acid sequence of PKS is shown in SEQ ID NO.90, the amino acid sequence of AROG is shown in SEQ ID NO.132, and the amino acid sequence of P450 is shown in SEQ ID NO.

155.

7. The coding gene of the amino acid mutant according to any one of claims 1-6, wherein, The gene sequence encoding TAL is shown in SEQ ID NO.181, the gene sequence encoding H3H is shown in SEQ ID NO.182, the gene sequence encoding LUZ is shown in SEQ ID NO.183, the gene sequence encoding PKS is shown in SEQ ID NO.184, the gene sequence encoding AROG is shown in SEQ ID NO.185, and the gene sequence encoding P450 is shown in SEQ ID NO.

186.

8. A recombinant vector, expression cassette, or recombinant strain comprising the coding gene of claim 7.

9. The use of any amino acid mutant or combination of mutants as described in any one of claims 1-6 in improving bioluminescence intensity or increasing the amount of 3-arylacrylic acid synthesized in biological cells; preferably, the 3-arylacrylic acid is selected from the group consisting of caffeic acid, cinnamic acid, p-coumaric acid, coumaric acid, umbelliferic acid, sinapic acid, and ferulic acid.

10. The application according to claim 9, wherein the combination of mutants is selected from the group consisting of: TAL W645 / H3H P200 ,TAL W645 / LIGHT V29 ,TAL W645 / PKS S366 ,TAL W645 / AROG K11 ,TAL W645 / P450 P95 、H3H P200 / LIGHT V29 、H3H P200 / PKS S366 、H3H P200 / AROG K11 、H3H P200 / P450 P95 ,LIGHT V29 / PKS S366 ,LIGHT V29 / AROG K11 ,LIGHT V29 / P450 P95 、PKS S366 / AROG K11 、PKS S366 / P450 P95 、GOOD K11 / P450 P95 ; TAL W645 / H3H P200 / LIGHT V29 ,TAL W645 / H3H P200 / PKS S366 ,TAL W645 / H3H P200 / RUDE K11 ,TAL W645 / H3H P200 / P450 P95 ,TAL W645 / LIGHT V29 / PKS S366 ,TAL W645 / LIGHT V29 / RUDE K11 ,TAL W645 / LIGHT V29 / P450 P95 ,TAL W645 / PKS S366 / AROG K11 ,TAL W645 / PKS S366 / P450 P95 ,TAL W645 / AROG K11 / P450 P95 、H3H P200 / LIGHT V29 / PKS S366 、H3H P200 / LIGHT V29 / RUDE K11 、H3H P200 / LIGHT V29 / P450 P95 、H3H P200 / PKS S366 / AROG K11 、H3H P200 / PKS S366 / P450 P95 、H3H P200 / AROG K11 / P450 P95 ,LIGHT V29 / PKS S366 / AROG K11 ,LIGHT V29 / PKS S366 / P450 P95 ,LIGHT V29 / AROG K11 / P450 P95 、PKS S366 / AROG K11 / P450 P95 ; TAL W645 / H3H P200 / LIGHT V29 / PKS S366 ,TAL W645 / H3H P200 / LIGHT V29 / AROG K11 ,TAL W645 / H3H P200 / LIGHT V29 / P450 P95 ,TAL W645 / H3H P200 / PKS S366 / AROG K11 ,TAL W645 / H3H P200 / PKS S366 / P450 P95 ,TAL W645 / H3H P200 / AROG K11 / P450 P95 ,TAL W645 / LIGHT V29 / PKS S366 / AROG K11 ,TAL W645 / LIGHT V29 / PKS S366 / P450 P95 ,TAL W645 / LIGHT V29 / AROG K11 / P450 P95 ,TAL W645 / PKS S366 / AROG K11 / P450 P95 、H3H P200 / LIGHT V29 / PKS S366 / AROG K11 / 、H3H P200 / LIGHT V29 / PKS S366 / P450 P95 、H3H P200 / LIGHT V29 / AROG K11 / P450 P95 、H3H P200 / PKS S366 / AROG K11 / P450 P95 ,LIGHT V29 / PKS S366 / AROG K11 / P450 P95 ; TAL W645 / H3H P200 / LUZ V29 / PKS S366 / AROG K11 、TAL W645 / H3H P200 / LUZ V29 / PKS S366 / P450 P95 、TAL W645 / H3H P200 / LUZ V29 / AROG K11 / P450 P95 、TAL W645 / H3H P200 / PKS S366 / AROG K11 / P450 P95 、TAL W645 / LUZ V29 / PKS S366 ]> / AROG K11 / P450 P95 、H3H P200 / LUZ V29 / PKS S366 / AROG K11 / P450 P95 ; or TAL W645 / H3H P200 / LIGHT V29 / PKS S366 / AROG K11 / P450 P95 。 11. The application according to claim 9, wherein the combination of mutants is selected from the group consisting of: TAL W645 / D571 / S108 / H3H F139 / P200 / E379 ,TAL W645 / D571 / S108 / LIGHT V29 / E181 / V28 ,TAL W645 / D571 / S108 / PKS L382 / L405 / Y327 ,TAL W645 / D571 / S108 / AROG K11 / E174 / G350 ,TAL W645 / D571 / S108 / P450 K50 / P95 、H3H F139 / P200 / E379 / LIGHT V29 / E181 / V28 、H3H F139 / P200 / E379 / PKS L382 / L405 / Y327 、H3H F139 / P200 / E379 / AROG K11 / E174 / G350 、H3H F139 / P200 / E379 / P450 K50 / P95 ,LIGHT V29 / E181 / V28 / PKS L382 / L405 / Y327 ,LIGHT V29 / E181 / V28 / AROG K11 / E174 / G350 / 、LIGHT V29 / E181 / V28 / P450 K50 / P95 、PKS L382 / L405 / Y327 / AROG K11 / E174 / G350 、PKS L382 / L405 / Y327 / P450 K50 / P95 、GOOD K11 / E174 / G350 / P450 K50 / P95 ; TAL W645 / D571 / S108 / H3H F139 / P200 / E379 / LIGHT V29 / E181 / V28 ,TAL W645 / D571 / S108 / H3H F139 / P200 / E379 / PKS L382 / L405 / Y327 ,TAL W645 / D571 / S108 / H3H F139 / P200 / E379 / RUDE K11 / E174 / G350 ,TAL W645 / D571 / S108 / H3H F139 / P200 / E379 / P450 K50 / P95 ,TAL W645 / D571 / S108 / LIGHT V29 / E181 / V28 / PKS L382 / L405 / Y327 ,TAL W645 / D571 / S108 / LIGHT V29 / E181 / V28 / AROG K11 / E174 / G350 ,TAL W645 / D571 / S108 / LIGHT V29 / E181 / V28 / P450 K50 / P95 ,TAL W645 / D571 / S108 / PKS L382 / L405 / Y327 / AROG K11 / E174 / G350 ,TAL W645 / D571 / S108 / PKS L382 / L405 / Y327 / P450 K50 / P95 ,TAL W645 / D571 / S108 / RUDE K11 / E174 / G350 / P450 K50 / P95 、H3H F139 / P200 / E379 / LIGHT V29 / E181 / V28 / PKS L382 / L405 / Y327 、H3H F139 / P200 / E379 / LIGHT V29 / E181 / V28 / AROG K11 / E174 / G350 、H3H F139 / P200 / E379 / LIGHT V29 / E181 / V28 / P450 K50 / P95 、H3H F139 / P200 / E379 / PKS L382 / L405 / Y327 / AROG K11 / E174 / G350 、H3H F139 / P200 / E379 / PKS L382 / L405 / Y327 / P450 K50 / P95 、H3H F139 / P200 / E379 / AROG K11 / E174 / G350 / P450 K50 / P95 ,LIGHT V29 / E181 / V28 / PKS L382 / L405 / Y327 / AROG K11 / E174 / G350 ,LIGHT V29 / E181 / V28 / PKS L382 / L405 / Y327 / P450 K50 / P95 、PKS L382 / L405 / Y327 / AROG K11 / E174 / G350 / P450 K50 / P95 ; TAL W645 / D571 / S108 / H3H F139 / P200 / E379 / LIGHT V29 / E181 / V28 / PKS L382 / L405 / Y327 ,TAL W645 / D571 / S108 / H3H F139 / P200 / E379 / LIGHT V29 / E181 / V28 / AROG K11 / E174 / G350 ,TAL W645 / D571 / S108 / H3H F139 / P200 / E379 / LIGHT V29 / E181 / V28 / P450 K50 / P95 ,TAL W645 / D571 / S108 / H3H F139 / P200 / E379 / PKS L382 / L405 / Y327 / AROG K11 / E174 / G350 ,TAL W645 / D571 / S108 / H3H F139 / P200 / E379 / PKS L382 / L405 / Y327 / P450 K50 / P95 ,TAL W645 / D571 / S108 / H3H F139 / P200 / E379 / AROG K11 / E174 / G350 / P450 K50 / P95 ,TAL W645 / D571 / S108 / LIGHT V29 / E181 / V28 / PKS L382 / L405 / Y327 / AROG K11 / E174 / G350 ,TAL W645 / D571 / S108 / LIGHT V29 / E181 / V28 / PKS L382 / L405 / Y327 / P450 K50 / P95 ,TAL W645 / D571 / S108 / LIGHT V29 / E181 / V28 / AROG K11 / E174 / G350 / P450 K50 / P95 ,TAL W645 / D571 / S108 / PKS L382 / L405 / Y327 / RUDE K11 / E174 / G350 / P450 K50 / P95 、H3H F139 / P200 / E379 / LIGHT V29 / E181 / V28 / PKS L382 / L405 / Y327 / RUDE K11 / E174 / G350 、H3H F139 / P200 / E379 / LIGHT V29 / E181 / V28 / PKS L382 / L405 / Y327 / P450 K50 / P95 、H3H F139 / P200 / E379 / LIGHT V29 / E181 / V28 / AROG K11 / E174 / G350 / P450 K50 / P95 、H3H F139 / P200 / E379 / PKS L382 / L405 / Y327 / AROG K11 / E174 / G350 / P450 K50 / P95 ,LIGHT V29 / E181 / V28 / PKS L382 / L405 / Y327 / RUDE K11 / E174 / G350 / P450 K50 / P95 ; TAL W645 / D571 / S108 / H3H F139 / P200 / E379 / LUZ V29 / E181 / V28 / PKS L382 / L405 / Y327 / AROG K11 / E174 / G350 、TAL W645 / D571 / S108 / H3H F139 / P200 / E379 / LUZ V29 / E181 / V28 / PKS L382 / L405 / Y327 / P450 K50 / P95 、TAL W645 / D571 / S108 / H3H F139 / P200 / E379 / LUZ V29 / E181 / V28 / AROG K11 / E174 / G350 / P450 K50 / P95 、TAL W645 / D571 / S108 / H3H F139 / P200 / E379 / PKS L382 / L405 / Y327 / AROG K11 / E174 / G350 / P450 K50 / P95 、TAL W645 / D571 / S108 / LUZ V29 / E181 / V28 / PKS L382 / L405 / Y327 / AROG K11 / E174 / G350 / P450 K50 / P95 、H3H F139 / P200 / E379 / LUZ V29 / E181 / V28 / PKS L382 / L405 / Y327 / AROG K11 / E174 / G350 / P450 K50 / P95 ; or TAL W645 / D571 / S108 / H3H F139 / P200 / E379 / LIGHT V29 / E181 / V28 / PKS L382 / L405 / Y327 / AROG K11 / E174 / G350 / P450 K50 / P95 。 12. Methods for increasing bioluminescence intensity or increasing the amount of 3-arylacrylic acid synthesized in biological cells, including: Expressing LUZ, SKP, H3H, CPH proteins or amino acid mutants as described in any one of claims 1-6 or combinations thereof in plants; Preferably, the 3-arylacrylic acid is selected from the group consisting of caffeic acid, cinnamic acid, p-coumaric acid, coumaric acid, umbelliferic acid, sinapic acid, and ferulic acid.

13. Methods for increasing bioluminescence intensity or increasing the amount of 3-arylacrylic acid synthesized in biological cells, including: Expressing LUZ, SKP, H3H, CPH, TAL, P450 proteins or amino acid mutants as described in any one of claims 1-6 or combinations thereof in plants; Preferably, the 3-arylacrylic acid is selected from the group consisting of caffeic acid, cinnamic acid, p-coumaric acid, coumaric acid, umbelliferic acid, sinapic acid, and ferulic acid.

14. Methods for increasing bioluminescence intensity or increasing the amount of 3-arylacrylic acid synthesized in biological cells, including: Expressing LUZ, SKP, H3H, CPH, TAL, P450, AROG proteins or amino acid mutants as described in any one of claims 1-6 or combinations thereof in plants; Preferably, the 3-arylacrylic acid is selected from the group consisting of caffeic acid, cinnamic acid, p-coumaric acid, coumaric acid, umbelliferic acid, sinapic acid, and ferulic acid.

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