KR-20260065556-A - Optimal combination of 5′UTR intron, leader sequence, and terminator for highly efficient recombinant protein expression in rice

Abstract

The present invention relates to an optimized gene expression construct for inducing high expression of recombinant proteins in rice, a vector containing the same, a transgenic plant, and a method for producing recombinant proteins using the same. The gene expression construct of the present invention expresses recombinant proteins stably and at a high level in target tissues such as rice callus or seeds, thereby significantly improving productivity compared to existing systems and can be effectively utilized as an industrial platform for producing various recombinant proteins.

Inventors

• 황인환
• 엔테사리 메흐나즈
• 사부리로뱃 엘함
• 차마니 모하세 파테매

Assignees

• 포항공과대학교 산학협력단
• 주식회사 바이오인

Dates

Publication Date

20260508

Application Date

20251030

Priority Date

20241031

Claims (20)

1. Rice ubiquitin 3 (Rubi3) intron sequence; A sequence encoding an endoplasmic reticulum (ER) target leader sequence; A sequence encoding a target protein; and An expression construct containing a terminator sequence.
2. In claim 1, the expression construct is an expression construct for enhancing the expression of a target protein in rice.
3. In claim 1, the endoplasmic reticulum target leader sequence is an expression construct selected from the group consisting of GluB4, Glu13a and K33D.
4. In claim 1, the terminator is an expression construct selected from the group consisting of GluB1 Terminator (TB1), GluB4 Terminator (TB4), GluB1-GluB4 Terminator (TB1-TB4), and HSP-GluB4 Terminator (HSP-TB4).
5. In claim 1, the rice ubiquitin 3 (Rubi3) intron sequence comprises the nucleotide sequence of SEQ ID NO. 10, an expression construct.
6. In paragraph 3, the GluB4 leader sequence is an expression construct comprising the amino acid sequence of SEQ ID NO. 1.
7. In paragraph 3, the Glu13a leader sequence comprises the amino acid sequence of SEQ ID NO. 3, an expression construct.
8. In paragraph 3, the K33D leader sequence is an expression construct comprising the amino acid sequence of SEQ ID NO. 5.
9. In claim 4, the GluB4 Terminator (TB4) sequence comprises the nucleotide sequence of SEQ ID NO. 25, an expression construct.
10. In claim 4, the GluB1 Terminator (TB1) sequence is an expression construct comprising the nucleotide sequence of SEQ ID NO. 26.
11. In claim 4, the GluB1-GluB4 Terminator (TB1-TB4) sequence comprises the nucleotide sequence of SEQ ID NO. 27, an expression construct.
12. In claim 4, the above HSP-GluB4 Terminator (HSP-TB4) sequence is an expression construct comprising the nucleotide sequence of SEQ ID NO. 28.
13. The expression construct of claim 1, wherein the expression construct further comprises a 5' UTR translation enhancement sequence comprising the nucleotide sequence of SEQ ID NO. 9 between a sequence encoding a Rice ubiquitin 3 (Rubi3) intron sequence and an endoplasmic reticulum (ER) target leader sequence.
14. A recombinant expression vector comprising an expression construct of any one of claims 1 to 13.
15. Transformed plant cells or transformed plants transformed with the recombinant expression vector of paragraph 14.
16. In paragraph 15, the above plant is a rice plant, a transformed plant cell, or a transformed plant body.
17. In paragraph 15, the transformed plant cell is a rice callus cell, a transformed plant cell or a transformed plant body.
18. In paragraph 15, the above-mentioned transformed plant is a rice seed, a transformed plant cell, or a transformed plant.
19. A method for producing a transformed plant cell or a transformed plant, comprising the step of introducing the recombinant expression vector of claim 14 into a plant cell or a plant body.
20. A method for producing a transformed plant cell or a transformed plant, wherein the step of introducing the recombinant expression vector into a plant involves using one or more selected from the group consisting of an Agrobacterium sp.-mediated method, particle gun bombardment, silicon carbide whiskers, sonication, electroporation, and a PEG (polyethylene glycol)-mediated transformation method.

Description

Optimal combination of 5′UTR intron, leader sequence, and terminator for highly efficient recombinant protein expression in rice The present invention relates to an optimized gene expression construct for inducing high expression of a recombinant protein in rice, a vector containing the same, a transgenic plant, and a method for producing a recombinant protein using the same. Rice is one of the most 주목받는 plants in the field of molecular agriculture recently, as it can accumulate large amounts of protein when target proteins are stored within the seeds. In addition, rice seeds can be stored stably for a long period even under general conditions such as room temperature, so they are considered a highly suitable system for the mass production of target proteins. In particular, rice is an ideal model as a host plant for recombinant protein production because transformation technology using Agrobacterium is well-established and transformation efficiency is high, and various expression vectors have already been developed. Furthermore, rice cultivation and harvesting technologies have been systematically developed worldwide, and related equipment and processes are also well-established, making large-scale cultivation and harvesting economically feasible. The high yield per unit area is also highly advantageous for utilizing rice as a platform for mass protein production. Under this technical background, the inventors have perfected a technology to maximize the efficiency of recombinant protein expression by designing and constructing an expression construct capable of expressing a target protein at a high level in a target tissue in rice. Figure 1 shows the structure of an expression construct for comparing GFP expression efficiency by introducing a 5'UTR-intron-leader sequence combination under the Fmm promoter. Each expression construct is distinguished by whether it contains a rice ubiquitin 3 (Rubi3) intron and the type of signal peptide (GluB4 (B4), Glu13a (13a), K33D(K)). Figure 2 compares the degree of GFP expression in rice calluses transformed with the expression construct prepared in Figure 1. Figure 2A shows the results of Western blot with an anti-GFP antibody, and Figure 2B shows the band intensity of the Western blot. All results are expressed as the mean ± standard deviation (SD) of six independent samples, and statistical significance was analyzed using a t-test, confirming significance at the p < 0.001 (***) and p < 0.0001 (****) levels. Figure 3 compares the degree of GFP expression in rice seeds transformed with the expression construct prepared in Figure 1. Figure 3A shows the results of Western blot with an anti-GFP antibody, and Figure 3B shows the band intensity of the Western blot. All results are expressed as the mean ± standard deviation (SD) of six independent samples, and statistical significance was analyzed using a t-test, confirming significance at the p < 0.01 (**) and p < 0.0001 (****) levels. Figure 4 shows the structure of an expression construct to compare GFP expression efficiency by replacing the rice ubiquitin 3 (Rubi3) intron with a modified UBQ10 intron. gBIP was used as a positive control. Figure 5 compares the degree of GFP expression in rice seeds transformed with the expression construct prepared in Figure 4. Figure 5A shows the results of Western blot with an anti-GFP antibody, and Figure 5B shows the band intensity of the Western blot. All results are expressed as the mean ± standard deviation (SD) of multiple independent samples, and statistical significance was analyzed using a t-test, confirming significance at the p < 0.0001 (****) level. Figure 6 shows the structure of an expression construct for comparing GFP expression efficiency by introducing various terminator sequences under the Fmm promoter. Figure 7 compares the degree of GFP expression in rice calluses transformed with the expression construct produced in Figure 6. All results are expressed as the mean ± standard deviation (SD) of three independent samples, and statistical significance was analyzed using a t-test, confirming significance at the p < 0.01 level. Figure 8 compares the degree of GFP expression in rice seeds transformed with the expression construct produced in Figure 6. All results are expressed as the mean ± standard deviation (SD) of multiple independent samples, statistical significance was analyzed using a t-test, and significance was confirmed at the p < 0.05(*) level. Figure 9 shows the structure of an expression construct for comparing GFP expression efficiency by introducing a rice ubiquitin 3 (Rubi3) intron sequence, a Glu13a leader sequence, and a 3′terminator (HSP-TB4) sequence under an Fmm promoter. Figure 10 compares the levels of GFP expression in rice callus (A) and rice seeds (B) transformed with the expression construct prepared in Figure 9. All results are expressed as the mean ± standard deviation (SD) of multiple independent samples. Statistical significance was analyzed