Genetic engineering of transitory starch accumulation by knockdown of <i>OsSEX4</i> in rice plants for enhanced bioethanol production

Huang, Li; Liu, Yu-Kuo; Su, Sung-Chieh; Lai, Chih-Chang; Wu, Chia-Huang; Chao, Tai-Jang; Yang, Yung-Hsing

doi:10.1002/bit.27262

Cited by 15 publications

(6 citation statements)

References 53 publications

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“…Genomic DNA was extracted from calli using a previously reported method (Huang et al 2020 ). Briefly, rice samples were ground with liquid nitrogen using a mortar and pestle, and genomic DNA was extracted using an extraction buffer containing 100 mM Tris-HCl (pH 8.0), 50 mM EDTA (pH 8.0), 100 mM NaCl, 1% SDS, and 1% β-mercaptoethanol.…”

Section: Methodsmentioning

confidence: 99%

High-yield BMP2 expression in rice cells via CRISPR and endogenous αAmy3 promoter

Nguyen,

Wu,

Chang

et al. 2024

Appl Microbiol Biotechnol

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Plant cells serve as versatile platforms for the production of high-value recombinant proteins. This study explored the efficacy of utilizing an endogenous αAmy3 promoter for the expression of a bioactive pharmaceutical protein, specifically the mature region of human bone morphogenetic protein 2 (hBMP2m). Utilizing a refined CRISPR/Cas9-mediated intron-targeting insertion technique, which incorporates an artificial 3’ splicing site upstream of the target gene, we achieved a transformation efficiency of 13.5% in rice calli that carried the rice-codon optimized mature region of hBMP2 cDNA (rhBMP2m) in the αAmy3 intron 1. Both homozygous and heterozygous rhBMP2m knock-in rice suspension cell lines were generated. These lines demonstrated the endogenous αAmy3 promoter regulated rhBMP2m mRNA and rhBMP2m recombinant protein expression, with strongly upregulation in respond to sugar depletion. The homozygous rhBMP2m knock-in cell line yielded an impressive 21.5 μg/mL of rhBMP2m recombinant protein, accounting for 1.03% of the total soluble protein. The high-yield expression was stably maintained across two generations, indicating the genetic stability of rhBMP2m gene knock-in at the αAmy3 intron 1 locus. Additionally, the rice cell-derived rhBMP2m proteins were found to be glycosylated, capable of dimer formation, and bioactive. Our results indicate that the endogenous rice αAmy3 promoter–signal peptide-based expression system is an effective strategy for producing bioactive pharmaceutical proteins. Key points • The endogenous αAmy3 promoter-based expression system enhanced the yield of BMP2 • The increased yield of BMP2 accounted for 1.03% of the total rice-soluble proteins • The rice-produced BMP2 showed glycosylation modifications, dimer formation, and bioactivity

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Section: Methodsmentioning

confidence: 99%

High-yield BMP2 expression in rice cells via CRISPR and endogenous αAmy3 promoter

Nguyen,

Wu,

Chang

et al. 2024

Appl Microbiol Biotechnol

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“…Genomic DNA was isolated from 2-week-old seedlings or calli [ 41 ]. The rice samples were ground by mortar and pestle with liquid nitrogen.…”

Section: Methodsmentioning

confidence: 99%

Knockdown expression of a MYB-related transcription factor gene, OsMYBS2, enhances production of recombinant proteins in rice suspension cells

Sinaga

et al. 2021

Plant Methods

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Background Transgenic plant suspension cells show economic potential for the production of valuable bioproducts. The sugar starvation-inducible rice αAmy3 promoter, together with its signal peptide, is widely applied to produce recombinant proteins in rice suspension cells. The OsMYBS2 transcription factor was shown recently to reduce activation of the αAmy3 promoter by competing for the binding site of the TA box of the αAmy3 promoter with the potent OsMYBS1 activator. In this study, rice suspension cells were genetically engineered to silence OsMYBS2 to enhance the production of recombinant proteins. Results The mouse granulocyte–macrophage colony-stimulating factor (mGM-CSF) gene was controlled by the αAmy3 promoter and expressed in OsMYBS2-silenced transgenic rice suspension cells. Transcript levels of the endogenous αAmy3 and the transgene mGM-CSF were increased in the OsMYBS2-silenced suspension cells. The highest yield of recombinant mGM-CSF protein attained in the OsMYBS2-silenced transgenic suspension cells was 69.8 µg/mL, which is 2.5-fold that of non-silenced control cells. The yield of recombinant mGM-CSF was further increased to 118.8 µg/mL in cultured cells derived from homozygous F5 seeds, which was 5.1 times higher than that of the control suspension cell line. Conclusions Our results demonstrate that knockdown of the transcription factor gene OsMYBS2 increased the activity of the αAmy3 promoter and improved the yield of recombinant proteins secreted in rice cell suspension cultures.

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“…Arabidopsis mutations for these genes do not cause abnormal pollen phenotypes. In rice, pollen fertility appeared to be normal in the OsSEX4 mutant [42]. Nonetheless, it is valuable to determine whether other corresponding mutations in rice pollen that stores starch as a major reserve cause male sterility.…”

Section: Starch Degradation and Utilization In Rice Pollen Grainmentioning

confidence: 99%

Exploration of Sugar and Starch Metabolic Pathway Crucial for Pollen Fertility in Rice

Lee

et al. 2022

IJMS

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Starch is the primary storage carbohydrate in mature pollen grains in many crop plants, including rice. Impaired starch accumulation causes male sterility because of the shortage of energy and building blocks for pollen germination and pollen tube growth. Thus, starch-defective pollen is applicable for inducing male sterility and hybrid rice production. Despite the importance of pollen starch, the details of the starch biosynthesis and breakdown pathway in pollen are still largely unknown. As pollen is isolated from the maternal tissue, photoassimilate transported from leaves must pass through the apoplastic space from the anther to the filial pollen, where it is stored as starch. Several sugar transporters and enzymes are involved in this process, but many are still unknown. Thus, the current review provides possible scenarios for sucrose transport and metabolic pathways that lead to starch biosynthesis and breakdown in rice pollen.

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Genetic engineering of transitory starch accumulation by knockdown of OsSEX4 in rice plants for enhanced bioethanol production

Cited by 15 publications

References 53 publications

High-yield BMP2 expression in rice cells via CRISPR and endogenous αAmy3 promoter

High-yield BMP2 expression in rice cells via CRISPR and endogenous αAmy3 promoter

Knockdown expression of a MYB-related transcription factor gene, OsMYBS2, enhances production of recombinant proteins in rice suspension cells

Exploration of Sugar and Starch Metabolic Pathway Crucial for Pollen Fertility in Rice

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