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Influence on Accumulation Levels and Subcellular Localization of Prolamins by Fusion with the Functional Peptide in Transgenic Rice Seeds

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Abstract

To exploit the rice seed-based oral vaccine against Sjögren’s syndrome, altered peptide ligand of N-terminal 1 (N1-APL7) from its M3 muscarinic acetylcholine receptor (M3R) autoantigen was expressed as fusion protein with the representative four types of rice prolamins (16 kDa, 14 kDa, 13 kDa, and 10 kDa prolamins) under the control of the individual native prolamin promoter. The 10kD:N1-APL7 and 14kD:N1-APL7 accumulated at high levels (287 and 58 µg/grain), respectively, whereas production levels of the remaining ones were remarkably low. Co-expression of these fusion proteins did not enhance the accumulation level of N1-APL7 in an additive manner. Downregulation of endogenous seed storage proteins by RNAi-mediated suppression also did not lead to substantial elevation of the co-expressed prolamin:N1-APL7 products. When transgenic rice seeds were subjected to in vitro proteolysis with pepsin, the 10kD:N1-APL7 was digested more quickly than the endogenous 10 kDa prolamin and the 14kD:N1-APL7 deposited in PB-Is. This difference could be explained by the finding that the 10kD:N1-APL7 was unexpectedly localized in the PB-IIs containing glutelins. These results indicated that not only accumulation level but also subcellular localization of inherent prolamins were highly influenced by the liked N1-APL7 peptide.

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Abbreviations

ALP:

Altered peptide ligand

BiP:

Binding protein

CBB:

Coomassie brilliant blue

Cys:

Cysteine

ER:

Endoplasmic reticulum

M3R:

M3 muscarinic acetylcholine receptor

PAC vesicle:

Precursor accumulating vesicle

PB:

Protein body

PDIL:

Protein disulfide isomerase-like

PSV:

Protein-storage vacuole

RNAi:

RNA interference

SS:

Sjögren’s syndrome

SSPs:

Seed storage proteins

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Acknowledgements

The author thanks Ms. Y. Ikemoto and Y. Yajima for technical assistance.

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  1. Soul Signal Institute, Kojyohama, Shiraoi, Hokkaido, 059-0641, Japan

    Fumio Takaiwa

  2. National Institute of Agrobiological Sciences, Kannondai 3-1-3, Tsukuba, Ibaraki, 305-8602, Japan

    Fumio Takaiwa

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Takaiwa, F. Influence on Accumulation Levels and Subcellular Localization of Prolamins by Fusion with the Functional Peptide in Transgenic Rice Seeds. Mol Biotechnol 65, 1869–1886 (2023). https://doi.org/10.1007/s12033-023-00666-6

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