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芝麻菜籽 napin 结构的深入研究和全面功能表征。

Eruca sativa seed napin structural insights and thorough functional characterization.

机构信息

Botany Division, Institute of Pure and Applied Biology, Bahauddin Zakariya University, Multan, Pakistan.

Botany Division Institute of Molecular Biology and Biotechnology, The University of Lahore, Lahore, Pakistan.

出版信息

Sci Rep. 2021 Dec 15;11(1):24066. doi: 10.1038/s41598-021-02174-6.

DOI:10.1038/s41598-021-02174-6
PMID:34911985
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC8674280/
Abstract

A potent napin protein has been thoroughly characterized from seeds of rocket salad (Eruca sativa). Eruca sativa napin (EsNap) was purified by ammonium sulfate precipitation (70%) and size-exclusion chromatography. Single intact 16 kDa EsNap band was reduced to 11 and 5 kDa bands respectively on SDS-PAGE. Nano LC-MS/MS yielded two fragments comprising of 26 residues which showed 100% sequence identity with napin-3 of Brassica napus. CD spectroscopy indicated a dominant α-helical structure of EsNap. Monodispersity of EsNap was verified by dynamic light scattering, which also confirmed the monomeric status with a corresponding hydrodynamic radius of 2.4 ± 0.2 nm. An elongated ab initio shape of EsNap was calculated based on SAXS data, with an R of 1.96 ± 0.1 nm. The ab initio model calculated by DAMMIF with P1 symmetry and a volume of approx. 31,100 nm, which corresponded to a molecular weight of approximately 15.5 kDa. The comparison of the SAXS and ab initio modeling showed a minimized χ-value of 1.87, confirming a similar molecular structure. A homology model was predicted using the coordinate information of Brassica napus rproBnIb (PDB ID: 1SM7). EsNap exhibited strong antifungal activity by significantly inhibiting the growth of Fusarium graminearum. EsNap also showed cytotoxicity against the hepatic cell line Huh7 and the obtained IC value was 20.49 µM. Further, strong entomotoxic activity was experienced against different life stages of stored grain insect pest T. castaneum. The result of this study shows insights that can be used in developing potential antifungal, anti-cancerous and insect resistance agents in the future using EsNap from E. sativa.

摘要

从火箭菜（Eruca sativa）种子中彻底表征了一种有效的 napin 蛋白。通过硫酸铵沉淀（70%）和分子筛层析对 Eruca sativa napin（EsNap）进行纯化。SDS-PAGE 上的 EsNap 显示出单一完整的 16 kDa 条带，分别还原为 11 和 5 kDa 条带。Nano LC-MS/MS 产生了包含 26 个残基的两个片段，这些片段与 Brassica napus 的 napin-3 具有 100%的序列同一性。圆二色光谱（CD 光谱）表明 EsNap 具有主要的α-螺旋结构。动态光散射验证了 EsNap 的单分散性，同时也证实了单体状态，相应的流体力学半径为 2.4±0.2nm。根据 SAXS 数据计算了 EsNap 的长初始形状，R 为 1.96±0.1nm。用 P1 对称和近似 31100nm3 的体积的 DAMMIF 计算的初始模型，对应于约 15.5 kDa 的分子量。SAXS 和初始模型比较显示 χ 值最小为 1.87，证实了相似的分子结构。使用 Brassica napus rproBnIb（PDB ID：1SM7）的坐标信息预测了同源模型。EsNap 通过显著抑制禾谷镰刀菌的生长表现出强烈的抗真菌活性。EsNap 对肝细胞系 Huh7 也表现出细胞毒性，获得的 IC 值为 20.49µM。此外，对不同阶段的储粮害虫赤拟谷盗表现出强烈的杀虫活性。这项研究的结果表明，利用火箭菜中的 EsNap 可以在未来开发出具有潜在抗真菌、抗癌和抗虫性的新药剂。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0784/8674280/c280a48c881e/41598_2021_2174_Fig8_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0784/8674280/441d5b003671/41598_2021_2174_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0784/8674280/c61f0f6e3945/41598_2021_2174_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0784/8674280/7e4e5d7bf052/41598_2021_2174_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0784/8674280/d21027cd1c92/41598_2021_2174_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0784/8674280/1629abe31400/41598_2021_2174_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0784/8674280/56752705de6a/41598_2021_2174_Fig6_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0784/8674280/b3e8f22223c4/41598_2021_2174_Fig7_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0784/8674280/c280a48c881e/41598_2021_2174_Fig8_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0784/8674280/441d5b003671/41598_2021_2174_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0784/8674280/c61f0f6e3945/41598_2021_2174_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0784/8674280/7e4e5d7bf052/41598_2021_2174_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0784/8674280/d21027cd1c92/41598_2021_2174_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0784/8674280/1629abe31400/41598_2021_2174_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0784/8674280/56752705de6a/41598_2021_2174_Fig6_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0784/8674280/b3e8f22223c4/41598_2021_2174_Fig7_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0784/8674280/c280a48c881e/41598_2021_2174_Fig8_HTML.jpg

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