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基于 CRISPR/Cas9 的基因敲除揭示，CpRLP1 是 Closterium peracerosum-strigosum-littorale 复合体中性信息素 PR-IP 的负调控因子。

CRISPR/Cas9-based knockouts reveal that CpRLP1 is a negative regulator of the sex pheromone PR-IP in the Closterium peracerosum-strigosum-littorale complex.

机构信息

Graduate School of Science, Japan Women's University, 2-8-1 Mejirodai, Bunkyo-ku, Tokyo, 112-8681, Japan.

Faculty of Science, Japan Women's University, 2-8-1 Mejirodai, Bunkyo-ku, Tokyo, 112-8681, Japan.

出版信息

Sci Rep. 2017 Dec 19;7(1):17873. doi: 10.1038/s41598-017-18251-8.

DOI:10.1038/s41598-017-18251-8
PMID:29259295
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC5736609/
Abstract

Heterothallic strains of the Closterium peracerosum-strigosum-littorale (C. psl.) complex have two sexes, mating-type plus (mt) and mating-type minus (mt). Conjugation between these two sexes is regulated by two sex pheromones, protoplast-release-inducing protein (PR-IP) and PR-IP Inducer, which are produced by mt and mt cells, respectively. PR-IP mediates the release of protoplasts from mt cells during mating. In this study, we examined the mechanism of action of CpRLP1 (receptor-like protein 1), which was previously identified in a cDNA microarray analysis as one of the PR-IP-inducible genes. Using CRISPR/Cas9 technology, we generated CpRLP1 knockout mutants in mt cells of the C. psl. complex. When the knockout mt cells were mixed with wild-type mt cells, conjugation was severely reduced. Many cells released protoplasts without pairing, suggesting a loss of synchronization between the two mating partners. Furthermore, the knockout mutants were hypersensitive to PR-IP. We conclude that CpRLP1 is a negative regulator of PR-IP that regulates the timing of protoplast release in conjugating C. psl. cells. As the first report of successful gene knockout in the class Charophyceae, this study provides a basis for research aimed at understanding the ancestral roles of genes that are indispensable for the development of land plants.

摘要

异宗生殖的胶网藻属（C. peracerosum-strigosum-littorale）复合种群的菌株具有两种性别，分别为交配型 plus（mt）和交配型 minus（mt）。这两种性别的交配受两种性信息素调控，即原生质体释放诱导蛋白（PR-IP）和 PR-IP 诱导剂，它们分别由 mt 和 mt 细胞产生。PR-IP 介导 mt 细胞在交配过程中释放原生质体。在本研究中，我们研究了 CpRLP1（受体样蛋白 1）的作用机制，该蛋白在 cDNA 微阵列分析中被鉴定为 PR-IP 诱导基因之一。我们利用 CRISPR/Cas9 技术，在胶网藻属复合种群的 mt 细胞中生成了 CpRLP1 敲除突变体。当敲除 mt 细胞与野生型 mt 细胞混合时，交配严重减少。许多细胞释放原生质体而不配对，表明两个交配伙伴之间的同步性丧失。此外，敲除突变体对 PR-IP 更加敏感。我们得出结论，CpRLP1 是 PR-IP 的负调节剂，它调节胶网藻属细胞中原生质体释放的时间。作为 Charophyceae 类中首次成功的基因敲除报告，本研究为研究对于陆地植物发育不可或缺的基因的祖先作用提供了基础。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ec68/5736609/78614c0eb1dc/41598_2017_18251_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ec68/5736609/b36cba28645e/41598_2017_18251_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ec68/5736609/d0f585233e0c/41598_2017_18251_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ec68/5736609/a2b5653bee0f/41598_2017_18251_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ec68/5736609/d8ca2027b20f/41598_2017_18251_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ec68/5736609/78614c0eb1dc/41598_2017_18251_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ec68/5736609/b36cba28645e/41598_2017_18251_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ec68/5736609/d0f585233e0c/41598_2017_18251_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ec68/5736609/a2b5653bee0f/41598_2017_18251_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ec68/5736609/d8ca2027b20f/41598_2017_18251_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ec68/5736609/78614c0eb1dc/41598_2017_18251_Fig5_HTML.jpg

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