综合流式细胞术和蛋白质组学分析揭示了甘蔗原生质体响应融合的调控网络。

Integrated flow cytometric and proteomics analyses reveal the regulatory network underlying sugarcane protoplast responses to fusion.

机构信息

Guangxi Key Laboratory of Sugarcane Biology, College of Agriculture, Guangxi University, 100 Daxue Rd., Nanning, 530004, China; Key Laboratory of Crop Cultivation and Tillage, College of Agriculture, Guangxi University, 100 Daxue Rd., Nanning, 530004, China.

School of Biomedical Engineering, South-Central Minzu University, No. 182, Minzu Avenue, Wuhan, 430074, China.

出版信息

Plant Physiol Biochem. 2023 Sep;202:107918. doi: 10.1016/j.plaphy.2023.107918. Epub 2023 Aug 19.

DOI:10.1016/j.plaphy.2023.107918

PMID:37619268

Abstract

BACKGROUND

Somatic cell fusion is a process that transfers cytoplasmic and nuclear genes to create new germplasm resources. But our limited understanding of the physiological and molecular mechanisms that shape protoplast responses to fusion.

METHOD

We employed flow cytometry, cytology, proteomics, and gene expression analysis to examine the sugarcane (Saccharum spp.) protoplast fusion.

RESULTS

Flow cytometry analysis revealed the fusion rate of protoplasts was 1.95%, the FSC value and SSC of heterozygous cells was 1.17-1.47 times higher than that of protoplasts. The protoplasts viability decreased and the MDA increased after fusion. During fusion, the cell membranes were perforated to different degrees, nuclear activity was weakened, while microtubules depolymerized and formed several short rod like structures in the protoplasts. The most abundant proteins during fusion were mainly involved in RNA processing and modification, cell cycle control, cell division, chromosome partition, nuclear structure, extracellular structures, and nucleotide transport and metabolism. Moreover, the expression of key regeneration genes, such as WUS, GAUT, CESA, PSK, Aux/IAA, Cdc2, Cyclin D3, Cyclin A, and Cyclin B, was significantly altered following fusion.

PURPOSE AND SIGNIFICANCE

Overall, our findings provide a theoretical basis that increases our knowledge of the mechanisms underlying protoplast fusion.

摘要

背景

体细胞融合是一种转移细胞质和核基因以创造新种质资源的过程。但我们对原生质体对融合的反应的生理和分子机制的了解有限。

方法

我们采用流式细胞术、细胞学、蛋白质组学和基因表达分析来研究甘蔗（Saccharum spp.）原生质体融合。

结果

流式细胞术分析显示原生质体的融合率为 1.95%，杂合细胞的 FSC 值和 SSC 值比原生质体高 1.17-1.47 倍。融合后原生质体的活力下降，MDA 增加。在融合过程中，细胞膜穿孔程度不同，核活性减弱，微管解聚并在原生质体中形成几个短棒状结构。融合过程中最丰富的蛋白质主要参与 RNA 加工和修饰、细胞周期调控、细胞分裂、染色体分配、核结构、细胞外结构以及核苷酸运输和代谢。此外，关键再生基因（如 WUS、GAUT、CESA、PSK、Aux/IAA、Cdc2、Cyclin D3、Cyclin A 和 Cyclin B）的表达在融合后也发生了显著改变。