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Importance of messenger RNA stability of toxin synthetase genes for monitoring toxic cyanobacterial bloom

机译：毒素合成酶基因的信使RNA稳定性对监测有毒蓝藻繁殖的重要性

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Toxic cyanobacterial blooms, occurring frequently worldwide, have posed serious threats to human health and aquatic ecosystem. RNA-based quantitative PCR, which could detect potential toxin-producing cyanobacteria that are actively transcribing toxin genes, is a more reliable method, compared to DNA-based qPCR. However, single-stranded mRNA is labile, and their degradation may lead to an underestimate of gene expression level, even misleading toxic risk management, and thus impeding its application. Here, the mRNA stability of microcystin synthetase genes (mcyA-J) was systematically evaluated in unicellular and colonial Microcystis with various treatments (- 80 degrees C, -196 degrees C, 4 degrees C or 25 degrees C with RNases inhibitors). Results revealed the highly instability of toxin gene transcripts, affected by transcript structures and cell aggregation. The -196 degrees C treatment was the most effective for stabilizing these transcripts. RNAstore (R) (4 degrees C) could stabilize these transcripts effectively for a short time (less than 7 d), but their stability was strikingly reduced in colonial Microcystis. Furthermore, decay kinetics of mcyA-J transcripts in various treatments was developed, and showed that their decay rates were varied (0.0018-3.014 d(-1)), due to different molecular structures. The mcyH transcripts had the lowest decay rate (0.0018 d(-1) at -196 degrees C), attributed to the fewest AU sites and stem-loops involved in its secondary structure. Thus, mcyH was the most proper target gene for monitoring toxic cyanobacterial bloom. These findings provided new insight into mRNA stability of toxin genes, and contributed to monitoring toxic cyanobacterial blooms and water managements using RNA-based molecular techniques.
机译：在世界范围内频繁发生的有毒蓝藻绽放对人类健康和水生生态系统构成了严重威胁。与基于DNA的qPCR相比，基于RNA的定量PCR可以检测出正在主动转录毒素基因的潜在产毒素蓝藻细菌。但是，单链mRNA不稳定，其降解可能导致基因表达水平的低估，甚至会误导毒性风险管理，从而阻碍其应用。在这里，系统地评估了微囊藻毒素合成酶基因（mcyA-J）在单细胞和殖民地微囊藻中的各种处理（使用RNase抑制剂的-80摄氏度，-196摄氏度，4摄氏度或25摄氏度）的mRNA稳定性。结果显示，毒素基因转录物高度不稳定，受转录物结构和细胞聚集的影响。 -196°C处理对于稳定这些转录本最有效。 RNAstore（R）（4摄氏度）可以在短时间内（不到7天）有效稳定这些转录本，但在殖民地微囊藻中其稳定性显着降低。此外，开发了多种处理方法的mcyA-J转录物的衰变动力学，结果表明，由于分子结构不同，它们的衰变速率也有所不同（0.0018-3.014 d（-1））。 mcyH转录本具有最低的衰减率（在-196摄氏度下为0.0018 d（-1）），这归因于参与其二级结构的AU位点和茎环最少。因此，mcyH是监测毒性蓝藻水华的最合适靶基因。这些发现提供了对毒素基因的mRNA稳定性的新见解，并有助于使用基于RNA的分子技术监测有毒的蓝藻水华和水管理。

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