养殖水体三种大型爆发性海洋藻类孢子/配子实时荧光定量PCR检测方法的建立与应用初探

Development and Application of ITS-rDNA Targeted Real-Time Quantitative PCR Assays for Detecting and Quantifying the Spores/Gamates of Harmful Marine Macroalgae

一些大型海洋藻类在近海养殖环境中快速生长且容易形成青苔,严重威胁到养殖生物的安全并可能带来巨大的经济损失。检测青苔孢子的数量与动态,理解青苔爆发的环境生态学机制是防控与预警的基础。使用PCR方法,利用特异性引物对非爆发期的生物量进行检测,是监控有害藻类的有效方法。本工作针对海参养殖池塘三种常见的青苔藻(两种石莼Ulva cf.linza,Ulva sp.和萱藻Scytosiphon lomentaria)设计了以核糖体转录间隔区(ITS)为靶区域的种类特异性PCR引物,通过藻类和环境水样DNA对照实验检验其特异性和适用性。本工作建立了实时荧光定量PCR(qPCR)方法检测养殖水体中藻类孢子/配子体含量,并实验得出该方法在三种藻类中的最低检出量,可最低检出200~400ITS rDNA拷贝。使用qPCR方法对烟台海参养殖水体2014年12月—2016年3月6个季度环境水样中的萱藻孢子/配子进行了定量检测,发现样品中萱藻孢子/配子ITS拷贝数的季节变化与青苔爆发规律呈现较高的吻合度,萱藻爆发前期伴随水体中孢子/配子量的增高,初步显示藻类爆发可用qPCR方法预测。

Marine macroalgal blooms in coastal eutrophic waters affect the physiology and ecology of the cultured organisms,causing massive death of these animals sometimes.Knowing the quantity and variation of the algal spores or gamates in the aquatic systems is crucial in understanding the biological and ecological mechanisms underlying algal outbreaks and in developing strategies of prevention.In this study,we newly designed specific PCR primers targeting the internal transcript region（ITS）of the ribosomal RNA genes（rDNA）of three macroalgal species,Ulva cf.linza,Ulva sp.,and Scytosiphon lomentaria,which all are known to be potentially blooming species in mariculture ponds.The specificity of these primers was verified in silico,in vitro and in situ.Real-time quantitative PCR（qPCR）assays with these primers were developed and verified,showing a detection limitation as low as 200～400 ITS copy numbers of the algal genomic DNA.The newly developed assays were further applied to quantify the spores/gamates of S.lomentariain surface waters of a farming pond for sea cucumbers.The quantification of the spores of S.lomentariaduring a 16-month period（from December of 2014 to March of 2016）showed that the ITS rDNA copy number of this species was relatively high in December of 2015,which was in coincidence with the observed blooming of the species in March of 2016,suggesting a link between the high spore abundance in the winter and the bloom of the macroalga in the early spring of next year.