脱落酸在藻华甲藻锥状斯氏藻休眠孢囊形成和萌发过程中的调控作用研究

Resting cyst has been proven to be one of the most important facilities used by dinoflagellates to form harmful algal blooms (HABs) and expand their geographic distribution. However, gaps remain in our knowledge about the exocellular regulatory factors involved in the formation and germination of resting cysts. Therefore, it is proposed here that an approach combining physiological measurements and gene expression monitoring will be employed to examine the possible key roles played by abscisic acid (ABA), an endogenous phytohormone, in regulating the formation and germination resting cyst. The cosmopolitan, toxic, and resting cyst-producing dinoflagellate, Scrippsiella trochoidea, which frequently forms HABs in China and many other countries around the world will be used as the model organism. Firstly, the resting cyst formation and germination will be examined under different concentrations of exocellularly-originated ABA in order to assess the effect of exogenous ABA on the formation and germination of resting cyst; Secondly, the changes of intracellularly-originated ABA level will be tracked via high performance liquid chromatography (HPLC) method. The endogenous ABA level of resting cysts of dinoflagellate in sediments from areas where HABs of S. trochoidea have frequently occurred will also be determined. Regression analysis will be conducted to examine the correlation between endogenous ABA level and the process of resting cyst formation and germination; Finally, the full-length cDNA sequences of 9-cis-epoxycarotenoid dioxygenase (NCED) and ABA-8’-hydroxylase (ABAH) genes, which encode the crucial speed-limiting enzymes in the biosynthesis and catabolism of ABA, respectively, will be obtained using the rapid amplification of cDNA ends (RACE). Their expressions will be quantified by real-time quantitative PCR for the resting cysts at different stages of formation and germination. A comprehensive analysis will be then conducted to uncover whether or not ABA is involved in regulating the resting cyst formation and germination in S. trochoidea. It is anticipated that accomplishment of this project will help to reveal the regulatory mechanisms of resting cyst formation and germination of dinoflagellates, and also provide an important basis for further understanding the mechanisms of dinoflagellate blooms.

休眠孢囊已被证明在甲藻藻华生消和地理扩散过程中起着关键作用，但在分子水平对甲藻孢囊形成和萌发的内源调控因子的研究尚未见诸报道。本项目以世界广布性有毒藻华甲藻锥状斯氏藻（Scrippsiella trochoidea）为研究对象，采用室内培养结合分子生物学手段对可能调控该生物学过程的内源激素脱落酸（abscisic acid, ABA）进行研究。首先，通过外源ABA单因子梯度实验明确孢囊形成和萌发与外源ABA之间的响应关系；之后，利用高效液相色谱方法测定内源ABA的含量，明确其含量变化与孢囊形成和萌发的相关性；最后，克隆ABA合成和分解代谢途径中的关键限速酶编码基因，再通过实时定量PCR技术分析其表达规律，进而探讨其与孢囊形成和萌发的关系。预期结果将有助于探明调控甲藻休眠孢囊形成和萌发的内在生物学机制，从而为阐明由甲藻引起的有害藻华的发生发展机制提供重要的基础性认识。

对于许多甲藻类藻华原因种而言，其特殊生活史，即形成休眠孢囊，是一种对藻华发生和种群地理扩散具有重要意义的生态策略。休眠孢囊在孢囊类甲藻种群延续、藻华生消、年际复发和地理扩散等方面发挥着非常关键的作用,但在分子水平上对甲藻孢囊形成和休眠维持的内源调控因子的研究尚未见诸报道。本项目以世界广布性有毒藻华甲藻锥状斯氏藻（Scrippsiella trochoidea）为研究对象，采用室内培养结合分子生物学手段对可能调控该生物学过程的内源激素脱落酸（abscisic acid, ABA）进行研究。本项目获得主要结果如下：（1）对锥状斯氏藻休眠孢囊和营养细胞分别构建转录组文库进行高通量测序分析其生活史不同阶段转录谱基因表达变化，挖掘到一系列可能与甲藻生活史转换相关的基因；（2）以获得的转录谱基因序列信息为基础，利用RACE技术获得锥状斯氏藻ABA生物合成途径中3个关键酶，9-顺式-环氧类胡萝卜素双加氧酶（9-cis-epoxycarotenoid dioxygenase, NCED），玉米黄质环氧化酶（zeaxanthin epoxidase, ZEP），脱落酸-醛氧化酶（abscisic-aldehyde oxidase, AAO）和分解途径中最关键限速酶脱落酸-8’-羟化酶（ABA-8’-hydroxylase, ABAH）的全长cDNA编码基因序列，首次证实了甲藻中含有编码高等植物脱落酸合成和分解途径关键酶类的同源基因。利用qRT-PCR技术检测上述关键基因在锥状斯氏藻生活史不同阶段的表达规律，结果表明：在孢囊形成和休眠维持过程中，ABA合成途径相关基因表达量显著升高，分解途径最关键限速基因表达量显著降低，且低温和黑暗条件下这种趋势更为显著，暗示休眠孢囊较营养细胞中内源ABA积累更丰富。（3）建立适用于甲藻的内源ABA含量超高效液相色谱-串联质谱（UHPLC-MS/MS）检测方法，利用建立的UHPLC-MS/MS方法和酶联免疫法定量检测锥状斯氏藻，证实休眠孢囊内源ABA含量显著高于营养细胞内含量。综合以上结果，我们认为ABA在锥状斯氏藻休眠孢囊形成和休眠维持过程中可能具有重要的调控作用。相关研究结果有助于探明调控甲藻休眠孢囊形成和休眠维持的内在生物学机制，并为深入研究甲藻生活史不同阶段转换的分子生物学机理提供重要理论依据。