海带褐藻多酚合成量与苗期绿烂病爆发的相关性研究

Saccharina japonica is an important economic seaweed in China. In commercial farming, frequent outbreak of diseases at hatchery stages has become a great threat. The so called “green rotten” disease has been believed to be caused by insufficient supply of irradiance. While the Algnic Acid Decomposing Bacteria (AADB) are thought to be opportunistic pathogens. Pholorotannins has been discovered to be the chemical substance in the alga to defend the bacteria intrusion. Its synthesis is impacted by multiple external factors including the irradiance. In this proposal, we plan to investigate the co-relation between irradiance levels in culture and the corresponding levels of pholorotannins inside the alga tissue, and thus to elucidate whether changes of pholorotannins will lead to variation of the chemical defense against the AADB, causing the outbreak of “green rotten” disease. We will particularly pay attention to investigating changes of pholorotannins content and the healthy status of the algal seedings when grown under different irradiance levels, try to discover the gene regulation mechanism of biological synthesis of pholorotannins. The results out of this investigation are expected to let us understand the biological mechanism of “green rotten” disease, put forward a practical and scientifically reliable measure to prevent its outbreak, and further help us to breed more new varieties that are resistant to hostile conditions in the future.

海带是我国重要的经济海藻，其种苗培育过程中病害频发严重威胁着海带养殖业的健康发展。其中苗期绿烂病危害最为严重，给育苗企业造成了严重经济损失。光照不足被认为是引发苗期海带绿烂现象的关键因子，而褐藻酸降解菌则是海带苗绿烂的条件致病菌，结合褐藻多酚是海带抵抗病原体入侵的重要化学防御物质且其合成量受光强影响，本项目中，我们将通过实验及分子生物学方法分析海带人工育苗过程中，光照不足是如何影响苗期海带褐藻多酚合成量的变化，阐释这个变化是否会影响藻体对褐藻酸降解菌的防御能力，以及这些变化和海带苗绿烂病爆发的关系。将重点研究不同光照强度条件下培养的海带苗体内褐藻多酚含量变化及幼苗的健康状况，探究光照强度对褐藻多酚合成关键酶基因的分子调控机制。预期取得的结果将对阐明海带苗期绿烂病爆发的成因、提出科学有效的防治措施，进而培育抗病性强的新品种具有重要指导意义。

海带苗期绿烂病的爆发严重威胁着海带养殖业的健康发展。前期研究发现，光强不足是引发绿烂的关键因子，但具体的发病原因及分子机理尚不明确。本项目比较分析了海带苗绿烂初期健康部位及病烂部位附生菌群的差异，根据细菌丰度的变化预测了引起海带苗绿烂的潜在致病菌；完成了绿烂幼苗藻体上细菌的分离及鉴定，获得了一株具有较高褐藻酸降解活性的菌株；完成了不同光强条件下海带苗化学防御物质褐藻多酚含量的测定及幼苗健康状况的评估，明确了海带苗褐藻多酚含量随光强增高而增加，幼苗的生长及代谢受光强影响显著。其次，为完成光强对褐藻多酚合成关键酶基因（PKS1基因）的分子调控机制的探究，克隆并获得了PKS1基因序列，通过定量分析，发现海带苗PKS1基因的表达受光强调控，随光强的增强表达量显著上调；进一步克隆获得了PKS1基因的上游启动子区域，预测得到7处与光强有关的顺势作用元件，通过酵母单杂交筛选方式对调控 PKS1 基因表达的转录因子进行了筛选，获得约1000个阳性克隆，随机挑选150个阳性克隆菌落进行PCR得到cDNA插入片段，测序分析后得到8条编码区较长的序列。通过上述研究工作的开展，初步揭示了光强对海带苗健康状态的影响及防御物质褐藻多酚合成的分子调控机制。本项目的开展对于阐明海带苗绿烂病爆发的原因及分子机理，进而提出科学有效的防治措施具有重要指导意义。