雨生红球藻多拷贝基因bkts差异表达的转录调控机理

β -carotene ketolase gene (bkts) known as multicopy genes is a key enzyme gene for astaxanthin biosynthesis in Haematococcus pluvialis. It is still unclear about these multicopy genes how to deal with environmental stresses with regulation in transcription differentiation in Haematococcus pluvialis. Regulation of bkts transcription might play a key role in the astaxanthin biosynthesis because references confirmed that regulation of astaxanthin biosynthesis is controlled in transcriptional level. However, we still unknow regulatory mechanism of astaxanthin biosynthesis due to a long period of stagnation in study methods and shortage of the knowledge on bkts promoter in Haematococcus pluvialis. In our previous study, both bkt1 and bkt2 promoter were cloned from Haematococcus pluvialis genomic library and their functions were confirmed by molecular methods. Sequence analysis and data search revealed that there were a lot of cis-elements associated with light and salt stress-related responses though these two promoters existed with low identity. Here, transcription factors involving in regulating bkts transcription will be screened from Haematococcus pluvialis and Chlamydomonas reinhardtii by Yeast one-hybrid system, transcriptome analyse, etc. In order to analyze the interactions and functions of these transcription factors involving in regulation of bkts transcription, some methods including Yeast two -hybrid system, gene knock-out, gene over-expression will be employed. In conclusion, regulatory mechanism of bkts transcription will be illuminated based on our study results. It will provide a new way for increasing production of astaxanthin by artificial control.

β-胡萝卜素酮化酶基因（bkts）是雨生红球藻虾青素合成的关键酶基因，同时也是多拷贝基因，它们如何采取差异化转录调控来应对环境胁迫的机理还不清楚。虾青素合成的调控发生在转录水平，因此，bkts转录调控的机理也是揭示虾青素合成调控机制的关键。然而，由于雨生红球藻研究方法的滞后以及bkts启动子研究的不足，至今还没弄清虾青素合成调控的分子机制。我们前期研究克隆了bkt1和bkt2的启动子并证明了它们的功能，发现bkts各拷贝基因的启动子虽然差异很大，但都含有大量与光照和盐胁迫相关的顺式作用元件。本项目拟在前期研究基础上，以雨生红球藻和莱茵衣藻为材料，利用酵母单杂交等技术和转录组学研究筛选并鉴定出参与bkts转录调控的转录因子，再通过酵母双杂交、基因敲除和基因过表达等技术对它们的功能进行系统研究，最终阐明雨生红球藻bkts转录调控的分子机理，为利用人工调控并提高虾青素产量提供新的途径。

雨生红球藻因能在胁迫条件下大量合成虾青素而备受关注，它的合成调控发生在转录水平。β-胡萝卜素酮化酶基因（bkts）是虾青素合成的关键酶基因，因此，bkts转录调控的机理也是揭示虾青素合成调控机制的关键。本课题采用多种研究手段对bkts转录调控机理进行研究，获得的具体结果如下：（1）雨生红球藻在MIX培养基中培养至对数中期，可采用改良的Trizol法提取总RNA和小RNA，并筛选M-3为小RNA荧光定量PCR的内参；（2）在强光和醋酸钠胁迫条件下，雨生红球藻bkts等基因的转录调控发生在胁迫早期，且普遍存在基因多拷贝现象，以分别响应不同胁迫；（3）完成了多个不同处理的转录组测序，注释获得83869个unigene，发现4367个差异表达基因，构建了雨生红球藻转录因子数据库，共计获得476个转录因子，分别属于52转录因子家族；（4）采用酵母单杂交、DNA pulldown-质谱等先进技术研究bkts启动子结合蛋白，构建bkts启动子互作蛋白数据库，获得参与bkts转录调控的关键蛋白；（5）获得GNAT、MYB、MYB_related、NF-YC、C3H、Nin-like等参与bkts转录调控的转录因子，克隆其基因并对其功能进行研究，发现这些核心转录因子对bkts的调控主要发生在胁迫初期，它们的表达可响应强光、或响应醋酸钠，也可以同时响应强光和醋酸钠，与bkts的表达高度一致；（6）获得雨生红球藻259个已知miRNA和300个新miRNA，建立了5组miRNA-靶基因对，找到多个与虾青素合成相关的miRNA，并对功能进行深入研究，发现雨生红球藻miRNA既可以调控转录因子的活性，也可以直接调控bkts的转录活性；（7）通过对雨生红球藻共调控网络的深度运算，建立一个由5个核心转录因子参与的虾青素合成调控网络。以上研究成果为我们初步阐明了雨生红球藻响应环境胁迫的机理，弄清bkts的转录调控过程。