实验海洋生物学重点实验室知识库

几丁质是地球上储量第二、海洋中丰度最高的生物多聚物，影响海洋中的碳氮元素循环。几丁质及其衍生物如几丁聚糖在工业、农业和医疗业都有广泛用途。在几丁质的三种构型（α-、β-、γ-）中，β-几丁质因其同向平行的糖链结构，具有更高反应活性，因此也具有更高的应用价值。硅藻中的海链藻属Thalassiosira sp.是β-几丁质的重要生产者，研究海链藻中的几丁质代谢通路及关键基因有重要理论和实际意义。本研究以海链藻属的假微型海链藻Thalassiosira pseudonana和威氏海链藻Thalassiosira weissflogii为对象，对几丁质代谢相关基因进行挖掘和基因功能的初步验证。主要研究结果如下:

1. 对中心纲硅藻模式生物假微型海链藻T. pseudonana进行全基因组范围内的几丁质酶基因家族鉴定，一共得到了24个几丁质酶基因。根据系统进化分析，将这些基因划分为8个群组。通过生物信息学分析，解析了基因家族中基因的结构、蛋白理化性质、模体（motif）和结构域组成、系统进化关系、染色体定位、基因复制事件、顺式作用元件等。通过实时荧光定量实验和酶活检测探究了假微型海链藻几丁质酶基因在转录和蛋白水平对胁迫的响应，发现家族基因在非生物胁迫条件下的转录表达总体上调，而几丁质酶活力保持稳定。研究结果表明几丁质酶基因家族可能参与假微型海链藻对胁迫的响应过程，对于假微型海链藻适应多变的栖息环境发挥着重要作用。

2. 通过PacBio（Pacific BioSciences）的单分子实时测序（single-molecule real-time，SMRT）构建了威氏海链藻的全长转录本。共得到25,412个基因，其中包含23,362 个注释基因、710个长非编码RNA（long non-coding RNA，lncRNA）、363个转录因子（transcription factors，TF）、3,113个可变剪接（alternative splicing，AS）事件和3,295个简单重复序列（simple sequence repeat，SSR）。我们还鉴定了227个编码几丁质代谢相关酶的基因，其中丰度最高的为几丁质酶基因，共检索到124个，另包括30个几丁质合酶CHS（chitin synthase）基因、4个几丁质脱乙酰酶CDA（chitin deacetylase）基因等。最后构建了威氏海链藻中几丁质生物合成与降解的完整代谢通路。

3. 从威氏海链藻转录组数据库中获取了两条可能编码几丁质脱乙酰酶的基因序列，成功构建了包含威氏海链藻CDA的三角褐指藻Phaeodactylum tricornutum表达载体。通过基因枪方法将载体转化进三角褐指藻，进行威氏海链藻CDA的功能初探。我们实现了威氏海链藻CDA在三角褐指藻基因组中的整合，但没有实现CDA和增强绿色荧光蛋白（enhanced green fluorescence，eGFP）融合蛋白的表达。后续将通过电转化方法进行CDA转基因，并更换基因进一步筛选阳性藻落，以期获得稳定表达的转基因藻株。

本研究丰富了硅藻基因组及转录组相关信息，为硅藻分子生物学尤其是几丁质代谢通路研究提供参考，并为β-几丁质高值衍生物和几丁质代谢相关酶的开发利用奠定基础。

Chitin is the existing second most ample biopolymer in nature and the most in oceans, affecting the carbon and nitrogen circulation in oceans. Chitin and its derivatives such as chitosan are widely used in the industry, agriculture and medicine. Among the α-、β-、γ- allomorph of chitin, β-chitin displays higher reaction activity due to its parallel polysaccharide chain, and is therefore more economically promising. The Thalassiosira sp. of diatoms is an important producer of β-chitin. The investigation on the Thalassiosira chitin genes and metabolism in which is of both theoretical and practical significance. In this study, we used Thalassiosira pseudonana and Thalassiosira weissflogii as the materials to retrieve and validate the genes related to chitin metabolism. The main research results are as follows:

1. A chitinase gene family of the model centric diatom T. pseudonana was identified at genome scale, including 24 members which were clustered into eight phylogenetic groups. We analyzed the structure of the family genes, physical and chemical properties of proteins, motifs and domain composition, phylogenetic relationship, chromosomal localization, gene replication and cis-acting elements. We further investigated the responses of the T. pseudonana chitinase family genes at the transcription and translation levels under abiotic stresses via qRT-PCR and enzymatic assay, respectively. We found that the transcription profiles of the genes were broadly up-regulated, while the chitinase activity remained stable. This indicated that the chitinase gene family may be involved in the response of T. pseudonana to stresses and played a pivotal role in the adaption of the species in shifting environments.

2. A full-length transcriptome of T. weissflogii was constructed via single-molecule real-time (SMRT) sequencing on Pacific BioSciences (PacBio) platform. A total of 25,412 genes were obtained, including 23,362 annotated genes, 710 long non-coding RNAs (lncRNAs), 363 transcription factors (TFs), 3,113 alternative splicing (AS) events and 3,295 simple sequence repeats (SSRs). We also identified 227 genes encoding enzymes related to chitin metabolism, the most abundant among which were genes encoding chitinases (124). The number of genes encoding chitin synthases (CHSs) and chitin deacetylases (CDAs) were 30 and 4, respectively. A complete metabolic pathway network for chitin biosynthesis and degradation was also explored.

3. Two putative genes encoding CDAs of T. weissflogii were obtained from several databases, and were individually inserted into expression vectors. We transformed the vectors into Phaeodactylum tricornutum by the method of gene bombardment, to explore the potential function of T. weissflogii CDAs. The results showed that CDAs were both integrated into the genome of P. tricornutum. However, the fusion protein of CDA and eGFP (enhanced green fluorescence protein) was not detected. We would further use high-voltage electroporation method to conduct gene transformation and screen candidate genes. Further positive colonies screening will be carried out to obtain ideal mutants.

The information in this study will provide a genome and transcriptome reference for molecular biology studies of Thalassiosira, facilitate chitin metabolism studies in diatoms, and pave the way for exploiting high-value derivatives of β-chitin and the chitin-related enzymes.