实验海洋生物学重点实验室知识库

铁元素是海洋浮游植物生长繁殖的限制性因素，也是全球生物地球化学循环和气候变化的重要推动力。硅藻贡献了20%的全球初级生产力（约占海洋初级生产力的40%），极大地影响着海洋生态系统及全球气候。铁元素对硅藻的限制尤甚，在缺铁的环境中硅藻只能发挥其最大初级生产力的60%，所以铁元素在驱动大洋里硅藻种群动态变化中发挥关键作用。由此可见，铁元素可以增加大洋中硅藻的生物量，进而促进大气中的二氧化碳转为有机碳并向深海沉积，以减轻温室效应。

在铁浓度很低的海洋（深远海）环境中，硅藻进化获得了一系列独特的对低铁浓度的响应和调节机制。对此进行深入研究，可以认识大洋中硅藻的生消过程，解析全球的生物地球化学循环规律。为此，本文以海洋模式硅藻——三角褐指藻为研究对象，利用基因编辑技术，研究了硅藻在铁的吸收、转运、储存过程中关键基因的功能，重点关注了藻华爆发过程中细胞密度波动对铁吸收的影响及调控机制，并进一步探究了其生态效应。本文的主要研究结果如下：

（1） 揭示了三角褐指藻的细胞密度对负责铁吸收的蛋白——ISIP2a和ISIP1的影响及调控机制。研究结果表明，当三角褐指藻细胞密度从1.6×10⁶ 升高至6.4×10⁶ cells/mL时，单个细胞内铁含量下降，但ISIP2a和ISIP1基因的表达量上升。在高细胞密度下沉默ISIP2a，细胞内铁含量显著下降，只有野生型的42.74%，表明ISIP2a可能参与了高细胞密度下三角褐指藻的铁吸收。在细胞培养过程中加入高密度培养的细胞的培养液和浸提液，并没有增加ISIPs的表达，排除了化学因素的调控作用。此外，660–685 nm红光会显著诱导ISIP2a和ISIP1的表达。蓝光激发出的细胞叶绿素荧光强度，与细胞密度及ISIPs的表达量呈正相关。同时，我们进一步设计了“三明治”形式的处理装置以滤除杂散光，保证内层细胞只接收外层细胞发出的叶绿素荧光。结果表明，外层细胞密度的增加显著提高了内层细胞接收到的叶绿素荧光强度以及ISIP2a和ISIP1的转录水平。在种群生态层面，通过构建数学模型计算出大洋中硅藻爆发时叶绿素荧光强度为0.01-1.19 μmol/m²/s。另外，在大洋中来自Tara Ocean的58个站点的原位测量数据和宏转录组数据显示，ISIPs的表达与海水中叶绿素含量也呈正相关。然而，敲除三角褐指藻的红光/远红光受体——光敏色素之后，三角褐指藻ISIPs对于红光的响应依旧存在，说明光敏色素可能没有介导叶绿素荧光对于ISIPs的调控。以上结果表明，随着硅藻爆发时种群密度的升高，细胞间距离降低，细胞可以感知到邻近细胞的叶绿素荧光，从而诱导表达ISIP2a和ISIP1，加速铁吸收。

（2） 比较了低(1.5×10⁶ cells/mL)、中(3×10⁶ cells/mL)、高(6×10⁶ cells/mL)三种细胞密度下，三角褐指藻在白光、蓝光和黑暗条件下的转录组。结果显示，参与铁代谢的基因——ISIP2a，ISIP2b，ZIP9均呈现叶绿素荧光介导的密度依赖的表达模式。加权基因共表达网络分析显示，lightyellow模块的基因与ISIP2a表达模式最为一致，即在白光和蓝光下，基因随着细胞密度的升高表达上调，在黑暗条件下则无显著差异。这一结果说明，叶绿素荧光介导的信号途径对细胞内的生理生化过程有广泛的影响。此外，grey60和darkgrey模块的基因在三种光环境下均呈现密度依赖的表达模式，说明该模块的基因可能受到密度影响的化学信号的调控。利用模块内的基因表达网络鉴定到16个hub gene，在其中发现一个编码Na⁺/Ca²⁺渗透通道结构域的基因SLC24A3。该基因（SLC24A3）在模块内互作网络中处于中心位置，说明Ca²⁺信号可能在响应密度变化的过程中发挥重要作用。

（3） 鉴定到了一个缺铁诱导的新蛋白，Pt_CSP。序列分析结果显示，Pt_CSP与ISIP2b具有同源性，均存在多个铁离子结合位点。Pt_CSP在拟南芥原生质体中定位于囊泡和叶绿体表面。测定了Pt_CSP与其他编码铁饥饿诱导蛋白以及储存蛋白的基因在不同条件下的表达，结果显示Pt_CSP与ISIP2a和ISIP1表达模式相似，均在缺铁、缺硅及高细胞密度条件下被显著诱导表达。利用反向遗传学技术，分别构建了沉默Pt_CSP、ISIP2a和ISIP1的突变株。与WT相比，Pt_CSP-Si、ISIP2a-Si和ISIP1-Si突变株在铁充足条件下生长速度无差异，但是在缺铁条件下，Pt_CSP-Si、ISIP2a-Si和ISIP1-Si沉默株的生长速度、光合参数及细胞壁铁含量均显著下降。基于以上结果，我们认为Pt_CSP在三角褐指藻中具有胞内铁转运的功能。

（4） 探究了三角褐指藻的铁储存蛋白Ferritin与细胞形态转变之间的关系。通过构建三角褐指藻Ferritin过表达和沉默突变株（FTN-OE，FTN-Si），发现FTN-OE突变株由梭形转变为卵圆形，而WT和FTN-Si突变株仍然为梭形。这些FTN-OE卵圆形细胞表现出典型的休眠特征，如生长速率低、适应弱光、硅化的壳面、分泌粘液、上调抗逆性蛋白的表达和硝酸盐储备等。此外，转录组和蛋白质组结果显示，FTN-OE卵圆形细胞在脱落酸（ABA）合成和信号通路上调；利用LC-MS，仅在FTN-OE卵圆形细胞中检测到ABA(2.96 ng/g FW)，而在FTN-Si、FTN-OE梭形细胞或WT细胞中均未检测到ABA。以上研究结果表明，三角褐指藻中Ferritin过表达促进了细胞从梭形向卵圆形的转变，之后进入休眠阶段，这一过程伴随着ABA的积累。

综上所述，三角褐指藻的自发叶绿素荧光作为信号不仅诱导ISIP2a和ISIP1的表达，而且影响胞内的诸多生理过程；钙离子通道蛋白SLC24A3可能也参与细胞对种群密度的感知与信号传递；敲降缺铁诱导蛋白Pt_CSP降低了三角褐指藻应对缺铁胁迫的能力；过表达铁储存蛋白Ferritin对三角褐指藻细胞形态及深海碳沉积的影响。

As one of the most important photosynthetic organisms on the planet, diatoms contribute at least 20% of the global primary productivity, greatly affecting the global climate, atmospheric carbon dioxide concentration and marine ecosystem. However, the availability of iron governs diatoms growth in wide-ranging open oceans, causing 30-40% limitation of primary production. To cope with the chronically iron deficiency and take advantage of pulsed iron supplies in open oceans, diatoms have evolved multiple physiological strategies and regulation mechanisms. Exploring this response and regulation mechanism in diatoms is of great significance to the global biogeochemical cycle. In this study, we used transgenic and gene editing technology to explore the functions of key genes involved in the process of iron absorption, transport, and storage, in the model marine diatom, Phaeodactylum tricornutum. More attention was paid on the underlying mechanisms regulating iron uptake under fluctuating cell density, especially during diatom blooms. The main findings of this article are as follows:

(1) The effect of cell density on the two most abundance iron uptake proteins, ISIP2a and ISIP1, and the potential regulation mechanism were explored in P. tricornutum. We demonstrated that transcription of ISIP2a and ISIP1 were enhanced with increasing cell density from 1.6×10⁶ to 6.4×10⁶ cells/mL, whereas the cellular iron content showed the opposite trend. When compared with the wild-type strain, knockdown of ISIP2a resulted in 42.74% decrease in cellular iron content, implying the involvement of ISIP2a in iron uptake under high cell density conditions. Incubation of the diatom cells with sonicated cell lysate conditioned by different cell densities did not affect ISIP2a and ISIP1 expression, ruling out regulation via chemical cues. In contrast, ISIP2a and ISIP1 transcription was strongly induced by red light irradiance at 660–685 nm. Besides, chlorophyll fluorescence excited from blue light was also positively correlated with population density. Subsequently, a “sandwich” illumination incubator was designed to filter out stray light and ensure that the inner layer cells only receive the emitted chlorophyll fluorescence from outer layers. The results showed that the increase in outer cell density significantly elevated ISIP2a and ISIP1 transcription in inner layer cells. In situ evidence from Tara oceans also showed positively correlated between diatom ISIPs transcripts and chlorophyll content. However, after knocking down the red light far-red light receptor, phytochrome, ISIPs were still induced by red light, indicating that the phytochrome did not mediate the regulation of chlorophyll fluorescence on ISIPs. Overall, with the decrease in intercellular space to 66.7 μm at high cell density, chlorophyll fluorescence derived from neighboring cells upregulated ISIP2a and ISIP1 expression to facilitate iron assimilation.

(2) We compared the transcriptome of 27 samples of P. tricornutum at low (1.5×10⁶ cells/mL), medium (3×10⁶ cells/mL) and high (6×10⁶ cells/mL) cell density under white, dark, and blue conditions. The results showed that the iron metabolism-related genes ISIP2a, ISIP2b, and ZIP9 showed a density-dependent expression pattern mediated by chlorophyll fluorescence under white and blue light conditions. Gene-weighted co-expression network analysis revealed a lightyellow module in which gene’s expression was most consistent with ISIP2a, indicating that chlorophyll fluorescence has a wide mediation within cells. In addition, the genes involved in grey60 and darkgrey modules showed a density-dependent expression pattern under both white light, blue light and dark conditions, implying these genes may be regulated by chemical signals affected by cell density. A Na⁺/Ca²⁺ permeable channel domain gene, SLC24A3, was found in the 16 core hub genes identified using the network information in the module, which is at the center of the intra-module interaction network, suggesting that the Ca²⁺ signal channel may play an important role in signal transduction of density-dependent regulation.

(3) A new protein Pt_CSP induced by iron-deficiency was identified. Sequence analysis revealed that Pt_CSP contains multiple iron binding sites and is homologous with ISIP2b. Pt_CSP located on the surface of vesicles and chloroplasts. The expression of Pt_CSP and other iron starvation-induced proteins and iron storage proteins under different environmental conditions was determined. It was found that Pt_CSP, ISIP2a and ISIP1 showed similar expression patterns, both significantly induced under iron-deficiency and silicon-depleted conditions. Furthermore, silent mutants of Pt_CSP, ISIP2a and ISIP1 were construct (CSP-Si, ISIP2a-Si and ISIP1-Si). Compared with WT, CSP-Si, ISIP2a-Si and ISIP1-Si mutant strains have no significant difference under iron-sufficient conditions. However, under iron deficiency conditions, the growth rate, photosynthetic performance and cell wall iron content of CSP-Si, ISIP2a-Si and ISIP1-Si strains decreased significantly. Based on the above results, it is speculated that Pt_CSP may play the role of iron transport in P. tricornutum.

(4) The relationship between iron storage protein, ferritin, and the morphological transformation was performed in P. tricornutum. We constructed the ferritin overexpression and silence strains in P. tricornutum (FTN-OE and FTN-Si, respectively). The morphological transformation from fusiform to ovoid were observed among the FTN-OE strains but not the wide type and FTN-Si strains. These FTN-OE oval cells exhibited typical characteristics of resting cells and benthic adaptation, such as lower growth rates, low-light adaptation, silicified valves, the excretion of exopolymeric substances, upregulation of stress-resistant proteins and increased nitrate reserves. Furthermore, FTN-OE oval cells showed increased abscisic acid (ABA) synthesis and signaling pathways in the transcriptome and proteome. And the ABA signal was detected in FTN-OE oval cells exclusively (2.96 ng g^–1 FW) by LC-MS, but absent in the FTN-Si, FTN-OE fusiform or WT cells. Overall, our results demonstrate that ferritin overexpression in marine diatom stimulates morphological transformation from fusiform to ovoid morphotype, after which they enter a resting stage, which is accompanied by ABA accumulation.

In summary, the spontaneous chlorophyll fluorescence of P. tricornutum act as a signal, not only induces the expression of ISIP2a and ISIP1, but also affects multiple physiological processes. Calcium channel protein SLC24A3 may participate in the cellular perception and signal transmission of population density. Knockdown of the iron-deficiency-inducible protein Pt_CSP reduced the ability of P. tricornutum to cope with iron-deficiency stress. Overexpression the iron storage protein Ferritin facilitate the cell morphology transformation and have a close relationship in deep-sea carbon deposition.

目  录

第1章 引  言... 1

1.1 大洋中铁的来源及其生态效应... 1

1.1.1 铁的来源及存在形式... 1

1.1.2 铁的生态效应... 2

1.2 硅藻概述... 4

1.3 硅藻与铁的关系... 5

1.3.1 硅藻胞内与铁相关的生理过程... 5

1.3.2 硅藻应对缺铁环境的策略... 7

1.3.3 硅藻的铁吸收系统... 10

1.3.4 硅藻的铁储存策略... 13

1.4 三角褐指藻概述... 15

1.4.1 遗传背景清晰的代表性硅藻... 15

1.4.2 成熟的基因编辑平台... 16

1.5研究内容与技术路线... 17

1.5.1 研究内容... 17

1.5.2 技术路线... 19

第2章 铁吸收过程中叶绿素荧光的信号介导作用... 21

2.1 引言... 21

2.2 材料与方法... 22

2.2.1 藻细胞培养条件及处理... 22

2.2.2 ISIP2a沉默株构建... 23

2.2.3 实时荧光定量分析... 26

2.2.4 叶绿素荧光的测定... 26

2.2.5 色素提取和分析... 27

2.2.6 细胞内铁含量的测定... 27

2.2.7 数学建模计算叶绿素荧光... 28

2.2.8 Tara Ocean 数据分析... 28

2.2.9 顺式作用元件预测... 28

2.2.10 三角褐指藻光敏色素（DPH）敲除株构建... 29

2.3 结果... 32

2.3.1 三角褐指藻对不同细胞密度的生理响应... 32

2.3.2 ISIP2a及ISIP1在高细胞密度条件下促进铁吸收... 35

2.3.3 光对ISIP2a和ISIP1表达调控的影响... 38

2.3.4 大洋中叶绿素荧光强度的计算... 42

2.3.5 宏转录组中ISIPs表达量和浮游植物生物量的关系... 45

2.3.6 光敏色素对红光诱导的ISIP2a表达的调控作用... 47

2.4 讨论... 49

2.4.1 三角褐指藻中铁吸收相关基因的表达受细胞密度的诱导... 49

2.4.2 叶绿素荧光介导了高细胞密度下ISIP2a和ISIP1的调控表达... 50

2.4.3大洋中的叶绿素荧光及其生态意义... 51

2.4.4光敏色素（DPH）不介导三角褐指藻对红光响应... 52

第3章 密度依赖的铁代谢过程及关键调控基因挖掘... 55

3.1 引言... 55

3.2 材料与方法... 56

3.2.2 材料的培养与处理... 56

3.2.3 RNA提取，文库构建和测序... 56

3.2.4 组装和注释... 57

3.2.5 加权基因共表达网络分析(WGCNA) 57

3.2.6 实时荧光定量验证... 57

3.2.7 差异表达基因分析... 58

3.3 结果... 59

3.3.1 转录组的基本信息... 59

3.3.2 差异表达基因的鉴定与qRT-PCR的验证... 59

3.3.3 铁代谢相关基因的表达模式分析... 61

3.3.4 加权基因共表达网络分析... 62

3.3.5 密度依赖的表达模块的GO和KEGG富集分析... 66

3.3.6 调节密度响应的Hub基因的鉴定... 68

3.4 讨论... 69

3.4.1 铁代谢过程对细胞密度呈现不同的响应... 69

3.4.2 叶绿素荧光对藻细胞响应密度变化的介导... 70

3.4.3 化学信号对藻细胞响应密度变化的介导... 71

3.4.4 Ca²⁺信号在响应密度依赖的调节过程中的潜在作用... 72

第4章 敲降缺铁诱导蛋白Pt_CSP对铁吸收、转运过程的影响... 73

4.1 引言... 73

4.2 材料与方法... 74

4.2.1 材料处理... 74

4.2.2 信号肽和跨膜区预测... 74

4.2.3 Pt_CSP沉默藻株构建... 75

4.2.4 生长测定... 75

4.2.5 光合参数测定... 75

4.2.6 Chla含量测定... 75

4.2.7 细胞铁含量测定... 75

4.3 结果... 75

4.3.1 Pt_CSP序列分析与定位... 75

4.3.2 Pt_CSP基因的表达模式... 79

4.3.3 Pt_CSP沉默突变株构建... 81

4.3.4 Pt_CSP沉默突变株光合活性测定... 82

4.3.5 Pt_CSP沉默突变株Chla含量测定... 83

4.3.6 Pt_CSP沉默突变株铁含量测定... 84

4.4 讨论... 86

4.4.1 Pt_CSP沉默显著影响缺铁条件下三角褐指藻的生长及胞外铁结合... 86

4.4.2 Pt_CSP可能参与缺铁条件下三角褐指藻的胞内铁转运... 87

第5章 过表达铁储存蛋白Ferritin对细胞形态的影响... 89

5.1 引言... 89

5.2 材料与方法... 90

5.2.1 藻种与培养方法... 90

5.2.2 转基因藻株的构建... 91

5.2.4 扫描电镜观察细胞形态... 92

5.2.5 细胞铁含量测定... 92

5.2.5 光合参数测定... 92

5.2.6 转录组分析... 92

5.2.7 蛋白组分析... 94

5.2.8 ABA的提取以及LC-MS的定量分析... 94

5.3 结果... 94

5.3.1 三角褐指藻FTN-OE和FTN-Si突变株的构建及形态观察... 94

5.3.2三角褐指藻FTN-OE突变株的生长及光合活性测定... 98

5.3.3 转录组和蛋白组分析... 99

5.3.4 ABA含量测定... 106

5.4 讨论... 107

5.4.1 Ferritin过表达伴随着三角褐指藻细胞形态从梭形转化成卵圆形... 107

5.4.2 FTN-OE卵圆形细胞是一种休眠状态... 107

5.4.3 FTN-OE突变株从梭形向卵圆形细胞的转变伴随着ABA的积累... 108

5.4.4 Ferritin积累对促进深海碳沉积具有重要意义... 109

第6章 结论与展望... 111

6.1 主要结论... 111

6.2 主要创新点... 112

6.3 后续研究计划... 112

参考文献... 113

附录-缩略词表... 135

致  谢... 137

攻读学位期间发表或完成的学术论文与研究成果... 139

作者简历... 141