实验海洋生物学重点实验室知识库

条斑紫菜（Neopyropia yezoensis）不仅是重要的经济海藻，同时还是海藻学研究的模式物种。在其生活史中，单细胞阶段的壳孢子和单孢子分别介导了条斑紫菜孢子体的减数分裂和配子体的无性繁殖，同时也是生产中常用的种苗来源。然而，目前对孢子囊枝放散壳孢子的发育调控机制以及两种孢子发育的分子机制所知甚少，在一定程度上制约了条斑紫菜新型采苗技术的发展。此外，由于条斑紫菜功能基因遗传转化体系尚未完全建立，严重影响了条斑紫菜分子育种和分子机制的探究。针对上述条斑紫菜在种苗、种质和基础研究方面面临的“瓶颈”问题，本论文首先从孢子囊枝出发，深入探究了光照强度和培养密度调控自由孢子囊枝放散壳孢子的基本规律和分子基础。随后，通过单细胞测序深入解析了壳孢子与单孢子基因表达特点，分析了两种孢子在能量代谢、减数分裂以及形态建成等方面的差异。最后，从上述分析结果中挑选重要基因，构建和验证条斑紫菜功能基因遗传转化体系，筛选出多个条斑紫菜功能基因突变株，在此基础上进一步探究了条斑紫菜高光诱导蛋白（high light-inducible protein, NyHLIP）和腺苷酸脱氨酶（Adenosine 5’-monophosphate deaminase, NyAMPD）的功能。主要的研究结果如下：

（1） 自由孢子囊枝形态变化与壳孢子放散的规律：我们发现自由孢子囊枝在高密度下（≥ 5000根/mL）总是呈现出“空心态”。在低密度下（1-1000根/mL），多数孢子囊枝（> 70%）在第3-9天会形成双分细胞，并在第12-15天，双分细胞会消失而没有伴随壳孢子的放散。透射电子显微镜的观察显示，空心细胞中出现严重的胞内降解，并且这种降解可能是通过一种双层膜包裹的小体与液泡融合的方式发生的。在新生的双分细胞中，液泡和胞内降解现象消失，并在双分细胞的伸长过程中重新出现。形态观察结果表明那些消失的没有放散壳孢子的双分细胞以伸长生长的方式返回到了营养生长阶段。壳孢子放散率的统计和鲜重增长的实验表明促进营养生长的因素，如高光（40-100 µmol photons m⁻² s⁻¹）、氨苄青霉素加链霉素（相较于卡那霉素）、生长素吲哚乙酸（Indoleacetic acid, IAA），都会对壳孢子的放散有明显的抑制作用，而低浓度的抗生长素三碘苯甲酸（Triiodobenzoic acid, TIBA，0.1-10 mg/L）则对壳孢子放散有一定的促进作用。这些结果表明壳孢子的放散主要被两种因素所抑制，一个是高密度培养时基于液泡膜转运系统形成的“空心态”抑制了双分细胞的形成从而抑制壳孢子放散，另一个是在适宜生长的条件下，如高光，双分细胞本身返回到营养生长阶段而抑制了放散；

（2） 光照和密度调控自由孢子囊枝发育的分子基础：通过分析比较高光低密度（High light with low denisty, 简称“HL”）、低光高密度（Low light with high denisty, 简称“HD”）两种培养条件与对照组低光低密度（low light with low density, 简称“LD”）之间的转录组学差异，我们发现众多与碳浓缩、碳固定、光合作用、叶绿素合成和氮吸收相关的差异表达基因（Differentially expressed genes, DEGs）在HL组中上调，揭示高光促进孢子囊枝营养生长的分子基础。在HD组中，多个植物盐和渗透胁迫、盐离子运输等相关的DEGs上调表达，同时，液泡形成相关的膜转运关键基因—BIG（Brefeldin A inhibited guanine nucleotide exchange protein，布雷菲德菌素A抑制的鸟嘌呤核苷酸交换蛋白），在HD组中表达量最高，HL组中表达量最低。表明高密度下孢子囊枝呈现的空心态可能与液泡形成、盐和渗透胁迫、盐离子转运等有关，BFA处理结果进一步映证了空心态与液泡形成之间的直接关联；另外，在三个实验组中，我们推测在LD组中表达量最高的DEGs可能与壳孢子的放散和形成有关，包括锌指蛋白、ASPO1527、细胞壁组分、细胞周期、细胞骨架等相关的基因；

（3） 壳孢子与单孢子转录组学比较分析：我们分别挑取了微量壳孢子与单孢子（1-30 细胞/样），通过SMART (switch mechanism at the 5′ end of RNA templates，5’端RNA模板转换机制) -Seq2单细胞测序技术对样品进行RNA测序。比较分析结果表明，光捕获和碳固定相关DEGs在单孢子中上调表达，揭示单孢子快速发育和生长的分子基础。在壳孢子中，蛋白质的合成和降解，特别是分子伴侣，相关的DEGs上调表达，表明壳孢子中蛋白质更新活跃。壳孢子中有68个与DNA复制和修复相关的基因进行了表达，表明壳孢子中可能存在DNA的复制活动。此外，通过荧光定量PCR，我们还证实了一个壳孢子特异表达的DEGs（py04595: DNA解旋酶）仅在二倍体阶段（丝状体、孢子囊枝）表达，三个单孢子特异表达的DEGs仅在单倍体阶段（叶状体）表达，表明壳孢子可能更接近于二倍体。这些分子水平的结果表明壳孢子可能是条斑紫菜减数分裂的母细胞，即其萌发阶段的前两次细胞分裂为两次连续的减数分裂。此外，我们还发现与孢子体向配子体形态转化相关的打结状同源盒基因（knotted-like homeobox gene, NyKNOX）仅在孢子体世代（丝状体和孢子囊枝）表达，而在壳孢子、单孢子和叶状体中均不表达，这表明条斑紫菜配子体的形态发生可能需要NyKNOX基因的失活。

（4） 条斑紫菜功能基因遗传转化的探索：我们选择了条斑紫菜高光诱导蛋白（NyHLIP）、腺苷酸脱氨酶（NyAMPD）、核糖磷酸焦磷酸激酶（Ribose phosphate pyrophosphokinase, NyPRPS）和叶绿素合酶（Chlorophyll synthase, NychlG）等基因，构建了用于遗传转化的沉默与过表达质粒。通过基因枪方法和潮霉素筛选压力，成功筛选出了多株阳性突变株，在DNA验证层面，总体阳性率约为49%。RNA层面的验证结果表明约70%的过表达株目标基因发生了显著上调，最高能上调40多倍，大多数上调倍数在10倍以内；约37%的过表达株出现共抑制现象，有一株下调超过100倍，其余下调在10倍以内；约68%的突变株目标基因发生显著下调，但下调倍数均在4倍以内。这些结果表明通过基因枪的方式，可以在条斑紫菜叶状体中实现功能基因的遗传转化，包括功能基因的沉默与过表达，同时还会出现共抑制现象；

（5） 条斑紫菜NyHLIP功能探究：基于qRT-PCR和Western blot结果，我们发现在高光处理6 h后的低光恢复过程中，NyHLIP在RNA水平和蛋白水平能够存在较长时间。RNA恢复到高光处理之前的水平大约需要24 h左右，而蛋白至少可以大量存在24 h。通过蛋白水平的验证，我们从NyHLIP突变株中挑选出了OE32（共抑制型）和Ri32（RNAi型），这两株在高光胁迫后NyHLIP的蛋白含量明显低于WT株。光合活性测量结果显示高光处理6 h后，OE32和Ri32株光系统II的最大光化学量子产量（F_v/F_m）、实际光化学量子产量（YII）、电子传递速率（ETR）等值明显低于WT株，同时光系统I反应中心P700的还原速率也明显低于WT株，但在非光化学淬灭（Nonphotochemical quenching，NPQ）方面没有明显差异。这些结果表明NyHLIP可能在高光胁迫下维持光系统II的基本活性、电子传递以及维持光系统I的还原活性等方面起着重要的作用；

（6） 条斑紫菜NyAMPD功能探究：腺苷酸脱氨酶（AMPD）能够催化磷酸腺苷（adenosine monophosphate，AMP）分解为鲜味物质肌苷5’-单核苷酸（Inosine 5’- mononucleotide，IMP）。从NyAMPD突变株中，我们分别选择了过表达株（Overexpression，简写为“OE”）AMD2-2、共抑制株（Co-suppression, 简写为“Ci”）AMD1-2、沉默株（RNAi，简写为“Ri”）iAD2-5进行生理水平探究。生长实验表明，相较于WT，OE株生长更快，而Ci与Ri株生长更慢。光合参数测定显示突变株与WT之间无显著差异。腺嘌呤核苷酸相关代谢产物测定显示OE株中鲜味物质IMP含量是WT中的2倍多，ADP、AMP含量显著低于WT，表明OE株中过表达效果良好。但在Ci和Ri株中，IMP含量也升高了，ADP和AMP的含量也降低了。针对条斑紫菜另一个AMPD（即NyAMPD2）基因表达量的检测表明，与WT相比，Ci和Ri株中NyAMPD2显著上调表达，而在OE株中无明显差异。我们认为转基因导致的NyAMPD基因的下调可能会引起NyAMPD2基因的上调，从而使得Ci和Ri突变株中IMP、ADP、AMP的含量出现于OE株类似的变化。

上述结果解析了自由孢子囊枝放散壳孢子的发育调控过程和分子基础，揭示了壳孢子与单孢子基因表达水平差异，同时构建了条斑紫菜配子体功能基因遗传转化体系并验证了其效率，对条斑紫菜的养殖和基础研究具有重要指导和借鉴意义。

Neopyropia yezoensis is not only an important economic marine crop, but also a model species for the research of seaweed. In the life cycle of N. yezoensis, conchospores and archeospores in single cell stage mediate the meiosis of sporophyte and asexual reproduction of gametophyte, respectively. Both the two types of spores are common sources of seedlings in production of N. yezoensis. However, at present, little was known about the development regulation mechanism of conchospores releasing from sporangial filaments and the molecular mechanism in development of the two kinds of spores. These restricted the establishment of new seedling collection technology of N. yezoensis to a certain extent. In addition, the functional gene genetic transformation system has not been fully established, that affected the exploration of molecular breeding and molecular mechanism in N. yezoensis to a great extent. In view of the “bottleneck problems” in seedling, breeding and basic research of N. yezoensis, firstly, this paper deeply explored the development law and molecular basis of conchospore release in free-living sporangial filaments controlled by light intensity and culture density. Then, we analyzed the differences in energy metabolism, meiosis and morphogenesis between archeospores and conchospores through single cell transcriptome platform. Finally, we constructed and screened out many transgenic mutants of important functional genes, which were selected from the above analysis results, through genetic transformation system, and explored the function of high light inducible protein (NyHLIP) and Adenosine 5’-monophosphate deaminase (NyAMPD) in N. yezoensis based on the corresponding mutants. The main results were as follows:

(1) Morphological changes and conchospore release of free-living sporangial filaments: we found that, at high density (≥ 5000 fragments/mL), the free-living sporangial filaments always showed a “hollow morph”. At low density (1-1000 fragments/mL), most of sporangial filaments (> 70%) formed bipartite cells at 3-9 days, and the bipartite cells disappeared at 12-15 days without conchospore release. Serious intracellular degradation, which might through fusion of a double-membrane coated body with vacuole, was observed in hollow cells but disappeared in newly-formed bipartite cells and reappeared in their subsequent vegetative growth through transmission electron microscope. The results of morphological observation showed that the bipartite cells which disappeared without conchospore release would return to vegetative state in the form of elongation growth. The statistics of conchospore release rate and the changes of fresh weight showed that the factors promoting vegetative growth, such as high light (40-100 µmol photons m⁻² s⁻¹), ampicillin and streptomycin (comparing with kanamycin) and auxin (Indoleacetic acid, IAA), could significantly inhibit the conchospore release, while low concentration of anti-auxin (Triiodobenzoic acid, TIBA: 0.1-10 mg/L) promoted conchospore release to a certain extent. These results indicated that the conchospore release was mainly inhibited by two factors: one was the “hollow morph”, which might cause by intracellular degradation and vacuole expansion, inhibited bipartite cell formation and conchospore release under high culture density; the other factor was that under rapid growth conditions, such as high light, the bipartite cells returned to the vegetative growth stage and inhibited conchospore release.

(2) Molecular basis of free-living sporangial filaments development controlled by light intensities and culture densities: through comparing the transcriptome between high light with low density (simplified as “HL”) culture and low light with high density (simplified as “HD”) culture with low light with low density (simplified as “LD”) culture, we found that many differentially expressed genes (DEGs) related to carbon concentration, carbon fixation, photosynthesis, chlorophyll synthesis and nitrogen absorption were up-regulated in HL group, revealing the molecular basis of rapid vegetative growth of sporangial filaments under high light. In HD group, many DEGs related to plant salt and osmotic stress, and ion transport were up regulated comparing with low density group (LD and HL). The expression of brefeldin A (BFA) target gene—BFA inhibited guanine nucleotide exchange protein (BIG, py05721), which was a key gene in vacuole formation related membrane transport, was highest in HD group and lowest in HL group. These results indicating that the hollow morph of sporangial filaments under high density might be associated with vacuole formation, salt and osmotic stress, and ion transport. The results of BFA treatment further demonstrated the direct correlation between “hollow morph” and vacuole formation. In addition, we speculated that some of DEGs with the highest expression level in LD group among three groups might be related to conchospore release and formation, including DEGs associated with zinc finger proteins, ASPO1527, cell wall components, cell cycle and cytoskeleton.

(3) Comparative analysis of transcriptome of conchospore and archeospore: we picked out conchospores and archeospores (1-30 cells in each sample) and conducted RNA-seq using single cell sequencing technology ─ SMART (switch mechanism at the 5′ end of RNA templates) -seq2. Comparative analysis showed that light capture and carbon fixation associated DEGs were upregulated in archeospores, thus indicating that archeospores might be in a state of rapid vegetative growth. In conchospores, protein synthesis and degradation, especially molecular chaperone, associated DEGs were upregulated, indicating the complex life activities might be occurred in conchospores. There were 68 genes related to DNA replication and repair expressed in conchospores, showing that DNA replication activity might occur in conchospores. Moreover, we verified that one conchospores specifically expressed DEG (py04595: DNA helicase) was only expressed in diploid stages (conchocelis, sporangial filament) and three archeospores specifically expressed DEGs were only expressed in haploid stage (thalli), indicating conchospores were closer to diploid. These molecular level results indicated that conchospores might be the meiotic mother cells of N. yezoensis. In addition, we found the knotted-like homeobox gene (NyKNOX), which might relate to the transition of gametophyte from sporophyte, was expressed in sporophyte generation but not expressed in conchospore, archeospore and thalli, indicating the morphogenesis of gametophyte in N. yezoensis might require the inactivation of NyKNOX.

(4) Exploration of functional gene genetic transformation in N. yezoensis: we selected high light inducible protein (NyHLIP), adenylate deaminase (NyAMPD), ribose phosphate pyrophosphate kinase (NyPRPS) and chlorophyll synthase (NychlG), to construct transformation plasmids of overexpression-type and silence-type. Through gene gun method and hygromycin screening pressure, many positive mutants were successfully screened out. At DNA verification level, the total positive rate was about 49%. At RNA verification level, around 70% of the overexpression strains, the expression of target gene was significantly up-regulated comparing with wild strain (WT). The highest foldchange was about 40 folds, most were less than 10 folds. Around 37% of the overexpression strains occurred co-suppression phenomenon. The expression of target gene in one co-suppression strain was 100 folds lower than in WT, others were less than 10 folds lower than in WT. In 68% of RNAi mutants, the expression of target gene was significantly down regulated (less than 4 folds lower than in WT). These results indicated that the genetic transformation of functional genes, including gene silencing, overexpression and co-suppression, could be implemented in thallus of N. yezoensis through gene gun method.

(5) Functional exploration of NyHLIP: based on qRT-PCR and western blot, we found that, during low light recovery after 6 h of high light treatment, NyHLIP could exist for a long time both at RNA level (highly expressed for at least 12 h) and protein level (massively existed at least 24 h). Based on protein level verification of NyHLIP transgenic mutants, two strains (OE32: co-suppression type; Ri32: RNAi type) were selected out, in which the protein level of NyHLIP was obviously lower than that in WT strain after 6 h of high light treatment. Photosynthetic activity measurement after 6 h high light treatment showed that the maximum photochemical quantum yield (F_v/F_m), actual photochemical quantum yield (YII), electron transfer rate (ETR) of photosystem II, and the reduction rate of P700 (photosystem I reaction center) were obviously lower in OE32 and Ri32 strains than those in WT strain, but there was no obvious difference in nonphotochemical quenching (NPQ) between OE32/Ri32 strain and WT strain. These results indicated that the function of NyHLIP might involve in maintaining the basic activity and ETR in photosystem II, and maintaining the reduction activity in photosystem I under high light stress.

(6) Functional exploration of NyAMPD: adenosine 5’-monophosphate deaminase (AMPD) catalyzes the conversion of adenosine 5’-monophosphate (AMP) to fresh flavor—inosine 5’-mononucleotide (IMP). From NyAMPD transgenic strains, we selected overexpression strain (OE: AMD2-2), co-supression strain (Ci: AMD1-2) and silent strain (Ri: iAD2-5) to conduct phenotypic exploration. Growth experiment results showed that, comparing with WT strain, OE strain grew faster and Ri/Ci strains grew slower. Photosynthetic parameters measurement showed that there was no obvious difference between the three transgenic strains and WT strain. Measurement of adenine nucleotide related metabolites showed that in OE strain, IMP content was 2 folds higher, ADP and AMP contents was significantly lower (p < 0.05) than those in WT strain, showed a good overexpression effect in OE strain. However, in Ci and Ri strains, IMP content also higher, AMP and ADP content also lower than those in WT strain. Detection of gene expression of another AMPD (NyAMPD2) gene showed that, comparing with WT strain, NyAMPD2 was significantly (p < 0.05) up regulated in Ci and Ri strains, and was similar between OE strain and WT strain. We thought the down regulation of NyAMPD in transgenic strains (Ci and Ri strains) might promote the expression of NyAMPD2, hence, IMP, ADP and AMP content changes were similar between Ri/Ci strains and OE strain.

The above results analyzed the development regulation process and molecular basis of conchosprore releasing from free-living sporangial filaments, revealed the differences at gene expression level between conchospores and archeospores, constructed the functional gene genetic transformation system of gametophyte in N. yezoensis and verified the efficiency of transformation system. These results would be important in basic research and guiding the cultivation of N. yezoensis.