条斑紫菜抗高温和快速生长细胞株系HB的建立及栽培

本文主要应用单细胞培养技术培育紫菜新品系,以建立紫菜细胞工程快速育苗系统。通过酶解技术分离出紫菜叶状体细胞,并进行多克隆,从中获得4个细胞纯系植株(HA,HB,HC,HD)。对该4个细胞株系进行了纯系培养,并对其细胞苗和丝状体的生长速度和抗高温(19℃,21℃,23℃,25℃)性进行了测定。结果显示,HB株系具有较高的抗高温性和最快生长速度。在1998年到2000年间,在江苏省启东县海丰海区对HB株系进行了海上栽培实验,结果显示,实验组的产量高于当地对照组产量,证明了条斑紫菜HB株系不仅具有较高的抗高温性,而且也有较快生长速度。本实验说明应用细胞工程进行条斑紫菜育种是一条很好的快速育种途径。     

条斑紫菜粗多糖对淋巴细胞和支持细胞的增殖作用

目的:探讨条斑紫菜粗多糖的一些生物学功能,包括促进淋巴细胞增殖以及促进支持细胞生长的功能。方法:将原代培养的大鼠和小鼠脾淋巴细胞以及大鼠睾丸支持细胞分别接种于96孔板中,将不同浓度的条斑紫菜粗多糖(0.05、0.5、5、10mg/mL)加入培养,每种浓度设6个复孔,并设对照组孔。采用四氮甲唑蓝(MTT)法检测细胞的增殖率。结果:条斑紫菜粗多糖能够显著地促进大鼠、小鼠脾淋巴细胞和大鼠睾丸支持细胞的增殖,增殖率随多糖浓度的提高而升高;当多糖浓度为10mg/mL时,增殖率分别为137.3%、149%和454.5%。结论:条斑紫菜粗多糖具有提高免疫力及促进生殖功能的作用,其对睾丸支持细胞的增殖作用目前还未见报道。     

环境因子及藻体密度对条斑紫菜生长与氮磷去除效率的影响

以条斑紫菜为材料,系统探究实验室条件下光照强度、温度、光周期、盐度和干出等生态因子和藻体密度对条斑紫菜生长和氮磷去除的影响,为今后条斑紫菜大规模栽培的生态修复潜力研究及海洋富营养化治理奠定理论基础。实验室条件下不同藻体密度、光照强度、光周期、温度、盐度和干出时间对条斑紫菜生长及氮磷去除效率的影响。结果表明:条斑紫菜在藻体密度0.1 g/L、光照强度为120μmol m-2s-1时特定生长率最大,为38.1%。随着藻体密度增加,光照强度减小,其特定生长率逐渐减小,当藻体密度为1.6 g/L、光照强度为30μmol m-2s-1时,特定生长率最小为12.6%;藻体密度0.1—0.8 g/L时随着藻体密度的增加条斑紫菜对NO-3-N和PO3-4-P的去除效率显著增加,藻体密度为0.8—1.6 g/L时藻体对NO-3-N和PO3-4-P去除效率差异不大,其中藻体密度1.6 g/L、光照强度90μmol m-2s-1时条斑紫菜对NO-3-N和PO3-4-P去除效率均达到93%以上。为进一步探究其他环境因子的影响,因此在温度和光周期实验中选择氮磷去除率适中的藻体密度0.4 g/L和最适光照强度90μmol m-2s-1。温度为15℃、光周期为16L∶8D条件下条斑紫菜特定生长率最大,为36.9%,且氮和磷去除效率也最大,分别为91.9%和81.6%。条斑紫菜生长适宜盐度为10—35,最佳盐度为25,干出时间越长生长越慢,氮磷去效率也越低,且有较大交互作用,但干出后的紫菜光合作用更强。以上研究将为应用大型海藻条斑紫菜大规模栽培进行生态修复奠定基础。     

奶牛乳腺上皮细胞系的建立及高温对细胞超微结构的影响

应用差酶消化法和反复贴壁法在体外建立奶牛乳腺上皮细胞培养方法,以细胞流式术、免疫组化、免疫印迹、超微结构观察等方法对乳腺上皮细胞特性进行检测,并研究高温热刺激对乳腺细胞超微结构的影响。实验结果表明,运用本方法建立的奶牛乳腺细胞系上皮特性及遗传特征完备;41℃ 1h 的高温热刺激可使乳腺细胞染色质浓缩、线粒体肿胀、空泡化,形成凋亡小体,说明高温可以诱发乳腺细胞凋亡。     

奶牛乳腺上皮细胞系的建立及高温对细胞超微结构的影响

应用差酶消化法和反复贴壁法在体外建立奶牛乳腺上皮细胞培养方法,以细胞流式术、免疫组化、免疫印迹、超微结构观察等方法对乳腺上皮细胞特性进行检测,并研究高温热刺激对乳腺细胞超微结构的影响。实验结果表明,运用本方法建立的奶牛乳腺细胞系上皮特性及遗传特征完备;41℃1h的高温热刺激可使乳腺细胞染色质浓缩、线粒体肿胀、空泡化,形成凋亡小体,说明高温可以诱发乳腺细胞凋亡。     

条斑紫菜藻红、藻蓝蛋白逐级放大的纯化工艺

采用“破碎-盐析-层析”的方法纯化条斑紫菜藻胆蛋白,并在提取规模上逐步放大。首先在综合比较凝胶层析去盐效率后,从Sephadex G-25、G-100、S-300和CL-6B中选择G-25作为实验流程中的去盐填料,其次将提取流程的初试原料条斑紫菜量逐步放大,选取了1g、20g和400g三个量,结果表明随着初试紫菜量逐步放大,最终所得藻胆蛋白中吸收光谱纯度>3.2的蛋白产率依次提高,其中400g冻干紫菜的藻红蛋白产率为0.323％,藻蓝蛋白产率为0.148％。由此认为该实验工艺流程具有规模放大的潜力,这为高纯度藻胆蛋白的规模生产提供了一条可行的方案。     

条斑紫菜R-藻红蛋白提纯工艺研究

对条斑紫菜叶状体R-藻红蛋白提纯方法进行了研究和分析。首先分别采用冻融法、化学试剂法、溶胀法等单一及组合细胞破碎方法对条斑紫菜细胞进行破碎,结果表明溶胀 组织捣碎法效果最佳。其次用25%～60%饱和度范围内的硫酸铵沉淀法粗提藻红蛋白,发现25%～45%硫酸铵分步沉淀法效果最好,再经结晶法盐析纯度(D565nm/D280nm)达到2.088。盐析液用SephadexG-25层析柱脱盐,再经羟基磷灰石(HA)柱层析,纯度可达到4.98。R-藻红蛋白的最大吸收峰在565nm,其室温荧光发射峰为578nm。SDS-PAGE结果显示,R-藻红蛋白可分为α、β两个亚基,Mr分别为17.0×103和19.0×103。     

Effects of cell wall synthesis on cell polarity in the red alga Porphyra yezoensis

Polarity is a fundamental cell property essential for differentiation, proliferation and morphogenesis in unicellular and multicellular organisms. We have recently demonstrated that phosphatidylinositol 3-kinase (PI3K) activity is required for the establishment of anterior-posterior axis, leading to asymmetrical localization of F-actin in migrating monospores of the red alga Porphyra yezoensis. We also showed that the formation of the apical-basal axis via adhesion of monospores to the substratum after the cessation of migration requires newly synthesized proteins and does not depend on PI3K activity. However, little is known about the mechanism and regulation of axis conversion during development of monospores. In this addendum, we report our investigation as to the role of the cell wall in axis conversion. Our results indicate that inhibition of cell wall synthesis prevented the development of germlings. Also, defects in the cell wall disrupted the asymmetrical distribution of F-actin and inhibited the adhesion to the substratum that is required for establishment of apical-basal axis. Hence, we conclude that the cell wall is critical for the maintenance of cell polarity in migrating cells, which is indirectly involved in axis conversion via enabling monospores to adhere to the substratum.Key words: BFA, cell polarity, cell wall, F-actin, monospores, PI3K, Porphyra yezoensisThe initial establishment of cell polarity, which is exhibited in asymmetrical cell division and directional migration, depends on asymmetrical cues that lead to reorganization of the cytoskeleton and polarized distribution of cortical proteins and membrane lipids.^1–3 For directional migration of Dictyostelium cells and leukocytes, cells in the axialized form can rapidly change their body shape along with the formation of cell polarity in response to external impulses such as cAMP and cytokines, enabling them to migrate toward the external impulse with driving and contractile forces provided by asymmetrically distributed cytoskeletal elements.^4,5 Evidence is growing that in both asymmetrical cell division and migration, intracellular compartmentalization of phosphatidylinositol 3-kinase (PI3K) and phosphatidylinositol polyphosphate phosphatases is responsible for the asymmetrical and reciprocal distributions of PI(3,4,5)P₃ and PI(4,5)P₂ on plasma membranes. This helps cells to define their polarity by organizing polarized localization of F-actin and myosin.^6–8We used the monospores of the red alga Porphyra yezoensis to elucidate the molecular mechanisms involved in the establishment of cell polarity in plants. Migration and asymmetrical cell division are both observed during the early development of monospores released from monosporangia produced at the marginal region of the thallus.^9,10 Thus, monospores are thought to be unique and useful materials for investigating polarity determination in plant cells. In the early development of monospores, there are two different cellular axes: the anterior-posterior axis during migration and the apical-basal axis in asymmetrical cell division and upward growth of a thallus. The use of {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, a PI3K inhibitor, prevented the migration of monospores because the anterior-posterior axis cannot be established. This is evidence that the PI3K activity is essential for the establishment of cell polarity and asymmetric distribution of F-actin for migration of monospores.¹⁰ Thus, the formation of the former axis requires PI3K activity and asymmetrical distribution of F-actin.¹⁰ These results are similar to those observed in Dictyostelium cells and leukocytes, suggesting that the role of PI3K-dependent F-actin asymmetry in the establishment of cell polarity might be evolutionarily conserved in migrating eukaryotic cells. However, it is still unclear whether PI(3,4,5)P₃ corresponds to D3-phosphorylated phosphatidylinositol in P. yezoensis, since this phosphoinositide has not yet been detected in any plant cell.^11,12In addition to D3-phosphorylated phosphatidylinositols, it is well known that the cell wall plays an essential role in the establishment of cell polarity, a phenomenon documented in the brown algae Fucus.^13–15 It has been demonstrated that the cell wall is required for the fixation, but not the formation, of the apical-basal axis.^13,14 In this case, polarized secretion via Golgi apparatus is needed for synthesis of the cell wall.¹⁵ In fact, we observed that monospores whose migration was inhibited by treatment with PI3K and cytoskeleton inhibitors have no cell wall,¹⁰ suggesting the importance of the cell wall in formation and/or maintenance of the cell axis in P. yezoensis.To confirm this possibility, we used Brefeldin A (BFA), a specific inhibitor of polysaccharide biosynthesis required for cell wall formation via Golgi-derived vesicle trafficking. As shown in Figure 1A, part a, the cell wall was synthesized during migration of monospores. However, when freshly released monospores were treated with BFA for 3 h, there was no cell wall synthesis in monospores with a rounded shape or in migrating monospores with a tapered shape (Fig. 1A, part b). This evidence led us to conclude that Golgi-derived vesicle trafficking is responsible for cell wall formation in these monospores. These results also indicated that the anterior-posterior axis during migration can be established without cell wall synthesis. Indeed, F-actin accumulated at the leading edge in the migrating monospores in the presence of BFA (Fig. 1A, part d).Open in a separate windowFigure 1Effect of BFA on the cell wall synthesis, F-actin localization and development of monospores. (A) Freshly released monospores were treated with (parts b–d) or without (part a) BFA (MP Biomedicals) at 20 µM for 3 h incubation. The cell wall (parts a and b) and F-actin (parts c and d) were stained with 0.01% Fluorescent Brightener 28 (Sigma) and 5 U· mL⁻¹ Alex Flour 488 phalloidin (Molecular probe), respectively. Migrating monospores are indicated by arrowheads. (Part c) Cell with a round shape, (Part d) cell with a tapered shape during migration. Upper and lower photographs in each panel show bright field and fluorescent images, respectively. Direction of migrating monospores is indicated by an arrow. Scale bars: (Parts a and b) 10 µm; (Parts c and d) 5 µm. (B) Freshly released monospores were treated with (parts b–d) or without (part a) BFA at 20 µM for 24 h incubation. The cell wall (parts a and b) and F-actin (parts c and d) were stained with 0.01% Fluorescent Brightener 28 and 5 U· mL⁻¹ Alex Flour 488 phalloidin, respectively. Migrating monospores are indicated by arrowheads. (Part c) Cell with a round shape, (Part d) cell with a tapered shape during migration. Upper and lower photographs in each panel show bright field and fluorescent images, respectively. Direction of migrating monospores is indicated by an arrow. Scale bars: (Parts a and b) 10 µm; (Parts c and d) 5 µm. (C) Dose-dependent effect of BFA on early development of monospores. Freshly released monospores were treated with an increasing concentration (2–20 µM) of BFA for 24 h, and the number of germlings was counted. Data are presented as mean ± SD (n = 3).Next, we analyzed the relationship between cell wall synthesis and development of germlings to confirm the functional significance of cell wall synthesis in the establishment of apical-basal axis. In the control medium, the cell wall was observed in 2-celled germlings 24 h after monospores release (Fig. 1B, part a), while the BFA-treated monospores still retained the migrating form in which the cell wall was not synthesized (Fig. 1B, part b) and the adhesion of monospores to the substratum and development of germlings was prevented (Fig. 1B, parts b–d). Moreover, the rate of the development of germlings from monospores decreased in a dose-dependent manner after 24 h incubation with BFA (Fig. 1C). Notably, it is significant that the polarized localization of F-actin in migrating cells was destroyed during BFA treatment (Fig. 1B, part d), suggesting the involvement of the cell wall in the maintenance of the asymmetrical distribution of F-actin in migrating monospores. Thus, the cell wall is indispensable for maintenance of anterior-posterior axis in migrating cells. Moreover, since adhering is trigger of the formation of the apical-basal axis, synthesis of cell wall could enable cells to develop further into germlings. We therefore concluded that cell wall plays a role indirectly in axis conversion during the development of monospores. Future work should be focused on the nature of the cell wall factors involved in the maintenance of cell axis and the adhesion to the substratum and how the function and expression of these factors are regulated.In summary, the establishment and maintenance of cell polarity during development of monospores is under complex regulation. Dissecting the molecular mechanisms of this regulatory system could help in further understanding the interrelation among PI3K signaling, the actin-based system and cell wall formation, which can provide new insight into the machinery regulating the establishment and maintenance of cell polarity in plants.     

Protoplast fusion between Monostroma nitidum and Porphyra yezoensis and subsequent growth of hybrid plants

Protoplasts from Monostroma nitidum (Chlorophyta) and Porphyra yezoensis (Rhodophyta) were fused utilizing the PEG method. The heterofusants were easily identified by the color of the hybrid cells. The frequency of fusion between the different protoplasts was 1.4%. The heterofusants were transferred into ES medium and cultured. The survival rate of heterofusants after 30 days cultivation was 2–7%. Two plants exhibited hybridizable DNA fragments as compared to parental DNA fragments. Additionally, these two plus a third hybrid plant had characteristic fatty acid components of both the Chlorophyta and Rhodophyta. The results indicate that genetic recombination is possible between these two genera. The field for breeding by protoplasts fusion should be extended across the divisions. This revised version was published online in June 2006 with corrections to the Cover Date.     

Cloning and characterization of the HLIP gene encoding high light-inducible protein from Porphyra yezoensis

High light-inducible proteins are critical for photosynthetic organisms when responding to high light stress. We cloned and characterized the HLIP gene from Porphyra yezoensis, which was designated as PyHLIP. Sequence analysis revealed that the open reading frame of PyHLIP was 387?bp in length that encoded a 128 amino acid-polypeptide containing a plastid transit peptide and an obvious membrane-spanning α-helix region. No intron existed in the genomic DNA of PyHLIP. The putative promoter of PyHLIP was TATA-less, and the initiator element (Inr) existed upstream of ATG. Analysis of genomic organization indicated there were two copies of PyHLIP gene in the P. yezoensis nuclear genome. The expression of PyHLIP can be induced by various stress conditions and is especially sensitive to high light.     

Red rot resistance in interspecific protoplast fusion product progeny of Porphyra yezoensis and P. tenuipedalis (Bangiales, Rhodophyta)

Nine primary regenerants were recovered by interspecific protoplast fusion of Porphyra yezoensis Ueda T‐14 (Py) (cultivated Porphyra) and Porphyra tenuipedalis Miura (Pt). This combination is difficult to achieve with conventional sexual hybridization, yet is important in that non‐cultivated P. tenuipedalis is partially resistant (PR) to red rot disease, caused by the microbial pathogen, Pythium porphyrae Takahashi et Sasaki. Out of the nine primary regenerants, two strains (Py‐Pt‐4 and Py‐Pt‐7) were like the parent, P. tenuipedalis, while the rest were like the other cultivated parent P. yezoensis T‐14 in their life cycle. Red rot resistance was assessed in parents and interspecific fusion product progeny (FPP) by exposing the foliose thalli to equivalent infection and measuring two parameters of the host‐pathogen interactions: supported fungal biomass and amount of disease produced. Intermediate resistance between P. yezoensis T‐14 (1.00) and P. tenuipedalis (0.13) was observed in two of the Py‐type FPP, Py‐Pt‐2F₂ (0.25) and Py‐Pt‐5F₂ (0.23). Stable inheritance of resistance was observed through two subsequent generations. The morphologic and reproductive characteristics of the regenerated foliose thalli, and nature of host‐pathogen interactions were used to further verify the hybrid origin of the FPP. Host‐pathogen interactions were followed using epi‐fluorescence and scanning electron microscopy (SEM). The zoospores encysted at higher rates on the susceptible cultivated parent (P. yezoensis T‐14) germinated immediately and the short germ tubes formed appres‐soria and penetrated the algal cells near the site of encystment. While on the PR parental (P. tenuipedalis) and partially resistant FPP (PRFPP) progeny (Py‐Pt‐2F₂ and Py‐Pt‐5F₂) the low rate of zoospore encystment was followed by cyst germination, but only a few of the germ tubes formed appressoria and penetrated the thallus surface. Long germ tubes (with no appressoria) were seen growing on the thallus surface without host penetration. The minimal rate of encystment concomitant with low rate of appressorium formation on the PR parent and PRFPP was observed as the major factor responsible for the partial resistance in these thalli.     

Reporter gene introduction and transient expression in protoplasts of Porphyra yezoensis

The transient expression of foreign genes in the protoplasts of Porphyrayezoensis was examined using three recombinant vectors, pYez-Rub-GUS, pYez-Rub-GFP and pYez-Rub-LUC, which were constructed with the promoter sequence of the ribulose-bisphosphate-carboxylase / oxygenase (Rubisco) gene as a promoter and the bacterial β-glucuronidase (GUS), mutant of green fluorescent protein (S65T-GFP) and firefly luciferase (LUC) genes, respectively, as reporter genes. When the pYez-Rub-GUS was introduced into protoplasts by electroporation, cells stained dark blue by indigotin were observed after the histochemical GUS assay. GUS activity was also detected by quantitative enzyme assays with a chemiluminescent substrate. When the pYez-Rub-GFP was electroporated into protoplasts, the expression of GFP could be detected in vivo observations with fluorescence microscopy. However, the rates of gene expression cells to the total number of cells were different between the GUS and GFP genes. LUC activity was also detected by assay with a chemiluminescent substrate after the introduction of pYez-Rub-LUC into protoplasts, although the activity levels were considerably lower. Relatively high expression rates of introduced GUS genes were observed 3 to 5 days after electroporation. These results show that the promoter sequence of the chloroplast Rubisco gene functions as a promoter of foreign gene expression and that transient expression occurred in protoplasts of P. yezoensis after the introduction of foreign genes. This revised version was published online in August 2006 with corrections to the Cover Date.     

Establishment of growth factor-dependent MOPC 104E cell line in vitro

The MOPC 104E cell line has been adapted to grow in vitro using a combination of feeder layer and growth factor(s). The growth of this myeloma cell line is dependent on the presence of growth factor(s). Growth-promoting activity generated from T-cell-mitogen-stimulated, Corynebacterium parvum-stimulated spleen cell culture supernatant, and peritoneal adherent cell culture supernatants gives dose-dependent proliferation. Generation of growth factors in the serum-free bovine serum albumin-substituted media and a rapid assay system based on [3H]thymidine uptake for the quantitation of growth promoting activity are described.