Evolutionary Conservation of Dazl Genomic Organization and its Continuous and Dynamic Distribution Throughout Germline Development in Gynogenetic Gibel Carp

To investigate germline development and germ cell specification, we identified a Dazl homolog (CagDazl) from gynogenetic gibel carp (Carassius auratus gibelio). Its cDNA sequence and BAC clone sequence analyses revealed the genomic organization conservation and conserved synteny of the Dazl family members and their neighborhood genes among vertebrates, especially in fish. Moreover, a polyclonal antibody specific to CagDazl was produced and used to examine its expression and distribution throughout germline development at protein level. Firstly, ovary-specific expression pattern of CagDazl was confirmed in adult tissues by RT-PCR and Western blot. In addition, in situ hybridization and immunofluorescence localization demonstrated its specific expression in germ cells, and both its transcript and protein were localized to germ plasm. Then, co-localization of CagDazl and mitochondrial cloud was found, confirming that CagDazl transcript and its protein are germ plasm component and move via METRO pathway during oogenesis. Furthermore, the CagDazl is abundant and continuous throughout germline development and germ cell specification including primordial germ cell (PGC) formation, oogonium differentiation, oocyte development, and embryogenesis, and the dynamic distribution occurs at different development stages. The data suggest that maternal CagDazl might play an important role in gibel carp PGC formation. Therefore, CagDazl is a useful and specific marker for tracing germ plasm and germ cell development in the gynogenetic gibel carp. In addition, in comparison with previous studies in sexual reproduction species, the continuous and dynamic distribution of CagDazl protein in the germ plasm throughout the life cycle seems to have significant implication in sex evolution of vertebrates. J. Exp. Zool. (Mol. Deu. Euol.) 312B:855-871, 2009. (C) 2009 Wiley-Liss, Inc.

Isolation of Y- and X-linked SCAR markers in yellow catfish and application in the production of all-male populations

P>Sex controls have been performed in some farmed fish species because of significant growth differences between females and males. In yellow catfish (Pelteobagrus fulvidraco), adult males are three times larger than female adults. In this study, six Y- and X-linked amplified fragment length polymorphism fragments were screened by sex-genotype pool bulked segregant analysis and individual screening. Interestingly, sequence analysis identified two pairs of allelic genes, Pf33 and Pf62. Furthermore, the cloned flanking sequences revealed several Y- and X-specific polymorphisms, and four Y-linked or X-linked sequence characterized amplified region (SCAR) primer pairs were designed and converted into Y- and X-linked SCAR markers. Consequently, these markers were successfully used to identify genetic sex and YY super-males, and applied to all-male population production. Thus, we developed a novel and simple technique to help commercial production of YY super-males and all-male populations in the yellow catfish.

Histone H2A Has a Novel Variant in Fish Oocytes

Histone variants and their modification have significant roles in many cellular processes. In this study, we identified and characterized the histone H2A variant h2af1o in fish and revealed its oocyte-specific expression pattern during oogenesis and embryogenesis. Moreover, posttranslational modification of H2af1o was observed that results from phosphorylation during oocyte maturation. To understand the binding dynamics of the novel core histone variant H2af1o in nucleosomes, we cloned ubiquitous gibel carp h2afx as a conventional histone control and investigated the dynamic exchange difference in chromatin by fluorescence recovery after photobleaching. H2af1o has significantly higher mobility in nucleosomes than ubiquitous H2afx. Compared with ubiquitous H2afx, H2af1o has a tightly binding C-terminal and a weakly binding N-terminal. These data indicate that fish oocytes have a novel H2A variant that destabilizes nucleosomes by protruding its N-terminal tail and stabilizes core particles by contracting its C-terminal tail. Our findings suggest that H2af1o may have intrinsic ability to modify chromatin properties during fish oogenesis, oocyte maturation, and early cleavage.

Expression pattern, cellular localization and promoter activity analysis of ovarian aromatase (Cyp19a1a) in protogynous hermaphrodite red-spotted grouper

Aromatase plays a key role in sex differentiation of gonads. In this study, we cloned the full-length cDNA of ovarian aromatase from protogynous hermaphrodite red-spotted grouper (Epinephelus akaara), and prepared the corresponding anti-EaCyp19a1a antiserum. Western blot and immunofluorescence studies revealed ovary-specific expression pattern of EaCyp19a1a in adults and its dynamic expression change during artificial sex reversal. EaCyp19a1a was expressed by follicular cells of follicular layer around oocytes because strong EaCyp19a1a immunofluorescence was observed in the cells of ovaries. During artificial sex reversal, EaCyp19a1a expression dropped significantly from female to male, and almost no any positive EaCyp19a1a signal was observed in testicular tissues. Then, we cloned and sequenced a total of 1967 bp T-flanking sequence of EaCyp19a1a promoter, and showed a number of potential binding sites for some transcriptional factors, such as SOX5, GATA gene family, CREB, AP1, FOXL1, C/EBP, ARE and SF-1. Moreover, we prepared a series of 5' deletion promoter constructs and performed in vitro luciferase assays of EaCyp19a1a promoter activities. The data indicated that the CREB regulation region from -1010 to -898 might be a major cis-acting element to EaCyp19a1a promoter, whereas the elements GATA and SOX5 in the region from -1216 to -1010 might be suppression elements. Significantly, we found a common conserved sequence region in the fish ovary-type aromatase promoters with identities from 93% to 34%. And, the motifs of TATA box, SF-1, SOX5, and CREB existed in the region and were conserved among the most of fish species. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Zebrafish eaf1 and eaf2/u19 Mediate Effective Convergence and Extension Movements through the Maintenance of wnt11 and wnt5 Expression

Studies have attributed several functions to the Eaf family, including tumor suppression and eye development. Given the potential association between cancer and development, we set forth to explore Eaf1 and Eaf2/U19 activity in vertebrate embryogenesis, using zebrafish. In situ hybridization revealed similar eaf1 and eaf2/u19 expression patterns. Morpholino-mediated knockdown of either eaf1 or eaf2/u19 expression produced similar morphological changes that could be reversed by ectopic expression of target or reciprocal-target mRNA. However, combination of Eaf1 and Eaf2/U19 (Eafs)-morpholinos increased the severity of defects, suggesting that Eaf1 and Eaf2/U19 only share some functional redundancy. The Eafs knockdown phenotype resembled that of embryos with defects in convergence and extension movements. Indeed, knockdown caused expression pattern changes for convergence and extension movement markers, whereas cell tracing experiments using kaeda mRNA showed a correlation between Eafs knockdown and cell migration defects. Cardiac and pancreatic differentiation markers revealed that Eafs knockdown also disrupted midline convergence of heart and pancreatic organ precursors. Noncanonical Wnt signaling plays a key role in both convergence and extension movements and midline convergence of organ precursors. We found that Eaf1 and Eaf2/U19 maintained expression levels of wnt11 and wnt5. Moreover, wnt11 or wnt5 mRNA partially rescued the convergence and extension movement defects occurring in eafs morphants. Wnt11 and Wnt5 converge on rhoA, so not surprisingly, rhoA mRNA more effectively rescued defects than either wnt11 or wnt5 mRNA alone. However, the ectopic expression of wnt11 and wnt5 did not affect eaf1 and eaf2/u19 expression. These data indicate that eaf1 and eaf2/u19 act upstream of noncanonical Wnt signaling to mediate convergence and extension movements.

Molecular characterization of Chinese sturgeon gonadotropins and cellular distribution in pituitaries of mature and immature individuals

Chinese sturgeon (Acipenser sinensis) is a rare and endangered species, and also an important resource for the sturgeon aquaculture industry. To understand molecular characterization of Chinese sturgeon gonadotropins (GTHs), we cloned the full-length cDNAs of gonadotropin subunits common alpha (GTH-alpha), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) from a pituitary cDNA library of mature female. Two subtypes of GTH-alpha were identified. The nucleotide sequences of A. sinensis common alpha I (AsGTH-alpha I), common alpha II (AsGTH-alpha II), FSH beta (AsFSH beta) and LH beta (AsLH beta) subunit cDNAs are 345, 363, 387 and 414 bp in length, and encode mature peptides of 115, 121, 129 and 138 aa, respectively. Then, three polyclonal antibodies were prepared from the in vitro expressed AsGTH-alpha I, AsFSH beta and AsLH beta mature proteins, respectively. Significant expression differences were revealed between immature and mature sturgeon pituitaries. Western blot detection and immunofluoresence localization revealed the existence of three-gonadotropin subunits (AsGTH-alpha, AsFSH beta and AsLH beta) in mature sturgeon pituitaries, but only AsFSH beta was detected in immature individual pituitaries during early stages in the sturgeon life, and obvious difference was observed between males and females. In males, AsFSH beta was expressed in 4-year-old individuals, whereas in females, AsFSH beta was just expressed in 5-year-old individuals. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Predominant expression and cellular distribution of fish Agr2 in renal collecting system

Anterior gradient 2 (Agr2) genes encode secretory proteins, and play significant roles in anterior-posterior patterning and tumor metastasis. Agr2 transcripts were shown to display quite diverse tissue distribution in different species, and little was known about the cellular localization of Agr2 proteins. In this study, we identified an Agr2 homologue from gibe[ carp (Carassius auratus gibelio), and revealed the expression patterns and cellular localization during embryogenesis and in adult tissues. The full-length cDNA of CagAgr2 is 803 nucleotides (nt) with an open reading frame of 510 nt encoding 169 amino acids. The Agr2 C-terminus matches to the class I PDZ-interacting motif, suggesting that it might be a PDZ-binding protein. During embryogenesis, CagAgr2 was found to be transcribed in the mucus-secreting hatching gland from tailbud stage and later in the pharynx region, swim bladder and pronephric duct as revealed by RT-PCR and whole mount in situ hybridization. In the adult fish, its transcription was predominantly confined to the kidney, and lower transcription levels were also found in the intestine, ovary and gills. To further localize the Agr2 protein, the anti-CagAgr2 polyclonal antibody was produced and used for immunofluorescence observation. In agreement with mRNA expression data, the Agr2 protein was localized in the pronephric duct of 3dph larvae. In adult fish, Agr2 protein expression is confined to the renal collecting system with asymmetric distribution along the apical-basolateral axis. The data provided suggestive evidence that fish Agr2 might be involved in differentiation and secretory functions of kidney epithelium. (C) 2009 Elsevier Inc. All rights reserved.

Apo-14 is required for digestive system organogenesis during fish embryogenesis and larval development

Apo-14 is a fish-specific apolipoprotein and its biological function remains unknown. In this study, CagApo-14 was cloned from gibel carp (Carassius auratus gibelio) and its expression pattern was investigated during embryogenesis and early larval development. The CagApo-14 transcript and its protein product were firstly localized in the yolk syncytial layer at a high level during embryogenesis, and then found to be restricted to the digestive system including liver and intestine in later embryos and early larvae. Immunofluorescence staining in larvae and adults indicated that CagApo-14 protein was predominantly synthesized in and excreted from sinusoidal endothelial cells of liver tissue. Morpholino knockdown of CagApo-14 resulted in severe disruption of digestive organs including liver, intestine, pancreas and swim bladder. Moreover, yolk lipid transportation and utilization were severely affected in the CagApo-14 morphants. Overall, this data indicates that CagApo-14 is required for digestive system organogenesis during fish embryogenesis and larval development.

Molecular cloning and expression characterization of ApoC-I in the orange-spotted grouper

Endogenous yolk nutrients are crucial for embryo and larval development in fish, but developmental behavior of the genes that control yolk utilization remains unknown. Apolipoproteins have been shown to play important roles in lipid transport and uptake through the circulation system. In this study, EcApoC-I, the first cloned ApoC-I in teleosts, has been screened from pituitary cDNA library of female orange-spotted grouper (Epinephelus coioides), and the deduced amino acid sequence shows 43.5% identity to one zebrafish (Danio rerio) hypothetical protein similar to ApoC-I, and 21.2%, 21.7%, 22.5%, 20%, and 22.5% identities to Apo C-I of human (Homo sapiens), house mouse (Mus musculus), common tree shrew (Tupaia glis), dog (Canis lupus familiaris) and hamadryas baboon (Papio hamadryas), respectively. Although the sequence identity is low, amphipathic alpha-helices with the potential to bind to lipid were predicted to exist in the EcApoC-I. RT-PCR analysis revealed that it was first transcribed in gastrula embryos and maintained a relatively stable expression level during the following embryogenesis. During embryonic and early larval development, a very high level of EcApoC-I expression was in the yolk syncytial layer, indicating that it plays a significant role in yolk degradation and transfers nutrition to the embryo and early larva. By the day 7 after hatching, EcApoC-I transcripts were observed in brain. In adult, EcApoC-I mRNA was detected abundantly in brain and gonad. In transitional gonads, the EcApoC-I expression is restricted to the germ cells. The data suggested that EcApoC-I might play an important role in brain and gonad morphogenesis and growth.

Quantum phase transitions in the S=1/2 distorted diamond chain

By means of the second derivative of the ground-state and first-excited energy, the quantum phase transitions (QPTs) for the distorted diamond chain (DDC) with ferromagnetic and antiferromagnetic frustrated interactions and the trimerized case are investigated, respectively. Our results show the plentiful quantum phases owing to the spin interaction competitions in the model. Meanwhile, by using the transfer-matrix renormalization-group technique, we study the two-site thermal entanglement of the DDC model in the thermodynamic limit for a further understanding of the QPTs.

黄土高原沟壑区不同年限苹果园土壤碳、氮、磷变化特征

管理措施是影响土壤质量演变的重要因素。分析和讨论了5、101、5年苹果园耕层(0—20 cm)和0—200 cm土壤有机碳、全氮、全磷、有效磷和硝态氮含量及其影响因素。结果表明,5年、10年和15年的塬面苹果园表层土壤有机碳依次为7.5、6.7和6.7 g/kg;全氮依次为0.940、.85和0.83 g/kg;但土壤全磷和速效磷含量随着种植年限而增加,与5年苹果园相比,塬面10年苹果园土壤全磷、速效磷含量分别提高了11%、60%,并且磷素的变异性随年限而增加。坡地10年、15年和20年苹果园土壤有机碳依次为6.36、.2和6.5 g/kg,全氮依次为0.76、0.76和0.81 g/kg;与10年苹果园相比,15年苹果园土壤全磷、速效磷含量分别提高了20%、28%。土壤剖面0—80 cm内不同土地利用方式土壤碳、氮、磷含量随土层加深而降低,80 cm以下不同利用条件苹果园土壤碳、磷含量差异不大,氮素含量在100 cm土层下随苹果园种植年限增加而增加。

聚3-羟基烷酸酯异相成核结晶的研究

本论文以具有巨大应用前景的PHBV作为研究对象，针对PHBV结晶速率低，易产生二次结晶，从而严重影响其加工性能和力学性能的稳定性等方面的缺点，根据PHBV的分子结构和脆性的机理分析，试图采用添加成核剂的方法，提高PHBV的结晶速率和结晶度，从而缩短PHBV的加工成型周期，控制其聚集态结构，提高其制品的稳定性，改善材料的物理力学性能。并在此基础上，探讨PHBV异相成核结晶的机理，加深成核剂对聚合物有效成核机理的认识，更好的理解聚合物结晶过程。1．添加成核剂的量达到0.5wt％时，对苯二甲酸（TPA）对PHBV起到了显著的结晶成核作用。结晶起始温度提高了约20℃，结晶速率达到最大值所对应的温度T_p提高了30℃，结晶烙增加了15J/g，结晶速率提高了4.4倍。这些数据表明，TPA是一种对PHBV极为有效的成核剂。2．加入成核剂TPA的PHBV表现出典型的双熔融行为，主要原因是TPA对PHBV的结晶成核作用和PHBV的熔融再结晶。低温侧的熔融峰对应着PHBV自熔体降温过程形成的结晶，高温侧的对应着PHBV升温过程中形成的结晶。3．TPA的成核作用大大的改变了PHBV的形态结构，使PHBV的球晶尺寸明显减小，球晶数量增大。4．TPA使PHBV晶体在（110）和（020）方向上微晶尺寸变大，晶区和非晶区的电子密度差增大。5．添加0.5wt％的TPA后，PHBV的断裂伸长率从4％提高到10％。6．TPA、IPA、淀粉和山梨醇对PHBV的结晶都具有很明显的成核作用，其原因可能是化学结构上都具有极性基团。7．红外光谱研究没有能够有效的给出PHBV与TPA是否存在特殊相互作用，从而导致TPA对PHBV的结晶成核作用的证据，但是，PHBV拨基伸缩振动谱带随温度的变化却给出了TPA对PHBv的结晶成核作用始于160℃高温。

松辽平原农田土壤线虫多样性研究

本研究立足于松辽平原农田生态系统，研究土壤线虫物种多样性，营养类群多样性，生活史多样性和功能多样性的分布规律，为进一步揭示线虫主要功能类群与土壤C、N等土壤环境因子之间的相互关系提供基础性数据。 在松辽平原沿不同纬度梯度，选择北至黑龙江海伦，南至辽宁大石桥共7个地点的玉米地，按照0–20 cm、20–40 cm、40–60 cm、60–80 cm和80–100 cm五个层次分层采集土壤样品。研究发现，土壤线虫多样性的纬度分布格局在0–40 cm土层表现得比较明显。在不同农田生态系统中，线虫属的丰富度在0–100 cm的土壤剖面内，随着土层深度的加深而降低；在0–40 cm土层，线虫属的丰富度随纬度梯度的降低而表现出增加的趋势，最高值（18属）出现在大石桥的0–20 cm土层，最低值出现在海伦采样点的20–40 cm土层，仅观察到10个线虫属。通过对松辽平原农田土壤线虫的生活史策略组成进行研究发现，在不同采样地点，不同生活史的土壤线虫表现出不同的分布特征：在0–20 cm土层，cp-2类群线虫的相对多度随纬度的降低表现出降低的趋势，而cp-3-5的线虫则表现出增加的趋势。在不同土层研究发现， 线虫的营养多样性指数没有表现出明显的纬度分布格局，而在0–20 cm土层，食细菌线虫和捕食/杂食线虫的相对多度随纬度的降低表现出降低的趋势，而植物寄生线虫则随纬度的降低而呈增加的趋势。土壤线虫区系分析结果表明，海伦样点土壤食物网受到的扰动较小，而处于结构化状态；而哈尔滨，公主岭和沈阳采样点的土壤食物网处于退化状态，受到外界环境扰动较大。通过对松辽平原农田土壤线虫群落与理化因子的典型对应分析，可以看出相关的土壤理化性质如阳离子交换量、有机碳、全氮和粘粒含量等能够在一定程度上解释松辽平原农田土壤线虫群落的分布特征。

长期施肥黑土微生物群落特征及其季节变化

研究长期施肥黑土微生物群落特征及其季节变化，可准确揭示施肥对黑土肥力质量的影响。本文以黑土肥料试验基地10个有机肥和无机肥处理0—20 cm表层土壤为研究对象，以不施肥和休闲处理为对照，分别经过8次季节取样，分析长期施肥与季节更替条件下黑土微生物群落特定磷脂脂肪酸（PLFA）含量、微生物量碳（氮）含量、r-k策略菌群数量、土壤酸（碱）磷酸酶活力以及16S rDNA的PCR-DGGE条带构成等微生物特征及其变化规律，探讨长期施肥与季节更替对黑土微生物群落的影响，明确限制各微生物群落生长的主要养分因子。 不同季节、多种施肥处理之间的土壤微生物指标多重比较表明，施肥与季节更替显著影响黑土微生物各菌群生长与活力，且施肥×季节更替交互作用显著。 与无机肥处理相比，有机肥施用能显著提高黑土各菌群特征PLFA与微生物量碳（氮）含量、r-k策略细菌或真菌数量以及磷酸酶活力；在一定时期、一定程度上改变土壤细菌PCR-DGGE条带构型，提高细菌群落多样性。不同有机肥-化肥配施处理之间各微生物学指标比较发现，有机肥+磷肥（MP）处理中的可培养细菌数量、细菌与放线菌PLFA含量、微生物量碳含量以及碱性磷酸酶活力普遍较低；高量有机肥（M2CK）处理中的各菌群特征性PLFA、微生物量碳（氮）含量以及酸性磷酸酶活力等则有一定提高。单施化肥各处理，尤其是氮磷钾（NPK）处理，在一定程度上抑制了黑土各菌群生长与活力。各处理PLFA因子分析表明，施用有机肥对黑土放线菌、G+与G-细菌等菌群影响较大。另外，休闲处理真菌特征PLFA含量、r-策略真菌数量、碱性磷酸酶活力及微生物量碳（氮）含量等均处较高水平。 季节更替对微生物群落各指标影响亦达显著水平，且各指标季节变化趋势存在一定差异。黑土各菌群特征性PLFA含量、微生物量氮、G+/G-比值及r-策略细菌等指标，均自春至夏有显著升高趋势；而磷酸酶活力、真菌/细菌比值及k-策略细菌等指标则自春至夏有明显的下降趋势。各处理PLFA因子分析表明，夏秋季节对土壤单烯不饱和脂肪酸影响较大。 多元线性回归分析表明，碱解氮、有机碳、有效磷等养分几乎是影响所有已测微生物学指标的最关键养分因子，较次要的微生物生长限制因子是速效钾与pH值。

远紫外辐射对紫杉幼苗针叶膜脂过氧化及内源保护系统的影响

为了探讨远紫外辐射对植物的损伤机理和紫杉的濒杉机制，本文就远紫外辐射对紫杉幼苗针叶膜脂过氧化及内源保护物质的影响进行了模拟研究，初步得到如下结果：UV-C 辐射紫杉针叶离体叶绿体可使膜脂过氧化产物丙二醛（MDA）含量增加，类胡萝卜素含量和光系统II（PS II）电子传递少性下降。UV-BC 辐射紫杉幼苗针叶可使叶绿体超氧离子自由基（O~-_2)，单线态氧（~O_2)，针叶有机自由基产额和H_2O_2含量有不同程度增加。针叶MDA，组织自动氧化速率及质膜相对透性也随辐射时间进程面增加。UV-BC处理初期超氧化物歧化酶（SOD），过氧化氢酶（CAT）活性及谷胱甘肽（GSH）含量均被诱导增高，21d后SOD活性开始下降，而GSH 含量和CAT活性始终高于对照。维生素C（ASA），类胡萝卜素（Car)，叶绿素（chl），PS II 电子传递活性在处理期间始终呈下降趋势，其中ASA下降最明显。可溶性蛋白21d前变化不大，之后开始下降。外源活性氧清除剂苯甲酸钠和抗坏血酸对针叶膜脂过氧化有抑制作用；甲基紫精和DDC 对针叶膜脂过氧化有促进效果。根据上述结果推测，紫杉的UV-BC伤害可能是由于活性氧产生过剩和清除系统水平下降，而引起的膜脂过氧化损伤。