花生种间杂种胚胎发育及内源激素变化

以花生栽培品种豫花15为母本分别与野生种A. correntina和A. macedoi杂交,以栽培品种间杂交组合豫花15×白沙1016为对照,采用石蜡切片技术观察花生杂种胚胎发育进程,酶联免疫吸附分析法(ELISA)测定花生杂种胚胎内源激素含量, 分析胚胎发育进程与胚胎内源激素含量的关系。结果显示,3个杂交组合的下针率无明显差异,表明栽培种与2个野生种杂交不存在受精前障碍;与对照相比,豫花15×A. correntina表现杂交可亲和,其胚胎发育中激素含量的变化与对照组合基本一致;而豫花15×A. macedoi杂交授粉后12 d内胚胎发育基本正常,17~27 d发育缓慢,之后珠被迅速向内增生抑制了胚的进一步发育并最终败育,在同一时期的杂种胚胎中,3-吲哚乙酸(IAA)、赤霉素(GA₃)和玉米素核苷+二氢玉米素核苷(ZR+DHZR)等促进生长的激素维持在较低的水平,而脱落酸(ABA)含量以及脱落酸与某一促进生长激素或3类促进生长激素之和的比值保持了较高的水平,推测可能是激素代谢的失衡导致种间杂种胚胎的败育。用IAA、激动素(KT)单独或混合处理不亲和组合豫花15×A. macedoi杂交授粉后的果针基部表明,外施促进生长的激素能够明显增加胚珠的长度,一定程度上验证了上述推测。     

改良大豆胚尖再生体系的研究

为了研究利用Thidiazuron(TDZ)建立大豆胚尖高效再生体系的可行性。跃进10号的成熟种子浸泡16 h后,收集胚尖转入添加了不同浓度的TDZ芽诱导培养基中,4周后计算出芽率,并对拔高培养基和生根培养基进行优化。TDZ浓度为0.08 mg/L时,出芽率最高,达92.6%,平均每个外植体产生的丛生芽的数目也最多。适宜大豆胚尖的芽诱导分化培养基为MS+0.08 mg/L TDZ,拔高培养基为MS+2.0 mg/L KT+0.2 mg/L NNN,生根培养基为1/2MS+1.0 mg/L IBA。利用TDZ建立大豆胚尖高效再生体系是可行的,提高了丛生芽的分化率,为快繁和提高遗传转化效率奠定了坚实的基础。     

Construction of mouse ESC line stably expressing Pdx1 by Tet-On system

AIM To construct the mouse embryonic stem cell （ESC） line with stable pancreatic and duodenal homeobox 1 （Pdx1） expression by Tet-On system, which may lay a foundation for further research on the differentiation of Pdx1⁺ definitive endoderm cells into pancreatic cells. METHODS The Pdx1-overexpressing lentiviral vector with green fluorescent protein marker and puromycin resistance was constructed by Tet-On system and was used to infect the mouse ESC. The cells were divided into 3 groups： blank control group （ESC group）, empty lentivirus control group （PDX1^- ESC group） and Pdx1 lentivirus transfection group （PDX1⁺ ESC group）. Flow cytometry was used to detect the transfected cells after screening by doxycycline （DOX）. The function of Tet-On system and the expression of Pdx1 gene were detected. The transfected cells in PDX1^- ESC group and PDX1⁺ ESC group were sorted by flow cytometry, and constructed ESC line with stable expression of Pdx1 and negative control ESC line were verified. RESULTS （1） The positive rates of transfected cells in PDX1^- ESC group and PDX1⁺ ESC group were 90.72% and 94.01% after screening by DOX, respectively. The positive rates of transfected cells in PDX1^- ESC group and PDX1⁺ ESC group was 97.84% and 98.13% after sorting by flow cytometry, respectively. （2） With DOX, green fluorescence was observed in PDX1^- ESC group and PDX1⁺ ESC group. The mRNA and protein expression of Pdx1 was significantly increased in PDX1⁺ ESC group （P<0.05）. Without DOX, no green fluorescence was observed in the cells of the 3 groups, and no significant difference in the mRNA and protein expression of Pdx1 was observed （P>0.05）. （3） After 3 months of cryopreservation, the cell lines still survived in resuscitation culture and were regulated by DOX. CONCLUSION Using Tet-On system, the mouse ESC line with inducible Pdx1 expression were successfully established and could be used as an effective cell model to research the differentiation of Pdx1⁺ definitive endoderm cells into pancreatic cells.     

Na?ve-like conversion enhances the difference in innate in vitro differentiation capacity between rabbit ES cells and iPS cells

Quality evaluation of pluripotent stem cells using appropriate animal models needs to be improved for human regenerative medicine. Previously, we demonstrated that although the in vitro neural differentiating capacity of rabbit induced pluripotent stem cells (iPSCs) can be mitigated by improving their baseline level of pluripotency, i.e., by converting them into the so-called “naïve-like” state, the effect after such conversion of rabbit embryonic stem cells (ESCs) remains to be elucidated. Here we found that naïve-like conversion enhanced the differences in innate in vitro differentiation capacity between ESCs and iPSCs. Naïve-like rabbit ESCs exhibited several features indicating pluripotency, including the capacity for teratoma formation. They differentiated into mature oligodendrocytes much more effectively (3.3–7.2 times) than naïve-like iPSCs. This suggests an inherent variation in differentiation potential in vitro among PSC lines. When naïve-like ESCs were injected into preimplantation rabbit embryos, although they contributed efficiently to forming the inner cell mass of blastocysts, no chimeric pups were obtained. Thus, in vitro neural differentiation following naïve-like conversion is a promising option for determining the quality of PSCs without the need to demonstrate chimeric contribution. These results provide an opportunity to evaluate which pluripotent stem cells or treatments are best suited for therapeutic use.     

Status of Biological Studies and Aquaculture Development of the Mud Crab, <Emphasis Type="Italic">Scylla serrata</Emphasis>, in China: An Experimental Ecological Studies

This is an initial paper in a series of overviews of biological research and aquaculture development of the mud crab, Scylla serrata, in China. Results of experimental ecological studies on mud crabs are reported here. As a result of these experimental studies, results that are important for mud crab culture were also discovered and these include, methods to condition and manage broodstock, determination of ecological conditions that are suitable for embryonic development, and the influence of temperature, salinity, diet and larval density on development and survival of larvae. Results of this work will be useful in establishing a good method for artificial mass culture of larvae.     

皖南烟区可持续发展研究

结合皖南烟区烟叶生产的实际情况,从多个方面分析了烟区可持续发展过程中存在的制约因素,并从烟叶生产人力资源、生产种植规模化、科技支撑和服务、烟叶标准化生产管理和烟叶生产保障体系等几个方面,提出了皖南烟区生产可持续发展的若干对策及建议,为皖南烟区实现可持续发展提供参考。     

家蚕胚胎伴性温敏性的遗传研究

试验用新九与伴1等4个温敏性的品种杂交,将其F1代蚕种用高温干燥条件催青,结果雄蚕正常孵化,雌蚕几乎不孵化,表明伴1等品种具有控制催青温敏性表达的基因存在。用华1与伴1组配的F1、F2及F1与亲本回交的14个组合作遗传分析,认为催青敏感性状由位于Z性染色体上的一个主基因控制,呈隐性的伴性遗传。     

猪胚胎干细胞的分离克隆

本文从分化抑制物、培养基的选择内细胞团的分离,胚胎干细胞的分离及鉴定方法等方面系统地综述了猪胚胎干细胞分离克隆的研究进展;并对其未来的应用前景进行展望。     

妊娠免疫维持机制及其失调与胚胎死亡的关系

本文讨论了妊娠免疫维持机制及其失调与胚胎死亡的关系,并指出了免疫失调引起的早期胚胎死亡的预防措施。     

Research on Isolation and Clone of Embryonic Stem Cell-Like in Bovine

Bovine embryonic stem cell would be invaluable for researching the aspect of animal cloning, production transgenic animal and discussion of gene function in vitro. With the object of establishing an effective culture system for isolation and clone of bovine pluripotent stem cell, we cultured bovine embryos and mouse embryos including morula blastula and hatached blastula and obtained animal ICM on Primary marine embryonic fibroblast (Primary murine embryonic fibroblast, PMEF) feeder layer with tissue medium(DMEM supplemented with 15ml/100ml NBS ,0.1μmol/L Na2SeO3, 0. 1mmol/L β-mercaptoethanol, 1 000ng/ml LIF,10 ng/ml IGF, 1mmol/L necessary amino acid and 1mmol/L L-glutamine), then, we obtained mouse ICM and bovine ICM. Moreover, we isolated and cloned the 6 passage bovine ES like cells(12 cell lines) and 9 passage marine ES like cells (52 cell lines) deriving from bovine ICM and murine ICM respectively on the feeder layer of PMEF by disaggregating ICM and ES cell clones of bovine and murine into smaller clumps through digesting with 0. 125g/100ml trypsin and 0.02g/100ml EDTA and scattering with a glass needle. The pluripotency of both murine and bovine ES like cells was identified with morphological character, histochemistry identification, karyotype analysis and differentiation of ES cells in vitro or in vivo. This result showed that bovine embryonic stem cell and murine embryonic stem cell had developmental pluripotency.