狗蔷薇PaWUS基因的超量表达诱导转基因烟草根尖形成不定芽

WUSCHEL是植物干细胞命运的决定基因,在植物发育过程中WUSCHEL基因的表达决定着干细胞的形成和器官的发生.从狗蔷薇的类原球茎中克隆出其同源基因；经过对其进行蛋白质序列比对和系统发育分析,将其命名为Ra WUS基因.构建在XVE系统下的化学调控的表达载体.在该系统下,Ra WUS基因的超量表达能诱导转基因烟草根尖形成不定芽；同时显微观察表明,根部的皮层中有圆球形的分生组织干细胞团的产生.结果表明:Ra WUS基因的过量表达能够促使根的皮层中薄壁细胞转变成为分生组织干细胞而形成不定芽在根尖异位发生,说明其能够在植物的根尖中诱导茎尖分生组织干细胞的活性和发育的可塑性.     

利用甜高粱秸秆汁产单细胞蛋白的分批补料发酵研究

本文以甜高粱秸秆汁为主要的发酵原料,对热带假丝酵母SG-1在15-L罐上产单细胞蛋白(SCP)的分批补料发酵过程进行研究。首先对分批发酵模式下的菌体代谢过程变化进行研究。研究发现,接种后菌体快速进入繁殖阶段,但是总糖在12 h处于耗竭状态,使得菌体量和SCP产量过早的在18 h就达到峰值。基于此,在15-L罐上建立了热带假丝酵母SG-1产SCP的分批补料发酵模式。结果表明:菌体代谢因基质的补加而得到进一步的延续,放罐时(42 h)其菌体干重和SCP产量分别达19.86 g/L和9.93 g/L,显著高于分批发酵模式的10.45 g/L和4.70 g/L。     

NPFFR1基因对鹅卵泡颗粒细胞激素分泌和细胞凋亡的影响研究

神经相关肽受体（RFamide-related peptide receptor,NPFFR1）是促性腺激素抑制激素的主要亲和受体,它在调控动物繁殖方面起着重要作用。为了解NPFFR1对鹅卵巢卵泡发育的作用,本研究以42周龄健康产蛋四川白鹅为试验材料（n=9）,利用RT-qPCR法检测NPFFR1基因在等级前和等级卵泡颗粒细胞中的mRNA表达规律;在颗粒细胞中过表达NPFFR1基因,酶联免疫吸附法检测颗粒细胞上清液（n=9）中雌二醇（estradiol,E2）、孕酮（progesterone,P4）和抗缪勒管激素（anti-Mullerian hormone,AMH）的浓度变化,剩余贴壁细胞作一步法TUNEL检测细胞凋亡情况;转录组测序方法筛选大黄卵泡（8~10 mm）颗粒细胞过表达NPFFR1前后表达差异显著基因,并对差异表达基因进行功能聚类分析。结果显示,除F1等级外,其余等级卵泡颗粒细胞NPFFR1表达量均极显著高于等级前卵泡（P<0.01）;过表达NPFFR1后,等级颗粒细胞上清液中的E2和等级前颗粒细胞上清液AMH的含量显著（P<0.05）降低,但孕酮P4含量变化不显著（P>0.05）;转录组测序共筛选到267个差异表达基因（119个下调,148个上调）,这些基因主要富集在生物节律过程、繁殖进程等生物学过程中;同时,与对照组相比,差异基因AMH显著下调表达（P<0.05）,Clock（clock circadian regulator）、FOS（proto-oncogene,AP-1 trans-cription factor subunit）、Per（period circadian regulator）和ANTXR2（cell adhesion molecule 2）分别极显著（P<0.01）或显著（P<0.05）上调表达。上述试验结果提示,NPFFR1可从激素、细胞凋亡和生物节律等多个环节影响卵泡颗粒细胞,参与调控卵泡的时序等级发育。     

Mesh-like vascular changes in copper deficiency-induced rat cardiomyopathy

The pathological effects of copper deficiency (COD) are well known. However, the pathogenesis of cardiomyopathy resulting from COD remains unclear. In this study, aimed to elucidate the pathogenesis of COD-induced cardiomyopathy by examining the morphology of the cardiovascular system in copper-deficient rats using histopathology, immunohistochemistry, and scanning and transmission electron microscopy. Changes detected in the myocardium and interstitium were consistent with those reported for COD. Morphological changes included mesh-like changes in the capillary endothelial cells that appear to be a novel finding in COD-induced cardiomyopathy. These changes are hypothesized to result from abnormal vascular remodeling following damage to the basement membrane and due to the mechanical effects of myocardial contractions. Although cardiomyopathy may be associated with microcirculatory disorders arising from these lesions, further investigations are necessary to demonstrate a causal relationship between the pathogenesis of cardiomyopathy and the contribution of these lesions to disease progression.     

AR regulates porcine immature Sertoli cell growth via binding to RNF4 and miR-124a

Sertoli cells are the only somatic cells in the seminiferous epithelium which directly contact with germ cells. Sertoli cells exhibit polarized alignment at the basal membrane of seminiferous tubules to maintain the microenvironment for growth and development of germ cells, and therefore play a crucial role in spermatogenesis. Androgens exert their action through androgen receptor (AR) and AR signalling in the testis is essential for maintenance of spermatogonial numbers, blood–testis barrier integrity, completion of meiosis, adhesion of spermatids and spermiation. In the present study, we demonstrated that AR gene could promote the proliferation of immature porcine Sertoli cells (ST cells) and the cell cycle procession, and accelerate the transition from G1 phase into S phase in ST cells. Meanwhile, miR-124a could affect the proliferation and cell cycle procession of ST cells by targeting 3′-UTR of AR gene. Furthermore, AR bound to the RNF4 via AR DNA-binding domain (DBD) and we verified that RNF4 was necessary for AR to regulate the growth of ST cells. Above all, this study suggests that AR regulates ST cell growth via binding to RNF4 and miR-124a, which may help us to further understand the function of AR in spermatogenesis.     

The influence of progesterone and prostaglandin F2α on decorin and the adhesion of caruncular epithelial cells of bovine placenta at early-mid pregnancy—Part II

One of the most important processes determining the proper course of gestation and its physiological termination in cows is the adhesion of epithelial cells allowing for direct contact of maternal and foetal parts of the placenta. Throughout pregnancy, placental cells are under strict hormonal control, which among others regulates the concentration and activity of specific proteins participating in the extracellular matrix remodelling of foetal membranes. The aim of the study was to evaluate the influence of progesterone and prostaglandin F_2α on the adhesion of epithelial cells at early-mid pregnancy in cows. Additionally, the impact of selected hormones on anti-adhesive properties of decorin was evaluated. Caruncular epithelial cells were isolated from healthy cows during pregnancy, immediately after slaughter. Primary cell cultures derived from the 2nd and 4th month of gestation were used in the experiments. The viability of cells was assessed by MTT assay. The adhesion of cells to fibronectin was measured spectrophotometrically. The activity of metalloproteinases was confirmed by the metalloproteinase assay. Progesterone (10^–5 and 10^–7 mol/L) and prostaglandin F_2α (10^–4_, 10^–5 and 10^–7 mol/L) increased the viability of bovine caruncular epithelial cells in the 2nd month of pregnancy. The treatment with prostaglandin F_2α significantly reduced the number of adherent cells from the 2nd month of gestation at the doses of 10^–4 and 10^–5 mol/L. Both progesterone and prostaglandin F_2α were shown to have an effect of decorin resulting in both a decrease in metalloproteinase activity and an increase in adhesion of cells to fibronectin.     

Production and Identification of Clone Sheep Using Bone Marrow Mesenchymal Stem Cells as Donor Cell

In order to obtain the cloned sheep using bone marrow mesenchymal stem cells（BMSCs）as the donor cells,the BMSCs of sheep were chosen and reconstructed embryos were built to transfer.10 published microsatellite markers were chosen,and the DNA samples from clone sheep,donor cells and surrogate ewes were amplified,and the relationship of father-son（RCP）was analyzed using the Quantity One for genotyping.The results showed that the reconstructed embryos were successfully built for electric fusion using sheep BMSCs as nuclear donor,and making nuclear transplantation into enucleated mature oocytes of which the fusion rate was 80.62%.20 surrogate ewe were chosen to be implanted with the reconstructed embryos at morula stage by implant surgery,and 5 lambs were born and only 3 were survived.The genotype of cloned sheep was in line with the dornor cell and the RCP were more than 99.999%.In conclusion,the first clone sheep were obtained successfully by using BMSCs as a nuclear donor in this experiment.     

Effects of Donor Cells Treated with Egg Extracts on the Development Competence of Cloned Embryos

Japanese Balck cattle fetal fibroblasts (JBCFF) were induced with Xenopus leavis egg extracts and somatic cell nuclear transfer (SCNT) was carried out with the reprogrammed JBCFF as donor cells in order to investigate their effects on SCNT efficiency.Three samples of egg extracts were acquired from different Xenopus laevis.The protein contents and kinds in extracts were evaluated with BCA Protein Quantification Kit and SDS-PAGE.Concentration of Digitonin to permeabilize JBCFF was optimized and assessed with PI staining.Reprogrammed cells treated with egg extract were used as donor in SCNT.Additionally the reconstructed embryos were activated with ionomycin+6-DMAP and A23187+6-DMAP to compare their effects on the development competence.The protein contents of extracts samples were 56.2255,64.6570 and 71.2158 μg/mL,respectively,the each extract had the same composition about 40-55 and 70-100 ku.The optimal concentration of Digitonin was 7 μg/mL and the permeabilization rate was 55.44%.After extracts treatment and continuous culture for 6-7 d,JBCFF formed well-defined colony structures.No significant composition difference was found in rates of fusion (92.83% vs 96.04%),cleavage (89.64% vs 89.78%) and blastocyst formation (24.06% vs 23.12%) of cloned embryos when the colony cells and JBCFF without extracts treatment were used as donor cells (P>0.05).Similarly,the two activation methods had no significant effect on the developmental competence of cloned embryos (cleavage rate 92.16% vs 92.28%,blastocyst rate 23.21% vs 24.18%).Conclusively,Xenopus leavis,egg extracts could induce JBCFF reprogramming to a low differentiated state.However donor cells with reprogramming partially could not improve the development of cloned embryos and its mechanism requires further research.