A Cathepsin B Inhibitor,E-64, Improves the Preimplantation Development of Bovine Somatic Cell Nuclear Transfer Embryos

Bovine somatic cell nuclear transfer (SCNT) is an important and powerful tool for basic research and biomedical and agricultural applications, however, the efficiency of SCNT has remained extremely low. In this study, we investigated the effects of cathepsin B inhibitor (E-64) supplementation of culture medium on in vitro development of bovine SCNT embryos. We initially used three concentrations of E-64 (0.1, 0.5, 1.0 μm), among which 0.5 μm resulted in the highest rate of blastocysts production after in vitro fertilization (IVF), and was therefore used for further experiments. Blastocyst development of SCNT embryos in the E-64 treatment group also increased relative to the control. Moreover, the cryosurvival rates of IVF and SCNT blastocysts were increased in E-64 treatment groups when compared with the control. On the other hand, we found that IVF and SCNT blastocysts derived from E-64-treated groups had increased total cell numbers and decreased apoptotic nuclei. Furthermore, assessment of the expression of apoptosis-related genes (Bax and Bcl-xL) in bovine IVF and SCNT blastocysts treated with E-64 by real-time RT-PCR analysis revealed suppressed expression of the pro-apoptotic gene Bax and stimulated expression of the anti-apoptotic gene Bcl-xL. Taken together, these finding indicate that addition of E-64 to embryo culture medium may have important implications for improving developmental competence and preimplantation quality in bovine IVF and SCNT embryos.     

体细胞数对发酵乳品质的影响研究

为确定用于生产发酵乳的原料乳质量标准,以提高发酵乳产品品质,选用蒙牛自有牧场30头不同健康状况、不同泌乳期的荷斯坦牛原料乳,按体细胞数分为低于20万个/mL组、20万-80万个/mL组及大于80万个/mL组,在发酵乳制备的当天及样品冷藏后的第1、7、14、21天分别测定发酵的产酸曲线、发酵乳的质构特性。结果表明,随着原料乳中体细胞数的增加,产品黏度下降,弹性降低,硬度增加,pH值降低,品质下降。     

基于ISSR分子标记技术的山核桃幼胚DNA甲基化初步研究

用简单序列重复区间扩增(inter-simple sequence repeat,ISSR)DNA分子标记技术对山核桃Carya cathayensis不同发育阶段幼胚(第9周至13周)的DNA进行甲基化状态检测。结果表明:17个引物在各个发育时期山核桃幼胚样品中共扩增检测到的128个甲基化位点。甲基化比例随着幼胚的发育逐渐升高,5个时期幼胚甲基化比例依次为23.44%,25.78%,28.90%,40.62%和42.19%。进一步研究表明:5个时期均存在全甲基化位点和半甲基化位点,其中,全甲基化位点所占比例分别为7.81%,8.59%,16.40%,24.22%和25.00%;半甲基化位点所占分别为15.62%,17.19%,12.50%,16.41%,17.19%。因此,山核桃幼胚5个发育时期在基因组DNA甲基化水平上存在着较大差异(P<0.01)。     

Lipid-based transfection as a method for gene delivery in zebrafish (Danio rerio) embryos

A major challenge to the widespread production of transgenic, knockout and knockdown zebrafish has been the absence of a simple and effective procedure for introducing macromolecules into the fertilized egg. None of the existing techniques for gene transfer in fish embryos has proven to be a major advance over cytoplasm microinjection, which is a technically demanding and time‐consuming procedure. This report addresses this need, considering that the development of protocols for lipid‐based transfection with fish embryos would considerably simplify gene transfer in this complex biological model. In this study, lipid‐based transfection with two different reporter vectors was carried out in zebrafish embryos at different developmental stages. The parameters tested included different plasmid/transfection reagent ratios as well as the influence of an added transfection enhancer reagent. When embryos were transfected in the blastula stage with a pEGFP‐N1 vector, more than 35% successfully incorporated the plasmid and expressed the fluorescent protein 24 h after transfection. The transfection enhancer did not show any significant effect in our experiments. This work presents an approach to implement this technique as a faster, cheaper and more practical alternative than microinjection.     

AgNO3对银杏胚愈伤组织诱导及分化的影响

为探讨AgNO3对不同发育期银杏胚愈伤组织诱导及分化的影响,以银杏3个不同发育程度合子胚为外植体,添加0～5 mg/L AgNO3做处理。结果表明：AgNO3能显著提高胚的愈伤诱导率：早期心形胚鱼雷形胚,早期子叶胚,成熟胚子叶分别在浓度1,0．5,0．1 mg/L时达到最大诱导率,为87％,77％,98％。隔代添加AgNO3促进了早期子叶胚愈伤的分化：1．0 mg/L处理在第90天分化出愈伤化银杏叶和胚类似物,3．0 mg/L处理分化出短小叶片;常规添加AgNO3的处理在0．1 mg/L分化出芽点。遮光处理表明：黑暗培养对愈伤诱导率影响不明显;添加活性炭时,AgNO3促进胚轴愈伤而抑制子叶愈伤,胚轴愈伤组织白色透明。综合分析表明：AgNO3能提高银杏胚的愈伤组织诱导率并促进其分化。     

杂交鹅掌楸体细胞胚萌发培养的研究

以杂交鹅掌楸体细胞胚为材料,研究体胚不同发育阶段、大量元素和维生素C、基因型、激素组合以及移栽基质对体胚苗萌发和移栽存活的影响。结果表明：鱼雷型胚萌发效果最好;3/4 MS培养基较适合体胚苗的萌发;Vc有利用保持体胚苗维持正常形态。不同基因型体胚苗萌发差异较大,基因型1×5002萌发效果最好,成苗率为84.26%;而1×4088仅有13.24%。KT 0.1 mg/L＋IBA 0.1 mg/L有利于促进体细胞胚的萌发。不同基因型体胚苗移栽存活率有差异,黄心土较混合泥炭土更适合体胚苗移栽。     

奶牛乳体细胞数变化及影响因素调查

调查分析了大庆地区四个规模化牧场近3年奶牛DHI数据。结果表明,乳体细胞评分均值为5.05(其乳体细胞数均值为49万·mL-1)。75.75%的奶牛个体,其生鲜乳体细胞数小于50万·mL-1。胎次、泌乳阶段、自然月份等因素影响奶牛生鲜乳体细胞数。调查结果为牛场奶牛综合管理及制定科学可行的乳房炎防治措施提供依据。     

马尾松体细胞胚胎发生相关基因PmSERKl的克隆与表达分析

体细胞胚胎发生不仅是植物微繁、种质保存的有效手段，同时也是研究高等植物胚胎发育和遗传转化的模式系统之一。本研究从马尾松未成熟合子胚诱导的胚性细胞团中成功克隆了体胚发生受体激酶基因PmSERK1。该基因全长2303bp，包含完整的开放阅读框，编码626个氨基酸，其蛋白分子量约为69kD。序列分析发现PmSERK1与其它植物的SERK基因高度同源。系统进化树重建表明，PmSERK1与已知参与体胚发生的关键SERK基因聚为一类，如MtSERK1、CuSERK1、AtSERK，和DcSERK。表达分析显示：PmSERK1在主要的组织器官（根、茎、针叶等）略有表达；但在诱导培养的非胚性愈伤中没有检测到表达，而在经继代培养的胚性愈伤中表达很高且逐渐增加，且在培养20d时达到最高。另外，在再生的子叶胚中，PmSERK1的表达量也很高。综上所述表明，PmSERK1是与体胚发生相关的关键SERK基因的直系同源基因，可以作为愈伤组织胚性的标记基因。     

Bovine Immunodeficiency Virus in Relation to Embryos Fertilized In Vitro

The association of bovine immunodeficiency virus (BIV) with embryos derived by in vitro fertilization from oocytes of experimentally infected heifers or oocytes/embryos exposed to the virus in vitro was investigated. Using a nested-PCR assay, proviral DNA of BIV was not detected in follicular fluid or in embryos derived from BIV-infected donors. In vitro exposure of oocytes to BIV during maturation or insemination with BIV-infected semen resulted in zona pellucida-intact embryos testing negative for BIV provirus. However, exposure of zona pellucida-free day-7 embryos to the virus resulted in a positive BIV assay for 28% of the batches of embryos, suggesting that the zona pellucida has a role in protecting against BIV infection. The presence of BIV in the IVF system had no apparent effect on the development of bovine embryos to the blastocyst stage.