香榧体细胞胚发生、发育的形态与细胞学观察

将香榧(Torreya grandis‘Merrillii’)的未成熟合子胚置于SH+0.1 mg·L-1 NAA+500 mg·L-1AC+3%蔗糖+0.5 g·L-1 Gln培养基上暗培养45 d,诱导产生半透明颗粒状胚性愈伤组织;将胚性愈伤组织转入SH+20 g·L-1 PEG+10 mg·L-1 ABA培养基中暗培养3个月诱导体细胞胚。采用碘—碘化钾染色和石蜡切片技术对体胚起源、形态发育与细胞组织学进行了观察。结果表明:胚性愈伤组织起源于合子胚胚轴表皮或皮层细胞的对称分裂。胚性愈伤组织含有两类细胞,一种是细胞质浓厚、细胞核大、体积小的圆形胚性细胞,另一种是高度液泡化拉长的细胞。胚性愈伤组织包含由这两种细胞构成的原胚团Ⅰ、原胚团Ⅱ和原胚团Ⅲ,以及一些游离细胞。原胚团Ⅲ在无植物生长调节剂的SH基本培养基上形成原胚,原胚接入成熟培养基,历经球形、棒状、心形、鱼雷形胚后发育成子叶胚。将子叶胚转入萌发培养基后胚根伸长,胚芽发育长出针叶,形成完整的再生植株。同时在离体培养中合子胚胚柄处退化的裂生多胚也能重新发育形成胚体。在初生体胚发育过程中表面常伴有次生体细胞胚的形成。     

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.     

激素对白三叶愈伤组织形成的影响及体细胞胚的组织学观察

以白三叶3个品种瑞文德、铺地和海法的子叶和下胚轴为外植体,采用正交设计试验研究2,4-D、6-BA和KT三种生长调节剂的不同浓度水平对白三叶愈伤组织诱导及继代的影响,筛选最佳激素组合并进行体细胞胚的组织学观察.结果表明:同一品种同一时期不同外植体愈伤诱导的最佳激素组合不相同,不同品种间也不一致;瑞文德子叶胚性愈伤诱导的最佳培养基为改良SH+2,4-D 2mg/L+6-BA 0.35mg/L+KT 0.5mg/L,下胚轴为改良SH+2,4-D 2mg/L+6-BA 0.2mg/L+KT 0.35mg/L;铺地子叶胚性愈伤诱导的最佳培养基为改良SH+2,4-D 2mg/L+6-BA 0.5mg/L+KT 0.5mg/L,下胚轴为改良SH+2,4 D 5mg/L+6-BA 0.35mg/L+KT 0.5mg/L;海法子叶胚性愈伤诱导的最佳培养基为改良SH+2,4-D 2mg/L+6-BA 0.2mg/L+KT 0.35mg/L,下胚轴为改良SH+2,4-D5mg/L+6-BA 0.5mg/L+KT 0.5mg/L.组织学观察表明,体细胞胚的发育经历球形胚、心形胚、鱼雷胚及子叶胚四个阶段后形成一个完整的体细胞胚.     

Cell-free extract from porcine induced pluripotent stem cells can affect porcine somatic cell nuclear reprogramming

Pretreatment of somatic cells with undifferentiated cell extracts, such as embryonic stem cells and mammalian oocytes, is an attractive alternative method for reprogramming control. The properties of induced pluripotent stem cells (iPSCs) are similar to those of embryonic stem cells; however, no studies have reported somatic cell nuclear reprogramming using iPSC extracts. Therefore, this study aimed to evaluate the effects of porcine iPSC extracts treatment on porcine ear fibroblasts and early development of porcine cloned embryos produced from porcine ear skin fibroblasts pretreated with the porcine iPSC extracts. The Chariot^TM reagent system was used to deliver the iPSC extracts into cultured porcine ear skin fibroblasts. The iPSC extracts-treated cells (iPSC-treated cells) were cultured for 3 days and used for analyzing histone modification and somatic cell nuclear transfer. Compared to the results for nontreated cells, the trimethylation status of histone H3 lysine residue 9 (H3K9) in the iPSC-treated cells significantly decreased. The expression of Jmjd2b, the H3K9 trimethylation-specific demethylase gene, significantly increased in the iPSC-treated cells; conversely, the expression of the proapoptotic genes, Bax and p53, significantly decreased. When the iPSC-treated cells were transferred into enucleated porcine oocytes, no differences were observed in blastocyst development and total cell number in blastocysts compared with the results for control cells. However, H3K9 trimethylation of pronuclear-stage-cloned embryos significantly decreased in the iPSC-treated cells. Additionally, Bax and p53 gene expression in the blastocysts was significantly lower in iPSC-treated cells than in control cells. To our knowledge, this study is the first to show that an extracts of porcine iPSCs can affect histone modification and gene expression in porcine ear skin fibroblasts and cloned embryos.     

中国荷斯坦牛部分乳房结构特性对泌乳性能的影响

本研究对规模化场奶牛乳房结构部分性状进行评价和测定,旨在分析其影响因素及其与泌乳性能的关系。现场评价和测定我国北方6个规模化奶牛场荷斯坦牛的乳房均衡(2 030条)、乳头长度(1 551条)和乳头位置评分(分前、后乳头,2 839条),使用SAS 9.2 GLM过程采用固定效应模型分析影响上述3个乳房性状的因素,同时分析3个单项性状对校正日产奶量和体细胞评分的影响。结果表明:场和胎次对3个乳房性状影响极显著(P0.01);前、后乳头位置和后乳头长度对校正日产奶量有显著影响(P0.05),校正日产奶量与前乳头位置评分值呈正比,与后乳头位置评分值呈反比;前乳头位置和长度对体细胞评分有显著影响(P0.05),体细胞评分随前乳头位置评分的增大而减小;5~5.5 cm的乳头长度对于校正日产奶量和体细胞评分都最佳。本研究结果为通过乳房性状选择、淘汰并提高奶牛的生产性能、乳房健康提供了参考。     

水稻OsLEA5c基因的克隆、表达和抗逆性分析

为了研究水稻胚胎发育后期丰富蛋白基因OsLEA5c 的抗逆性,利用 RT PCR技术从水稻中克隆到OsLEA5c的 cDNA。序列分析表明,OsLEA5c基因的读码框为456 bp,编码1个由151个氨基酸残基组成的蛋白。蛋白分子量为16.55 kD,等电点为5.07。OsLEA5c蛋白的平均亲水系数（GRAVY）为0.020,为疏水蛋白。OsLEA5c蛋白的44－140位氨基酸残基形成 LEA_2结构域。进化树分析表明,OsLEA5 c属于第5组LEA蛋白的C亚组。OsLEA5 c与5 C亚组LEA蛋白的氨基酸一致性为45.14％～61.59％。利用基因重组技术构建的原核表达载体 pET32a Os-LEA5c,转化到E．coli BL21 pLysS菌株中,诱导得到34 kD的融合蛋白。OsLEA5c在大肠杆菌中的表达增加了大肠杆菌对高温、高盐、高渗透压和反复冻融的抗性。OsLEA5c 基因在水稻抗逆和种子成熟脱水过程中发挥重要作用。     

月月红月季体细胞胚胎植株再生关键技术研究

以带1 mm叶柄的月季叶片为外植体诱导出胚性愈伤和体细胞胚胎,对月季体细胞胚胎植株再生进行研究。结果表明,月季体细胞胚胎再生与培养基种类、培养基坡度和体细胞胚胎自身类型等密切相关。月季体细胞胚胎在SP/R培养基(含1.0 mg/L 6-BA+0.05 mg/L NAA+3.0mg/L GA3)上比在EM培养基(含1.0 mg/L 2,4–D+0.1 mg/L TDZ)上能再生出更多小植株,在EP培养基(含3.0 mg/L 2,4–D+0.5 mg/L TDZ)上则逐步褐化死亡;将培养基放置成坡面,有利于体细胞胚胎分化出芽和根,提高植株再生率;在体细胞胚胎的子叶尚未完全开张(呈90°~150°角)前转入倾斜的SP/R培养基可以再生出正常植株,体细胞胚胎子叶充分伸展甚至向外翻转时(大于150°角)再转入倾斜的SP/R培养基,体细胞胚胎则会回到愈伤组织形态,丧失植株再生能力。     

取材时期和母树来源对水曲柳体细胞胚诱导的影响

以水曲柳(Fraxinus mandshurica Rupr.)不同母树、不同发育阶段的未成熟合子胚切离子叶为外植体,研究了外植体发育时期和母树来源对该树种体细胞胚诱导的影响。结果表明:外植体的取材时期、母树来源及二者的交互作用对水曲柳体胚诱导均具有极显著影响(p0.01),影响大小依次为取材时期采种母树交互作用;7月下旬到8月上旬是水曲柳体胚诱导的最佳采种时期,此时种子的胚乳已固化,合子胚形态分化完成,胚大小达到胚腔的2/3左右,47株母树的平均诱导率分别达到15.86%和16.77%,最高的4株母树的诱导率达到53.33%~66.67%。