Knockout of targeted gene in porcine somatic cells using zinc‐finger nuclease

Targeted genome editing is a widely applicable approach for efficiently modifying any sequence of interest in animals. It is very difficult to generate knock‐out and knock‐in animals except for mice up to now. Very recently, a method of genome editing using zinc‐finger nucleases (ZFNs) has been developed to produce knockout rats. Since only injection of ZFNs into the pronuclear (PN) embryo is required, it seems to be useful for generating gene‐targeted animals, including domestic species. However, no one has reported the successful production of knockout pigs by direct injection of ZFNs into PN embryos. We examined whether ZFN works on editing the genome of porcine growth hormone receptor in two kinds of cell lines (ST and PT‐K75) derived from the pig as a preliminary study. Our data showed that pZFN1/2 vectors were efficiently transfected into both ST and PT‐K75 cells. In both cell lines, results from Cel‐I assay showed that modification of the targeted gene was confirmed. We injected ZFN1/2 mRNAs into the nucleus of PN stage embryos and then they were transferred to the recipients. However, pups were not delivered. Taken together, ZFN can be an available technology of genome editing even in the pig but further improvement will be required for generating genome‐modified pigs.     

Vitrification procedure decreases inositol 1,4,5‐trisphophate receptor expression,resulting in low fertility of pig oocytes

Although cryopreservation of mammalian oocytes is an important technology, it is well known that unfertilized oocytes, especially in pigs, are highly sensitive to low temperature and that cryopreserved oocytes show low fertility and developmental ability. The aim of the present study was to clarify why porcine in vitro matured (IVM) oocytes at the metaphase II (MII) stage showed low fertility and developmental ability after vitrification. In vitro matured cumulus oocyte complexes (COCs) were vitrified with Cryotop and then evaluated for fertility through in vitro fertilization (IVF). Although sperm‐penetrated oocytes were observed to some extent (30–40%), the rate of pronuclear formation was low (9%) and none of them progressed to the two‐cell stage. The results suggest that activation ability of cryopreserved oocytes was decreased by vitrification. We examined the localization and expression level of the type 1 inositol 1,4,5 trisphosphate receptor (IP₃R1), the channel responsible for Ca²⁺ release during IVF in porcine oocytes. Localization of IP₃R1 close to the plasma membrane and total expression level of IP₃R1 protein were both decreased by vitrification. In conclusion, our present study indicates that vitrified‐warmed porcine COCs showed a high survival rate but low fertility after IVF. This low fertility seems to be due to the decrease in IP₃R1 by the vitrification procedure.     

不同品种茶树新梢响应"倒春寒"的转录组分析

为探究“倒春寒”冻害对不同品种茶树新梢转录水平的影响,本研究以茶园“倒春寒”发生时受冻害和未受冻害的龙井43和中茶126新梢为研究材料,对两组样品进行转录组分析,分别鉴定到1 012个和1 079个差异基因,以及284个共同差异基因。从中选取18个差异基因进行实时荧光定量PCR验证,结果与转录组测序结果基本一致,证明转录组数据可靠。利用GO和KEGG数据库,对差异基因进行代谢通路富集分析,发现两个品种的差异基因主要集中在光合作用、碳代谢和细胞色素P450等代谢进程,说明“倒春寒”对新梢生长发育相关的基础代谢造成了严重损伤,抑制了相关基因的正常表达;随后对284个共同差异基因进行表达聚类分析,结果表明,其中99个差异基因在两个茶树品种中的表达模式完全相反,涉及的生物过程包括MAPK信号通路、谷胱甘肽和苯丙烷代谢等,推测这些差异基因与龙井43和中茶126在受到低温胁迫后产生的不同信号传导模式有关。     

Generation of rats from vitrified oocytes with surrounding cumulus cells via in vitro fertilization with cryopreserved sperm

The objective of this study was to evaluate fertility and full‐term development of rat vitrified oocytes after in vitro fertilization (IVF) with cryopreserved sperm. Oocytes with or without surrounding cumulus cells were vitrified with 30% ethylene glycol + 0.5 mol/L sucrose + 20% fetal calf serum by using the Cryotop method. The warmed oocytes were co‐cultured with sperm. Although the denuded/vitrified oocytes were not fertilized, some of the oocytes vitrified with cumulus cells were fertilized (32.7%) after IVF with fresh sperm. When IVF was performed with cryopreserved sperm, vitrified or fresh oocytes with cumulus cells were fertilized (62.9% or 41.1%, respectively). In addition, to confirm the full‐term development of the vitrified oocytes with surrounding cumulus cells after IVF with cryopreserved sperm, 108 vitrified oocytes with two pronuclei (2PN) were transferred into eight pseudopregnant females, and eight pups were obtained from three recipients. The present work demonstrates that vitrified rat oocytes surrounded by cumulus cells can be fertilized in vitro with cryopreserved sperm, and that 2PN embryos derived from cryopreserved gametes can develop to term. To our knowledge, this is the first report of successful generation of rat offspring derived from vitrified oocytes that were fertilized in vitro with cryopreserved sperm.     

基于GSM的精准农业嵌入式远程实时测控系统

给出了基于GSM无线技术的精准农业嵌入式远程实时测控系统框架,介绍了系统的硬件组成和基于嵌入式实时操作系统的软件实现方法.测试结果表明,GSM无线通信技术能够很好地满足农业远程测控的要求.     

番木瓜环斑病毒CP基因同源区段的克隆及植物表达载体的构建

采用RT-PCR技术对番木瓜环斑病毒CP基因的同源区段进行了克隆和鉴定分析。序列分析结果表明,获得PRSV-CP基因3′端的278bp DNA片段同源率最高。构建了目的基因包含278bp正义链、内含子(PDK内含子)及278bp反义链的RNA介导的植物表达载体p2301-CPU,并通过电激法导入农杆菌中。经PCR及酶切鉴定,证实质粒已被导入获得了农杆菌工程菌株。利用该植物表达载体对番木瓜的遗传转化工作目前正在进行中。     

P and N deficiency change the relative abundance and function of rhizosphere microorganisms during cluster root development of white lupin (Lupinus albus L.)

We studied microbe-plant interactions of white lupin, a cluster root-forming plant, under low P and N conditions to examine increased nutrient acquisition by plants either by a shift to a more specialized microbial community or changes in microbial enzyme production. White lupin plants were grown in rhizoboxes filled with either P- or N-deficient soil; fertilized soil was used as control. After cultivation of plants in a glasshouse for 41 d, plant growth (shoot and roots) and P and N accumulation in shoots were measured. Microbial functions were analyzed by P- and N-cycling enzymes. The microbial community structure was estimated by fingerprinting (denaturing gradient gel electrophoresis) and sequencing techniques. P deficiency induced the released citrate and acid phosphomonoesterases from cluster roots and stimulated the production of microbe-derived alkaline phosphomonoesterase in the rhizosphere. P deficiency decreased microbial diversity in the cluster root rhizosphere. Increased relative abundance of Burkholderiales in the rhizosphere of P deficient plants might be responsible for the degradation of different organic P fractions such as phytates. N deficiency induced an increase of the number of nodules and P concentration in shoot as well as roots of white lupin. We clarified that high release of citrate from cluster roots might be the preferred mechanisms to meet the P demand of nodulated plants under N deficiency. In addition, the high abundance of Rhizobiales and Rhodospirillales in the rhizosphere of cluster roots showed that the importance of N-fixing microorganisms under N deficiency. The contribution of rhizosphere microorganisms due to similar activities of N-cycling enzymes under the two different N treatments is less important for N nutrition of plants. Further understanding of the regulation of cluster roots under N-deficiency will provide new information on the interactions between P and N nutrition.     

Effects of alcohol extract of defatted soybean meal on growth performance and digestive physiology of yellowtail <Emphasis Type="Italic">Seriola quinqueradiata</Emphasis>

The effects of alcohol extract of soybean meal on growth performance, digestibility, and pancreatic enzyme and bile acid status of yellowtail Seriola quinqueradiata were studied. Fish were fed one of the following for 8 weeks: a fish meal (FM) control diet (FMD), a defatted soybean meal diet (SBMD), an alcohol-extracted SBM diet (ExSBMD), an ExSBMD with alcohol-extract diet (ExSBM + ExtD), and an FMD with alcohol-extract diet (FM + ExtD). Growth performance was significantly inferior in fish fed with SBM, ExSBM + ExtD, and FM + ExtD compared to FMD- or ExSBMD-fed fish. Total bile acid levels, and trypsin and lipase activities in the anterior intestine were significantly lower in fish fed with SBM, ExSBM + ExtD, and FM + ExtD than in fish fed with ExSBMD and FMD, despite similar values in gallbladder or pyloric caeca. Cholecystokinin mRNA levels of the former diet-fed fish were significantly lower than those of FMD- or ExSBMD-fed fish. Lipid and protein digestibility of fish fed with SBMD, ExSBM + ExtD, and FM + ExtD was significantly reduced in comparison with that of fish fed with FMD or ExSBMD. These findings indicate that the alcohol extract of soybean meal inhibited the secretion of bile acids and pancreatic enzymes by a decrease of cholecystokinin stimulation. This inhibition seemed to be responsible for a low growth performance through impairment of the assimilation of lipid and protein.     

肉用西门塔尔牛与和牛杂交群体的肉品质分析

本研究旨在对肉用西门塔尔牛与杂交和牛两试验群体进行肉质性状比较,以期为肉品质性状遗传机制剖析及优质肉牛培育提供理论指导。收集肉用西门塔尔牛与杂交和牛两试验群体肉质性状表型数据,包括眼肌面积、剪切力、系水力、pH、肉色、脂肪颜色和大理石花纹等指标,进行相关性及差异性分析,从而综合评价两群体的肉品质。结果显示,两群体的脂肪颜色、系水力及剪切力呈现两两正相关;肉色与脂肪颜色、pH呈正相关,且肉色与pH呈显著相关（P < 0.05）,而系水力与pH、肉色和大理石花纹呈负相关。在两试验群体间,和牛杂交群体的大理石花纹、眼肌面积、pH性状表现为极显著优于肉用西门塔尔牛（P < 0.01）。从性别比较来看,肉用西门塔尔牛母牛在大理石花纹、pH、剪切力等性状上极显著或显著优于公牛（P < 0.01;P < 0.05）;而和牛杂交群体母牛的肉色和剪切力性状表现为显著或极显著高于公牛（P < 0.05;P < 0.01）。总体而言,和牛杂交群体的肉质性能优于肉用西门塔尔牛;群体内比较表明,肉用西门塔尔牛群体母牛肉质性能优于公牛,而和牛杂交群体母牛肉质性能低于公牛。     

重复序列在牛亚科中的研究进展

重复序列是在整个基因组中以多个拷贝出现的核酸序列,是真核生物基因组中的重要组成部分,对染色体重排、基因组及生物进化具有重要意义。它在模式动物、人类、植物等物种中都有报道,但在牛亚科中的研究进展较少,因此本文对重复序列的分类和特点进行综述,重点关注串联重复序列和散在重复序列在牛亚科中的研究进展,并分析了这两大类重复序列在牛亚科物种进化中的作用。