Ether–zymolyase ascospore isolation procedure: an efficient protocol for ascospores isolation in Saccharomyces cerevisiae yeast

Here we describe a new procedure for ascospore isolation from cultures containing a majority of unsporulated vegetative cells of Saccharomyces cerevisiae. The EZ ascospore isolation procedure relies on the combination of two conventional protocols, diethyl ether treatment and modified zymolyase treatment, allowing a significant increase in the efficiency of ascospore isolation and consequently enabling a large number of meiotic offspring to be efficiently obtained and screened, thus improving the efficacy of genetic research and the genetic selection of S. cerevisiae strains. Copyright © 2010 John Wiley & Sons, Ltd.     

双单倍体牙鲆的性腺发生、性别分化及早期发育的研究

本文利用组织切片的方法研究了双单倍体牙鲆的性腺发生、性别分化及早期发育。结果显示:双单倍体牙鲆中除雌性外,还有雄性和未分化鱼出现,所占比例分别为56%、38%和6%。双单倍体牙鲆雌性的性别分化晚于普通牙鲆。雌鱼至100日龄,卵巢腔仍未闭合;110日龄雌鱼的产卵板上存在大量向卵母过渡的卵原细胞,未见Ⅰ时相卵母细胞出现。双单倍体牙鲆雄鱼精巢的分化在时间上和普通牙鲆的基本相同。随着发育的进行,双单倍体牙鲆雌鱼的卵巢出现部分异常的卵母细胞以及卵巢退化现象;部分雄鱼的精巢也有退化现象出现。9～11月龄,双单倍体牙鲆雄鱼的精巢发育到Ⅲ期,而普通牙鲆性腺在同期进入了Ⅳ期;11月龄开始双单倍体牙鲆雄鱼精巢有退化的迹象;13～15月龄,精巢退化成精小叶,其中只含有精原细胞,初级精母细胞消失不见,似Ⅱ期精巢。     

MFN1 and MFN2 Are Dispensable for Sperm Development and Functions in Mice

MFN1 (Mitofusin 1) and MFN2 (Mitofusin 2) are GTPases essential for mitochondrial fusion. Published studies revealed crucial roles of both Mitofusins during embryonic development. Despite the unique mitochondrial organization in sperm flagella, the biological requirement in sperm development and functions remain undefined. Here, using sperm-specific Cre drivers, we show that either Mfn1 or Mfn2 knockout in haploid germ cells does not affect male fertility. The Mfn1 and Mfn2 double knockout mice were further analyzed. We found no differences in testis morphology and weight between Mfn-deficient mice and their wild-type littermate controls. Spermatogenesis was normal in Mfn double knockout mice, in which properly developed TRA98+ germ cells, SYCP3+ spermatocytes, and TNP1+ spermatids/spermatozoa were detected in seminiferous tubules, indicating that sperm formation was not disrupted upon MFN deficiency. Collectively, our findings reveal that both MFN1 and MFN2 are dispensable for sperm development and functions in mice.     

毕赤酵母单倍体制备方法的研究

毕赤酵母(Pichia pastoris)是子囊孢子型的出芽酵母,以单倍体或二倍体形式存在,能够进行无性繁殖和有性繁殖,在酵母育种或基因功能研究方面,单倍体细胞起着重要的作用.本研究以P.pastoris X33二倍体细胞为研究材料,探讨了子囊孢子诱导、单倍体制备方法.结果表明,P pastoris X33二倍体细胞在氮源限制培养基上能够诱导形成子囊.随后可经2种方法获得单倍体:(1)3％蜗牛酶30℃处理2h,58℃处理12min,单倍体获得率为60％;(2)50％乙醚涡旋处理5min,单倍体获得率为37％.     

酿酒酵母单倍体制备方法的优化

研究了酿酒酵母单倍体的制备方法。对酿酒酵母BY-6的产孢条件进行了优化，产孢培养基为McClary培养基，培养时间为7d，培养温度为25-28℃，在此条件下，酿酒酵母的产孢率可达98．5％。蜗牛酶浓度及酶解时间是影响孢子释放的关键因素，最适酶浓度为3％，酶解时间为60min。酶解后，50℃热处理10min杀死营养体，有利于单倍体的检出。     

甘蓝型油菜DH植株创建及其倍性的流式鉴定

以甘蓝型油菜大粒品系和小粒品系的小孢子为试验材料,研究取样时期、花蕾大小、培养基类型及添加成分对小孢子产胚率的影响,利用流式细胞仪对小孢子再生植株及加倍植株进行了准确、快速的倍性分析。结果表明,始花期为最佳取蕾时期,花蕾大小在2.5~3.5mm为宜,培养基以液体培养基最好。培养基中加入0.1mg/L6-BA可以促进小孢子胚的发生,而活性炭对小孢子培养没有明显的促进作用。流式细胞仪倍性检测显示小孢子再生植株加倍后的双单倍体（doubled haploid,DH）占49%,还存在少量的三倍体（11.3%）、四倍体（1.1%）及嵌合体（27.3%）植株。本研究为油菜材料的快速纯合及其DH系群体的构建等提供了新的经验。     

Advances and Perspectives in Tissue Culture and Genetic Engineering of Cannabis

For a long time, Cannabis sativa has been used for therapeutic and industrial purposes. Due to its increasing demand in medicine, recreation, and industry, there is a dire need to apply new biotechnological tools to introduce new genotypes with desirable traits and enhanced secondary metabolite production. Micropropagation, conservation, cell suspension culture, hairy root culture, polyploidy manipulation, and Agrobacterium-mediated gene transformation have been studied and used in cannabis. However, some obstacles such as the low rate of transgenic plant regeneration and low efficiency of secondary metabolite production in hairy root culture and cell suspension culture have restricted the application of these approaches in cannabis. In the current review, in vitro culture and genetic engineering methods in cannabis along with other promising techniques such as morphogenic genes, new computational approaches, clustered regularly interspaced short palindromic repeats (CRISPR), CRISPR/Cas9-equipped Agrobacterium-mediated genome editing, and hairy root culture, that can help improve gene transformation and plant regeneration, as well as enhance secondary metabolite production, have been highlighted and discussed.     

Genome Editing Using Mammalian Haploid Cells

Haploid cells are useful for studying gene functions because disruption of a single allele can cause loss-of-function phenotypes. Recent success in generating haploid embryonic stem cells (ESCs) in mice, rats, and monkeys provides a new platform for simple genetic manipulation of the mammalian genome. Use of haploid ESCs enhances the genome-editing potential of the CRISPR/Cas system. For example, CRISPR/Cas was used in haploid ESCs to generate multiple knockouts and large deletions at high efficiency. In addition, genome-wide screening is facilitated by haploid cell lines containing gene knockout libraries.     

酿酒酵母子囊孢子单倍体形成和制备的研究

实验研究了二倍体酿酒酵母(Saccharomyces cerevisiae)菌株GJ2008和GGFS16的孢子形成和分离条件,并获得了相应的单倍体细胞.实验结果表明,酿酒酵母GJ2008和GGFS16在McClary培养基上22℃培养7d,产孢率分别为91.46％和87.20％.在此条件下,以浓度为0.3g/L的蜗牛酶37℃水浴处理,孢子释放率分别为66.15％和67.35％.对获得的单倍体菌株进行发酵实验,并测定其酒精发酵性能,为融合实验做好铺垫.