Novel gene expressed during early embryogenesis of zebrafish identified by mRNA differential display

Following the revelation of the molecular mechanism of morphogenesis in fruitfly, research on the molecular mechanism of morphogenesis in vertebrate becomes the focus of developmental biology. The isolation of genes controlling the embryogenesis of zebrafish, a vertebrate model animal, is considered as an initial step toward investigating this issue. There are several approaches that can be used to isolate developmental genes, each of which is suited to a particular situation. In this note, mRNA differential display was utilized to demonstrate the mRNA differences among zebrafish embryos at 4, 5 and 6 h post fertilization (28.5℃, corresponding to oblong, dome and shield stages, respectively, called blastula, gastrula and neurula in this note). One cDNA tag that was specific to embryos at neurula stage was cloned and sequenced. After sequence comparison in Genbank, we found that this cDNA tag represents a novel gene. The expression of this gene in the developing zebrafish embryos was examined by whole mount in situ hybridization. The hybridization results confirmed that this gene was specifically expressed in zebrafish neurula embryos.     

用mRNA差异显示技术分离盐胁迫下小麦耐盐相关cDNA

目的用mRNA差异显示技术分离小麦盐胁迫应答cDNA,为进一步研究小麦耐盐机理奠定基础。方法将小麦(Triticum aesticum L.)耐盐品系宝丰7228r种子分别置于含NaC l 0‰和12‰的Hoagland培养液中生长,7d后分别取叶片,提取总RNA并分离出其中的mRNA,通过锚锭引物O ligo dT10GC反转录和9个10核苷酸随机引物进行PCR扩增。结果DDRT-PCR结果显示,有4个差异DNA片段只在盐胁迫的小麦耐盐品系基因组中表达,而在对照中没有出现。这4个差异cDNA片段分别命名为ts01(260 bp),ts02(330 bp),ts03(420 bp),ts04(600 bp)。4个差异cD-NA片段的RNA杂交结果显示,只有ts04存在明显差异,在盐胁迫条件下有杂交斑点而在对照中没有杂交斑点,其余3个cDNA片段在盐胁迫和对照中都没有杂交斑点。进一步将ts04克隆并进行DNA序列测定及同源性分析。结论ts04可能是耐盐相关cDNA片段,该片段核酸序列与麦属抗性相关的肌动蛋白基因有23%同源。     

mRNA differential display on gene expression in settlement metamorphosis process of Ruditapes philippinarum larvae

The mRNA differential display （DDRT-PCR） technique was adopted to find out the genes related to settlement metamorphosis development process of Ruditapes philippinarum larvae. In this study, we have obtained three hundred and forty-six amplification bands in total from pediveliger larvae, veliger larvae, eye spot larvae and post-larvae. Sixty-five out of three hundred and forty-six bands are distinctly differential display from band pattern, which can be put into four groups, standing for different expression characters. Sixteen differential display bands were cloned, sequenced and analyzed and nine different sequences are obtained in the study. Three sequences have higher similarity to the cDNAs deposited in database and three are very similar to the rDNA of other species, considered as the rDNA of Ruditapes philippi narum. The rest three sequences are found to be novel sequences after analyzed. Their accession numbers are AY916799, AY916798, and AY916797 respectively. We thought the novel sequences are possibly relevant to the early embryo development of Ruditapes philippinarum larvae and can provide some fundamental understandings that are helpful for the improvement of scallop seed raising industry.     

用差异显示技术分离空心莲子草抗旱相关基因

采用mRNA差异显示技术,分离空心莲子草在干旱胁迫1 d后差异表达的基因,获得139个基因片段.经过Reverse Northern检测,初步证实有两个片段受干旱诱导表达上调.对其中一个片断克隆测序并进行核甘酸序列比对,显示与其他基因同源性都很低,表明可能是新的基因.半定量PCR技术证实该基因受干旱诱导表达上调.     

Differential display of carrot radicle genes in different developmental states

mRNA differential display was used in analyzing the expression of carrot somatic embryo radicle gene in different developmental stages brought about by controlled cultivation. As shown by experiment, marked differences in electrophoresis patterns of DNA exist between the normally developing carrot radicle and those with their development inhibited. 12 series of specific bands have been cloned, and structural analysis of them is under way.     

mRNA差异显示PCR技术在水稻研究中的应用

mRNA差异显示PCR技术是在基因转录水平上研究基因差异表达和性状差异的有效方法之一,该方法在生物的发育、分化和对各种生物、理化因子作用时应答过程基因表达的研究中应用十分广泛。就mRNA差异显示PCR技术的基本原理、优点与不足、改进与完善等作了简要归纳,综述了该方法在水稻的发育分化、激素调控、抗逆性和抗病性等方面所取得的研究成果,并对该方法在水稻方面的应用前景作了简单分析。     

Isolation of a vernalization-related cDNA clone (VRC) using mRNA differential display in winter wheat

Vernalization is an essential factor which affects the flowering development in cold-requiring plants. There is a key stage of nucleic acid and protein metabolism in the process of vernalization in winter wheat. To probe into the molecular determinants of vernalization, we examined mRNA populations in differently-treated plumules of winter wheat (Triticum aestivum L. cv Yanda 1817) using mRNA differential display. One vernalizationrelated cDNA clone (VRC), VRC54, was identified and was only expressed at the key stage of 20 d vernalization, rather than at other stages of nonvernalization, 4 d vernalization and devernalization. Northern blot and sequence analysis indicated that VRC54 was a novel vernalization-related clone found in higher plant which not only might play an important role in the floral induction in vernalization-requiring plants but also was different from the cold-acclimatized genes.     

辽宁产中国林蛙早期胚胎发育研究

在恒温条件下，观察了辽宁产中国林蛙的正常胚胎发育。根据基外形变化和胚胎主要特征的出现以及胚胎的行为，将其胚胎发育划分为２５个期。从卵子受精开始至蝌蚪的鳃盖闭合，出水孔形成止。１８℃时共历时１５１．５６ｈ。     

mRNA差异显示技术研究低温条件下深黄被孢霉基因的表达差异

mRNA差异显示技术已经广泛应用于研究植物、动物和微生物在各种环境条件下基因的差异表达研究.研究以15℃和30℃培养的深黄被孢霉M6-22菌体为材料,利用mRNA差异显示技术研究两种培养条件下基因的表达差异.经实时荧光定量PCR验证,共获得7条差异片段,相似性搜索结果表明它们是6-磷酸葡萄糖异构酶、单糖核苷酸转运蛋白、Ras1鸟苷酸转移因子、依赖于NAD的苹果酸脱氢酶、Δ12-脂肪酸脱氢酶、CLK4关联的丝氨酸/精氨酸丰富蛋白和假定蛋白,涉及糖酵解、蛋白质修饰、信号传导、脂肪酸合成和mRNA加工等生命过程,表明深黄被孢霉M6-22低温适应性是多种途径协同调控的结果.     

以mRNA差异显示法分离牙鲆白化相关基因的初步研究

以正常和白化牙鲆表皮组织总RNA为模板,用Oligo d(T)13M(M分别为A、C、G)3种锚定引物和26种差异显示随机引物组合进行差异显示,PAGE凝胶电泳后,用新型高灵敏度核酸荧光染料SYBR GREEN I显示差异DNA条带,得到区分度较好的差异表达图谱,共分离49条牙鲆白化相关DNA片段,片段长度260～800bp左右,经克隆测序后将可作为新的ESTs进行同源序列比较.     

隆线溞孤雌卵胚胎发育的形态学研究

采用组织学方法较为系统地研究了隆线溞(Daphnia carinata)孤雌卵(夏卵)胚胎发育的全过程.隆线溞夏卵为中黄卵,室温24℃下,整个胚胎发育过程需45h左右.根据隆线溞胚胎内部结构特征及外部形态学变化,将其胚胎发育分为卵裂期、囊胚期、原肠期、前无节幼体期、后无节幼体期、复眼色素期和准备孵化期7个时期.卵排至孵育囊约40min后开始表面卵裂.卵裂至256细胞时,胚胎发育进入囊胚阶段,在卵表形成一薄层细胞层,囊胚腔则全被卵黄颗粒所充塞.囊胚后期,囊胚层细胞分裂加快,相互挤压向囊胚腔中移入形成原肠胚.随后,胚胎外部形态开始出现变化.首先在胚胎前端出现头部的三对附肢原基(两对触角原基及一对大颚原基),胚胎发育进入前无节幼体期;随后胸节分化,胚胎发育进入后无节幼体期,并形成胸肢、壳瓣和肠道等结构.复眼在复眼色素期的基础上,逐渐发育形成完整的复眼结构,同时其他各组织器官也不断发育完善.至准备孵化期的胚胎结构与幼体已基本相同.以上研究结果可为深入研究枝角类胚胎发育的机理积累基础生物学资料.     

Isolation of cDNA fragment of fertility-related gene in photoperiod-sensitive genic male sterile rice

The photoperiod_sensitive genic male sterile rice (PGMR) is particularly useful to take advantage of heterosis in rice. mRNA differential display was used to isolate the fertility_relative genes in rice. After establishing an optimized mRNA differential display system, one of the differential cDNA fragments that maybe related to the development and maturation of rice panicle was cloned from a PGMR Nongken 58S.     

Preliminary study on human fibroblasts as feeder layer for human embryonic stem cells culture in vitro

To avoid the direct contact with mouse cells and possible heterogeneous pathogen in future application ,we need to replace mouse embryonic fibroblasts with human fibroblasts as the feeder layer to maintain human embryonic stem cells growth in the undifferentiated state,We Success-fully use human fibroblasts derved from aborted fetus and adult prepuces as feeder layer to maintain human embryonic stem cells growth ,During the passage and growth on this feeder layer,the human embryonic stem cells can keep their undifferentiated state.     

涪江下游唇胚胎发育研究

涪江下游唇鱼骨每年3～4月,在水温12～24℃范围内均可产卵繁殖.成熟卵圆形,橘黄色或橙黄色,卵径160～190mm,平均175mm,遇水能普遍激动,但始终不分裂.受精卵具粘性,卵膜吸水膨胀后达240～270mm,并在卵膜外形成一层胶膜.受精卵在125～185℃下历时约195h孵化,胚胎的孵化与孵化腺细胞的出现密切相关.低温下可出现“宕延孵出”的现象,发育的下限温度为102℃.初孵仔鱼全长650～680mm,平均662mm,眼色素和胸鳍原基已出现.