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.     

The expression of vasa gene during zebrafish ( Danio rerio ) oogenesis

vasa gene expression pattern during oogenesis of zebrafish was examined usingin situ hybridization and fluorescent quantitative RT-PCR. During zebrafish oogensis,vasa mRNA is expressed strongly and uniformly distributed in the cytoplasm in stage II oocytes, followed by a distribution among vacuome in stage III. Later in stage IV and V,vasa mRNA is enriched at the cortex and finally localized at the cortex. The fluorescent quantitative RT-PCR shows that the quantity ofvasa mRNA decreases from stage II to stage III, but remains relatively invariable from stage III to stage V. The observed differences invasa mRNA expression in the different stages of zebrafish oogenesis suggest thatvasa gene plays an important role during oogenesis. Foundation item: Supported by the National Natural Science Foundation of China (30370744, 30150005) Biography: XIANG Fang (1979-), male, Master candidate, research direction: molecular development of animals.     

文昌鱼Pax1/9基因上游调控区的克隆及分析

脊椎动物Pax1/9是一重要的发育调控基因亚家族,文昌鱼基因组中仅有单一的该亚家族直系同源基因Amphi-Pax1/9,为检验此基因上游调控元件的功能在脊椎动物体内是否具有通用性,将文昌鱼Pax1/9基因上游约4.6 kb的侧翼序列与绿色荧光蛋白(GFP)报告基因连接,构建重组质粒表达载体(pAmphiPax1/9-AcGFP),显微注射斑马鱼胚胎,并以斑马鱼Pax1和Pax9的上游同源序列为阳性对照.结果表明,阳性对照斑马鱼胚胎中GFP能够表达,但注射pAm-phiPax1/9-AcGFP质粒的斑马鱼胚胎中无明显的GFP表达,这一现象可能是由于Pax1/9上游调控序列在二物种间存在较大的进化差异,启动元件具有物种特异性.     

Isolation and cloning of a novel cDNALDB1 encoding human LIM domain binding protein

Primers for screening cDNA library have been designed according to EST AA453734 which is corresponding to the mouse LIM domain binding protein Ldbl. Arrayed human fetal brain cDNA library has been screened by PCR and routine hybridization method. A 2398 bp-cD-NA clone has been obtained. The cDNA encodes a 347 amino acids protein highly homologous to the mouse Ldbl,Xenopus Xldbl andDrosophila Chip. It also contains an LIM binding domain and a nuclear localization signal. It has been namedLDB1 ( LIM domain binding protein 1), GenBank accession number is AF052389. Northern blot showed a 2.4 kb band, and the expression amounts ofLDBI in heart, brain and lung were considerably higher than those in other tissues.     

Nuclear transplantation in different strains of zebrafish

Single later blastula nuclei from AB strain of zebrafish (Danio rerio) were transplanted into enucleated unfertilized eggs of Long fin strain. Of 1119 cloning embryos, 14 reconstructed embryos developed into fry. DNA fingerprinting systems of the cloned fish were similar to those of the nuclear donor fish, but were distinctly different from those of the nuclear recipient fish. It confirmed that the genetic material originated from nuclear donor cell other than from nuclear recipient egg. The research suggested that the basic technique for nuclear transplantation performed with different strains of zebrafish has made a breakthrough. It should be helpful for the study of some important developmental problems such as gene function, the regulation ogene expression during animal development, the developmental potential of a nucleus and the interactions between the donor nucleus and the recipient cytoplasm, etc.     

Cloning the interferon regulatory factor I gene in lungfish （Protopterus annectens） and its molecular evolution among sarcopterygians

Sarcopterygians is an important vertebrate clade that includes crossopterygians and tetrapods. Crossopterygians are lobe-finned fish that include lungfish and coelacanths. Tetrapods include amphibians, reptiles, avians and mammals. To compare the interferon regulatory factor 1 (irf-1) gene structure and to explore phylogenetic relationships among sarcopterygians, we cloned the cDNA sequence of irf-1 from lungfish and compared it with irf-1 orthologs in other sarcopterygian species. The lungfish is a primitive sarcopterygian that occupies a very important position in vertebrate phylogeny. Interferon regulatory factors (IRFs) are a family of proteins involved in innate immunity. To date, 11 IRF family members have been reported. All IRFs share homology in the first 115 amino acids, which encompasses a DNA binding domain containing a characteristic repeat of 5 tryptophan residues separated by 10–18 amino acids. IRF-1 and IRF-2 were the first members of this family to be reported and they have a very important role in innate immunity. However, studies of the irf-1 and irf-2 genes are mostly confined to mammals; very few non-mammalian irf-1 genes have been reported. Consistent with the irf-1 gene sequences already published, the first 345 nucleotides of lungfish irf-1 are highly conserved. At the carboxyl terminal a C-terminal transactivating region motif and an interferon associated domain (IAD2) were identified. 417 million years separate the present from the closest common ancestor of lungfish and tetrapods; however, the irf-1 genes among sarcopterygians are highly conserved and have very obvious phylogenetic relationships. Also the interrelationship tree of sarcopterygians, based on IRF-1 amino acid sequences, is identical with trees produced using other data, such as morphological characteristics or mitochondrial gene sequences.     

Isolation and characterization of soybean NBS analogs

Isolation of plant resistance genes is greatly helpful to crop resistance breeding and the insight of resistance mechanism. The cloned plant resistance genes are classified into four classes according to their putative structural domain, of which the majority possesses nucleotide-binding site (NBS) domain that consists of P-loop, kinase2a and kinase3a. The conservation of this domain affords the potential possibility of cloning the plant resistance genes, which is homology-based cloning technique. In the present study, the degenerate oligonucleotide primers were designed according to the tobaccoN andArabidopsis RPS2, and 358 clones were isolated from the genomic DNA of resistance soybean cultivar Kefengl, resistant to soybean mosaic virus, and 4 open-reading NBS analogs were finally characterized and designated asKNBS1, KNBS2, KNBS3 andKNBS4. Southern hybridization suggested that they were present with multicopy in the soybean genome;KNBS4 was mapped to F linkage group andKNBS2 co-located J linkage group with the SCAR marker ofRsa resistant to soybean mosaic virus by RFLP analysis. Northern analysis suggested thatKNBS2- related sequence was low and constitutively expressed in the root, stem and leaves of soybean. The detailed characterization of NBS analogs is very helpful to ultimately cloning the soybean resistance gene.