Cloning and expression analysis of p26 gene in Artemia sinica

The protein p26 is a small heat shock protein that functions as a molecular chaperone to protect embryos by preventing irreversible protein damage during embryonic development. A 542 bp fragment of the p26 gene was cloned and sequenced. The fragment encoded 174 amino acid residues and the amino acid sequence contained the α-crystallin domain. Phylogenetic analysis showed that eight Artemia populations were divided into four major groups. Artemia sinica (YC) belonged to the East Asia bisexual group. Expression of the p26 gene at different developmental stages ofA. sinica was quantified using real-time quantitative polymerase chain reaction followed by cloning and sequencing. The relationship between the quantity of p26 gene expression and embryonic development was analyzed. The results indicated that massive amounts of p26 were expressed during the development of A. sinica. At the developmental stage of 0 h, A. sinica expressed the highest level of p26. As development proceeded, expression levels of the p26 gene reduced significantly. There was a small quantity of p26 gene expression at the developmental stages of 16 h and 24 h. We concluded that p26 might be involved in protecting the embryo from physiological stress during embryonic development.     

Tomato Fruit Development and Ripening Are Altered by the Silencing of LeEIN2 Gene

Loss-of-function ethylene insensitive 2 （EIN2） mutations showed ethylene insensitivity in Arabidopsis, which indicated an essential role of EIN2 in ethylene signaling. However, the function of EIN2 in fruit ripening has not been investigated. To gain a better understanding of EIN2, the temporal regulation of LeEIN2 expres- sion during tomato fruit development was analyzed. The expression of LeEIN2 was constant at different stages of fruit development, and was not regulated by ethylene. Moreover, LeEIN2-silenced tomato fruits were developed using a virus-induced gene silencing fruit system to study the role of LeEIN2 in tomato fruit ripening. Silenced fruits had a delay in fruit development and ripening, related to greatly descended expression of ethylene-related and ripening-related genes in comparison with those of control fruits. These results suggested LeEIN2 positively mediated ethylene signals during tomato development. In addition, there were fewer seeds and Iocules in the silenced fruit than those in the control fruit, like the phenotype of parthenocarpic tomato fruit. The content of auxin and the expression of auxin-regulated gene were declined in silenced fruit, which indicated that EIN2 might be important for crosstalk between ethylene and auxin hormones.     

小鼠早期胚胎发育过程中TGF-α和EGF-R mRNA的表达

This study was conducted to examine the activity of TGF α and EGF R mRNA using the RT PCR technique in the early embryonic development of the mouse. Expression of TGF α mRNA was initially observed in the 8 cell stage embryo of the mouse. The level of expression gradually increased in the morula and blastula. Expression of EGF R mRNA was initially observed in the 4 cell stage embryo of the mouse and 8 cell stage embryo;morula and blastula all expressed EGF R mRNA and the levels of expression had not changed. These results confirm that epidermal growth factors play a key role in early embryonic development of the mouse, especially TGF α which can stimulate and regulate embryonic development via the autocrine system.     

Cloning and characterization of Methoprene-tolerant(Met)and Kruppel homolog 1(Kr-h1)genes in the wheat blossom midge,Sitodiplosis mosellana

Juvenile hormone(JH),a growth regulator,inhibits ecdysteroid-induced meta-morphosis and controls insect development and diapause.Methoprene-tolerant(Met)and Krippel homolog I(Kr-h1)are two proteins involved in JH action.To gain some insight into their function in development of Sitodiplosis mosellana,an insect pest undergoing obligatory larval diapause at the mature 3rd instar stage,we cloned full-length complemen-tary DNAs of Met and Kr-h1 from this specics.SmMet encoded a putative protein,which contained three domains typical of the bHLH-PAS family and eight conserved amino acid residues important for JH binding.SmKr-h1 encoded a protein showing high sequence homology to its counterparts in other specics,and contained all eight highly conserved Zn-finger motifs for DNA-binding.Expression patterns of SmMet and SmKr hl were de-velopmentally regulated and JH III responsive as well.Their mRNA abundance increased as larvae entered carly 3rd instar,pre-diapause and maintenance stages,and peaked during post-diapause quiescence,a pattern correlated with JH titers in this species.Different from reduced expression of SmMer,SmKr-h1 mRNA increased at mid-to-late period of post-diapause development.Topical application of JH II on diapausing larvac also induced the two genes in a dose-dependent manner.Expression of SmuMer and SmKr-h1 clearly declined in the pre-pupal phase,and was significantly higher in female adults than male adults.These results suggest that JH-responsive SmMet and SmKr-h1 might play key roles in diapause induction and maintenance as well as in post-diapause quiescence and adult reproduction,whereas metamorphosis from larvae to pupac might be correlated with their reduced expression.     

Molecular Characterization of Thyroid Hormone Receptors(TRs) and their Responsiveness to T3 in Microhyla fissipes

To explore and enrich the molecular mechanisms of thyroid hormone receptors(TRs) in the metamorphosis of amphibians, the c DNA sequences of TRα and TRβ in Microhyla fissipes were cloned and characterized. TRα was 1 706 bp in length with an open reading frame(ORF) of 1 257 bp encoding a predicted protein of 418 amino acids and TRβ was 1 422 bp with an ORF of 1 122 bp encoding a predicted protein of 373 amino acids. Their protein sequences contained 4 conserved domains of the nuclear receptor superfamily with two highly conserved cysteine-rich zinc fingers in the DNA-binding domain, whereas TRβ was 42 amino acids shorter in its A/B domain than TRα. Highly-conserved sequences and structures indicated their conserved functions during metamorphosis. TRα expression reached peak at 12 h and then decreased from 12 h to 48 h. While dramatically up-regulated TRβ was observed after exposure of T3 within 24 h, and it was down-regulated from 24 h to 48 h. The expression pattern of TRβ is similar to that in the natural metamorphosis. Furthermore, tadpoles treated 24 h also resembled the climax of metamorphosis tadpoles and TRβexpression had higher responsiveness than TRα to T3 in M. fissipes. These results suggest M. fissipes may serve as the model to assay environmental compounds on TH signaling disruption.     

Flexible manipulation of Omb levels in the endogenous expression region of Drosophila wing by combinational overexpression and suppression strategy

Manipulating an exogenous or endogenous gene of interest at a defined level is critical for a wide variety of experiments.The Gal4/UAS system has been widely used to direct gene expression for studying complex genetic and biological problems in Drosophila melanogaster and other model organisms.Driven by a given tissue-specific Gal4,expressing UAS-transgene or UAS-RNAi(RNA interference)could be used to up-or down-regulate target gene expression,respectively.However,the efficiency of the Gal4/UAS system is roughly predefined by properties of transposon vector constructs and the insertion site in the transgenic stock.Here,we describe a simple way to modulate optomotor blind(omb)expression levels in its endogenous expression region of the wing disc.We co-expressed UAS-omb and UAS-omb-RNAi together under the control of dpp-Gal4 driver which is expressed in the omb expression region of the wing pouch.The repression effect is more sensitive to temperature than that of overexpression.At low temperature,overexpression plays a dominant role but the efficiency is attenuated by UAS-omb-RNAi.In contrast,at high temperature RNAi predominates in gene expression regulation.By this strategy,we could manipulate omb expression levels at a moderate level.It allows us to manipulate omb expression levels in the same tissue between overexpression and repression at different stages by temperature control.