Cloning of the full-length gene of activin receptor-interacting protein2a and characterization of its interaction with ActRⅡA

To investigate the mechanism of intracellular signal transduction mediated by activin receptors, the full-length gene encoding a novel activin receptor-interacting protein2a (ARIP2a) was identified from a mouse brain cDNA library. The sequences of ARIP2a and ARIP2, distribution of ARIP2a and ARIP2 mRNA in mouse tissues, and expression of ARIP2a and ARIP2 in activin-induced RAW264.7 cell were compared, and the interaction between ARIP2a and ActRIIA was confirmed. The sequence analysis revealed that the full-length gene of ARIP2a, which composed of 1008 bp and encoded 153 amino acid residues, shared high sequence identity with ARIP2 except the position of the 99th amino acid. RT-PCR assay showed that ARIP2a mRNA was highly expressed in brain, pituitary and testis, and moderately in pancreas and ovary, but undetectable in other tissues. Whereas, ARIP2 mRNA was widely distributed in all mouse tissues that we tested. Moreover, expression of ARIP2a mRNA was significantly decreased in activin-stimulated RAW264.7 cells; however, the expression of ARIP2 mRNA was increased. Additionally, the interaction between ARIP2a and activin type IIA receptor (ActRIIA) was further demonstrated by mammalian two-hybrid assays and pull-down assays. Taken together, those results indicate that although ARIP2a is homologous to ARIP2, they are different in tissue distribution and responses to activin. ARIP2a could also interact with activin type II receptor as a novel member of ARIP family.     

Cloning and characterization of a gene encoding cysteine proteases from senescent leaves of Gossypium hirsutum

A gene encoding a cysteine proteinase was isolated from senescent leave of cotton (Gossypium hirsutum) cv liaomian No. 9 by utilizing rapid amplification of cDNA end spolymerase chain reaction (RACE-PCR), and a set of consensus oligonucleotide primers was designed to anneal the conserved sequences of plant cysteine protease genes. The cDNA, which designated Ghcysp gene, contained 1368 bp terminating in a poly(A)^ trail, and included a putative 5‘(98 bp) and a 3‘(235 bp) non-coding region. The opening reading frame (ORF) encodes polypeptide 344 amino acids with the predicted molecular mass of 37.88 kD and theoretical pl of 4.80. A comparison of the deduced amino acid sequence with the sequence in the GenBank database has shown considerable sequence similarity to a novel family of plant cysteine proteases. This putative cotton Ghcysp protein shows from 67% to 82% identity to the other plants. All of them share catalytic triad of residues, which are highly conserved in three regions. Hydropaths analysis of the amino acid sequence shows that the Ghcysp is a potential membrane protein and localizes to the vacuole, which has a transmembrane helix between resides 7-25. A characteristic feature of Ghcysp is the presence of a putative vacuole-targeting signal peptide of 19-amino acid residues at the N-terminal region. The expression of Ghcysp gene was determined using northern blot analysis. The Ghcysp mRNA levels are high in development senescent leaf but below the limit of detection in senescent root, hypocotyl, faded flower, 6 d post anthesis ovule, and young leaf.     

Controlled expression of enhanced green fluorescent protein and hepatitis B virus precore protein in mammalian cells

A novel tetracycline regulation expression system was used to regulate the expression of enhanced green fluorescent protein (EGFP) and hepatitis B virus precore protein in the mammalian cell lines with lipofectAMINE. Flow cytometry assays showed that application of the system resulted in about 18-fold induction of EGFP expression in CHO cell lines and 5-fold induction in SSMC-7721 cells and about 2-fold in the HEK293 cells. Furthermore, the effective use of this system for the controlled expression of HBV precore protein gene in hepatocellular carcinoma cells was tested.     

Cloning of a down-regulated gene encoding small GTP binding protein in hybrid wheat

Previous studies showed that differential gene expression between wheathybrids and their parents was responsible for the heterosis. To provide an insight into the molecular basis of wheat heterosis, one cDNA, designated TaRab, was identified from the cDNA library of wheat seedling leaves. The sequence comparison in GenBank revealed that TaRab is homologous to a group of genes encoding Rab-GTP binding protein. Semi-quantitative RT-PCR analysis indicated that TaRab was expressed in all plant tissues examined, but at slightly higher level in leaves. Further analysis exhibited that TaRab displayed lower expression in hybrid than in its patents in both roots and leaves, which was in agreement with the original results of suppression subtractive hybridization. TaRab was located on chromosome 7B and C-7DS5-0.36 by in silico mapping. The relationship between differential expression of TaRab and the molecular basis of wheat heterosis was also discussed.     

Cloning,sequencing of the HSC70 gene in Ctenopharyngodon idella

A cDNA encoding heat shock cognate protein 70(HSC70)was cloned from liver of grass carp(Ctenopharyngodon idella)(GenBank JF436930).This cDNA was found out to contain2 346 bp in length,including 1 950 bp of complete coding sequence encoding 649 amino acids(aa),plus 89 bp of 5′-UTR and 307 bp of3′-UTR.Analysis of its genomic structure revealed that its corresponding gene contained seven exons and six introns.Homology analysis indicated that it shared 99%of identity with HSC70 of breams and 86%of identity with HSP70 of Drosophila.Fluorescent RT-PCR analysis revealed that at 28℃,this gene was expressed in abdominal fat,muscle,intestines,brain,middle kidney,head kidney,gonads,swim bladder,liver,heart,spleen,gills,and fins with expression level in liver being the highest(p0.05),followed by that in the gonads;at 36℃,its mRNA expression level was increased at first but then decreased thereafter under heat shock stress,indicating that its expression can be regulated by heat shock.In conclusion,cloning and expression analysis identified a cDNA encoding a constitutive HSP70 gene that is expressed in many tissues of Ctenopharyngodon idella and its expression was down-regulated by heat shock.     

A suppressed gene in integument cells of a fiberless seed mutant in upland cotton

A fiberless seed mutant(fl) was identified in a commercial cotton(Gossypium hirsutum L.) variety Xu-Zhou 142(FL).THis phenotype is associated with lack of fiber cell initiation in the outer integument of the ovule,as was characterized by analysis of genes related to fiber differentiation and development.Two genes,fl-E6 and FL-E6,were cloned from fl-integument cells and FL-fiber or integument cells,respectively.Compared with FL-E6,fl-E6 showed a dramatic change in nucleotide sequence:(1) FL-E6 contained a tandem repetitive sequence in which GGCTCA( Gly-Ser) is repeated five times between the 82nd and the 93rd codon from the first ATG codon,while in fl-E6 the same sequence is repeated four times;(2) The fl-E6 gene encodes a polypeptide of 241 amino acids but lacks two codons between the 90th and 93rd codon and three between the 171st and 174th relative to FL-E6;(3) There are also 12 nucleotide substitutions which would result in 7 amino acid differences between fl-E6 and FL-E6.Analysis of RT-PCR and Northern Blot showed that expression of the fl-E6 gene is suppressed in the fl-integument cells.but highly expressed in FL-fiber cells.The difference between fl-E6 and FL-E6 may be associated with lower expression of fl-E6 in the fli-inbackbones of two arabinogalactan-proteins(AGPs),one from the filtrate of suspension-cultured cells of Pyrus communis (AGPPc2) and the other from Nicotiana alata(AGPNa2),Although the function of the FL-E6 protein in differentation and development of cotton fiber cells is not known,the data indicate that the mutation of fl-E6 gene from FL-E6 gene may inhibit the fiber cell initiation from epidermal cells of the outer integument of the ovule.     

cDNA cloning and expression of erythropoietin in the plateau zokor （Myospalax baileyi） from the Qinghai-Tibet Plateau

All organisms respond to variation in their environments and manage environmental stress through metabolic adjustments.The plateau zokor(Myospalax baileyi) is an endemic and keystone subterranean rodent species that inhabits the Qinghai-Tibet Plateau between 2800 and 4200 m above sea level.It is a hypoxic-tolerant mammal with a high ratio of oxygen utilization that enables it to cope with its harsh surroundings.To explore the molecular mechanism of altitude acclimatization of the plateau zokor,we cloned the zokor erythropoietin(Epo) gene and used real-time PCR to compare Epo mRNA levels in zokors inhabiting 16 different altitudes.The full-length zokor Epo open reading frame was 579 bp that encoded a precursor peptide of 192 amino acids with a signal peptide of 26 residues.The Epo gene of the plateau zokor was 81%-95% homologous to that of human,mouse,rat,root vole and the Golan Heights blind mole rat,with the highest homology(95%) to species of the genus Spalax.Epo mRNA was detected mainly in the zokor kidney and spleen among 8 selected tissues.The level of Epo mRNA increased in the liver and kidney with increases in altitude.The increase in the kidney was 5 times that in the liver.Remarkably,expression of Epo mRNA in the kidney of zokors living at the highest altitude(4268 m) was 12-fold higher than that of zokors living at the lowest(2492 m) altitude.These findings provide essential information for understanding the possible role of Epo in adaptation to hypoxia in the plateau zokor.     

cDNA cloning and functional analysis of 4-coumarate --CoA: ligase (4CL) gene in Chinese white aspen

cDNA encoding 4-coumarate: CoA ligase (4CL) was isolated from the secondary developing xylem of Chinese white aspen (Populus tomentosa) by RT-PCR for the first time, which was 1619 bp in length. The coding sequence and putative amino acid sequence showed 97.53% and 97.00% identity to that of Pt4CL1 in quaking aspen (P. tremuloids), respectively. Molecular analysis indicated that 4CL was encoded by multiple genes in P.tomentosa, its mRNA was highly accumulated in xylem and the expression of 4CL revealed a biphasic pattern in one growing season, almost in phase with the expression of other related enzymes in lignin biosynthesis. Transgenic research showed that expression of antisense 4CL cDNA led to the decreasing of lignin content in transgenic tobaccos, among which the average reduction was 10.3% and the highest could be up to 18.9%. These data suggested that 4CL gene was a potential gene used in altering lignin biosynthesis by biotechnology for producing new materials of papermaking.     

Tissue-specific expression of glutathione S-transferases induced by 2-tridecanone or quercetin in cotton bollworms, Helicoverpa armigera (Hübner)

The tissue-specific expression of glutathione S-transferases (GSTs) in the cotton bollworm and the expression level induced by 2-tridecanone and quercetin were examined using the methods of biochemistry and the quantitative PCR. The relative expression level of GST mRNA was unanimous with the GSTs activity conjugaging with 1-chloro-2, 4-dimitro-benzene (CDNB) in fat bodies, midguts, heads and integuments of cotton bollworms. The GSTs activity in fat bodies was the highest, then midguts, heads and integuments in turn, which was in consistent with the relative expression level of GST mRNA. The specific activity of GSTs and the relative expression level of GST mRNA could be significantly induced by 2-tridecanone and quercetin, and after the induction the order of the GSTs activity and the relative expression level of GST mRNA in the above four tissues in cotton bollworms was not different from the control. The induction of GSTs by 2-tridecanone was stronger than by quercetin in all four tissues, which was in accordance with the relative expression level of GST mRNA. It suggested that the increase of GSTs activity induced by plant allelochemicals was associated with the elevated expression of GST mRNA in cotton bollworms.     

Tissue-specific expression of glutathione S-transferases induced by 2-tridecanone or quercetin in cotton bollworms, Helicoverpa armigera (Hübner)

The tissue-specific expression of glutathione S-transferases (GSTs) in the cotton bollworm and the expression level induced by 2-tridecanone and quercetin were examined using the methods of biochemistry and the quantitative PCR. The relative expression level of GST mRNA was unanimous with the GSTs activity conjugaging with 1-chloro-2, 4-dimitro-benzene (CDNB) in fat bodies, midguts, heads and integuments of cotton bollworms. The GSTs activity in fat bodies was the highest, then midguts, heads and integuments in turn, which was in consistent with the relative expression level of GST mRNA. The specific activity of GSTs and the relative expression level of GST mRNA could be significantly induced by 2-tridecanone and quercetin, and after the induction the order of the GSTs activity and the relative expression level of GST mRNA in the above four tissues in cotton bollworms was not different from the control. The induction of GSTs by 2-tridecanone was stronger than by quercetin in all four tissues, which was in accordance with the relative expression level of GST mRNA. It suggested that the increase of GSTs activity induced by plant allelochemicals was associated with the elevated expression of GST mRNA in cotton bollworms.     

Deregulation of c-myc and SV40Tag causing brain tumor in mice

Deregulated expressions of both c-myc and simian virus 40 large T antigen （SV40Tag） are consistent features of lots of tumors. To investigate whether the expression of c-myc and SV40Tag in mouse might help develop a model of human tumor, we generated c-myctransgenics by inserting human c-mycgene into pTRE2 of Tet-On system. We obtained conditional expression of SV4OTag transgenics by the Tet-On system from Yangzhou University. Crossing the c-myc transgenic mouse with the SV40Tag transgenic mice to generate bitransgenics we got double-transgenic mice expressing c-myc and SV40Tag by the Tet-On system. After being treated with doxycycline continuously, single-transgenic SV40Tag mice developed brain tumor infrequently （3 of 84, 3.6%） with a long onset （185 d on average）. In contrast, double-transgenic c-mydSV4OTag mice developed brain tumor with a short onset （96 days on average） and a 41% brain tumor incidence rate （7 of 17, 41%）. This tumor was assumed to be medulloblastoma. Our experiments suggest that deregulated expression of c-myc and SV4OTag in brain might generate a mouse model of human brain tumor that recapitulates some features of human medulloblastoma.     

Isolation of tomato proteinase inhibitor Ⅱ gene and the function of its intron

The genomic DNA sequence of tomato proteinase inhibitor Ⅱ gene (named tin2i, whose accession number in GenBank is AF007240) was isolated by PCR techniques. The intron sequence (TPI), with a length of 109 bp, owns typical structures of GT/AG dinucleotides at both ends and high content of AT base pairs which accounts for 80.7% of the total nucleotides. As shown by recombination experiment, the TPI sequence could efficiently promote the expression of the reporter gene gusA and this effect was independent of the position and orientation of the intron, thus showing its role as an enhancer. Such experiments as gel retardation assays, GUS histochemical staining and GUS fluorometric assays further demonstrated that TPI sequence maybe has promoter-like activity.