Molecular cloning and expression of interleukin Ibeta (IL-1β) from red seabream (Pagrus major)

The interleukin 1β (IL-1β) cDNA was cloned from the red seabream (Pagrus major) by homology cloning strategy. A cDNA fragment was amplified by PCR using two degenerated primers, which were designed according to the conserved regions of other known IL-1β sequences, and elongated by 3' ends and 5' ends RACE PCR to get the full length coding sequence of red seabream IL-1β (RS IL-1β). The sequence contained 1252 nucleotides that included a 5' untranslated region (UTR) of 84bp, a 3' UTR of 410 bp and an open reading frame (ORF) of 759 nucleotides which could be translated into a putative peptide of 253 amino acids with molecular weight of 28.6 kD and putative isoelectric point pI of 5.29. The deduced peptide contained two potential N-glycosylation sites and an identifiable IL1 family signature, but lacked the signal peptide and the clear ICE cut site, which were common in other nonmammalian IL-1β genes. The RS IL-1β had the highest homology with piscine IL-1β according to phylogenetic tree analysis.     

Genomic cloning and sequencing of betaine aldehyde dehydrogenase gene in spinach

A genomic library derived from leaves of spinach was constructed with the λGem11_BamHI Arms as the vector. The library was screened using the BADH cDNA of mountain spinach as a probe and six positive clones were obtained through three rounds of screening. One of the positive clones named D, which was hybridized with the 5′600 bp fragment of mountain spinach BADH cDNA, was selected and further analyzed. The size of the insert in clone D was about 12 kb. 8 856 nucleotides of the insert were sequenced which contained 2 459 nucleotides of 5′ noncoding region, 6 111 nucleotides of the complete sequence of the BADH gene, and 286 nucleotides of a 3′ noncoding region. The result of sequence analysis indicated that the BADH gene contained 14 introns and the junction sequences at splicing sites followed the GT_AG rule basically.     

Isolation and ectopic expression of a bamboo MADS-box gene

A cDNA named DIMADS18 was isolated from the young spikelets of the sweet bamboo, Dendrocalamus latiflorus by RACE. DNA sequence analysis showed that DIMADS18 was composed of full ORF and 3UTR, but without 5UTR. The cDNA contained 1039 nucleotides and encoded a putative protein of 249 amino acid residues. The gene displayed the structure of a typical plant MADS box gene, which consisted of an MADS domain, K domain, a short I region, and the C-terminal region. Phylogenetic analysis of plant MADS box genes based on amino acid sequences revealed that DlMADS18 was grouped into the AGAMOUS-LIKE 6 (AGL6)-like subfamily. It was most likely homologous to the OsMADS6 of rice (Oryza sativa), with 88% sequence identity for the entire amino acid sequences. The DlMADS18 also showed relatively high amino acid sequence identity (59%) to AGL6 ofArabidopsis thaliana. To study the functions of DlMADS18, DlMADS18 cDNA clone driven by the CaMV 35S promoter was transformed into Arabidopsis plants. Transgenic plants of DlMADS18 exhibited the phenotypes of curled leaves, dwarfism, and early flowering with clustered terminal flowers. These results indicated that DlMADS18 may probably be involved in controlling the flowering time of D.latiflorus.     

Emergence of a new satellite RNA from cucumber mosaic virus isolate P1

The cucumber mosaic virus (CMV) isolate P1 caused very mild symptoms on many plant species.After serial passages by mechanical inoculation over five years, CMV P1 caused severe symptoms on several tobacco cultivars and tomato. A specific band of approximately 0.3 kb in length was amplified by RT-PCR with primers synthesized based on reported CMV satellite RNA (satRNA) sequences. Sequence analysis showed there were two satRNAs (Sat-Pl-1 and Sat-P1-2). Sat-Pl-1 contained 335 nucleotides, and Sat-P1-2 contained 394 nucleotides. These two satRNAs shared 64% overall nucleotide sequence homology, and differences between the two satRNAs included mutations as well as deletions. Sat-Pl-1 was identical to a satRNA (Z96099) reported in 1995 in CMV P1. Based on differences in the sequence and secondary structure between these two satRNAs, we conclude that Sat-P1-2 represents the emergence of a new satellite ( necrotic satellite) from attenuated satRNA populations. The possible effect of the emergence of this new satRNA is discussed.     

A complete sequence and comparative analysis of a SARS-associated virus （Isolate BJO1）

The genome sequence of the Severe Acute Respiratory Syndrome (SARS)-assoclated virus provides essential information for the identification of pathogen(s), exploration of etiology and evolution, interpretation of transmission and pathogenesis, development of diagnostics, prevention by future vaccination, and treatment by developing new drugs.We report the complete genome sequence and comparative analysis of an isolate (B J01) of the coronavirus that has been recognized as a pathogen for SARS. The genome is 29725 nt in size and has 11 ORFs (Open Reading Frames). It is composed of a stable region encoding an RNA-dependent RNA polymerase (composed of 20RFs) and a variable region representing 4 CDSs (coding sequences) for viral structural genes (the S, E, M, N proteins) and 5 PUPs (putative uncharacterized proteins). Its gene order is identical to that of other known coronaviruses. The sequence alignment with all known RNA viruses places this virus as a member in the family of Coronaviridae. Thirty putative substitutions have been identified by comparative analysis of the 5 SARS-associated virus genome sequences in GenBank. Fifteen of them lead to possible amino acid changes (non-synonymous mutations) in the proteins. Three amino acid changes, with predicted alteration of physical and chemical features, have been detected in the S protein that is postulated to be involved in the immunoreactions between the virus and its host.Two amino acid changes have been detected in the M protein,which could be related to viral envelope formation. Phylogenetic analysis suggests the possibility of non-human origin of the SARS-associated viruses but provides no evidence that they are man-made. Further efforts should focus on identifying the etiology of the SARS-associated virus and ruling out conclusively the existence of other possible SARS-related pathogen(s).     

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.     

Complete Nucleotide Strain Sequence of a Newly Avirulent Newcastle Disease Virus Hubei 92（HB92） Strain

A new avirulent, heat-resistance HB92 strain of newcastle Disease Virus (NDV) was acquired from Australia V4 strain. Its complete nucleotides sequence was first determined. The entire genome of NDV HB92 consists of 15 186 nucleotides (GenBank accession no. AY225110). It was demonstrated by sequence analysis that nucleotides homology of HB92 strain with virulent strain ZJ1 were 83.9%, and the homology compared with avirulent vaccine strain La Sota and B1 were 94. 0% and 93. 5%, respectively. These results might be contributive to the study of the molecular mechanism of evolution of the NDV strain HB92 and to develop the engineered recombinant vaccine.     

Expression of the intact C<Subscript>4</Subscript> type<Emphasis Type="Italic">pepc</Emphasis> gene cloned from maize in transgenic winter wheat

Maize intact C4-pepc gene was amplified through LA-PCR and successfully sub-cloned into modified vector pGreen0029 to form a stable expression construct named as pBAC214 (12 kb), which contains CaMV 35S promoter driven bar gene as selection marker. Comparing the cloned DNA sequences (6.7 kb) with published maize C4-pepc gene (GenBank accession E17154) sequences, the identity of DNA sequence alignment is 98.96%. There are only 49 differences between these two intact DNA sequences, of which 13 occur in the region of promoter, 18 in introns, and 18 in exons. The homology of mRNA sequence alignment is 99.38%, and the putative amino acids sequence identity is 99.38%. There are only 15 differences between these two mRNA, and these differences bring 4 sites mutant on the putative amino acids of PEPC protein. Through biolistic bombardment of PDS1000/He system, expression vector pBAC214 has been transformed into winter wheat. Southern blotting results show that the intact C4-pepc gene has been integrated into genome of winter wheat. SDS-PAGE analysis of leaf soluble protein in transgenic wheat showed that the intact C4opepc gene was well transcribed, spliced and translated as in maize. The enzyme activity of leaf PEPC in transgenic wheat has been detected. The activities of leaf PEPC increased over 3-5 times in some transgenic plants. The data of photosynthesis rate and transpiration rate of transgenic wheat flag leaves showed that the C4-pepc gene can increase the photosynthesis rate and transpiration rate of transgenic wheat.     

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.     

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.