Full-length Cloning and Protein Expression Analysis of Glycosyltransferases Gene SaUGT1/SaUGT2 in Sorbus aucuparia / 中国实验方剂学杂志

Objective: To obtain the glycosyltransferase gene involved in modification reaction of phytoalexin from Sorbus pohuashanensis suspension cell,and conduct sequence analysis and prokaryotic expression analysis. Method: Based on the transcriptome data,specific primers were designed to obtain 2 cDNA sequences of SaUGTs genes,construct prokaryotic expression vector HIS-MBP-pET28a-SaUGTs and induce the expression of recombinant SaUGTs protein. Result: SaUGT1 and SaUGT2 sequences were cloned and obtained from glycosyltransferases,then bioinformatic analysis of the sequence and prokaryotic expression analysis were conducted. SaUGT1 gene contained 1 458 bp open reading frame (ORF),encoding a polypeptide of 485 amino acids,with a relative molecular weight of 54.27 kDa and theoretical isoelectric point (pI) of 5.50.SaUGT2 gene contained 1 431 bp ORF,encoding a polypeptide of 476 amino acids,with a relative molecular weight of 53.49 kDa and theoretical pI of 5.63. Bioinformatics analysis indicated that SaUGT1 and SaUGT2 protein had no signal peptide,and the conserved domains of glycosyltransferase family were detected. Phylogenetic results showed that SaUGT1 and SaUGT2 proteins had the closest relationship with the UGT85 family of A. thaliana. Differential expression analysis revealed that the relative expression levels of SaUGT1 and SaUGT2 were increased significantly after being induced by yeast extract (YE), with the highest expression level found at 24 h and 12 h. The recombinant SaUGT1 and SaUGT2 proteins were successfully expressed in Escherichia coli DE3 cells and finally,the recombinant SaUGT1 and SaUGT2 proteins were purified through Ni2+ affinity chromatography. Conclusion: The glycosyltransferase gene was cloned from the S. aucuparia for the first time,and the prokaryotic expression vector was successfully constructed,laying foundation for further study of the function of this gene.

Construction of full-length cDNA library of Sarcandra glabra and sequences analysis on EST / 中草药

Objective: Sarcandra glabra was recognized as an important research material attributing to its high medicinal value and economic value. However, little information was known about its genomics and regulatory pathway participating in reproductive development. For the first step to understand the molecular basis and further explore genes which related to metabolism and resistance in S. glabra. Methods: A SMART full-length complementary DNA library from the leaves tissue was constructed and characterized to providing the experimental basis for discovery of functional genes of S. glabra. The assembly expressed sequence tag (EST) data were completed by ABI3730 DNA program. A high quality full-length cDNA library was constructed successfully from S. glabra leaves. Results: The titer of library was 1.14×107 pfu/mL and the average length of inserted fragments was 1 000 bp. A total of 221 clones were sequenced from the cDNA library and obtained 177 EST sequences. The EST sequences were assembled into 151 unigenes including 12 contigs and 119 singletons (79%). EST exhibited significant similarity with known putative functional nucleotide sequences in the GenBank database. These genes were mostly involved in cell development, signal transduction, protein synthesis, transcription, stress tolerance response, energy metabolism based on molecular function of GO annotation. Conclusion: This report constructs a full-length-cDNA library and analyzes the bioinformatics of the related EST sequences, and then offers a reference to genomic research of S. glabra.

Cloning and expression analysis of squalene synthase gene in Tripterygium wilfordii / 药学学报

In this paper, we cloned the full-length cDNA of TwSQS from Tripterygium wilfordii suspension cells (GenBank:KR401220) and performed the bioinformation and mRNA expression analysis. The expression after methyl jasmonate (MJ) treatment of the gene was detected by RT-PCR. The full-length cDNA of TwSQS was 1800 bp containing a 1242 bp open reading frame (ORF) encoding a polypeptide of 413 amino acids. The theoretical isoelectric point (pI) was 7.94 and the calculate molecular weight was about 47.20 kD. The relative expression level of TwSQS was deduced by MJ and reached the highest at 4 h after the treatment. The gene information we got in this study enriched the biosynthesis pathway of triterpenoids in Tripterygium wilfordii and laid foundation for further studies.

Construction and identification of infectious full-length cDNA clone of enterovirus 71 / 中华微生物学和免疫学杂志

Objective To construct an infectious full-length cDNA clone of enterovirus 71(EV71)and develop a technological platform for study on vaccine development as well as molecular virology of EV71.Methods According to the nucleotide sequence of EV71 strain 085 isolated in China,four pairs of primers were designed for amplification of four end to end overlapping subgenomic cDNA fragments,the cDNA fragments were directional cloned into pBluescript SK(+)vector,and the virus genome cDNA clone was obtained by ligation orderly.The rescued virus of parental strain 085 from RNA transfected host cells was identified by RT-PCR,IFA,titration as well as transmission electron microscope(TEM)after the transcription of the full-length cDNA clone in vitro.Results The full-length cDNA clone was constructed successfully,and the typical CPE was observed after its transcription into Vero cells.The rescued virus with 20-30 nm in diameter can not only be neutralized by EV71 special anti-serum but also react with anti-EV71 monoclonal antibody that virus infected cells stained with FITC can be detected by IFA.After amplification from the total RNA extraction of virus infected cells by RT-PCR with EV71 special primers,the 226 bp products can be detected.The growth curve showed that the rescued virus can propagate in Vero cells stably with a titer of 4.5 ～6.0 lgCCID50/ml during 8 passages.The plaque formed by rescued virus is identical as parental virus in morphology but smaller in size.Conclusion An infectious full-length clone of EV71 was developed successfully,which will be used for further study on pathogenesis and vaccine development of EV71.

Identification of 1,531 cSNPs from Full-length Enriched cDNA Libraries of the Korean Native Pig Using in Silico Analysis

Sequences from the clones of full-length enriched cDNA libraries serve as valuable resources for functional genomics related studies, genome annotation and SNP discovery. We analyzed 7,392 high-quality chromatograms (Phred value >30) obtained from sequencing the 5' ends of clones derived from full-length enriched cDNA libraries of Korean native pigs including brainstem, liver, cerebellum, neocortex and spleen libraries. In addition, 50,000 EST sequence trace files obtained from GenBank were combined with our sequences to identify cSNPs in silico. The process generated 11,324 contigs, of which 2,895 contigs contained at least one SNP and among them 610 contigs had a minimum of one sequence from Korean native pigs. Of 610 contigs, we randomly selected 262 contigs and performed in silico analysis for the identification of cSNPs. From the results, we identified 1,531 putative coding single nucleotide polymorphisms (cSNPs) and the SNP detection frequency was one SNP per 465 bp. A large-scale sequencing result of clones from full-length enriched cDNA libraries and identified cSNPs will serve as a useful resource to functional genomics related projects such as a pig HapMap project in the near future

KBUD: The Korea Brain UniGene Database

Human brain EST data provide important clues for our understanding of the molecular biology associated with the function of the normal brain and the molecular pathophysiology with brain disorders. To systematically and efficiently study the function and disorders of the human brain, 45,773 human brain ESTs were collected from 27 human brain cDNA libraries, which were constructed from normal brains and brain disorders such as brain tumors, Parkinson's disease (PD) and epilepsy. An analysis of 45,773 human brain ESTs using our EST analysis pipeline resulted in 38,396 high-quality ESTs and 35,906 ESTs, which were coalesced into 8,246 unique gene clusters, showing a significant similarity to known genes in the human RefSeq, human mRNAs and UniGene database. In addition, among 8,246 gene clusters, 4,287 genes (52%) were found to contain full-length cDNA clones. To facilitate the extraction of useful information in collected these human brain ESTs, we developed a user-friendly interface system, the Korea Brain Unigene Database (KBUD). The KBUD web interface allows access to our human brain data through three major search modes, the BioCarta pathway, keywords and BLAST searches. Each result when viewed in KBUD offers comprehensive information concerning the analyzed human brain ESTs provided by our data as well as data linked to various other public databases. The user-friendly developed KBUD, the first world-wide web interface for human brain EST data with ESTs of human brain disorders as well as normal brains, will be a helpful system for developing a better understanding of the underlying mechanisms of the normal brain well as brain disorders. The KBUD system is freely accessible at http://kugi.kribb.re.kr/KU/cgi-bin/brain.pl.

Sequence analysis of full length cDNA of Schistosoma japonicum egg miracidia genes harboring signal sequence / 中国血吸虫病防治杂志

Objective To analyze the full length cDNA sequence of Schistosoma japonicum egg miracidia genes harboring signal sequence.Methods The gene specific primers were designed and synthesized according to S.japonicum egg miracidia cDNA fragment containing signal sequence identified by signal sequence trapping method previously. The 5′and 3′ end cDNA fragments of each egg miracidia cDNA fragment harboring signal sequence were amplified by nest PCR using the first strand cDNA of S.japonicum as the template. The specific PCR fragments were cloned by TA clone method and sequenced. The full length cDNA sequence of each gene with signal sequence was constructed by comparing the cDNA sequence identified with signal sequence trapping method and the 5′ end sequence, the 3′ end sequence and deleting the overlapping fragments. The splicing model between mRNA of signal sequence and one of mature portions of S.japonicum egg miracidia gene was checked by analyzing the genomic DNA sequence structure of some genes with signal sequence. Results The 5′ and 3′ end cDNA fragments of sixteen among thirty cDNA fragment with signal sequence were amplified successfully, and their DNA sequences were determined. The full length cDNA sequences of sixteen egg miracidia genes were obtained by sequence matching and splicing. The results of deduced amino acid analysis found that the signal peptide of gene SjP4001 was the same to the one of SjP1531 and the signal peptide of gene SjP1183 was similar to the one of gene SjP3742. It confirmed that different genes could share the same or similar signal peptide. The data of S.japonicum genomic DNA sequence analysis showed that the S.japonicum could obtain its signal sequence by alternative splicing model or trans-splicing model. Conclusions The full length cDNA sequences of sixteen S.japonicum egg miracidia genes with signal sequence have been defined, it indicated that the S.japonicum egg miracidia genes could get its signal sequence by alternative splicing model or trans-splicing model was found in this study.