Cloning and expression analysis of RcUDPGTs genes in Tibetan Rhodiola crenulata / 药学学报

UDP glucosyltransferase (UDPGT) catalyzes the synthesis of secondary metabolites and plant hormones to regulate plant growth and development, pathogen defense and environmental adaptability. In this study 18 members of the RcUDPGT gene family were cloned from Tibetan Rhodiola crenulata and analyzed using bioinformatics. The tissue-specific expression, abiotic stresses and plant hormones (abscisic acid, auxin, methyl jasmonate) induced expression patterns were identified by real-time quantitative PCR. The bait vector of RcUDPGT (JX228125.1) was constructed to select interacting proteins from an Arabidopsis yeast library. The results of the bioinformatics analysis revealed that RcUDPGT nucleotide sequences were about 1 400 bp and encoded 452-498 amino acids. In the primary protein sequences, C-terminal sequences were more conserved compared with N-terminal regions, which held a PSPG (plant secondary product glycosyltransferase) domain. In the tertiary structures, RcUDPGTs contained a UDP sugar donor recognition binding site. In addition, all genes had multiple phosphorylation sites. The results of qRT-PCR showed that RcUDPGTs genes were expressed in root, stem and leaf. The expression levels were regulated by low temperature/ultraviolet light and various plant hormones (ABA, IAA, MeJA), but the expression patterns were quite different among them. For example, RcUDPGT6, RcUDPGT11, and RcUDPGT17 had the highest expression in leaves and were induced by all three hormones, suggesting that the functions of these genes might be to respond to environmental changes. RcUDPGT9, RcUDPGT10, RcUDPGT14 were most abundantly expressed in roots and were significantly induced by ABA and MeJA hormones, indicating that these genes may be involved in the synthesis and accumulation of salidroside. Yeast two-hybrid results showed that RcUDPGT did not exhibit autoactivation and cell toxicity, and two significant interactional genes were identified, AtKCR1 (AT1G67730.1) and AtSNL4 (AT1G70060). The AtKCR1 gene encodes a β-ketoacyl reductase (KCR) involved in synthesis of very long chain fatty acids. The AtSNL4 gene encodes a homolog of the transcriptional repressor SIN3, which could participate in the ABA hormone signaling pathway and enhance the transcriptional repression of AP2/EREBP class factors in Arabidopsis. These results suggest that the accumulation of the secondary metabolite salidroside in Rhodiola crenulata might be affected by several regulatory mechanisms. The above results may lay the foundation for understanding the adaptive mechanism of Rhodiola crenulata in a high altitude environment and stimulate an in-depth study of the synthesis and accumulation of secondary metabolites in this species.

Bio-molecular Mechanism of Celastrol Anti-diabetes and Its Complications Based on Biological Information Database / 中国药学杂志

OBJECTIVE: To investigate the biomolecular anti-diabetes mechanism and its complications of celastrol based on bioinformatics database. METHODS: The related genes of celastrol were obtained from the bioinformatics database, the cross-related genes of celastrol and diabetes mellitus were searched by Veen diagram software, the protein-protein interaction network was constructed by STRING software, the related KEGG pathway was identified by the DAVID software and KOBAS software, and the molecular network of anti-diabetes "compound-target gene-pathway-disease" was further constructed by R program software. RESULTS: There were 50 genes related to diabetes in celastrol, and 11 related pathways (P<0.001), which mainly affected the AGE-RAGE signaling pathway in diabetic complications. The mechanism was to regulate AGE-RAGE-NF-κB axis by inhibiting NF-κB, which reduced the production of downstream inflammatory effectors, insulin resistance and fibrotic damage. CONCLUSION: Celastrol can improve the symptoms of diabetes and its complications by regulating the AGE-RAGE-NF-κB axis-related signaling pathway, which is expected to be a potential drug for the treatment of diabetes and its complications.

Cloning, bioinformatics, and expression analysis of squalene epoxidase in Antrodia cinnamomea / 中草药

Objective To clone the squalene epoxidase gene of Antrodia cinnamomea (AcSE) and analyze the bioinformatics and expression of the gene. Methods AcSE was cloned by rapid-amplification of cDNA ends (RACE) from cDNA of A. cinnamomea. The physical and chemical properties of AcSE protein were analyzed, and its secondary structure, tertiary structure, and function were predicted by using bioinformatics analysis. The expression of AcSE in mycelium and fruit body of A. cinnamomea at different culture time was detected by using quantitative real-time PCR (qRT-PCR). Results The full-length cDNA sequence of AcSE were 1 446 bp (Genbank: KT070558), encoding a 481-amino-acid polypeptide. The molecular weight of AcSE was 53 300 and pI was 6.36. Domain analysis results showed that AcSE had three transmembrane domains without coiled-coil structure, and hydrophobic and hydrophilic regions existed alternately. The gDNA sequence of AcSE was 1 607 bp, contained four exons and three introns. A gene expression analysis by relative qRT-PCR showed that the highest expression level of AcSE was in mycelia incubated for 7 d of A. cinnamomea, and it was 7.89 times than that in fruiting body, and a gradual decline was observed with the extension of the culture time. Conclusion Gene AcSE was firstly cloned from A. cinnamomea, and it would lay a foundation for exploring the mechanism of terpenoid biosynthesis in A. cinnamomea.

Cloning of flavanone 3-hydroxylase gene from Lithocarpus polystachyus and its sequence analysis / 中草药

Objective: To clone flavanone 3-hydroxylase (F3H) gene from Lithocarpus polystachyus, and to understand its gene characteristics and initially investigate its expression level in different organs. Methods: The total RNA and genomic DNA from blade of L. polystachyus were extracted. Based on the result of RNA-seq, a pair of specific primers were designed. cDNA and DNA sequences of F3H gene from L. polystachyus were amplified by PCR, then bioinformation analysis was performed after sequencing. The expression level of F3H gene in different organs of L. polystachyus was detected by qRT-PCR. Results: The full length of cDNA of F3H gene was 1 340 bp containing a 1 092 bp open reading frame that encoded 393 amino acids, and F3H was located in the cytoplasm. The result of qRT-PCR showed that F3H gene expressed in different organs of L. polystachyus, and the expression levels of F3H gene were significantly different in different organs (P < 0.05). Conclusion: The F3H gene of L. polystachyus was cloned and its bioinformation was analyzed for the first time, proving that the expression level of F3H gene in different organs of L. polystachyus was different. This finding lays a foundation for the studies on secondary metabolism of flavonoids in L. polystachyus.

Cloning and expression analysis of 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase gene in Cinnamomum camphora / 中国中药杂志

The 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase was the fourth key enzymes in plant terpenoid biosynthesis pathway of methyl erythritol phosphate pathway(MEP). According to the study of Cinnamomum camphora transcriptome data,we abtained the 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase gene using RT-PCR,and named CcCMK1,then deposited it in GeneBank(Accession number： Ku376098).Bioinformatics analysis showed the open reading frame (ORF) of the CcCMK1 was 1 212 bp.The putative protein encoded 403 amino acids,and its molecular weight was 44.46 kDa and theoretically isoelectric point was 4.99.Transmembrane structure analysis showed that there was no transmembrane structure. Signal peptide analysis showed that it was a non secretory protein, and there was no signal peptide. The subcellular localization showed that the chloroplast was located in the chloroplast.Analysis of the expression of CcCMK1 gene in five chemotypes of C. camphora using Real-time PCR showed its expression level was highest in C. longepaniculatum, and the lowest in Borneol camphor.This research provided a basis for characterizing the key enzyme genes of terpenoid biosynthetic pathway in C. camphora.

Characterization and gene function analysis of SSR sequences in Poria cocos transcriptome / 中草药

Objective: To develop new molecular markers for Poria cocos, and to characterize the SSR in P. cocos transcriptome. Methods: The transcriptome Ungenes and genomic scaffolds were examined by the tool of MISA. The gene annotation and gene function cluster were obtained by blasting the Unigenes which contained SSR to the nr and KEGG databases with BlastX. Results: A total of 2 075 SSRs were identified in 4.57% Unigene sequences, the density of distribution was average one SSR per 17.01 kb, and the average length of SSR was 19.59 bp. Meanwhile, those were 54.00 SSRs per Mb, and 20.74 bp in genomic sequences. Among all 241 SSR motifs found in transcriptome, (CG/CG)n which accounted for 10.97% was the most frequent repeat motif. And hexa-nucleotide repeats which accounted for 35.64% was the most group among mono- to hexa-nucleotide repeats. (ACCACG/CGTGGT)14 with the length of 84 bp was the longest SSR. Only 115 Unigenes of 1 887 Unigenes containing SSR were annotated to cellular metabolic process or nucleotide binding, etc, with GO classification. On the other hand, 1 223 Unigenes containing SSR annotated into 219 KEGG pathway maps. 314 and 297 Unigenes of them were annotated into metabolism pathways and genetic information processing pathways, respectively. Conclusion: The SSR in the transcriptome of P. cocos is rich in type, and has a high potential of polymorhpism. Associating gene function, SSR might be applied in marker-assisted breeding with the aim of specific traits.

Analysis of differential microRNA expression in patient with gallbladder stones through high-throughput sequencing technologies / 天津医药

Objective To detect the differential expression profile of microRNAs between patients with or without gall?bladder stone. Methods Samples from 30 patients with gallbladder stones (GS) and 30 without gallbladder stones (GP) were collected, in which microRNAs expression profiles were examined using high-throughput sequencing instrument Illumi?na HiSeq 2500. MicroRNA sequences were obtained and compared to Genebank and Rfam database for classification. Differ?entially expressed microRNAs were screened, and their target genes were predicted. Significant enrichment analysis of GO and KEGG were performed. Real-time quantitative PCR was performed on selected miRNAs in order to validate their expres?sion. Results Clean tags were obtained from both GS group (n=2 215 832) and GP group (n=1 424 770). A total of 17 mi?croRNAs were differentially expressed between GS and GP groups with statistical significance, among which 9 were up-regu?lated and 8 were down-regulated in GS group compared to those in GP group. GO (Gene ocology) analysis showed that target genes were enriched in ion binding and transport, apolipoprotein binding, calcium channel activity, protein kinase activity, steroid hormone biosynthesis and metabolism. KEGG(Kyoto Encyclopedia of Genes and Genomes)analysis is shown for the target genes enriched in cancer related pathways, including WNT, HIPPO pathways. qRT-PCR validation of some differen?tially expressed miRNAs confirmed the result of high-throughput data analysis. Conclusion The differential expression levels of microRNAs may play an important role in occurrence and development of gallbladder stones.

Cloning, bioinformatic analysis, and expression of chalcone synthase gene in Carthamus tinctorius / 中草药

Objective: To clone the chalcone synthase (CHS) gene in Carthamus tinctorius, to analyze the bioinformation of CHS, to compare the expression of CHS during the florescence, and to provide the foundation for composition and regulation mechanism of the active ingredients in C. tinctorius. Methods: RNA was obtained from fresh safflower corolla, cDNA was reversely transcriped, specific primers were designed, and then CHS was cloned. The protein characteristics was analyzed using bioinformatics, the phylogenetic tree of CHS was constructed using MEGA5.1, the expression of CHS during the florescence was analyzed using real time-PCR. Results: The 1 149 bp CHS sequence in C. tinctorius was obtained, which has a 1 041 bp ORF, encoding 346 amino acids. This protein belongs to the CHS family according to Blastp in NCBI. The CHS in safflower was similar to that in above 100 plants, and the similarities to Silybum marianum, Callistephus chinensis, Chrysanthemum x morifolium, and Gynura bicolor were respectively reaching 95%, 95%, 94%, and 94%. It has the closest relationship to S. marianum according to the phylogenetic tree using MEGA5.1. The CHS formula was C1678H2693N451O493S20 and the molecular weight was 37 700, with the isoelectric point of 6.10. The number of negatively charged amino acid residues (Asp + Glu) was 42, and the number of positively charged amino acid residues (Arg + Lys) was 38. The gene expression analysis showed that the highest expression of CHS in safflower was on day 3 of florescence, much higher than that on the other days. Conclusion: The CHS in safflower is successfully cloned, analyzed, and expressed, which provides the foundation for composite and regulation mechanism of the active ingredients in C. tinctorius.

Review on the application of drug target database / 中国药学杂志

OBJECTIVE: To help researchers learn and utilize drug target database for new drug research, and to provide important information for the management and construction of Drug Target Database.

Cloning and bioinformation analysis of chalcone synthase gene from Lycium barbarum in Ningxia / 中草药

Objective: To obtain cDNA of chalcone synthase (CHS, EC 2.3.1.74) involved in the flavonoid/isoflavonoid biosynthesis pathway. Methods: The partial sequence of CHS in Lycium barbarum (LyCHS) was successfully cloned by RT-PCR and using a sequence homology strategy, and the bioinformation analysis was carried out. Results: The cDNA fragment of CHS gene was 1148 bp in length, which encoded a protein of 382 amino acids with the predicted relative molecular weight of 4.168 × 104. The estimated isoelectric point (pI) of the putative protein is 6.22. According to the amino acid sequence and structural analysis, it showed that this protein contained one conserved active site, namely chalcone and stilbene synthases active site. Subcellular localizations of LyCHS proteins were likely in the cytoplasm. The secondary and tertiary structures of LyCHS were abundant in α-helixs (166) and random coils (126), while were less in β-turns (25) and extended strains (65). Phylogenetic analysis showed that the genes of LyCHS were closely related to CHS in Solanum pinnatisectum, S. tuberosum, and Lycopersicon esculentum. The sequences had been registered in GenBank with the accession numbers JQ964237. Conclusion: The present results provide the foundation for the next study of CHS gene about its expression and function in L. barbarum.

Caracterización de cepas cubanas de VIH-1 por ensayos biológicos y métodos bioinformáticos / Characterization of Cuban strains of HIV-1 by bioassays and bio-information methods

Mediante ensayos biológicos clásicos se ha podido determinar el uso del correceptor por las cepas de VIH-1, las cuales se han clasificado en R5, X4 o R5/X4, características que guardan relación con el fenotipo NIS o IS y con la evolución clínica. Por métodos bioinformáticos se han relacionado cambios aminoacídicos en la región del lazo V3 del env con el uso del correceptor. En este trabajo se presentan los resultados del estudio de caracterización de once cepas provenientes de individuos infectados con VIH-1, cinco de ellos seropositivos asintomáticos y seis progresores rápidos al SIDA. Se compararon los resultados del estudio fenotípico realizado por dos ensayos biológicos clásicos, uso de correceptores y capacidad de inducir sincicios en MT2 y tres métodos bioinformáticos, regla 11/25, matrices de puntos en posiciones específicas (PSSM) y el programa geno2pheno. Las cinco cepas de seropositivos asintomáticos coincidieron por todos los métodos como R5/NIS. Cinco de las seis cepas de progresores rápidos se clasificaron R5/X4 e IS por los ensayos biológicos; mientras dos clasificaron R5/X4 por Geno 2 Pheno, una por PSSM y todas fueron X4 puras por la regla 11/25. Los ensayos utilizados en el estudio permitieron caracterizar las cepas aisladas y relacionar el fenotipo con la evolución de la infección, por lo que valoramos que por ensayos biológicos o métodos bioinformáticas se pueden hacer estudios de caracterización, su utilidad está en dependencia del objetivo que se persiga

By the classic biological assays it has been possible to determine the use of HIV-1 strains co-receptor, which have been classified in R5, X4 or R5/X4, characteristic related to the NIS or IS phenotype and to the clinical course. By bio-information methods the amino acid changes in the region of V3 loop of the env using the co-receptor. In present paper are showed the results of a characterization study of eleven strains from HIV-1 infected subjects, five seropositive and six with a fast progression to AIDS. Authors compared the results of phenotype study conducted by two classic assays, use of co-receptors and ability to induce syncytia in MT2 and three bio-information methods, rule 11/25, point matrices in specific locations (PSSM) and the geno2pheno program. The five strains from asymptomatic seropositive patients coincided according all methods used as R5/NIS. Five of the six strains from fast progression patients were classified as R5/X4 and IS by biological assays; whereas two were classified as R5/X4 by Geno 2 Pheno, one by PSSM and all were pure X4 by the rule 11/25. The assays used in the study allowed us to characterize the isolated strains and to relate the phenotype to the infection course, thus, we considered that using biological assays or bio-information methods it is possible to conduct characterization studies, its usefulness depends on the pursued objectives