[Cloning,subcellular localization and spatio-temporal expression analysis of a flavonoid 3-O-glucosyltransferase gene( SmUF3GT) in Salvia miltiorrhiza].

The family of flavonoid 3-O-glucosyltransferase catalyzes the modification of anthocyanin from unstable-structure to stable-structure. In this study,based on homology cloning and transcriptome library,we isolated the full-length c DNA of UDP-glucose: flavonoid 3-O-glucosyltransferase( named SmUF3GT) from the flower tissues of S. miltiorrhiza. This gene was consisted of 1 353 bp open reading frames( ORF) encoding 450 amino acids. And the SmUF3GT protein was performed for the bioinformatic analysis. Our results showed that the protein was preliminary localized in the Golgi and peroxisome of cytosol,as well as plasma membrane and cell nuclear.QRT-PCR analyses indicated that SmUF3GT expressed differently in all tissues and organs but roots of S. miltiorrhiza and S. miltiorrhiza f.alba. During floral development,the expression of SmUF3GT showed a trend of rising fist and then down in purple-flower Danshen,whereas decreasing sharply fist and then slowly in white-flower Danshen. The present study provides basic information for further research on the network of synthesis and accumulation of flavonoids in S.miltiorrhiza.

[Cloning and expression analysis of protein kinase SmSnRK2.4 from Salvia miltiorrhiza].

Sucrose non-fermenting 1-related protein kinase 2(SnRK2) plays a key role in abiotic stress signaling in plants. In this study, we cloned a SmSnRK2.4 gene belonging to subclass I of SnRK2 from Salvia miltiorrhiza by screening its transcriptome database. The SmSnRK2.4 gene contains 8 introns and 9 exons, with a 1 068 bp open reading frame encoding a polypeptide of 355 amino acids, the predicted molecular mass of which is 40.63 kDa. Prokaryotic expression of SmSnRK2.4 protein using pMAL-c2X as the expression vector displayed that the recombinant protein of SmSnRK2.4 gene in E. coli was consistent with the predicted size. A 3 000 bp promoter sequence of SmSnRK2.4 contained some stress-responsive elements and hormone-responsive elements. Quantitative real-time PCR analysis revealed that the expression of SmSnRK2.4 in root was much higher than that in stem and leaf, SmSnRK2.4 was strongly induced by PEG stress, weakly induced by ABA stress. This research provided a basis for further study of the SmSnRK2.4 gene playing the role in accumulate mechanism of secondary metabolites in S. miltiorrhiza under drought.

Targets for human encoded microRNAs in HBV genes.

MicroRNAs (miRNAs) are increasingly being shown to play vital roles in development, apoptosis, and oncogenesis by interfering with gene expression at the post-transcriptional level. miRNAs, in principle, can contribute to the repertoire of host-pathogen interactions during infection by the Hepatitis B virus (HBV). Using a consensus-scoring approach, high-scoring miRNA-target pairs were selected, which were identified by four well-established target-prediction softwares. The miRNAs miR-7, miR196b, miR433, and miR511 target the polymerase or S gene of HBV, miR205 targets the X gene, and miR345 targets the preC gene. The minimum free-energy values for the bound complexes were the lowest, and the rules so far observed for miRNA-target pairing, namely, (1) pairing at a continuous stretch of 6-7 bases toward the 5'-end of the miRNA and (2) incomplete complementarity with the target sequence, were found to be valid. The target regions were highly conserved across the various clades of HBV. miRNA expression profiles from previously reported Solexa-sequencing based experiments showed that the four human miRNAs are expressed in the liver. This is the first report of human miRNAs that can target crucial HBV genes.

HBV-encoded microRNA candidate and its target.

MicroRNAs (miRNAs) are a group of short (approximately 22 nt) noncoding RNAs that specifically regulate cellular gene expression at the post-transcriptional level. miRNA precursors (pre-miRNAs), which are imperfect stem loop structures of approximately 70 nt, are processed into mature miRNAs by cellular RNases III. To date, hundreds of miRNAs and their corresponding targets have been reported in kinds of species. Although only a few of these miRNA/target pairs have been functionally verified, some do play important roles in regulating normal development and physiology. Several viruses (e.g. the Epstein-Barr virus and human herpesvirus Kaposi's sarcoma-associated herpesvirus) has been reported to encode miRNAs. Here, we extend the analysis of miRNA-encoding potential to the Hepatitis B virus (HBV). Using computational approaches, we found that HBV putatively encodes only one candidate pre-miRNA. We then matched deduced mature miRNA sequence from this pre-miRNA against a database of 3' untranslated sequences (UTR) from the human genome. Surprisingly, none of cellular transcripts could potentially be targeted by the viral miRNA (vmiRNA) sequence. However, one viral mRNA was found to be targeted by the vmiRNA when we searched the target from viral mRNAs. We propose that HBV has evolved to use vmiRNAs as a means to regulate its own gene expression for its benefit.