Quan-Hong Yao

Microbial biodegradation of polyaromatic hydrocarbons

Polycyclic aromatic hydrocarbons (PAHs) are widespread in various ecosystems and are pollutants of great concern due to their potential toxicity, mutagenicity and carcinogenicity. Because of their hydrophobic nature, most PAHs bind to particulates in soil and sediments, rendering them less available for biological uptake. Microbial degradation represents the major mechanism responsible for the ecological recovery of PAH-contaminated sites. The goal of this review is to provide an outline of the current knowledge of microbial PAH catabolism. In the past decade, the genetic regulation of the pathway involved in naphthalene degradation by different gram-negative and gram-positive bacteria was studied in great detail. Based on both genomic and proteomic data, a deeper understanding of some high-molecular-weight PAH degradation pathways in bacteria was provided. The ability of nonligninolytic and ligninolytic fungi to transform or metabolize PAH pollutants has received considerable attention, and the biochemical principles underlying the degradation of PAHs were examined. In addition, this review summarizes the information known about the biochemical processes that determine the fate of the individual components of PAH mixtures in polluted ecosystems. A deeper understanding of the microorganism-mediated mechanisms of catalysis of PAHs will facilitate the development of new methods to enhance the bioremediation of PAH-contaminated sites.

Genome-wide analysis of the AP2/ERF gene family in Populus trichocarpa.

Populus is a model system for investigating the wood development, crown formation, and disease resistance in perennial plants. AR2/ERF is a large family of transcription factors in plant, encoding transcriptional regulators with a variety of functions involved in the developmental and physiological processes. Here, starting from database of Populus genome, we identified 200 AP2/ERF genes by in silico cloning method using the AP2/ERF conserved domain amino acid sequence of Arabidopsis thaliana as probe. Based on the number of AP2/ERF domains and the function of the genes, those AP2/ERF genes from Populus were classified into four subfamilies named the AP2, DREB, ERF, RAV, and a soloist. Among these genes, the number genes of total AP2/ERF family genes, DREB subfamily, and ERF subfamily from Populus trichocarpa were about 1.4-1.6-fold than those from A. thaliana. The rates were very similar for the putative homologs between Populus and Arabidopsis.

PCR-based accurate synthesis of long DNA sequences

Here we describe a simple and rapid method for assembly and PCR-based accurate synthesis (PAS) of long DNA sequences. The PAS protocol involves the following five steps: (i) design of the DNA sequence to be synthesized and of 60-bp overlapping oligonucleotides to cover the entire DNA sequence; (ii) purification of the oligonucleotides by PAGE; (iii) first PCR, to synthesize DNA fragments of 400–500 bp in length using 10 inner (template) and two outer (primer) oligonucleotides; (iv) second PCR, to assemble the products of the first PCR into the full-length DNA sequence; and (v) cloning and verification of the synthetic DNA by sequencing and, if needed, error correction using an overlap-extension PCR technique. This method, which takes ∼1 wk, is suitable for synthesizing diverse types of long DNA molecule. We have successfully synthesized DNA fragments from 0.5 to 12.0 kb, with high G+C content, repetitive sequences or complex secondary structures. The PAS protocol therefore provides a simple, rapid, reliable and relatively inexpensive method for synthesizing long, accurate DNA sequences.

AtCPK6, a functionally redundant and positive regulator involved in salt/drought stress tolerance in Arabidopsis

The calcium-dependent protein kinase (CDPK) family is needed in plant signaling during various physiological pathways. The ArabidopsisAtCPK6 gene belongs to the subclass of stress-inducible CDPKs, which is stimulated by salt and osmotic stress. To elucidate the physiological function of AtCPK6, transgenic Arabidopsis plants under the control of double CaMV 35S promoter were obtained. AtCPK6 over-expressing plants showed enhanced tolerance to salt/drought stresses. The elevated tolerance of the AtCPK6 over-expressing plants was confirmed by the change of proline and malondialdehyde (MDA). Real-time PCR analyses revealed that the expression levels of several stress-regulated genes were altered in AtCPK6 over-expressing plants. However, cpk6 mutant displayed no obvious difference with control. These results are likely to indicate that AtCPK6 is functionally redundant and a positive regulator involved in the tolerance to salt/drought stress in Arabidopsis.

Chemical gene synthesis : strategies, softwares, error corrections, and applications

Chemical synthesis of DNA sequences provides a powerful tool for modifying genes and for studying gene structure, expression and function. Modified genes and consequently protein/enzymes can bridge genomics and proteomics research or facilitate commercial applications of gene and protein technologies. In this review, we will summarize various strategies, designing softwares and error correction methods for chemical gene synthesis, particularly for the synthesis and assembly of long DNA molecules based on polymerase cycling assembly. Also, we will briefly discuss some of the major applications of chemical synthesis of DNA sequences in basic research and applied areas.

Expression of the Human Hepatitis B Virus Large Surface Antigen Gene in Transgenic Tomato Plants

ABSTRACT The original hepatitis B virus (HBV) large surface antigen gene was synthesized. In order to optimize the expression of this gene in tomato plants, the tobacco pathogenesis-related protein S signal peptide was fused to the 5′ end of the modified gene and the sequence encoding amino acids S, E, K, D, E, and L was placed at the 3′ end. The gene encoding the modified HBV large surface antigen under the control of a fruit-specific promoter was constructed and expressed in transgenic tomato plants. The expression of the antigen from transgenic plants was confirmed by PCR and reverse transcriptase PCR. Enzyme-linked immunoassays using a monoclonal antibody directed against human serum-derived HBsAg revealed that the maximal level of HBsAg was about 0.02% of the soluble protein in transgenic tomato fruit. The amount of HBsAg in mature fruits was found to be 65- to 171-fold larger than in small or medium fruits and leaf tissues. Examination of transgenic plant samples by transmission electron microscopy proved that HBsAg had been expressed and had accumulated. The HBsAg protein was capable of assembling into capsomers and virus-like particles. To our knowledge, this is the first time the HBV large surface antigen has been expressed in plants. This work suggests the possibility of producing a new alternative vaccine for human HBV.

A direct and efficient PAGE-mediated overlap extension PCR method for gene multiple-site mutagenesis

A simple, two-step efficient method to perform multiple-site mutagenesis of a gene from bacterial genome was developed. The method was named polyacrylamide gel electrophoresis (PAGE)-mediated overlap extension polymerase chain reaction (PCR) (POEP). The first step involves synthesis of individual fragments containing mutant sites with 15- to 25-bp overlap between two adjacent fragments. Mutations were introduced into the overlapping oligonucleotide primers which ensured the particular primer-template annealing. PAGE was used to remove contaminating parental templates, mispriming fragments, and leftover primers. The second step involves synthesis of the mutant full-length fragment. All purified PCR products from the first step were combined and used as the template for a second PCR using high-fidelity DNA polymerase, with the two outermost flanking oligonucleotides as primers. Using the POEP method, we have successfully introduced eight EcoRI sites into the Escherichia coli β-galactosidase (Lac Z) gene. The overall rate of obtaining the multiple mutant sites was 100%. The POEP method is simple, involving only two steps, and reliable for multiple-site mutagenesis and is promising to be widely used in gene modification.

Discovery and expression profile analysis of AP2/ERF family genes from Triticum aestivum

Throughout its development, common wheat, Triticum aestivum responds to different kinds of adverse abiotic and biotic stress by expressing specific genes that allow it to adapt to these stresses. In this process, genes in the AP2/ERF family encode transcriptional regulators involved in diverse developmental and physiological processes play critical roles. Here, we established an extensive picture of the AP2/ERF family genes in wheat. From 960, 174 ESTs of T. aestivum, 117 putative AP2/ERF family genes were identified by in silico analysis based on the presence of the conserved AP2/ERF domain amino acid sequence of Arabidopsis thaliana. Based on the model species A. thaliana, the AP2/ERF TFs from T. aestivum were classified into five subfamilies with the following number of members: DREB (57), ERF (47), AP2 (9), RAV (3) and Soloist (1). Using the available EST information as a source of expression data, the putative AP2/ERF family genes from T. aestivum were detected in nine kinds of tissues. Transcripts of the genes were shown to be most abundant in leaves, followed by roots and seeds, and the least abundant in stem. Most of the T. aestivum AP2/ERF family genes showed some tissue specificity.

OsNAC52, a rice NAC transcription factor, potentially responds to ABA and confers drought tolerance in transgenic plants

A novel NAC (NAM, ATAF1/2, CUC2) homologous gene, OsNAC52, was isolated from Oryza sativa L using RACE (rapid amplification of cDNA ends). Analysis of the amino acid sequence of OsNAC52 revealed a highly conserved NAC domain and a potential nuclear localization sequence in its N-terminus, and the transcriptional activation motif in the C-terminal region. Transgenic plants over-expressing OsNAC52 were highly sensitive to ABA (Abscisic acid), and the growth of the 35S-OsNAC52 transgenic seedlings was significantly more restrained by ABA treatment than those of the wild-type seedlings. Furthermore, over-expression of OsNAC52 activated the expression of downstream genes in transgenic Arabidopsis, resulting in enhanced tolerance to drought stresses but not growth retardation. The results from this study indicate that this novel rice OsNAC52 gene functions as an important transcriptional activator in ABA-inducible gene expression and may be useful in improving plant tolerance to abiotic stress.

High level expression of a recombinant acid phytase gene in Pichia pastoris.

Aims:  To achieve high phytase yield with improved enzymatic activity in Pichia pastoris.