Biomedical subjects

Quan-Hong Yao

Publications and source records attributed to Quan-Hong Yao.

At least 19 recordsLinked to original sources

PubMed2006

Isolated and characterization of a cDNA encoding ethylene-responsive element binding protein (EREBP)/AP2-type protein, RCBF2, in Oryza sativa L.

A transcription factor RCBF2 which interacts with C-repeat/DRE was isolated from Oryza sativa L. by a yeast one-hybrid method. Analysis of the deduced RCBF2 amino acid sequence revealed that RCBF2 contained a conserved ethylene-responsive element binding protein (EREBP)/AP2 domain of 59 amino acids and a potential nuclear localization sequence. RCBF2 showed a high level of homology with other CBF family members only in AP2 domain. Phylogenetic analysis showed that RCBF2 might be different from other eight DRE-binding proteins on evolutionary relationship. The semi-quantitative RT-PCR (s-Q RT-PCR) analysis indicated the expression of RCBF2 gene was induced by cold, dehydration and high-salinity, but not by abscisic acid, and the transcription of RCBF2 gene accumulated primarily in rice immature seeds, growing point and shoots.

Base Sequence↗

PubMed2006

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 beta-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.

DNA, Bacterial↗

PubMed2006

High efficiency and throughput system in directed evolution in vitro of reporter gene.

In vitro directed evolution, especially with DNA shuffling, is a powerful means in biological studies of protein structure and function, and consequently for industrial applications. Escherichia coli beta-glucuronidase (gusA) gene, a versatile and efficient reporter gene, was the model for studying in vitro directed evolution because of its stability, easy analysis of the enzyme properties and conveniently visible phenotype. We developed a high efficiency, throughput system for in vitro directed evolution using gusA reporter gene as the model. The system consisted mainly of three aspects: a prokaryotic expression vector pYPX251, an easy method for obtaining the mutated gene from DNA shuffling and a suitable selected strategy. The vector pYPX251 carried the moderately strong aacC1 gene promoter and T1T2 transcription terminator that allowed expression in E. coli. Over 10,000 individuals could be selected individually in a 9 cm Petri dish after colonies were absorbed on a nitrocellulose filter. A library, which contained 100,000 individuals was screened by incubating ten filter papers with X-Glu. The polymerase chain reaction products of the gusA gene, the fragments of 50-100 bp, with high mutation rates were purified using a dialysis bag from 10% PAGE after electrophoresis. The possibility of obtaining desirable mutations was increased dramatically as the size of the library expanded. A GUS variant, named GUS-TR, was obtained through this system, which is significantly more resistant to high temperature than the wild type enzyme. GUS-TR maintained its high activity even when the nitrocellulose filter containing the variant colony was heated at 100 degrees C for 30 min.

Biotechnology↗

PubMed2006

High level expression of a synthetic gene encoding Peniophora lycii phytase in methylotrophic yeast Pichia pastoris.

Phytase is widespread in nature. It has been used as a cereal feed additive that can enhance the phosphorus and mineral absorption in monogastric animals to reduce the level of phosphorus output in manure. Phytase of Peniophora lycii is a 6'-phytase, which owns high specific activity. To achieve a high expression level of 6'-phytase in Pichia pastoris, the 1,230-bp phytase gene of P. lycii was synthesized and optimized for codon usage, G+C content, as well as mRNA secondary structures. The gene constructs containing wild type or modified phytase gene coding sequences under the control of the highly-inducible alcohol oxidase gene (AOX1) promoter, the synthetic signal peptide (designated MF4I), which is a codon-modified Saccharomyces cerevisiae mating factor alpha-prepro-leader sequence, were used to transform P. pastoris. The P. pastoris strain that expressed the modified phytase gene (phy-pl-sh) with MF4I sequence produced 12.2 g phytase per liter of fluid culture, with the phytase activity of 10,540 U ml(-1). The yield of the modified phytase gene, with bias codon usage and MF4I signal, is 4.4 times higher than that of the wild type gene with MF4I signal and 13.6 times higher than that of the wild type gene with wild type S. cerevisiae signal. The recombinant phytase had one optimum pH (pH 4.5) and an optimum temperature of 50 degrees C. The P. pastoris strain expressed the modified 6-phytase gene, with the MF4I signal peptide showing great potential as a commercial phytase production system.

6-Phytase↗

PubMed2006

Site-specific DNA excision in transgenic rice with a cell-permeable cre recombinase.

The removal of selected marker genes from transgenic plants is necessary to address biosafety concerns and to carry out further experiments with transgenic organisms. In the present study, the 12-amino-acid membrane translocation sequence (MTS) from the Kaposi fibroblast growth factor (FGF)-4 was used as a carrier to deliver enzymatically active Cre proteins into living plant cells, and to produce a site-specific DNA excision in transgenic rice plants. The process, which made cells permeable to Cre recombinase-mediated DNA recombination, circumvented the need to express Cre under spatiotemporal control and was proved to be a simple and efficient system to achieve marker-free transgenic plants. The ultimate aim of the present study is to develop commercial rice cultivars free from selected marker genes to hasten public acceptance of transgenic crops.

Blotting, Southern↗

PubMed2006

[Rational evolutionary design and modification of gene: a short-path of direct evolution].

Directed evolution is a powerful means in academic study and industrial application to modified and functionally improved proteins. There have been significant and impressive advances using the artificial method. Rational Evolutionary Design utilizes structural and sequence alignment information to create new genes and proteins. Rational Evolutionary Design has recently emerged as an attractive approach for studying function of proteins. The review mainly introduced four aspects: preferred codon usage, structural and sequence alignment, key domain analysis, site-directed recombination.

Directed Molecular Evolution↗

PubMed2006

[Progress in the study on conditional male sterility in plants by genetic engineering].

Conditional (reversible) male sterile line in plants would be fertile under special conditions, and thus could be used as two-line to produce hybrid seeds. This paper described the strategies for creating conditional male sterile line and correspondingly restoring it. Some related problems about the issue were commented on. In addition, this review also predicted the prospects of the study on conditional male sterility.

Genes, Plant↗

PubMed2005

Codon-modifications and an endoplasmic reticulum-targeting sequence additively enhance expression of an Aspergillus phytase gene in transgenic canola.

Transgenic plants offer advantages for biomolecule production because plants can be grown on a large scale and the recombinant macromolecules can be easily harvested and extracted. We introduced an Aspergillus phytase gene into canola (Brassica napus) (line 9412 with low erucic acid and low glucosinolates) by Agrobacterium-mediated transformation. Phytase expression in transgenic plant was enhanced with a synthetic phytase gene according to the Brassica codon usage and an endoplasmic reticulum (ER) retention signal KDEL that confers an ER accumulation of the recombinant phytase. Secretion of the phytase to the extracellular fluid was also established by the use of the tobacco PR-S signal peptide. Phytase accumulation in mature seed accounted for 2.6% of the total soluble proteins. The enzyme can be glycosylated in the seeds of transgenic plants and retain a high stability during storage. These results suggest a commercial feasibility of producing a stable recombinant phytase in canola at a high level for animal feed supplement and for reducing phosphorus eutrophication problems.

6-Phytase↗

PubMed2005

Isolation and characterization of a cDNA encoding two novel heat-shock factor OsHSF6 and OsHSF12 in Oryza sativa L.

As a crucial transcription factor family, heat-shock factors were mainly analyzed and characterized in tomato and Arabidopsis. In this study, we isolated two putative heatshock factors OsHSF6 and OsHSF12 that interact specifically with heat-shock element (HSE) from Oryza sativa L by yeast one-hybrid method. The full-length cDNA of OsHSF6 and OsHSF12 have 1074bp and 920bp open reading frame (ORF), respectively. Analysis of the deduced amino acid sequences revealed that OsHSF6 was a class A heat shock factor (HSF) with all the conserved sequence elements characteristic of heat stress transcription factor, while OsHSF12 was a class B HSF with C-terminal domain (CTD) lacking of AHA motif. Bioinformatic analysis showed that the sequences and structures of two HSFs' DNA binding domain (DBD) had a high similarity with LpHSF24. The results of RT-PCR indicated OsHSF6 gene was expressed immediately after rice plants exposure to heat stress, and the transcription of OsHSF6 gene accumulated primarily in immature seeds, roots and leaves. However, we did not find the transcription of OsHSF12 gene in different organs and growth periods. Our results implied that OsHSF6 might be function as a HSF regulating early expression of stress genes in response to heat shock, and OsHSF12 might be act as a synergistic factor to regulate the expression of down-stream genes.

Amino Acid Sequence↗

PubMed2005

Agrobacterium-mediated transformation of Malus robusta with tomato iron transporter gene.

The tomato iron transporter gene (LeIRT2) was introduced to Malus robusta Rehd. via Agrobacterium-mediated transformation to produce iron-deficiency tolerant apple rootstock. A total of 19 putative transformants were obtained, 11 of which were verified by PCR amplification to carry a fragment of the transgene. Among them, nine were confirmed to carry the transgene by Southern blot analysis with one to three copies of the transgene integrated into the plant genome. Two transgenic plants, one carrying one copy and the other three copies of the transgene, were hydroponically cultured to test their tolerance to iron-deficiency, which was found only in the transgenic plant with a single copy, which weighted 21%-4% greater than those of the control plants.

Biological Transport↗

PubMed2004

Vitreoscilla hemoglobin overexpression increases submergence tolerance in cabbage.

Agrobacterium tumefaciens was used to deliver the vhb gene into cabbage (Brassica oleracea var. Cabitata) cv. Xiaguang's parent line, 103. Using hypocotyls and cotyledon petioles as explants for infection, we obtained a transformation efficiency of 3-5% based on the number of transgenic shoots produced from the number of explants used for infection. Molecular analysis indicated that the vhb gene was stably integrated into the cabbage genome and that the vhb gene was expressed at the RNA level. Characterization of the vhb over-expressing transgenic plants revealed that transgenic seeds germinated faster than the wildtype controls. More importantly, the transgenic plants showed tolerance to a prolonged submergence treatment, suggesting that the vhb gene may provide an excellent tool for creation of submergence/flooding-tolerant cultivars of agriculturally important crops.

Adaptation, Physiological↗

PubMed2004

Different effects on ACC oxidase gene silencing triggered by RNA interference in transgenic tomato.

RNA interference (RNAi) is a potent trigger for specific gene silencing of expression in a number of organisms and is an efficient way of shutting down gene expression. 1-Aminocyclopropane-1-carboxylate (ACC) oxidase catalyzes the oxidation of ACC to ethylene, a plant growth regulator that plays an important role in the tomato ripening process. In this research, to produce double-stranded (ds)RNA of tomato ACC oxidase, we linked the sense and antisense configurations of DNA fragments with 1,002-bp or 7-nt artificially synthesized fragments, respectively, and then placed these under the control of a modified cauliflower mosaic virus 35S promoter. The dsRNA expression unit was successfully introduced into tomato cultivar Hezuo 906 by Agrobacterium tumefaciens-mediated transformation. Molecular analysis of 183 transgenic plants revealed that the dsRNA unit was integrated into the tomato genome. With respect to the construct with the 1,002-bp linker, the severity of phenotypes indicated that 72.3% of the transformed plants had non-RNA interference, about 18.1% had semi-RNA interference, and only 9.6% had full-RNA interference. However when the construct with the 7-nt linker was used for transformation, the results were 13.0%, 18.0%, and 69.0%, respectively, indicating that the short linker was more efficient in RNAi of transgenic tomato plants. When we applied this fast way of shutting down the ACC oxidase gene, transgenic tomato plants were produced that had fruit which released traces of ethylene and had a prolonged shelf life of more than 120 days. The RNA and protein analyses indicated that there was non-RNA interference, semi-RNA interference and full-RNA interference of ACC oxidase in the transgenic tomato plants.

Agrobacterium tumefaciens↗

PubMed2004

A simple, rapid, high-fidelity and cost-effective PCR-based two-step DNA synthesis method for long gene sequences.

Chemical synthesis of DNA sequences provides a powerful tool for modifying genes and for studying gene function, structure and expression. Here, we report a simple, high-fidelity and cost-effective PCR-based two-step DNA synthesis (PTDS) method for synthesis of long segments of DNA. The method involves two steps. (i) Synthesis of individual fragments of the DNA of interest: ten to twelve 60mer oligonucleotides with 20 bp overlap are mixed and a PCR reaction is carried out with high-fidelity DNA polymerase Pfu to produce DNA fragments that are approximately 500 bp in length. (ii) Synthesis of the entire sequence of the DNA of interest: five to ten PCR products from the first step are combined and used as the template for a second PCR reaction using high-fidelity DNA polymerase pyrobest, with the two outermost oligonucleotides as primers. Compared with the previously published methods, the PTDS method is rapid (5-7 days) and suitable for synthesizing long segments of DNA (5-6 kb) with high G + C contents, repetitive sequences or complex secondary structures. Thus, the PTDS method provides an alternative tool for synthesizing and assembling long genes with complex structures. Using the newly developed PTDS method, we have successfully obtained several genes of interest with sizes ranging from 1.0 to 5.4 kb.

Bacterial Proteins↗

PubMed2004

Isolation, characterization, and molecular cloning of the cDNA encoding a novel phytase from Aspergillus niger 113 and high expression in Pichia pastoris.

Phytases catalyze the release of phosphate from phytic acid. Phytase-producing microorganisms were selected by culturing the soil extracts on agar plates containing phytic acid. Two hundred colonies that exhibited potential phytase activity were selected for further study. The colony showing the highest phytase activity was identified as Aspergillus niger and designated strain 113. The phytase gene from A. niger 113 (phyI1) was isolated, cloned, and characterized. The nucleotide and deduced amino acid sequence identity between phyI1 and phyA from NRRL3135 were 90% and 98%, respectively. The identity between phyI1 and phyA from SK-57 was 89% and 96%. A synthetic phytase gene, phyI1s, was synthesized by successive PCR and transformed into the yeast expression vector carrying a signal peptide that was designed and synthesized using P. pastoris biased codon. For the phytase expression and secretion, the construct was integrated into the genome of P. pastoris by homologous recombination. Over-expressing strains were selected and fermented. It was discovered that ~4.2 g phytase could be purified from one liter of culture fluid. The activity of the resulting phytase was 9.5 U/mg. Due to the heavy glycosylation, the expressed phytase varied in size (120, 95, 85, and 64 kDa), but could be deglycosylated to a homogeneous 64 kDa species. An enzymatic kinetics analysis showed that the phytase had two pH optima (pH 2.0 and pH 5.0) and an optimum temperature of 60 degrees C.

6-Phytase↗

PubMed2004

Development of an efficient method for the isolation of factors involved in gene transcription during rice embryo development.

Summary An efficient yeast-based system was developed for the isolation of plant cDNAs encoding transcription factors (TFs) and proteins with transcription activation functions (co-activators). The system consists of two vectors: (i) a reporter vector (pG221) harboring the iso-1-cytochrome c (CYC1) core promoter and the beta-galactosidase (lacZ) gene; and (ii) a cDNA library construction vector (pYF503), which yields a library of plant peptides fused to the GAL4-binding domain (GAL4-BD). Expression of a peptide harboring the characteristics of a transcriptional activator leads to expression of lacZ, allowing for selection of relevant colonies. TFs during rice embryo development were isolated through this system. Approximately 200 confirmed positive colonies were obtained from screening 10(6) yeast colonies, and sequence analysis of conserved domains identified 75 independent cDNAs, 20 of which encoded plant TFs or co-activators, including members of the APETALA2 (AP2)/ethylene-responsive element-binding protein (EREBP), MYB and growth-regulating factor (GRF) families. Peptides encoded by 13 of the isolated cDNAs were classified as potential TFs or co-activators because of the presence of conserved TF-like domains. Additionally, 2, 11, and 13 clones encoded kinases, chromosome-related proteins, and unknown proteins, respectively, while the remaining 16 cDNAs were associated with specific functions seemingly unrelated to TFs. Expression pattern analysis of selected TF-encoding genes via RT-PCR revealed that these genes were expressed during seed development, with differential transcription observed during various stages. This work provides informative hints for further study of the regulatory mechanism of rice seed development and illustrates an identification strategy that will be of practical value for the isolation of TFs and co-activators associated with specific plant developmental processes.

Amino Acid Sequence↗

PubMed2004

[Endosperm-specific expression of the ferritin gene in transgenic rice (Oryza sativa L.) results in increased iron content of milling rice].

Iron deficiency is the most widespread micronutrient deficiency world-wild, and is estimated to affect about 30% of the world population. To increase the iron content of rice in Chinese, the 764 bp cDNA of ferritin gene was cloned from soybean (Phaseolus limensis), and constructed between the 1 353 bp rice glutelin GluB-1 promoter and NOS terminator in a binary vector pCAMIBA1301. The constructed pYF1067 vector was introduced into Agrobacterium strain EHA105, and used for transformation of the primary callus derived from immature embryos of a high-yielding rice ( Oryza sativa L. ssp. japonica) variety Wuxiangjin 9. Under the selection on hygromycin-containing medium, seventeen independent transgenic rice lines, total more than 80 transgenic plants, were finally regenerated, and most of these transgenic rice plants grew normally. PCR and Southern blot analysis of total DNA from primary transformants confirmed that one to three copies of the transgenes integrated into the genome of most of the transgenic plants, and they could be stably transmitted into the progeny of the transgenic rice. Northern blot analysis showed that the ferritin gene could specifically express in the endosperm of transgenic rice with high level, while no or low expression in leaves. The expression level varied among different independent transgenic rice plants. There was a significant effect of the high-expression of ferritin on the increased iron content in transgenic rice, the iron content in the milling rice of transgenic rice was up to 64% higher than that of the untransformed wild-type plant, whereas no significant alteration of the zinc level occurred between these two type rice plants.

Ferritins↗

PubMed2003

[Influence of signal peptide sequences on the expression of heterogeneous proteins in Pichia pastoris].

Pichia pastoris has been developed to be a very efficient expression host for the heterogeneous proteins since its alcohol promoter was isolated and cloned, and its transformation technique was established. For further improving the secretion expression of heterogeneous proteins, in this research, the signal sequences were studied. At first, the Saccharomyces cerevisiae mating factor alpha prepro-leader sequence was synthesized using successive PCR and designated as MF4I. Then, ten different signal sequences were constructed by adding the N-terminal residues of Pichia pastoris Aox1 protein to the N-terminal of the MF4I. These ten signal sequences were used for directing phytase gene secretion in Pichia pastoris, the secretion of phytase were increased in Pichia pastoris strains containing new signal sequence. Among these strains, the phytase secretion was highest in strain contain signal sequence added with A, I, P three Aox1 N-terminal residues; the phytase secretion of Pichia pastoris was 90 mg/L in flake. The secretion was six-fold of that with original Saccharomyces cerevisiae mating factor alpha prepro-leader sequence. In addition, insert of ten residues E E A E A E A E P K can further increase the phytase secretion by 35%, the secretion reach 120 mg/L.

6-Phytase↗

PubMed2002

[High expression of a heat-stable phytase in Pichia pastoris].

It is difficult to obtain naturally occurring phytase having the required thermostability for application in animal feeding. The 1.3 kb thermal stable phytase gene (fphy) of Aspergillus fumigatus was synthesized by using a successive PCR method for the optimal expression in Pichia pastoris. Though the nucleotides of synthetic fphy share only 74% homology with the natural gene, the amino acid sequences coded by both genes were identical. After being cloned into the yeast expression vector (pPIC9) and inserted into the chromosome of Pichia pastoris by homologous recombination, phytase was expressed in the yeast and secreted from the cell. The strains for phytase over-expression were selected out by SDS-PAGE and enzyme analysis. After fermentation in 5 L fermention tank and induced by 0.5% methanol for 60 h, about 5.6 g purified phytase was obtained per liter culture fluid. The activity of phytase was 130 000 u per microlitre fluid. The thermostable phytase remained 40% active after exposure at 90 degrees for 80 min.

6-Phytase↗