Biomedical subjects

Quan-Hong Yao

Publications and source records attributed to Quan-Hong Yao.

21 records · Page 2Linked to original sources

PubMed2002

[Molecular evolution of beta-glucuronidase in vitro: obtaining thermotolerant GUS gene].

The Escherichia coli beta-glucuronidase gene (gus) has been developed as a reporter gene for plants, and has been widely used for over a decade. Both chromogenic and fluorogenic GUS substrates have been synthesized, allowing rapid nonradioactive assays. The use of the Escherichia coli enzyme beta-glucuronidase (GUS) as a reporter in gene expression studies is limited by some plants and plant-associated bacteria express endogenous glucuronidase activities. The use of the enzyme as a reporter in transgenic plants is limited by high false positive. Laboratory evolution methods were used to enhance the thermostability and activity of the beta-glucuronidase. Using plasmid pBI121 as template, a 1.8 kb specific product was amplified and cloned into the vector pBluescript SK. The result of nucleotide sequence analysis was the same as reported. In vitro recombination (DNA shuffling), which involves DNase I digestion, primerless PCR, and primer PCR was used to generate mutant libraries. The mutant GUS3-3 gene was isolated after three rounds of mutation, DNA shuffling, and screening. The GUS3-3 enzyme can resistant high temperature up to 80 degrees C for 30 min. The nucleotide sequence analysis showed 99.2% homology between the GUS-ck gene from pBI121 and GUS3-3 gene. The deduced amino acid sequence demonstrated that 11 amino acid was changed. The Tm value of GUS3-3 is 80 degrees C and increased by 25 degrees C above GUS-ck (55 degrees C). The researches indicated the feasibility of the molecular evolution of beta-glucuronidase in vitro to improve enzymatic thermostability.

Amino Acid Sequence↗

PubMed2001

Expression of Choline Oxidase Gene (codA) Enhances Salt Tolerance of the Tobacco.

The codA gene for choline oxidase, which converts choline into betaine. This enzyme, cloned from a soil bacterium Arthrobactor globiformis, has been transferred and expressed in tobacco by the Agrobatcterium-method transformation through the binary plasmid pGAH/codA. The pGAH/codA carried with Km(R) and Hyg(R),and coding sequence for a transit peptide from Rubisco small subunit gene (rbcS) was inserted between 35 S promoter and codA, so that the COD could be introduced into the chloroplast by this transit peptide. The transformed plants were screened on the medium containing the Km and Hyg. PCR, Western and gold immunolocalization tests showed that the codA gene has integrated into the tobacco DNA genome and its protein was expressed and the mature peptide has gone into the chloroplasts by the transit peptide. The results of salt-tolerance measuring for transgenic plants showed that the transgenic plants were more tolerance to the salt than the control plants. The young transgenic plants (1.0--1.5 cm) could survive at 400 mmol/L NaCl MS medium for more than 30 days. From them the higher tolerance plant T4-400 was obtained, which could grow at the 300 mmol/L NaCl MS medium well. The transgenic plants (6--8cm) could grow normally at the 400 mmol/L NaCl MS medium while wild plants failed to do so. So the transferred plant with codA enhanced its tolerance to the salt stress.

Journal Article↗

PubMed2001

PCR-aided Synthesis and Stable Expression in E.coli of the cryIA (c)Bt Gene.

Insecticidal crystal proteins, known as delta-endotoxins, from a gram-positive bacterium Bacillus thuringiensis have been used as bio-pesticides for over 3 decades. By using a successive PCR method, the 1.8 kb cryIA (c)Bt gene coding for the fragment of protoxin was synthesized. Different from B. thuringiensis subsp. kurstaki Hd 73 cry gene, the synthesized gene has the codon usage pattern of an average Pseudomonas spp gene. 614 nucleotides were changed in the synthesized cryIA (c)Bt gene and the G C content was increased from 37.2% to 64%. The synthesized cryIA (c)Bt gene was cloned into pUT56 vector under the tac promoter and T1-T2 terminator. SDS-PAGE analysis revealed that 66 kD protein of the modified cryIA (c)Bt gene was expressed in E.coli and accounted for about 30% of total protein in the bacterial cells. Bioassays using crude expression products from host strains indicated that they had high toxicity to third instar larvae of the cabbage butterfly (Pieris brassicae). The LD(50) was calculated to be 0.024 &mgr;g/cm(2).

Journal Article↗