Jin-Ge Liu

Isolation, optimization, and functional analysis of the cDNA encoding transcription factor OsDREB1B in Oryza Sativa L.

A previous study had indicated that the transcription factors DREB/CBF (DRE-binding protein/C-repeat binding factor) play important roles in the expression of many stress inducible genes under cold-temperature, dehydration and high-salt conditions. In this study, we have isolated a cDNA clone that encoded a DRE-binding protein from rice cDNA library using the yeast one-hybrid system with DRE cis-acting element in the promoter region of rd29A gene as bait. Sequence analysis of the deduced amino acid sequence showed this protein was a putative AP2/EREBP transcription factor with a conserved AP2/EREBP domain and a potential nuclear localization signal (NLS). Expression pattern studies of this DRE-binding protein revealed that this gene was not only strongly induced by cold-temperature as reported by previous study but also induced by high-temperature as well. For the purpose of analyzing this gene conveniently, we attempted to apply the codon optimization method to reconstruct the gene of transcription factor in plants. A new sequence having decreasing GC contents, secondary structures, optimized codons and identical amino acid sequence with native gene was synthesized, which named OsDREB1BI, and then this optimized gene was transformed into Arabidopsis thaliana cv. Columbia by floral dip method. Results indicated that the OsDREB1BI gene was over-expressed in transgenic plants under cold and high-temperature, meanwhile, those transgenic plants also revealed freezing and heat tolerance. These might lay a strong foundation for exploiting the freezing and heat tolerance of rice and other species.

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.

Directed evolution of a beta-galactosidase from Pyrococcus woesei resulting in increased thermostable beta-glucuronidase activity

We performed directed evolution on a chemically synthesized 1,533-bp recombinant beta-galactosidase gene from Pyrococcus woesei. More than 200,000 variant colonies in each round of directed evolution were screened using the pYPX251 vector and host strain Rosetta-Blue (DE3). One shifted beta-galactosidase to beta-glucuronidase mutant, named YG6762, was obtained after four rounds of directed evolution and screening. This mutant had eight mutated amino acid residues. T29A, V213I, L217M, N277H, I387V, R491C, and N496D were key mutations for high beta-glucuronidase activity, while E414D was not essential because the mutation did not lead to a change in beta-glucuronidase activity. The amino acid site 277 was the most essential because mutating H back to N resulted in a 50% decrease in beta-glucuronidase activity at 37°C. We also demonstrated that amino acid 277 was the most essential site, as the mutation from N to H resulted in a 1.5-fold increase in beta-glucuronidase activity at 37°C. Although most single amino acid changes lead to less than a 20% increase in beta-glucuronidase activity, the YG6762 variant, which was mutated at all eight amino acid sites, had a beta-glucuronidase activity that was about five and seven times greater than the wild-type enzyme at 37 and 25°C, respectively.

Isolation and characterization of a novel cDNA encoding ERF/AP2-type transcription factor OsAP25 from Oryza Sativa L.

Using a yeast one-hybrid method, a transcription factor, OsAP25, which interacts specifically with a GCC box was isolated from rice. The OsAP25 protein contained a conserved ethylene-responsive element binding factor (ERF) domain which shared identity with other reported ERF domains. Phylogenetic analysis showed that OsAP25 could be categorized into class III ERF of the previously characterized ERF proteins on an evolutionary relationship. The semi-quantitative RT-PCR analysis revealed that OsAP25 gene was constitutively expressed in leaves, roots, growing points, flower, bolting stage and grain filling stage. In addition, OsAP25 gene was induced by NaCl, cold, drought, abscisic acid and exogenous ethylene.

A semi-rational design strategy of directed evolution combined with chemical synthesis of DNA sequences

Abstract Directed evolution in vitro is a powerful molecular tool for the creation of new biological phenotypes. It is unclear whether it is more efficient to mutate an enzyme randomly or to mutate just the active sites or key sites. In this study, the strategy of a semi-rational design of directed evolution combined with whole sequence and sites was developed. The 1553 bp gene encoding the thermostable β-galactosidase of Pyrococcus woesei was chemically synthesized and optimized for G+C content and mRNA secondary structures. The synthesized gene product was used as a template or as a wild-type control. On the basis of the first round of DNA shuffling, library construction and screening, one mutant of YH6754 was isolated with higher activity. Eight potential key sites were deduced from the sequence of the shuffled gene, and 16 degenerate oligonucleotides were designed according to those eight amino acids. Two variants of YG6765 and YG8252 were screened in the second part of DNA shuffling, library construction and screening. For comparison, one mutant of YH8757 was screened through the same routine rounds of directed evolution with YH6754 as template. The purified β-galactosidase from YH8757 exhibited a lower specific activity at 25°C than those purified from mutated YG6755 and YG8252.