Isolation and expression characteristic of cyp71av1 promoter from Artemisia annua / 中草药

Objective: Trying to find the ways to enhance the expression of cyp71av1 gene encoding cytochrome P450 mono-oxygenase which is a key enzyme in artemisinin biosynthesis pathway accelerating the artemisinin synthesis, the promoter of cyp71av1 was isolated and characterized. Methods: 5′ untranslated regions of cyp71av1 were isolated from Artemisia annua with thermal asymmetric interlaced PCR. For functional characterization, the isolated fragments were fused with β-glucuronidase GUS reporter gene and introduced into Nicotiana tabacum by Agrobacterium-mediated transformation. The GUS expression regulated by 5′ untranslated regions of cyp71av1 in transgenic N. tabacum under the normal or stressed conditions were detected by histochemical staining and quantitative spectrophotometry assay. Results: Two DNA fragments upstream of cyp71av1 coding sequence, a long fragment and a truncated fragment, were isolated from A. annua and introduced into N. tabacum respectively. Histochemical staining showed that two isolated fragments confered stable GUS expression in transgenic plants, and no significant difference was found between the two fragments on the GUS activity. The quantitative results also showed that the GUS activity in transgenic tobacco plants treated by dehydration, low-temperature (4 °C), and ultraviolet irradiation were 1.4 to 2.7 folds higher than that in the controls. Conclusion: It suggests that the isolated fragments has promoter activity and may be responsive to adverse environmental stresses.

Cloning, soluble expression and mutant activity analysis of lactate dehydrogenase gene from Plasmodium falciparum / 生物工程学报

To establish a platform for high throughput screening and in vitro evaluating novel metabolic enzyme-targeted inhibitors towards anti-malarial drugs, a lactate dehydrogenase gene of Plasmodium falciparum (PfLDH) was amplified from the Hainan isolate FCC1/HN. The fusion expression vectors, pGEX-2TK and pET-29a( + ), were utilized to introduce the PfLDH gene into strains of Escherichia coli, BL21 and BL21 (DE3), for over-expression. Consequently, the enzymatic activity of PfLDH was successfully detected in the suspension of lytic bacteria. The PfLDH gene cloned in pGEX-2TK was mainly expressed as inclusion bodies, while the same gene cloned in pET-29a( + ) was nearly expressed in a soluble form of PfLDH, demonstrating the latter vehicle might be more suitable for the large-scale preparation of recombinant PfLDH. Furthermore, according to the electrophoregram of SDS-PAGE and the sequencing data, a series of truncated PfLDH sequences generated randomly from gene amplification were screened and cloned, from which four pre-matured genes with a terminator mutation, PfLDH-delta271, -delta236, -delta167 and -delta53 coding for 45, 80, 149 and 263 amino acid residues, were individually recovered. Through the gene expression and enzymatic activity measurement, the effect of pre-matured terminator mutation on the activity of PfLDH was evaluated, which should pave the way for probing the relationship between structure and function of PfLDH.

Establishment of stably expressed human RANTES gene in prunella vulgaris cell clone / 生物工程学报

To express interesting human genes in herbal cells for boosting their specific pharmacological activities, RANTES gene cloned from human peripheral blood lymphocyte (PBL) mRNA was introduced into A. tumefaciens strain LBA4404 harboring pAL4404 plasmid via tumor-inducing (Ti) plasmid-derived intermediate expression vector pROKII. In vitro cultured P. vulgaris cells were transformed by leaf-disk cocultivation procedure. Integration of RANTES gene in the genome of transformed cells was confirmed by Southern blotting, and expression of RANTES gene in transformed cells was analyzed by RT-PCR amplification, Western blotting and enzyme-linked immunosorbent assay (ELISA). The peroxidase activity of PBL was utilized as a detection index of cellular chemotropism induction by recombinant RANTES. The results have shown the RANTES gene was integrated in transgenic P. vulgaris cells, and RANTES gene-stably expressed cell clones were available, which could pave the way to obtain transgenic P. vulgaris plants demonstrating specific pharmacological activities.