Effect of the rolC gene on biosynthesis of tropane alkaloids in Atropa belladonna / 药学学报

Hair roots induced by Agrobacterium rhizogene produce higher levels of secondary metabolites than non-induced plants, and the enhanced metabolic capacity is driven by the rol gene. We hypothesized that rol genes can be utilized to improve the biosynthesis of tropane alkaloids (TAs) in Atropa belladonna. In this study, the rolC gene from Agrobacterium rhizogene pRiA4 plasmid, driven by a CaMV35S promoter, was overexpressed in A. belladonna. The phenotypes, TAs content and transcriptional expression of key genes in TAs biosynthesis were analyzed in transgenic A. belladonna plants. Results show that transgenic A. belladonna exhibited a well-developed root system, male sterility, higher stamen column length than pistil, early flowering, internode shortening, smaller but more flowers, increased axillary buds and lateral buds, decreased apical dominance, and long and narrow leaves as compared to wild-type plants. Transgenic A. belladonna produced more TAs than wild-type plants, with the content of hyoscyamine, anisodamine and scopolamine reaching 2.58, 3.59 and 15.77-fold that of the control group, respectively. The gene expression of putrescine N-methyltransferase (PMT), tropinone reductase I (TRⅠ) and hyoscyamine 6-β-hydroxylase (H6H), key enzymes in TAs biosynthesis, were up-regulated compared with the control group. The above results indicate that the rolC gene enhances TAs biosynthesis in A. belladonna by up-regulating the expression of key enzymes in the TAs biosynthesis pathway, laying a foundation for genetic manipulation of A. belladonna to increase TAs content by increasing rolC gene expression.

DNA and RNA random amplification polymorphism in 5-flurocytosine-resistant strains of Candida albicans from revul-vaginal candidasis / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the DNA genome and RNA expression in 5-flurocytosine-resistant strains of Candida albicans from vaginal candidasis.</p><p><b>METHODS</b>Sixteen strains of Candida albicans were selected from clinically diagnosed revul-vaginal candidasis. Eight 5-flurocytosine-sensitive isolates and 8 resistant isolates were examined by France Media FUNGUS sensitive test. DNA genome was detected with random amplification polymorph DNA. RNA expression was detected with random amplification polymorph RNA method.</p><p><b>RESULTS</b>There were no distinct differences between 5-flurocytosine-sensitive and resistant Candida albicans in DNA genome, while RNA expression showed significant differences between 5-flurocytosine-resistant and sensitive strains.</p><p><b>CONCLUSION</b>Clinical 5-flurocytosine-resistant strains of Candida albicans from revul-vaginal candidasis may be related to phenotype changes.</p>

Application of differential display-PCR technique in fluconazole-resistance gene expression of Candida / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the application of differential display-2PCR(DD-PCR) in research on gene expression of Candida.</p><p><b>METHODS</b>Resistance to fluconazole was induced in a Candida albicans isolate 435 from vagina by culturing in YEPD broth with increasing fluconazole concentration in vitro, and the resistant isolate 435-2 (MIC=128 microg/ml ) was obtained after 80 days of incubation. Comparisons between 435 and 435-2 either in fluconazole-containing medium or in drug-free medium were performed with the modified DD-PCR including amplification with long primers, silver staining, reverse dot blot and non-radiographic labeling techniques.</p><p><b>RESULTS</b>Three differential displayed bands were found which showed high homology to alcohol dehydrogenase 1 (ADH1), TOP2 and CDR1, respectively. The up-regulating expression of ADH1 and CDR1 associated with fluconazole resistance was further identified by RT-PCR.</p><p><b>CONCLUSION</b>The up-regulating expression of ADH1 and CDR1 was associated with fluconazole resistance in Candida albicans, ADH1 might be a candidate of novel fluconazole resistant gene.</p>