Cloning, Heterologous Expression, and Characterization of a Neutral Uricase from Arthrobacter sp. CSAJ-16 in Cangshan Mountain.

Uricase (or Urate oxidase), a key enzyme involved in purine metabolism, is commonly used in treating conditions such as gout, hyperuricemia, and tumor lysis syndrome. In this study, a uricase-producing strain (named CSAJ-16) was isolated from the soil sample of Cangshan Mountain, Yunnan Province, China. This strain was identified as Arthrobacter sp. CSAJ-16. Based on the gene sequence alignment, the uricase gene (named aruox) of Arthrobacter sp. CSAJ-16 was amplified and heterologously expressed. The recombinant uricase (ArUOX) was about 32 kDa. The optimal pH and temperature of ArUOX were pH 7 and 20°C, respectively. The ArUOX remained above 50% relative activity after incubation at 37°C for 100 min or at pH 6.0-8.6 for 24 h. Moreover, metal ions such as K+, Mg2+, Ca2+, Ba2+ and Pb2+ can significantly enhance the activity of ArUOX (> 200%). These enzymatic properties indicate that ArUOX has potential applications in pharmaceutical enzymes and uric acid detection kits.

Assessment of the Genetic Diversity of Different Job's Tears (Coix lacryma-jobi L.) Accessions and the Active Composition and Anticancer Effect of Its Seed Oil.

Job's tears (Coix lachryma-jobi L.) is an important crop used as food and herbal medicine in Asian countries. A drug made of Job's tears seed oil has been clinically applied to treat multiple cancers. In this study, the genetic diversity of Job's tears accessions and the fatty acid composition, triglyceride composition, and anti-proliferative effect of Job's tears seed oil were analyzed using morphological characteristics and ISSR markers, GC-MS, HPLC-ELSD, and the MTT method. ISSR analysis demonstrated low genetic diversity of Job's tears at the species level (h = 0.21, I = 0.33) and the accession level (h = 0.07, I = 0.10), and strong genetic differentiation (GST = 0.6702) among all accessions. It also clustered the 11 accessions into three cultivated clades corresponding with geographical locations and two evidently divergent wild clades. The grouping patterns based on morphological characteristics and chemical profiles were in accordance with those clustered by ISSR analysis. Significant differences in morphological characteristics, fatty acid composition, triglyceride composition, and inhibition rates of seed oil were detected among different accessions, which showed a highly significant positive correlation with genetic variation. These results suggest that the seed morphological characteristics, fatty acid composition, and triglyceride composition may be mainly attributed to genetic factors. The proportion of palmitic acid and linoleic acid to oleic acid displayed a highly significant positive correlation with the inhibition rates of Job's tears seed oil for T24 cells, and thus can be an important indicator for quality control for Job's tears.

Genetic diversity and distribution patterns of host insects of Caterpillar Fungus Ophiocordyceps sinensis in the Qinghai-Tibet Plateau.

The caterpillar fungus Ophiocordyceps sinensis is one of the most valuable medicinal fungi in the world, and it requires host insects in family Hepialidae (Lepidoptera) to complete its life cycle. However, the genetic diversity and phylogeographic structures of the host insects remain to be explored. We analyzed the genetic diversity and temporal and spatial distribution patterns of genetic variation of the host insects throughout the O. sinensis distribution. Abundant haplotype and nucleotide diversity mainly existed in the areas of Nyingchi, ShangriLa, and around the edge of the Qinghai-Tibet Plateau, where are considered as the diversity center or micro-refuges of the host insects of O. sinensis. However, there was little genetic variation among host insects from 72.1% of all populations, indicating that the host species composition might be relatively simple in large-scale O. sinensis populations. All host insects are monophyletic except for those from four O. sinensis populations around Qinghai Lake. Significant phylogeographic structure (NST>GST, P<0.05) was revealed for the monophyletic host insects, and the three major phylogenetic groups corresponded with specific geographical areas. The divergence of most host insects was estimated to have occurred at ca. 3.7 Ma, shortly before the rapid uplift of the QTP. The geographical distribution and star-like network of the haplotypes implied that most host insects were derived from the relicts of a once-widespread host that subsequently became fragmented. Neutrality tests, mismatch distribution analysis, and expansion time estimation confirmed that most host insects presented recent demographic expansions that began ca. 0.118 Ma in the late Pleistocene. Therefore, the genetic diversity and distribution of the present-day insects should be attributed to effects of the Qinghai-Tibet Plateau uplift and glacial advance/retreat cycles during the Quaternary ice age. These results provide valuable information to guide the protection and sustainable use of these host insects as well as O. sinensis.

Comparative phylogenetic relationships and genetic structure of the caterpillar fungus Ophiocordyceps sinensis and its host insects inferred from multiple gene sequences.

Ophiocordyceps sinensis (Ascomycota: Ophiocordycipitaceae) is a native fungal parasite of Hepialidae caterpillars and one of the most economically important medicinal caterpillar fungi in China. However, little is known about the phylogenetic and evolutionary relationships between O. sinensis and its host insects. In this study, nuclear ITS and ß-tubulin sequences from O. sinensis and mitochondrial COI, COII, and Cytb sequences from its hosts were analyzed across 33 populations sampled from five regions in China. Phylogenetically, both O. sinensis and its hosts were divided into three geographically correlated clades, and their phylogenies were congruent. Analysis of molecular variance and calculated coefficients of genetic differentiation revealed significant genetic divergence among the clades within both O. sinensis (F(ST)= 0.878, N(ST)=0.842) and its hosts (F(ST)=0.861, N(ST)=0.816). Estimated gene flow was very low for O. sinensis (Nm=0.04) and the host insects (Nm=0.04) among these three clades. Mantel tests demonstrated a significant correlation (P<0.01) between the genetic distances for O. sinensis and its hosts, as well as a significant association (P<0.05) between geographic and genetic distances in both. The similar phylogenetic relationships, geographic distributions, and genetic structure and differentiation between O. sinensis and its hosts imply that they have coevolved.

[Identification of four medicines of Panax L. revealed by mitochondrial nad 1 gene b/c intron].

OBJECTIVE: To study the identification method and phylogenetic relationships of four medicines of Panax L genus: Panax ginseng C. A. Meyer, P. quinquefolicum L. , P. notoginseng (Burk.) F. H. Chen and P. japonicus C. A. Meyer. METHODS: The mitochondrial nad 1 gene was amplified. Their sequence differences were analyzed after sequencing and alignment. RESULTS: The sequence lengths of P. ginseng (including Chinese transplanted ginseng and Korean ginseng), P. japonicus were 1 290 bp, and those of P. quinquefolicum and P. notoginseng were 1 269 bp and 1 522 bp respectively. The main difference among these sequences was in nad 1 gene b/c intron. The NJ phylogenetic tree showed that P. ginseng was most closely related to P. japonicus, next was closer to P. quinquefolicum, and P. notoginseng was comparatively distantly related to P. ginseng. CONCLUSION: P. quinquefolicum and P. notoginseng can be identified from the 4 medicines of Panax L. based on the sequence difference in mitochondrial nad 1 gene. The mitochondrial nad 1 gene b/c intron can provide some evolutionary information, therefore, it is useful to identify and phylogenetically analyse for the medicines of Panax L. genus.

[Tissue culture and rapid propagation of Trichosanthes kirilowii].

OBJECTIVE: To study tissue culture of Trichosanthes kirilowii, and establish the technique of rapid propagation. METHODS: Cluster buds were induced from stem tip and stem with axillary bud, calluses were induced from stems and leaves. RESULTS: Cluster buds could be induced on both the bud and axillary bud from 2-years-old tuber of Trichosanthes kirilowii with MS medium containing 2 mg/L BA and 0.5 -0.05 mg/L NAA. The roots could be induced with MS + 0.1 mg/L NAA + 0.2 mg/L IBA. The seedling with roots could be transplanted after domestication, and achieved the rapid seedling propagation. Calluses could be induced from the stems and leaves with MS + 4 mg/L BA + 0.5 mg/L NAA, and the calluses then could be differentiated into seedlings without root. CONCLUSION: The male and female seedlings of Trichosanthes kirilowii can be propagated largely using stem tip or axillary bud in short period. The technique of rapid propagation on Trichosanthes kirilowii have a high benefit and low costs.