Effect of Different Edible Trichosanthes Germplasm on Its Seed Oil to Enhance Antioxidant and Anti-Aging Activity in Caenorhabditis elegans.

The seeds of various Trichosanthes L. plants have been frequently used as snacks instead of for traditional medicinal purposes in China. However, there is still a need to identify the species based on seeds from Trichosanthes germplasm for the potential biological activities of their seed oil. In this study, 18 edible Trichosanthes germplasm from three species were identified and distinguished at a species level using a combination of seed morphological and microscopic characteristics and nrDNA-ITS sequences. Seed oil from the edible Trichosanthes germplasm significantly enhanced oxidative stress tolerance, extended lifespan, delayed aging, and improved healthspan in Caenorhabditis elegans. The antioxidant activity of the seed oil exhibits a significant positive correlation with its total unsaturated fatty acid content among the 18 edible Trichosanthes germplasm, suggesting a genetic basis for this trait. The biological activities of seed oil varied among species, with T. kirilowii Maxim. and T. rosthornii Harms showing stronger effects than T. laceribractea Hayata.

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.

The phylogeny and divergence time of Ophiocordyceps sinensis and its host insects based on elongation factor 1 alpha.

Ophiocordyceps sinensis Berk. is a fungal parasite that parasitizes the larvae of Hepialidae and is endemic to the Qinghai-Tibet Plateau (QTP). The phylogeny and divergence time of O. sinensis and its host insects were analyzed for 137 individuals from 48 O. sinensis populations based on the elongation factor 1 alpha (EF-1α) gene. Lower nucleotide variation, with only 7 and 16 EF-1α haplotypes, was detected in O. sinensis and its host insects, respectively. The isolated and broad distribution patterns coexisted in both O. sinensis and its host insects on the QTP. The divergence time estimates show that O. sinensis and its host insects originated later than 14.33 million years (Myr) and earlier than 23.60 Myr in the Miocene period, and the major differentiation occurred later than 4 Myr. Their origin and differentiation match well with the second and third uplifts of the QTP, respectively. The host insects from the O. sinensis populations distributed around Qinghai Lake are inferred as an ancient and relict species that has survived various geological events of the QTP. It is suitable to estimate the divergence times of both O. sinensis and its host insects from the same individuals using one gene: EF-1α. Our findings of the origin, phylogeny, and evolution of the endemic species also support the epoch of geological events on the QTP.

Haplotype Diversity of NADPH-Cytochrome P450 Reductase Gene of Ophiocordyceps sinensis and the Effect on Fungal Infection in Host Insects.

Ophiocordyceps sinensis Berk. is a fungal parasite that parasitizes the larvae of Hepialidae and is used as a traditional Chinese medicine. However, it is not clear how O. sinensis infects its host. The encoding gene haplotype diversity and predicted function of the nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome P450 reductase (CPR) related to the fungal pathogenicity was analyzed for 219 individuals from 47 O. sinensis populations. Two NADPH CPR genes of O. sinensis were detected and their dominant haplotypes were widely distributed throughout the entire distribution range in Western China. Only 5.43% of all O. sinensis individuals possessed the specific private haplotypes of NADPH CPR-1 and CPR-2 genes. Bioinformatic analyses predicted that the phosphorylation sites, motifs, and domains of NADPH CPR of O. sinensis were different between those encoding by the dominant and private gene haplotypes. The one-to-one match fungus-host correspondence of the same individual suggested that the widely distributed O. sinensis with the dominant NADPH CPR gene haplotypes may strongly infect almost all host insects through a random infection by oral or respiratory pores. Conversely, O. sinensis with the specific private NADPH CPR gene haplotypes is likely to infect only a few corresponding host insects by breaching the cuticle, due to the changed NADPH CPR structure and function.

Effects of fungus-host associations on nucleoside differences among Ophiocordyceps sinensis populations on the Qinghai-Tibet Plateau of China.

Nucleosides are active components in Ophiocordyceps sinensis. All individual nucleoside contents in both fruiting and caterpillar bodies were significantly different among the 24 O. sinensis populations that are distributed across the Qinghai-Tibet Plateau of China. The nucleoside contents of the fruiting and caterpillar bodies show significant positive correlations with the geographical distances of O. sinensis populations, and the nucleoside contents in the O. sinensis populations with the non-dominant fungus-host haplotype associations from isolated geographical areas show significant positive correlations with the genetic distances among those populations based on nrDNA ITS and COI sequences, respectively. The fungus-host associations play important roles in the nucleoside differences of O. sinensis populations.

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.

A comparative study of Lonicera japonica with related species: Morphological characteristics, ITS sequences and active compounds.

Morphological characteristics, ITS sequences, and active compounds have been used to differentiate between species of Lonicera used in the traditional Chinese medicines Flos Lonicerae Japonicae (FLJ) and Flos Lonicerae (FL). FLJ includes L. japonica whereas FJ includes Lonicera macranthoides, Lonicera hypoglauca, Lonicera confusa and Lonicera fulvotomentosa. FLJ could be distinguished from FL using four quantitative and 10 qualitative characters, ITS sequences, chlorogenic acid and luteoloside contents. Analyses revealed that L. japonica was very different from the other species. The results have implications for the identification and quality control of species of Lonicera used medicinally, suggesting that species should not be interchanged in medicinal preparations.

[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.

Metabolism of reactive oxygen species in cotton cytoplasmic male sterility and its restoration.

To elucidate reactive oxygen species (ROS) metabolism of cotton cytoplasmic male sterility and the effects of restorer gene on the metabolism of ROS, the metabolism changes in the production and scavenging of ROS and gene expression related to ROS-scavenging enzymes were investigated in the anther mitochondria of CMS line, maintainer line and hybrid F(1). During the abortion preliminary stage (sporogenous cell division stage), anthers of CMS line had a little higher superoxide (O(2)(-)) production rate and hydrogen peroxide (H(2)O(2)) and malondialdehyde (MDA) contents than those of maintainer or hybrid F(1. )Simultaneously, a little higher ROS contents might serve as a signal to increase the activity of superoxide dismutase (SOD) in anthers of CMS line to reduce the ROS damage to the anther development. But at the abortion peak (pollen mother cell meiosis stage), anthers of CMS line had extraordinarily higher ROS contents and lower ROS-scavenging enzymic activities compared with the hybrid F(1), during which the ROS contents and ROS-scavenging enzymic activities in hybrid F(1) were approximate to those of maintainer line. The expression of Mn-sod and apx mRNA in anther of CMS line was obviously inhibited when ROS produced with a great deal during anther abortion, however the gene expression in hybrid F(1) kept normal with the maintainer. Excessive accumulation of O(2)(*-) , H(2)O(2 )and MDA, significant reduction of ROS-scavenging enzymic activities and lower gene expression level of ROS-scavenging enzyme were coinstantaneous with male cells death in anthers of CMS line. But when the restorer gene was transferred into CMS line, excessive production of ROS could be eliminated in the anthers of hybrid F(1). The restorer gene likely plays an important role in keeping the dynamic balance between the production and elimination of ROS.

[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.