Molecular characterization of a Fus3/Kss1 type MAPK from Puccinia striiformis f. sp. tritici, PsMAPK1

Puccinia striiformis f. sp. tritici (Pst) is an obligate biotrophic fungus that causes the destructive wheat stripe rust disease worldwide. Due to the lack of reliable transformation and gene disruption method, knowledge about the function of Pst genes involved in pathogenesis is limited. Mitogen-activated protein kinase (MAPK) genes have been shown in a number of plant pathogenic fungi to play critical roles in regulating various infection processes. In the present study, we identified and characterized the first MAPK gene PsMAPK1 in Pst. Phylogenetic analysis indicated that PsMAPK1 is a YERK1 MAP kinase belonging to the Fus3/Kss1 class. Single nucleotide polymerphisms (SNPs) and insertion/deletion were detected in the coding region of PsMAPK1 among six Pst isolates. Real-time RT-PCR analyses revealed that PsMAPK1 expression was induced at early infection stages and peaked during haustorium formation. When expressed in Fusarium graminearum, PsMAPK1 partially rescued the map1 mutant in vegetative growth and pathogenicity. It also partially complemented the defects of the Magnaporthe oryzae pmk1 mutant in appressorium formation and plant infection. These results suggest that F. graminearum and M. oryzae can be used as surrogate systems for functional analysis of well-conserved Pst genes and PsMAPK1 may play a role in the regulation of plant penetration and infectious growth in Pst.     

Richness and composition of herbaceous species in restored shrubland and grassland ecosystems in the northern Loess Plateau of China

Restored grasslands and shrublands are integral parts of the semi-natural landscape and are of major importance for biodiversity in the northern Loess Plateau. Determining the underlying factors that control the richness and composition of herbaceous species in restored grasslands and shrublands is urgently needed. Thus, the specific objective of this study was to evaluate the relative importance of soil, plant, and topographic explanatory variables affecting the richness and composition of herbaceous species in restored shrubland and grassland ecosystems in a typical watershed within the northern Loess Plateau. In this study, 27 restored grassland sites and 16 restored shrubland sites were sampled during September 2009. Using variation partitioning (partial canonical correspondence analysis), we determined the individual and shared effects of these three sets of explanatory variables on herbaceous biodiversity in the two restored habitats. Most of the explained variation in plant diversity was related to the pure effect of soil, plant, and topographic variables. Restored shrublands had significantly more species than grasslands, and abandoned dam farmlands had significantly more species than other grassland sites. Moreover, botanical diversity responded differently to the explanatory variables in different plant communities. The pure effects of soil properties, soil moisture in particular, accounted for the largest fractions of explained variation in species diversity in restored grasslands. Both plant and topographic variables had balancing pure effects on species diversity in restored shrublands, in particular the shrub density and slope angle. We conclude that the maintenance of a moderate density of shrubs (less than 3600 shrubs per ha), construction of check-dams, and grazing at a low stocking rate, taking conditions of soil and topographic site into account, may help to conserve biodiversity in the northern Loess Plateau.     

Anti-Bacterial Activity of Recombinant Human β-Defensin-3 Secreted in the Milk of Transgenic Goats Produced by Somatic Cell Nuclear Transfer

The present study was conducted to determine whether recombinant human β-defensin-3 (rHBD3) in the milk of transgenic goats has an anti-bacterial activity against Escherichia coli (E. coli), Staphylococcus aureus (S. aureus) and Streptococcus agalactiae (S. agalactiae) that could cause mastitis. A HBD3 mammary-specific expression vector was transfected by electroporation into goat fetal fibroblasts which were used to produce fourteen healthy transgenic goats by somatic cell nuclear transfer. The expression level of rHBD3 in the milk of the six transgenic goats ranged from 98 to 121 µg/ml at 15 days of lactation, and was maintained at 90–111 µg/ml during the following 2 months. Milk samples from transgenic goats showed an obvious inhibitory activity against E. coli, S. aureus and S. agalactiae in vitro. The minimal inhibitory concentrations of rHBD3 in milk against E. coli, S. aureus and S. agalactiae were 9.5–10.5, 21.8–23.0 and 17.3–18.5 µg/mL, respectively, which was similar to those of the HBD3 standard (P>0.05). The in vivo anti-bacterial activities of rHBD3 in milk were examined by intramammary infusion of viable bacterial inoculums. We observed that 9/10 and 8/10 glands of non-transgenic goats infused with S. aureus and E. coli became infected. The mean numbers of viable bacteria went up to 2.9×10³ and 95.4×10³ CFU/ml at 48 h after infusion, respectively; the mean somatic cell counts (SCC) in infected glands reached up to 260.4×10⁵ and 622.2×10⁵ cells/ml, which were significantly higher than the SCC in uninfected goat glands. In contrast, no bacteria was presented in glands of transgenic goats and PBS-infused controls, and the SSC did not significantly change throughout the period. Moreover, the compositions and protein profiles of milk from transgenic and non-transgenic goats were identical. The present study demonstrated that HBD3 were an effective anti-bacterial protein to enhance the mastitis resistance of dairy animals.     

Ultrastructure of the larval Malpighian tubules in <Emphasis Type="Italic">Terrobittacus implicatus</Emphasis> (Mecoptera: Bittacidae)

The larvae of Bittacidae, a cosmopolitan family in Mecoptera, have an interesting habit of spraying the body surface with soil through the anus after hatching, and each molts. The fine structure of Malpighian tubules, however, remains largely unknown in the larvae of Bittacidae to date. Here, we studied the ultrastructure of the larval Malpighian tubules in the hangingfly Terrobittacus implicatus (Huang & Hua) using scanning and transmission electron microscopy. The larvae of T. implicatus have six elongate Malpighian tubules at the junction of the midgut and hindgut. The tubule comprises a basal lamina, a single-layered epithelium, and a central lumen. The basal plasma membranes of the epithelial cells are conspicuously infolded and generate a labyrinth. The epithelium consists of two types of cells: large principal cells and scattered stellate cells. Mitochondria and cisterns of rough endoplasmic reticulum are numerous in the principal cells but are sparsely distributed in the stellate cells, indicating that the principal cells are active in transport. On the other hand, spherites are only abundant in the principal cells and are likely associated with the soil-spraying habit of the larvae.     

Molecular phylogeny and host-plant use (Lamiaceae) of the Thymogethes pollen beetles (Coleoptera)

The 24 members of the Euro-Asiatic genus Thymogethes are highly specialized pollen beetles associated as larvae with flowers of Lamiaceae Nepetoideae. All members of the genus were analysed in within the framework of an integrative taxonomy approach, which was aimed to reconstruct the phylogenetic relationships, as well as the possible pattern of evolution of their larval-host-plant association. Evidence from multiple molecular markers [COI; 16S; H3], combined with an estimation of divergence times using an average rate of 0.0177 substitutions/site/My among branches, placed the origin of the genus at a minimum of 9–10 Mya. This date of origin approximates the known evolution of the host plants in Euro-Mediterranean areas. Evidence from combined molecular and cladistic morphological analyses resulted in suitable agreement with the previously established morphology-based systematics of the genus, although members of the exilis species-group were split into three clades. The only disagreement between results of this new combined phylogeny and previous classification is in the exclusion of “Thymogethes” grenieri. This species is herein positioned outside the genus, based on molecular evidence. Our analysis depicts several Thymogethes species differentiating in the last few Mys, specifically those included in the T. lugubris species-group. Combined evidence from DNA, morphology and ancestral state parsimony reconstruction of larval-host-plant associations suggests that subtribe Menthinae likely represents the ancestral host plants, with a series of independent host shifts during the radiation of the clade, in association first with Menthinae and subsequently with Lavandulinae and Nepetinae. Steno-oligophagy is the most frequent (86%) condition, while strictly monophagous species are less numerous (14%).     

Feeding Fitness and Quality of Domesticated and Feral Predators: Effects of Long-Term Rearing on Artificial Diet

Predatory geocorids, Geocoris punctipes Say (Geocoridae: Hemiptera), that had been reared (domesticated) for over 6 years (60 continuous generations) on an artificial diet were compared with feral (F₁) counterparts to determine possible domestication-associated losses in predatory capabilities. Using adult female predators provided with either tobacco budworm larvae, Heliothis virescens F., or pea aphids, Acyrthosiphon pisum Harris, as prey, I measured predator weights, handling time with a single prey, amount extracted, consumption rate, and feeding (gut) capacity. Domesticated females were significantly smaller than ferals, weighing 4.53 mg versus 5.09 mg, respectively. Domestication did not significantly influence handling times, which averaged 131 (domesticated) and 122 min (feral) for predators feeding on H. virescens larvae and 106 (domesticated) and 94 min (feral) for G. punctipes feeding on A. pisum. Although there were significant differences in the weights of the two kinds of prey (H. virescens larvae being about twice as heavy as the A. pisum), both prey species exceeded the ingestion capacity of the predators. Amounts extracted by predators were 1.12 to 1.20 mg and were not significantly influenced by rearing background, prey biomass, or prey type. Consumption rates of 11.86 and 12.91 μg/min were nearly identical for both domesticated and feral predators regardless of prey species.     

GnRHR gene polymorphisms and their effects on reproductive performance in Chinese goats

In this study, polymorphisms in the goat GnRHR gene exon 1 were detected by PCR-SSCP and DNA sequencing methods in 786 individuals from two different goat breeds. Two haplotypes (A and B), two observed genotypes (AA and AB), and two single nucleotide polymorphisms (SNPs) were detected, which resulted in five amino acid substitutions. The frequencies of haplotypes A and B in the two goat breeds were 0.78–0.83 and 0.17–0.22, respectively. The SNP locus was in Hardy–Weinberg disequilibrium in the two goat breeds (P < 0.05). Polymorphisms of the GnRHR gene were shown to be associated with litter size in the two goat breeds. The SNPs in the goat GnRHR gene had significant effects on litter size (P < 0.05). Therefore, these results suggest that the GnRHR gene is a strong candidate gene that affects litter size in goat.     

Swainsonine promotes apoptosis in human oesophageal squamous cell carcinoma cells <Emphasis Type="BoldItalic">in vitro</Emphasis> and <Emphasis Type="BoldItalic">in vivo</Emphasis> through activation of mitochondrial pathway

Swainsonine, a natural indolizidine alkaloid, has been reported to have antitumour effects, and can induce apoptosis in human gastric and lung cancer cells. In the present study, we evaluated the antitumour effects of swainsonine on several oesophageal squamous cell carcinoma cells and investigated relative molecular mechanisms. Swainsonine treatment inhibited the growth of Eca-109, TE-1 and TE-10 cells in a concentration-dependent manner as measured by MTT assay. Morphological observation, DNA laddering detection and flow cytometry analysis demonstrated that swainsonine treatment induced Eca-109 cell apoptosis in vitro. Further results showed that swainsonine treatment up-regulated Bax, down-regulated Bcl-2 expression, triggered Bax translocation to mitochondria, destructed mitochondria integrity and activated mitochondria-mediated apoptotic pathway, followed by the release of cytochrome c, which in turn activated caspase-9 and caspase-3, promoted the cleavage of PARP, resulting in Eca-109 cell apoptosis. Moreover, swainsonine treatment inhibited Bcl-2 expression, promoted Bax translocation, cytochrome c release and caspase-3 activation in xenograft tumour cells, resulting in a significant decrease of tumour volume and tumour weight in the swainsonine-treated xenograft mice groups compared with that in the control group. Taken together, this study demonstrated that swainsonine inhibited Eca-109 cells growth through activation of mitochondria-mediated caspase-dependent pathway.     

Use of protocorm-like bodies for studying alkaloid metabolism in <Emphasis Type="Italic">Pinellia ternata</Emphasis>

Highly differentiated tissue masses known as protocorm-like bodies (PLBs) have been commonly used for plant regeneration. In this study the potential use of PLBs for studying alkaloid metabolism in the Chinese medicinal herb Pinellia ternata (Thunb.) Breit. was investigated. Tuber, leaf, and petiole explants of P. ternata were incubated on Murashige and Skoog (MS) (1962) basal medium containing different combinations of α-naphthaleneacetic acid (NAA) and 6-benzyladenine (BA). It was observed that 0.5 mg/L NAA and 1.0 mg/L BA induced the highest frequency of undifferentiated PLBs from tuber explants; whereas, a combination of 0.2 mg/L NAA and 1.0 mg/L BA was best suited for inducing undifferentiated PLBs from leaf and petiole explants. When these PLBs were subcultured on solid MS medium containing 0.6 or 1.2 mg/L abscisic acid (ABA), ABA promoted proliferation of PLBs, but inhibited their germination. To elicit alkaloid biosynthesis, suspension cultures of PLBs were established in half-strength MS (1/2 MS) liquid medium supplemented with 0.6 mg/L ABA. Water extracts of PLBs collected from suspension cultures contained guanosine and inosine, two important alkaloids of P. ternata. Levels of guanosine concentrations were tenfold higher in tuber-derived PLBs compared to those in field-grown tubers; whereas, those of inosine were slightly lower in PLBs compared to those from field-grown tubers.     

黄土高原不同土壤结构体有机碳库的分布

根据不同植被类型和土壤类型,分别从黄土高原不同地域分层（0～20 cm、20～40 cm和40～60 cm）采集22个土壤剖面样品,研究土壤有机碳库在不同结构体中的分布特征.结果表明,所有结构体,从表层向下有机碳含量和贮量皆呈递减趋势;而各土层,从＞5 mm、2～5 mm、1～2 mm到0.25～1 mm结构体有机碳含量呈递增趋势,而从0.25～1 mm到＜0.25 mm呈下降趋势,以0.25～1 mm结构体中有机碳含量最高.由于不同大小结构体所占比例不同,因此不同结构体中的有机碳贮量与含量并不完全一致：从＞5 mm、2～5 mm到1～2 mm结构体中有机碳贮量呈递减趋势,而从1～2 mm、0.25～1 mm到＜0.25 mm呈递增趋势,以1～2 mm结构体有机碳贮量最低.有机碳含量除土垫旱耕人为土在＜0.25 mm结构体中最大外,干湿砂质新成土、黄土正常新成土和简育干润均腐土在0.25～1 mm结构体中最高;但有机碳贮量在干湿砂质新成土和黄土正常新成土中以＜0.25 mm结构体所占比例最大,在简育干润均腐土和土垫旱耕人为土中以＞5 mm结构体所占比例最大.在不同植被下有机碳含量、贮量不同,表现为自然林地＞裸地＞人工林地＞农地.