[Show abstract][Hide abstract] ABSTRACT: Thlaspi arvense is a well-known annual farmland weed with worldwide distribution, which can be found from sea level to above 4000 m high on the Qinghai-Tibetan Plateau (QTP). In this paper, a phylogeographic history of T. arvense including 19 populations from China was inferred by using three chloroplast (cp) DNA segments (trnL-trnF, rpl32-trnL and rps16) and one nuclear (n) DNA segment (Fe-regulated transporter-like protein, ZIP). A total of 11 chloroplast haplotypes and six nuclear alleles were identified, and haplotypes unique to the QTP were recognized (C4, C5, C7 and N4). On the basis of molecular dating, haplotypes C4, C5 and C7 have separated from others around 1.58 Ma for cpDNA, which corresponds to the QTP uplift. In addition, this article suggests that the T. arvense populations in China are a mixture of diverged subpopulations as inferred by hT/vT test (hT ≤ vT, cpDNA) and positive Tajima's D values (1.87, 0.05 < p < 0.10 for cpDNA and 3.37, p < 0.01 for nDNA). Multimodality mismatch distribution curves and a relatively large shared area of suitable environmental conditions between the Last Glacial Maximum (LGM) as well as the present time recognized by MaxEnt software reject the sudden expansion population model.
International Journal of Molecular Sciences 06/2015; 16(6):13339-55. DOI:10.3390/ijms160613339 · 2.86 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Astragalus membranaceus Bge. var. mongolicus (Bge.) Hsiao (A. mongolicus, family Leguminosae) is one of the most important traditional Chinese herbs. Among many secondary metabolites it produces, the effective bioactive constituents include isoflavonoids and triterpene saponins. The genomic resources regarding the biosynthesis of these metabolites in A. mongolicus are limited. Although roots are the primary material harvested for medical use, the biosynthesis of the bioactive compounds and its regulation in A. mongolicus are not well understood. Therefore, a global transcriptome analysis on A. mongolicus tissues was performed to identify the genes essential for the metabolism and to profile their expression patterns in greater details.
RNA-sequencing was performed for three different A. mongolicus tissues: leaf, stem, and root, using the Illumina Hiseq2000 platform. A total of 159.5 million raw sequence reads were generated, and assembled into 186,324 unigenes with an N50 of 1,524bp. Among them, 129,966 unigenes (~69.7%) were annotated using four public databases (Swiss-Prot, TrEMBL, CDD, Pfam), and 90,202, 63,946, and 78,326 unigenes were found to express in leaves, roots, and stems, respectively. A total of 8,025 transcription factors (TFs) were identified, in which the four largest families, bHLH, MYB, C3H, and WRKY, were implicated in regulation of tissue development, metabolisms, stress response, etc. Unigenes associated with secondary metabolism, especially those with isolavonoids and triterpene saponins biosynthesis were characterized and profiled. Most genes involved in the isoflavonoids biosynthesis had the lowest expression in the leaves, and the highest in the stems. For triterpene saponin biosynthesis, we found the genes in MVA and non-MVA pathways were differentially expressed among three examined tissues, indicating the parallel but compartmentally separated biosynthesis pathways of IPP and DMAPP in A. mongolicus. The first committed enzyme in triterpene saponin biosynthesis from A. mongolicus, cycloartenol synthase (AmCAS), which belongs to the oxidosqualene cyclase family, was cloned by us to study the astragalosides biosynthesis. Further co-expression analysis indicated the candidate CYP450s and glycosyltransferases (GTs) in the cascade of triterpene saponins biosynthesis. The presence of the large CYP450 families in A. mongolicus was further compared with those from Medicago truncatula and Arabidopsis thaliana, and the diversity and phylegenetic relationships of the CYP450 families were established.
A transcriptome study was performed for A. mongolicus tissues to construct and profile their metabolic pathways, especially for the important bioactive molecules. The results revealed a comprehensive profile for metabolic activities among tissues, pointing to the equal importance of leaf, stem, and root in A. mongolicus for the production of bioactive compounds. This work provides valuable resources for bioengineering and in vitro synthesis of the natural compounds for medical research and for potential drug development.
[Show abstract][Hide abstract] ABSTRACT: Human papillomaviruses (HPVs) including high-risk (HR) and low-risk (LR) subtypes have distinguishable variation on both genotypes and phenotypes. The co-infection of multiple HR-HPVs, headed by HPV16, is common in cervical cancer in female. Recently accumulating reports have focused on the interaction between virus and host, particularly the role of human microRNAs (miRNAs) in anti-viral defense by targeting viral genome. Here, we found a well-conserved target site of miRNAs in the genomes of most HR-HPVs, not LR-HPVs, by scanning all potential target sites of human miRNAs on 24 HPVs of unambiguous subtypes of risk. The site is targeted by two less common human miRNAs, miR-875 and miR-3144, and is located in E6 oncogene open reading frame (ORF) and overlap with the first alternative splice exon of viral early transcripts. In validation tests, miR-875 and miR-3144 were identified to suppress the target reporter activity markedly and inhibit the expression of both synthetically exogenous E6 and endogenous E6 oncogene. High level of two miRNAs can inhibit cell growth and promote apoptosis in HPV16-positive cervical cancer cells. This study provides a promising common target of miRNAs for most HR-HPVs and highlights the effects of two low expressed human miRNAs on tumour suppression.
Protein & Cell 04/2015; 6(8). DOI:10.1007/s13238-015-0142-8 · 3.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Lycium ruthenicum (Solananeae), a spiny shrub mostly distributed in the desert regions of north and northwest China, has been shown to exhibit high tolerance to the extreme environment. In this study, the phylogeography and evolutionary history of L. ruthenicum were examined, on the basis of 80 individuals from eight populations. Using the sequence variations of two spacer regions of chloroplast DNA (trnH-psbA and rps16-trnK), the absence of a geographic component in the chloroplast DNA genetic structure was identified (G(ST) = 0.351, N-ST = 0.304, N-ST<G(ST)), which was consisted with the result of SAMOVA, suggesting weak phylogeographic structure of this species. Phylogenetic and network analyses showed that a total of 10 haplotypes identified in the present study clustered into two clades, in which clade I harbored the ancestral haplotypes that inferred two independent glacial refugia in the middle of Qaidam Basin and the western Inner Mongolia. The existence of regional evolutionary differences was supported by GENETREE, which revealed that one of the population in Qaidam Basin and the two populations in Tarim Basin had experienced rapid expansion, and the other populations retained relatively stable population size during the Pleistocene. Given the results of long-term gene flow and pairwise differences, strong gene flow was insufficient to reduce the genetic differentiation among populations or within populations, probably due to the genetic composition containing a common haplotype and the high number of private haplotypes fixed for most of the population. The divergence times of different lineages were consistent with the rapid uplift phases of the Qinghai-Tibetan Plateau and the initiation and expansion of deserts in northern China, suggesting that the origin and evolution of L. ruthenicum were strongly influenced by Quaternary environment changes.
Pakistan Journal of Botany 12/2014; 46(6):2121-U201. · 0.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Ultraviolet-B (UV-B) is a natural component of the solar radiation. Due to its high energy, low dosages of UV-B can bring huge potential damage effect to organisms. Despite much research that has analyzed the gene expression changes of plants that under UV-B radiation, the transcriptome response of Lycium ruthenicum under the UV-B induction is still un-available. The aim of our study was to identify UV-B responsive genes and gain an insight into the underlying genetic basis of the pathobiology of UV-B related damage. We collected leaf samples from L. ruthenicum with and without UV-B exposure, and then performed a transcriptome profiling to comprehensively investigate their expression signatures. By employing the high throughput RNA-sequencing analysis of samples with and without UV-B exposure, we identified 1,913 up-regulated and 536 down-regulated genes at least by twofold changes. The activity of antioxidant enzyme related genes, including the superoxide dismutase, was decreased, genes related to the synthesis of secondary metabolites and defense responses, such as cinnamyl alcohol dehydrogenase, chalcone-flavanone isomerase and dihydroflavonol reductase were also downregulated. The expression patterns of 14 randomly selected genes resulted from quantitative real-time PCR were basically consistent with their transcript abundance changes identified by RNA-sequencing. We found that several biological pathways related to biotic and abiotic stresses, including cell defense, photosynthesis processes, energy metabolism, were involved in the process of UV-B stress response. A genome-wide screening of gene deregulation under UV-B induction would provide an insight into the understanding of the molecular bases and pathogenesis of UV-B responses.
[Show abstract][Hide abstract] ABSTRACT: Mir-181 is an ancient microRNA (miRNA) gene family that originated in urochordata. Although their functions were subjected to extensive studies in recent years, their evolutionary process remains largely unknown. Here we systematically investigated the homologous genes of the mir-181 family by a sequence similarity search. Representative sequences of the mir-181 gene family were used to reconstruct their evolutionary history. Our results indicated that this family could have derived from multiple duplications, which include two rounds of whole genome duplications and one round of segmental replication. Functional annotation of the target genes of the mir-181 family suggested that this family could participate in some important biological processes including transcriptional and translational regulation, signaling transduction etc. This analysis presented a complex evolutionary dynamics for the origination of a miRNA gene family.
Computers in Biology and Medicine 09/2014; 52. DOI:10.1016/j.compbiomed.2014.06.004 · 1.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Ubiquitin belongs to an important class of protein modifier and gene expression regulator proteins that participates in various cellular processes. A large number of ubiquitin-related proteins have been identified during the last two decades. However, the evolutionary history of this ancient gene family remains largely unknown. We analyzed the members of the superfamily using both sequence- and structure-based methodology to better understand the evolution of ubiquitin-related proteins. As a part of these analyses we used the MEME algorithm to extract common sequence motifs across the superfamily, and we inferred the phylogeny and distribution of the superfamily members across multiple species. A total of 23 families were identified in the gene family. Several common sequence motifs were revealed and evaluated. We also found that the number of genes for ubiquitin-related proteins encoded within a specific genome correlates with the biological complexity of that particular species. This analysis should provide valuable insight into the sequence/function relationships and evolutionary history of ubiquitin and ubiquitin-related proteins.
[Show abstract][Hide abstract] ABSTRACT: Microrefugia at high altitudes or high latitudes are thought to play an important role in the post-glacial colonization of species. However, how populations in such microrefugia have responded to climate changes in alternating cold glacial and warm interglacial stages remain unclear. Here we present evidence to indicate the Rongbuk Valley of the Mt. Qomolangma (Mt. Everest) area, the highest region on earth, had microrefugia for Hippophae tibetana and discuss how this low shrub was adapted to the extreme climate fluctuations of the last 25,000 years by shifts. By integrating geological, glaciological, meteorological, and genetic information, we found that the Rongbuk Valley was not only a glacial microrefugium but also an interglacial microrefugium for H. tibetana: the former was located on the riverbank below 4800 m above sea level (asl) or lower area and the latter at ∼5000 m asl. Our results show that after the Last Glacial Maximum (LGM), H. tibetana in the valley has undergone upward and downward migrations around ∼5000 m driven by climate fluctuations and the population in the glacial microrefugium has suffered extinction or extreme contraction. Moreover, with the rise of temperature in the last four decades, the upper limit of H. tibetana has shifted at least 30 m upward. Combining population history and recent range shift of this species is important in predicting the fate of this endemic species to future climate changes.
PLoS ONE 05/2014; 9(5):e97601. DOI:10.1371/journal.pone.0097601 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cerbera manghas L. (Apocynaceae), a mangrove species, is fragile and susceptible to human pressure. In this study, 26 polymorphic microsatellite markers were developed across 64 C. manghas individuals from four populations in South China. The number of alleles varied from 2 to 11 alleles with an average of 5.54 per locus. The observed and expected heterozygosities per population ranged from 0.063 to 0.874, respectively. These markers will be useful for genetic studies toward the strategies for conservation and management of this important mangrove resource.
[Show abstract][Hide abstract] ABSTRACT: Dactylorhiza hatagirea (D. Don) Soo is a critically endangered orchid distributed in eastern China and central Asia. In order to investigate its genetic diversity within and between populations and design effective conservation strategies, we developed microsatellite marker for this species in the present study. A total of 14 polymorphic markers were isolated and characterized using 68 individuals from three natural populations. The number of alleles per locus for a single population ranged from 2 to 15, and the polymorphism information content ranged from 0.043 to 0.908. No genotypic linkage disequilibrium within populations was detected. These microsatellite markers are the first characterized for D. hatagirea and they are expected to be useful for population level studies in this species.
[Show abstract][Hide abstract] ABSTRACT: The Qinghai-Tibetan Plateau (QTP) is the highest and one of the most extensive plateaus in the world. Phylogenetic, phylogeographic, and ecological studies support plant diversifications on the QTP through multiple mechanisms such as allopatric speciation via geographic isolation, climatic oscillations and divergences, pollinator-mediated isolation, diploid hybridization and introgression, and allopolyploidy. These mechanisms have driven spectacular radiations and/or species diversifications in various groups of plants such as Pedicularis L., Saussurea DC., Rhododendron L., Primula L., Meconopsis Vig., Rhodiola L., and many lineages of gymnosperms. Nevertheless, much work is needed toward understanding the evolutionary mechanisms of plant diversifications on the QTP. Well-sampled biogeographic analyses of the QTP plants in the broad framework of the Northern Hemisphere as well as the Southern Hemisphere are still relatively few and should be encouraged in the next decade. This paper reviews recent evidence from phylogenetic and biogeographic studies in plants, in the context of rapid radiations, mechanisms of species diversifications on the QTP, and the biogeographic significance of the QTP in the broader context of both the Northern and Southern Hemisphere biogeography. Integrative multidimensional analyses of phylogeny, morphological innovations, geography, ecology, development, species interactions and diversifications, and geology are needed and should shed insights into the patterns of evolutionary assembly and radiations in this fascinating region.
Frontiers in Genetics 02/2014; 5:4. DOI:10.3389/fgene.2014.00004
[Show abstract][Hide abstract] ABSTRACT: Although molecular phylogenetics is a strong tool for reconstructing the tree of life, many problems persist due to systematic errors caused by model mis-specifications. Resolving misconstructed trees should lead us to better understand the processes of molecular evolution. Mammalian mitogenomes provide us with a good opportunity in this respect, because the mammalian tree is well established on the basis of multiple nuclear genes, and mitogenome trees are sometimes in conflict with it, for example concerning the positions of tarsiers and colugos. The utility of mitogenomes as a phylogenetic marker is therefore sometimes questioned, and an important problem is whether any method can overcome the misleading phylogenetic signals of mitogenomes. Here we show that the maximum likelihood tree of 463 eutherian mitogenomes reconstructed from nucleotide sequences of protein-encoding genes gives positions of tarsiers and colugos that are consistent with the well-established nuclear tree; this is the first study to obtain a consistent tree with respect to the positions of tarsiers and colugos using mitogenomes. Furthermore, our mitogenome tree of the 463 eutherians is mostly consistent with the nuclear gene tree. Previous mitogenomic studies have been hampered by sparse taxon sampling, and our analysis demonstrates the importance of dense taxon sampling to relieve the misleading phylogenetic signals of mitogenomes. However, because there are many convergent and parallel substitutions in the amino acid sequences, the effect of dense taxon sampling on the accuracy of tree reconstruction seems to be very limited. We further show the importance of using synonymous substitutions with dense taxon sampling as well as with appropriate modeling in recovering the well-established tree from lower to even higher levels of eutherian phylogeny.
[Show abstract][Hide abstract] ABSTRACT: The schizothoracine fish, also called snow trout, are members of the Cyprinidae, and are the most diversified teleost fish in the Qinghai-Tibetan Plateau (QTP). Clarifying the evolutionary history of the schizothoracine fish is therefore important for better understanding the biodiversity of the QTP. Although morphological and molecular phylogenetic studies have supported the monophyly of the Schizothoracinae, a recent molecular phylogenetic study based on the mitochondrial genome questioned the monophyly of this taxon. However, the phylogenetic analysis of that study was on the basis of only three schizothoracine species, and the support values were low. In this report, we inferred the phylogenetic tree on the basis of mitochondrial genome data including 21 schizothoracine species and five closely related species, and the polyphyletic origins of the Schizothoracinae were strongly supported. The tree further suggests that the Schizothoracinae consists of two clades, namely the "morphologically specialized clade" and the "morphologically primitive clade", and that these two clades migrated independently of each other to the QTP and adapted to high altitude. We also detected in their mitochondrial genomes strong signals of positive selection, which probably represent evidence of high-altitude adaptation. In the case of the morphologically specialized clade, positive selection mainly occurred during the Late Paleocene to the Early Oligocene. Its migration also seems to have occurred in the Early Eocene, and this timing is consistent with the drastic uplifting of the QTP. On the other hand, positive selection in the morphologically primitive clade has mainly occurred since the Late Miocene. Because its members are thought to have migrated to the QTP recently, it is possible that they are now undergoing high-altitude adaptation.
[Show abstract][Hide abstract] ABSTRACT: The schizothoracine fish, also called snow trout, are members of the Cyprinidae, and are the most diversified teleost fish in the Qinghai-Tibetan Plateau (QTP). Clarifying the evolutionary history of the schizothoracine fish is therefore important for better understanding the biodiversity of the QTP. Although morphological and molecular phylogenetic studies have supported the monophyly of the Schizothoracinae, a recent molecular phylogenetic study based on the mito-chondrial genome questioned the monophyly of this taxon. However, the phylo-genetic analysis of that study was on the basis of only three schizothoracine species, and the support values were low. In this report, we inferred the phylo-genetic tree on the basis of mitochondrial genome data including 21 schizothora-cine species and five closely related species, and the polyphyletic origins of the Schizothoracinae were strongly supported. The tree further suggests that the Schizothoracinae consists of two clades, namely the " morphologically specialized clade " and the " morphologically primitive clade " , and that these two clades migrated independently of each other to the QTP and adapted to high altitude. We also detected in their mitochondrial genomes strong signals of positive selection, which probably represent evidence of high-altitude adaptation. In the case of the morphologically specialized clade, positive selection mainly occurred during the Late Paleocene to the Early Oligocene. Its migration also seems to have occurred in the Early Eocene, and this timing is consistent with the drastic uplifting of the QTP. On the other hand, positive selection in the morphologically primitive clade has mainly occurred since the Late Miocene. Because its members are thought to have migrated to the QTP recently, it is possible that they are now undergoing high-altitude adaptation. Despite being a harsh environment due to its high elevation , the Qinghai Tibetan Plateau (QTP) is known as a hot spot of biodiversity because of its high species richness and abundant endemic species (Myers et al., 2000; Qi et al., 2012). The Schizothoracinae, also called " snow trout " , is a subfamily of the Cyprinidae, and comprises 10–13 genera with about 100 species (Mizra, 1991). Schizothoracinae is diagnosed by two lines of enlarged scales along both sides of the anus and urogenital opening (Chen and Chen, 2001). Distributed in the QTP and its surrounding areas, they are the most diversified teleost fish in the QTP, and play important roles in its riverine ecosystems. Therefore, clarifying the evolutionary history of this taxon is important to better understand how the biodiversity of QTP has been configurated. The schizothoracine fish have been classified into three groups on the basis of morphological characters: the " primitive group " , " specialized group " and " highly specialized group " (Cao et al., 1981). The primitive group consists of Schizothorax and Aspiorhynchus. They exhibit more traits similar to the outgroup Barbodes hexagonolepis, and are characterized by having uroneuralia, three or
Genes & Genetic Systems 01/2014; 89:187-191. · 0.93 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The composition and the bioactivity of the essential oil of Artemisia anomala, a traditional medicinal herb in China, were investigated. The composition of the essential oil was analyzed by gas chromatography–mass spectometry (GC–MS), and a total of 39 components representing 98.1% in this plant were identified. The main constituents were camphor (18.3%), 1, 8-cineole (17.3%), β-caryophyllene oxide (12.7%), and borneol (9.5%). The antimicrobial activities of the oil were separately evaluated against six micro-organisms, Bacillus cereus (CMCCB 63301), Micrococcus flavus (ATCC 14698), Escherichia coli (ATCC 11775), Pseudomonas aeruginosa (ATCC 10145), Staphylococcus epidermidis (ATCC 12228), and a clinical-isolated strain Staphylococcus aureus. The results showed that the oil possessed inhibitory activity against B. cereus, M. flavus, E. coli, S. epidermidis, and S. aureus, among which the best inhibition was observed against B. cereus with a MIC of 0.313 μL/mL. The antiproliferative activities of the oil were assessed against four human tumor cell lines, A459 (human lung adenocarcinoma), BRO (human melanoma), MCF7 (human breast cancer), and PC3 (human prostate). The oil showed the ability to inhibit proliferation of human cancer cells. And the MCF7 cell line was the most sensitive to the oil with an IC50 of 0.09.
Journal of Essential Oil Research 12/2013; 25(6):520-525. DOI:10.1080/10412905.2013.820670 · 0.79 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In the field of drug discovery, it is particularly important to discover bioactive compounds through high-throughput virtual screening. The maximum common substructure-based (MCS) algorithm is a promising method for the virtual screening of drug candidates. However, in practical applications, there is always a trade-off between efficiency and accuracy. In this paper, we optimized this method by running time evaluation using essential drugs defined by WHO and FDA-approved small-molecule drugs. The amount of running time allocated to the MCS-based virtual screening was varied, and statistical analysis was conducted to study the impact of computation running time on the screening results. It was determined that the running time efficiency can be improved without compromising accuracy by setting proper running time thresholds. In addition, the similarity of compound structures and its relevance to biological activity are analyzed quantitatively, which highlight the applicability of the MCS-based methods in predicting functions of small molecules. 15-30s was established as a reasonable range for selecting a candidate running time threshold. The effect of CPU speed is considered and the conclusion is generalized. The potential biological activity of small molecules with unknown functions can be predicted by the MCS-based methods.
[Show abstract][Hide abstract] ABSTRACT: Our study, based on a survey of permanent residents of Beijing or residents with Beijing hukou (residential registration), explores the mood and feelings of ordinary people in China regarding levels of political interest and democratic values, attitudes toward economic reform, evaluations of government performance in different policy areas, and general levels of satisfaction and confidence. The survey had two purposes: first, we intended to find out what ordinary Beijing residents thought about political issues; second, we tried to map China's political future.We believe that public opinion affects public policy in China. In a recent book, Allen Liu (1996) argues that post-Mao economic and political reforms came about more as a result of public opinion-which grew too strong for the Communist Party leaders to ignore after the death of Mao-and less as a result of the political motivations of individual leaders. The fact that the Chinese government has made noticeable efforts to control inflation and combat official corruption is evidence that the leaders and their policies are constrained to a certain degree by public opinion.
Political Science and Politics 09/2013; 30(3). DOI:10.2307/420126 · 0.48 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Intercontinental biotic disjunctions have been documented and analyzed in numerous Holarctic taxa. Patterns previously synthesized for animals compared to plants suggest that the timing of animal disjunctions are mostly Early Tertiary and were generated by migration and vicariance events occurring in the North Atlantic, while plant disjunctions are mostly Mid-Late Tertiary and imply migration and vicariance over Beringia. Melaphidina aphids (Hemiptera: Aphididae: Fordini) exhibit host-alternating life cycles comprising an obligate seasonal shift between Rhus subgenus Rhus species (Anacardiaceae) and mosses (Bryophyta). Similar to their Rhus hosts, melaphidines are distributed disjunctly between Eastern Asia and Eastern North America. We examined evolutionary relationships within Melaphidina to determine the position of the North American lineage, date its divergence from Asian relatives, and compare these results to a previous historical biogeographic study of Rhus. We sampled nine species and three subspecies representing all six genera of Melaphidina. Data included sequences of mitochondrial cytochrome c oxidase subunits I and II + leucine tRNA, cytochrome b, and nuclear elongation factor 1α genes. Phylogenetic analyses (Bayesian, maximum-likelihood, parsimony) of the combined data (3282 bp) supported the monophyly of all genera except Nurudea and Schlechtendalia, due to the position of N. ibofushi. While the exact position of the North American Melaphis was not well resolved, there was high support for a derived position within Asian taxa. The divergence of Melaphis from Asian relatives centered on the Eocene-Oligocene boundary (∼33-35 Ma), which coincides with closure of Beringian Land Bridge I. This also corresponded to the Asian-North American disjunction previously estimated for subgenus Rhus spp. We suggest the late-Eocene Bering Land Bridge as the most likely migration route for Melaphis ancestors, as was also hypothesized for North American Rhus ancestors. Results for the Melaphidina disjunction depart from the modal pattern in animal lineages, and present a case where insect and host-plant taxa apparently responded similarly to Tertiary climate change.
[Show abstract][Hide abstract] ABSTRACT: Key message
The gene and cDNA sequence encoding PAL from Chinese medicinal plant Rhus chinensis were cloned and analyzed, furthermore the biochemical properties, kinetic parameters, differential expression and key sites were studied. Abstract Rhus chinensis is a well-known Chinese medicinal plant. Phenylalanine ammonia-lyase (PAL) is the first enzyme of phenylpropanoid pathway. Several recent studies suggested that PAL also play an important role in plant–aphid interaction. In this study, both the cDNA and the genomic sequence encoding PAL from Rhus chinensis (designated as RcPAL) were cloned and analyzed. The 3,833 bp gene contained a 1,342 bp intron and two extrons. The ORF was 2,124 bp and predicted to encode a 707-amino acid polypeptide. The results of real-time PCR showed that RcPAL expressed in all tested tissues and followed the order: stems > young leaves > petioles > roots > seeds > mature leaves. RcPAL was successfully expressed in E. coli with the pET-28a-RcPAL recombinant vector. The recombinant protein exhibited a high level of PAL activity. Biochemical properties and kinetic parameters of recombinant RcPAL were further studied. The results showed that the optimal temperature and pH for RcPAL activity were 45 °C and 9.0, and the K m and K cat values were 7.90 mM and 52.31 s−1, respectively. The active sites and substrate selectivity site were also investigated with site-directed mutagenesis methods, suggesting that Phe126 is responsible for the substrate selectivity. To our knowledge, this was the first full-length PAL gene cloned and characterized from the family Anacardiaceae so far.