The regulatory mechanism of flavonoids, which synergise anti-malarial and anti-cancer compounds in Artemisia annua, is still unclear. In this study, an anthocyanidin-accumulating mutant callus was induced from A. annua and comparative transcriptomic analysis of wild-type and mutant calli performed, based on the next-generation Illumina/Solexa sequencing platform and de novo assembly. A total of 82,393 unigenes were obtained and 34,764 unigenes were annotated in the public database. Among these, 87 unigenes were assigned to 14 structural genes involved in the flavonoid biosynthetic pathway and 37 unigenes were assigned to 17 structural genes related to metabolism of flavonoids. More than 30 unigenes were assigned to regulatory genes, including R2R3-MYB, bHLH and WD40, which might regulate flavonoid biosynthesis. A further 29 unigenes encoding flavonoid biosynthetic enzymes or transcription factors were up-regulated in the mutant, while 19 unigenes were down-regulated, compared with the wild type. Expression levels of nine genes involved in the flavonoid pathway were compared using semi-quantitative RT-PCR, and results were consistent with comparative transcriptomic analysis. Finally, a putative flavonol synthase gene (AaFLS1) was identified from enzyme assay in vitro and in vivo through heterogeneous expression, and confirmed comparative transcriptomic analysis of wild-type and mutant callus. The present work has provided important target genes for the regulation of flavonoid biosynthesis in A. annua.

SummaryExotic plants can compete well with native species because many invasive species are considered better nutrient users in both low‐ and high‐resource environments. However, whether invasive plants can outperform native plants at all stages of invasion is not very clear. We investigated the nitrogen (N), phosphorus (P) and N:P homeostasis of an invasive Eupatorium adenophorum and a co‐occurring native plant Artemisia argyi in an area across the five invasion stages of E. adenophorum. The N homeostasis (HN) of E. adenophorum was higher than that of A. argyi, whereas the P and N:P homeostasis (HP and HN/P) were higher for A. argyi. For E. adenophorum, HN decreased, but HP and HN/P increased with the invasion time. For A. argyi, HN/P increased, HP and HN remained stable with the invasion time. The results demonstrated that E. adenophorum could maintain higher HN during invasion stages when N was limited and could maintain higher HN and HP at invasion stages when P was more limited. This rapid nitrogen and phosphorus homeostasis transformation of invasive E. adenophorum during its invasion stages guarantees its stronger competitive ability over native species and promotes its invasion success.

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Hemalin, initially described in Haemaphysalis longicornis, is a protein with anticoagulant activity. We retrieved a gene fragment functionally annotated as hemalin from H. flava salivary gland transcriptomic library, but its full‐length complementary DNA (cDNA) and antithrombogenicity have not been investigated in the species. Here we cloned the full length of hemalin (Hf‐hemalin) by 3′‐end rapid‐amplification of cDNA ends, and the open reading frame (ORF) of Hf‐hemalin was expressed in Escherichia coli. The recombinant protein (rHf‐Hemalin) was tested for antithrombogenicity. The full‐length of Hf‐hemalin was 607 bp with an ORF of423 bp. Protein encoded by Hf‐hemalin was predicted to contain 2 Kunitz domains and a signal peptide. The expression of Hf‐hemalin in salivary glands, midguts and ovaries was higher in the semi‐engorged than the fully engorged. Prokaryotic expression yielded a product of 40 kDa containing a glutathione S‐transferase (GST) tag. Incubation of rHf‐Hemalin with rat plasma significantly extended prothrombin time and activated partial thromboplastin time compared with normal saline and GST controls. Our data demonstrated that Hemalin from H. flava shared a similar primary structure with that from H. longicornis, and was also anticoagulant. Further investigations are needed to test its feasibility to be an antigen candidate for the development of vaccines against ticks.

The heat shock protein 70 (HSPA) family and their genes have been studied in ticks and are considered as possible antigen candidates for the development of anti-tick vaccines. However, knowledge about their members, structure and function in ticks is incomplete. Based on our transcriptomic data, the full length of four HSPA genes in Haemaphysalis flava (Acari: Ixodidae) was cloned via rapid amplification of cDNA ends. The open reading frame of HSPA2A, HSPA2B, HSPA5 and HSPA9 was 1920, 1911, 1983 and 2088 bp in length, respectively. Three family signatures and one localization motif were in the encoding proteins. HSPA2A and HSPA2B were predicted to be located at cytoplasm/nucleus, whereas HSPA5 and HSPA9 were at endoplasmic reticulum and mitochondria, respectively. In silico simulation demonstrated that those proteins had distinct numbers of -helixes, extended strands and coils, and different antigenic epitopes. Expression of HSPA5 and HSPA9 in the salivary gland was significantly higher in partially-engorged female adult ticks than the fully-engorged (P < 0.01) as shown by a quantitative polymerase chain reaction. Our data indicated that H. flava ticks had at least four HSPA genes encoding proteins with different cellular locations, structures and expression profiles, suggesting their diverse roles in tick biology.