WRKY Transcription Factors Actively Respond to Fusarium oxysporum in Lilium regale.

The WRKY transcription factors form a plant-specific superfamily important for regulating plant development, stress responses, and hormone signal transduction. In this study, many WRKY genes (LrWRKY1-35) were identified in Lilium regale, which is a wild lily species highly resistant to Fusarium wilt. These WRKY genes were divided into three classes (I to III) based on a phylogenetic analysis. The Class-II WRKY transcription factors were further divided into five subclasses (IIa, IIb, IIc, IId, and IIe). Moreover, the gene expression patterns based on a quantitative real-time PCR analysis revealed the WRKY genes were differentially expressed in the L. regale roots, stems, leaves, and flowers. Additionally, the expression of the WRKY genes was affected by an infection by Fusarium oxysporum as well as by salicylic acid, methyl jasmonate, ethephon, and hydrogen peroxide treatments. Moreover, the LrWRKY1 protein was localized to the nucleus of onion epidermal cells. The recombinant LrWRKY1 protein purified from Escherichia coli bound specifically to DNA fragments containing the W-box sequence, and a yeast one-hybrid assay indicated that LrWRKY1 can activate transcription. A co-expression assay in tobacco (Nicotiana tabacum) confirmed LrWRKY1 regulates the expression of LrPR10-5. Furthermore, the overexpression of LrWRKY1 in tobacco and the Oriental hybrid 'Siberia' (susceptible to F. oxysporum) increased the resistance of the transgenic plants to F. oxysporum. Overall, LrWRKY1 regulates the expression of the resistance gene LrPR10-5 and is involved in the defense response of L. regale to F. oxysporum. This study provides valuable information regarding the expression and functional characteristics of L. regale WRKY genes.

[Functional analysis of a chitinase gene PnCHI1 from Panax notoginseng].

Chitinases, a glycosidase enzyme that hydrolyzes chitin to N-acetylglucosamine, are widely found in plant cells, and they are an important part of plant antifungal defense system. The function of a Panax notoginseng chitinase gene PnCHI1 was characterized in this paper. Expression vector of PnCHI1 was constructed and transiently expressed in onion epidermal cells, and laser scanning confocal microscopy demonstrated that PnCHI1 was localized in the cell wall. Prokaryotic expression vector of PnCHI1 was also constructed, and recombinant protein of PnCHI1 was induced and purified. In vitro antibacterial assay showed that recombinant PnCHI1 protein had strong inhibitory activity on the mycelium growth of Fusarium solani, F. oxysporum and F. verticillioide. The function of PnCHI1 was further verified by reverse genetics. PnCHI1 expression vector was transferred into tobacco by Agrobacterium tumefaciens and expression of PnCHI1 was confirmed by qRT-PCR. It was found by leaf inoculation experiment that resistance of transgenic tobacco to F. solani was significantly increased. It is conclnded that: PnCHI1 is a chitinase localized in the cell wall, which inhibits several fungi which cause the root rot disease of P. notoginseng. Overexpression of this chitinase gene in tobacco greatly increased resistance to F. solani. PnCHI1 may be an important resistance gene in P. notoginseng that participates in the defense against root rot disease.

[Cloning and expression analysis of a chitinase gene PnCHI1 from Panax notoginseng].

Chitinases(EC3.2.1.14), which are present in various organisms, catalyze the hydrolytic cleavage of chitin and play a vital role in plant defense mechanisms against fungal pathogens.In addition, the chitinases are well known to regulate plant growth and development and are involved in programmed cell death(PCD).A chitinase expressed sequence tag(EST) was isolated from Panax notoginseng, and the full-length cDNA of this EST was cloned with the method of rapid amplification of cDNA ends and named as PnCHI1. PnCHI1 was 1 022 bp in length and contained an intact open reading frame(ORF) of 822 bp, a 26 bp 5'-untranslated region(UTR), and a 174 bp 3'-UTR.The predicted protein of PnCHI1 with 273 amino acid residues belongs to glycoside hydrolase family 19 and fell into the class IV of chitinases through phylogenetic analysis.QRT-PCR analysis showed that the expression of PnCHI1 was induced by methyl jasmonate, ethylene, H2O2, and salicylic acid.PnCHI1 was quickly induced after inoculation with Alternaria panax.Moreover, the expression level of PnCHI1 was increased after pretreatment with methyl jasmonate, and then the transcription level of PnCHI1was sharp increased after inoculation with Fusarium solani,and the highest transcription level was achieved at 4 h post inoculation.But the expression level of PnCHI1 in the sterile water pretreated P.notoginseng was increased gradually after inoculation with F.solani, and the highest expression level was achieved at 48 h post inoculation.All the results of present study indicated that PnCHI1 was involved in defense response of P.notoginseng against the F.solani and A.panax.

[Application of ICP-AES to the chemical speciation of heavy metals in flyash].

Chemical speciation of seven heavy metals of flyashes in incinerator was quantitatively tested using ICP-AES. Results showed that ICP-AES procedure could carry out quick, exact and high precision experiments. RSD ratio for most detected metals was lower than 3% while few metals present a comparatively high RSD when whose content was near the detection limits. The recovery ratio was 85.7%-100.63% flyashes were found to have high content of Zn, Pb. Cd, Cu, Mn, Pb and Zn existed mostly as carbonates and were leachable, while Cr and Ni were combined to metal oxides substrates and present immobilization characteristics.