Identification and expression analysis of QM-like gene from Spodoptera litura after challenge by the entomopathogenic fungus Nomuraea rileyi.

A partial sequence of QM homologue was isolated from a Spodoptera litura fatbody suppression subtractive hybridization library. The full-length Spodoptera litura QM (SpLQM) cDNA of 838 bp contains a 5' untranslated region (UTR) of 112 bp, a 3' UTR of 66 bp, and an open reading frame of 660 nucleotides coding for a 219 amino acid peptide with a molecular weight of 25.5 kDa. Analysis of SpLQM sequence revealed the presence of characteristic motifs, including the ribosomal protein L10 signature and SH3-binding motif. Multiple alignment analysis revealed that SpLQM shares an overall identity of 57.1-99.1% with other members of QM family. Phylogenetic analysis confirmed that SpLQM is closely related to other insect QMs. Analysis of the tissue expression pattern showed that the SpLQM mRNA was expressed in all tissues tested, with highest levels measured in hemocytes, followed by fat bodies. Upon Nomuraea rileyi challenge, SpLQM showed significant upregulation in fat bodies and hemocytes, while slightly upregulation in midguts. The results suggest that SpLQM might play an important role in the innate immunity of S. litura in response to N. rileyi infection. SpLQM was also successfully overexpressed in Escherichia coli, and the recombinant fusion protein SpLQM-His has a molecular weight of 32 kDa.

Identification of suitable reference genes for gene expression studies by qRT-PCR in the blister beetle Mylabris cichorii.

The blister beetle Mylabris cichorii L. (Coleoptera: Meloidae) is a traditional medicinal insect recorded in the Chinese Pharmacopoeia. It synthesizes cantharidin, which kills cancer cells efficiently. Only males produce large amounts of cantharidin. Reference genes are required as endogenous controls for the analysis of differential gene expression in M. cichorii. Our study chose 10 genes as candidate reference genes. The stability of expression of these genes was analyzed by quantitative PCR and determined with two algorithms, geNorm and Normfinder. We recommend UBE3A and RPL22e as suitable reference genes in females and UBE3A, TAF5, and RPL22e in males.

RacA and Cdc42 regulate polarized growth and microsclerotium formation in the dimorphic fungus Nomuraea rileyi.

Small GTPases, RacA and Cdc42, act as molecular switches in fungi, regulating cell signaling, cytoskeletal organization, polar growth and reactive oxygen species (ROS) generation, the latter by influencing the activity of the NADPH oxidase complex. In this study, the racA and cdc42 genes from Nomuraea rileyi were cloned and shown to encode 218 and 184 amino acid proteins, respectively. To determine the functions of racA and cdc42, gene-silencing mutants (racARM, cdc42RM and racA&cdc42RM, respectively) were generated using RNA silencing technology. In racARM and cdc42RM, the conidial and microsclerotium (MS) yields, ROS production and virulence were reduced, the hyphal extension rate was decreased and the dimorphic switch was delayed. On the other hand, the double-silencing mutants showed growth retardation and virtually no conidia, MS or ROS production. The transcription levels of the noxA and noxR genes that regulate ROS generation were reduced in the three RNAi-silenced strains. Interestingly, when compared with the controls, racARM exhibited thicker hyphae and bigger conidia; moreover, the MS produced by racARM were bigger than those of the control and smaller than those of cdc42RM. Thus RacA and Cdc42 appear to share some essential functions in N. rileyi, including hyphal growth, conidiation, MS formation, ROS generation and virulence. Yet RacA appears to play a more pivotal role in the polar growth of N. rileyi.

Isolation of single chain variable fragments against six esters of pyrethrins by subtractive phage display.

Recombinant antibodies (rAbs) are a new diagnostic test for immulogical detection. To date, there are no reports about anti-pyrethrins rAbs. Here we describe the generation of monomeric and dimeric single chain variable fragments (scFvs) with affinity for six esters of pyrethrins using a subtractive phage display technology. First, scFv libraries with long-linker (Ger(4)Ser)(3) and short-linker (Ger(4)Ser) were established to contain 1.04 x 10(7) or 6.07 x 10(6) transformants. After four rounds of panning, phage ELISA demonstrated that three clones (E2, F2, and H7) showed higher affinity from the long-linker library, and clones (h6, a5) exhibited better antibody activity to pyrethrin I and II from the short-linker library. The scFv candidates were sequenced to identify the specific antibody response against pyrethrins. Isolated scFvs constitute valuable tools for real-time detection of pyrethrins. In addition, the subtractive phage display provides a simple approach for isolation of scFvs.

[Selection of differential DNA fragment of Tilletia controversa Kühn and establishment of molecular detection approach].

A reliable and simple polymerase chain reaction method for TCK pathogen was established firstly. A 1322bp unique fragment of TCK was amplified and identified by the technique of semi-specific random amplified polymorphism (RM-PCR). Two pairs of species-specific primers CQUK2/CQUK3 and CQUK4/CQUK5 were designed according to the unique fragment of TCK. The first pair primers were capable to stably amplify target DNA band of 747bp from chromosomal DNA of 18 strains of TCK isolates without any DNA bands obtained from 29 strains of TCT. The second pair primers could produce a 200bp target DNA band stably, while no band was amplified from teliospore or mycelium DNA of TCT of strains. Tilletia genus primers were used as internal control of molecular detection system, which can detect whether the PCR inhibitors exist in testing sample or avoid pseudo-negative and pseudo-positive of PCR reaction. The molecular detection approach could rapidly, accurately detect and identify the DNA of teliospore or mycelium of TCK from wheat tissues.

[High efficient expression and bio-activity assay of recombinant antibody for citrus bacterial canker disease].

Citrus bacterial canker disease caused by the gram negative bacterium Xanthomonas axonopodis pv. citri (XAC) is a severe bacterial disease of most commercial citrus species and cultivars around the world. Single chain variable fragment (ScFv) is artificial construction small molecular antibody produced by genetic engineering which may be used to identify target pathogens and prevent plant diseases including XAC. To express ScFv against XAC and obtain functional ScFv, single-chain antibody fragments (ScFv 95) selected by ribosome display was amplified using an assembly of polymerase chain reaction (PCR), and a recombined plasmid pET30a( + )-XAC-ScFv was constructed by inserting the single chain Fv gene into bacterial expression vector pET30a( + ). PET30a( + )-XAC-ScFv was transformed into Escherichia coli BL21 (DE3) and expressed which was induced by IPTG. Products were purified though Ni-NTA His Bind resin. The collected antibodies were refolded by gel filtration chromatography, and activity assaying process was done. The results showed that ScFv recombined antibody of XAC with a molecular of 32kDa was expressed successfully as inclusion bodies and the functional ScFv was obtained through purification and renaturation. Meanwhile, the Biacore analysis indicates that XAC-ScFv-95 showed significant affinity to LPS of Xac, which paves a new way for immunization diagnosis and exploration of integrated control of citrus bacterial canker disease.

Role of hunchback in segment patterning of Locusta migratoria manilensis revealed by parental RNAi.

In long germ embryos, all body segments are specified simultaneously during the blastoderm stage. In contrast, in short germ embryos, only the anterior segments are specified during the blastoderm stage, leaving the rest of the body plan to be specified later. The striking embryological differences between short and long germ segmentation imply fundamental differences in patterning at the molecular level. To gain insights into the segmentation mechanisms of short germ insects, we have investigated the role of the homologue of the Drosophila gap gene hunchback (hb) in a short germ insect Locusta migratoria manilensi by paternal RNA interference (RNAi). Phenotypes resulting from hb knockdown were categorized into three classes based on severity. In the most extreme case, embryos developed the most anterior structures only, including the labrum, antennae and eyes. The following conclusions were drawn: (i) L. migratoria manilensis hb (Lmm'hb) controls germ band morphogenesis and segmentation in the anterior region; (ii) Lmm'hb may function as a gap gene in a wide domain including the entire gnathum and thorax; and (iii) Lmm'hb is required for proper growth of the posterior germ band. These findings suggest a more extensive role for L. migratoria manilensis hunchback in anterior patterning than those described in Drosophila.

[Cloning and characterization of the neutral trehalase gene in Metarhizium anisopliae CQMa102].

The partial fragment of the neutral trehalase (NTL) in Metarhizium anisopliae CQMa102 was amplified by the polymerase chain reaction (PCR) with primers designed according to the sequences of the NTL in GenBank. The amplified fragment was cloned and sequenced. Based on the known sequence of NTL gene, the 5' and 3' rapid amplification of cDNA ends (RACE) were used to amplify the 5' and 3'regions of the NTL cDNA, then the whole cDNA sequence of NTL gene in M. anisopliae CQMa102 was obtained by combining the above sequences and its whole genomic DNA was amplified by PCR. In order to obtain more regulatory information of the NTL, panhandle polymerase chain reaction strategy was used to amplify the 5' flanking sequences adjacent to the known sequence of the neutral trehalase gene. The sequence analysis shows that the DNA sequence is 3484bp in size, includes three introns, and the cDNA sequence is 2385 bp in size, encodes a protein of 737 amino acid residues with a calculated Mr of 83.1kD, in which there is a cyclic adenosine 3', 5'-monophosphate-dependent phosphorylation consensus site and a putative calcium binding site, which is consistent with a regulatory enzyme. Comparison of this sequence with the NTL of M. anisopliae ME1 in GenBank (GenBank Accession: AJ298019, AJ298020) shows that the nucleotide homology is as high as 93%, and the amino acid homology comes up to 99%. Southern analysis indicated that NTL gene was present as a single copy in this M. anisopliae strain. A single transcript of approximately 2.5 kb was detected by Northern blot analysis. The NTL mRNA was present throughout spore germination in rich medium, but accumulated to its highest level at the early stage of exponential growth. Thereafter, the mRNA level declined at the late stage of exponential growth, but began to show an increase during the stationary phase of growth. A 982bp upstream sequence of NTL was amplified using panhandle PCR method, which contains one stress response element (STRE). The NTL nucleotide sequence and its amino acid sequence have been accessed by GenBank (Accession: AY557613, AY557612).

[Cloning and characterization of cuticle degrading enzyme CDEP-1 from Beauveria bassiana].

Beauveria bassiana extracellular subtilisin-like serine endoprotease is a potential virulence factor by virtue of its activity against insect cuticles. A cDNA library was constructed using mRNA from mycelia of Beauveria bassiana grown on cuticle/chitin cultures. A cDNA clone of the protease, designated CDEP-1, was isolated from cDNA library. CDEP-1 contained an 1,134 bp ORF that predicted a protein of 377 amino acids with M(r) = 38,616 and PI = 8.302. The amino acid sequence of the gene shows 57.9%, 83.3% and 54.7% identity to Metarhizium nisopliae Pr1, Beauveria bassiana Pr1 and proteinase K, respectively. Southern analysis indicated that CDEP-1 was present as singly copy in Beauveria bassiana.