Identifying PCR primers to facilitate molecular phylogenetics in Caddisflies(Trichoptera)

The molecular phylogenetics of the Lepidoptera （butterflies and moths） is well studied, but that of Trichoptera （caddisflies）, the sister clade of Lepidoptera, is less studied. The PCR primer libraries developed for lepidopteran phylogenetics might work in Trichoptera. DNA from 8 caddisfly species （Asynarchus nigriculus （Banks, 1908）, Grammotaulius lorettae Denning, 1941, Hesperophylax occidentalis （Banks, 1908）, Limnephilus externus Hagen, 1861, Limnephilus picturatus McLachlan, 1875, Limnephilus secludens Banks, 1914, Limnephilus sublunatus Provancher, 1877 and Agrypnia deflata （Milne, 1931）） was used to screen for amplification. 107 primer pairs for 45 nuclear and 3 mitochondrial genes were tested. Primers for 1 new gene （40S ribosomalprotein $2 （RPS2）） and 8 genes previously used in Trichopteran phylogenetics were recovered （16S rRNA, 18S rRNA, carbamoyl-phosphate synthetase （CAD）, cytoehrome oxidase I （CO1）, cytochrome oxidase 11 （COIl）, elongation factor-1 alpha （EF-1 alpha）, isoeitrate dehydrogenase （IDH）, and RNA polymerase-II （POL-I1））. New primer pairs extended the genomic region sampled for many genes. Evolution rates among loci varied by 2 orders of magnitude. Differences among evolution rates and modes of inheritance offer flexible tools for resolving phylogenetic questions and examining genome evolution in the Trichoptera. Screening libraries of PCR primers is a useful approach for identifying PCR primers in related taxa with limited molecular genetic resources.

Species-specific PCR-based assays for identification and detection of Botryosphaeriaceae species causing stem blight on blueberry in China

Botryosphaeriaceae species are important causal agents of blueberry stem blight worldwide. Blueberry stem blight has become an important disease, potentially affecting the quality and production of blueberries in China. It is difficult and time-consuming to identify at the species level using morphological methods. The aim of this study was to develop polymerase chain reaction（PCR） assays for the diagnosis and early detection of latent infections of blueberry stems by Botryosphaeria spp. Species-specific primers, based on the ribosomal DNA internal transcribed spacer region and β-tubulin gene, were designed and selected for use in PCR assays. Three primer pairs, Lt347-F/R for Lasiodiplodia theobromae, Np304-F/R for Neofusicoccum parvum and FaF/Bt2b for Botryosphaeria dothidea, successfully amplified specific PCR fragments of different sizes on pure cultures or from blueberry stems inoculated and naturally infected blueberry plants with three pathogens, respectively. These primers did not amplify any PCR fragments from other blueberry stem disease-associated pathogens, such as Phomopsis spp. and Pestalotiopsis spp. This PCR protocol could detect as low as 1 00 pg to 1 ng of purified fungal DNA. This PCR-based protocol could be used for the diagnosis and detection of these pathogens from pure cultures or from infected blueberry plants.

Effect of Subcellular Localization of P21 on Proliferation and Apoptosis of HepG2 Cells

This study examined the effect of subcellular localization of P21 on the proliferation and apoptosis of HepG2 cells. The coding genes of the wild and the mutant P21 were amplified by mega primer PCR from the plasmid pCEP-WAF1 which contains human P21 cDNA in the nuclear localizational signal （NLS） sequence, and then inserted into the eukaryotic expression vector pDsRedl-C1. The recombinants were transfected into HepG2 cells. The transcription and expression of P21 were determined by RT-PCR and fluorescence microscopy. The cell proliferation was measured by MTT, and the cell cycle and apoptosis of HepG2 cells by flow cytometry. The results of restriction analysis, DNA sequencing and fluorescence microscopy confirmed the construction of the wild and the mutant P21 in the eukaryotic expression plasmid. The plasmid containing the mutant P21 was found to accelerate cell proliferation and the wild P21 plasmid to inhibit cell proliferation. Cell cycle analysis showed that the cell ratio of GeYG1 in the wild type group was significantly increased as compared with that in the mutant type group, and cell apoptosis analysis revealed that the apoptosis rate in the wild type group was much higher than that in the mutant type group. It was concluded that the subcellular localization of P21 may contribute to the develonment of heoatic cancer.

Identification and initial characterization of the 3＇ end of gene transcripts encoding putative members of the pheromone receptor subfamily in Lepidoptera

Semiochemicals, including pheromones and kairomones, used in pest man- agement programs reduce the need for chemical insecticides, and understanding their interactions with their membrane receptors may help make them more effective in the field. Identification of odorant receptors in the Lepidoptera has mainly been achieved us- ing bioinformatics to search DNA sequences generated by genome or expressed sequence tag （EST） sequencing projects. This study reports a rapid method to identify members of the pheromone receptor subfamily in Lepidoptera. Degenerate oligonucleotide primers were designed against a conserved amino acid sequence in the carboxyl terminus of known lepidopteran pheromone receptors, and the primers were used in a 3＇ rapid amplifica- tion of complementary DNA （cDNA） ends procedure. Polymerase chain reaction products generated from seven different lepidopteran species were TA cloned and sequenced. The cDNA sequences of 25 transcripts were determined to encode potential members of the pheromone receptor subfamily. These cDNAs ranged from 238 to 642 bp and encoded 49-54 amino acids of the carboxyl terminus. Analysis of the 3＇ untranslated region reveals that most of the transcripts contain multiple polyadenylation signal sequences, and in the case ofManduca sexta, an alternate polyadenylation signal appears to be used in transcript processing. The 3＇ untranslated region was also useful in determining unique receptors en- coded by transcripts having highly similar nucleotide and amino acid sequences. Overall, this technique provides a complementary method of pheromone receptor identification in EST sequencing projects, or can be used as a stand-alone method in conjunction with 5＇ rapid amplification of cDNA ends procedures.