Preparation of chicken IgY against recombinant E2 protein of bovine viral diarrhea virus (BVDV) and development of ELISA and ICA for BVDV detection

Bovine viral diarrhea virus (BVDV) infects cattle and may lead to persistent infection (PI). The PI animals harbor BVDV throughout their life and become immune tolerant against BVDV. Thus, diagnosis of this virus in herd is highly important. Recombinant E2 protein expression (using pET-32a in Escherichia coli) was confirmed by SDS-PAGE and Western blotting; then purified by Ni⁺ affinity chromatography. Chickens were immunized with BVDV-E2 protein, and IgY antibodies were extracted from egg yolk by PEG-6000. The peak titer of anti-BVDV-E2-IgY was 1:128,000 after the fifth immunization. IgY-based enzyme-linked immuno sorbent assay (ELISA) and immunochromatographic assay (ICA) were further developed. Coincidence of ELISA and ICA test with RT-PCR was 95.45 and 90.91%, respectively. The anti-BVDV-E2 IgY could be used in routine screening of BVDV infection. Besides, it can also be applicable while licensing and/or using live vaccines; screening of imported products containing bovine serum and strong surveillance of BVDV outbreaks.     

Complete Genome Sequence of a Novel Wild tomato mosaic virus Isolate Infecting Nicotiana tabacum in China

Virus particles of approximately 740–760 nm in length and 13 nm in diameter were observed from a diseased Nicotiana tabacum (tobacco) plant in Sichuan Province, China. The complete genomic sequence of the virus isolate XC1 was determined to contain 9659 nucleotides without 3′ terminal poly(A) tail. XC1 has a genome typical of members of the genus Potyvirus, encoding a large polyprotein of 3075 amino acids. Putative proteolytic cleavage sites and a number of well characterized functional motifs were identified by sequence comparisons with those of known potyviruses. Sequence comparison revealed that XC1 shared the highest level of nucleotide sequence identity (76.5%) with Wild tomato mosaic virus (WTMV). Phylogenetic analysis showed that XC1 was closely related to the WTMV Guangdong isolate with an identity of 94.3% between CP gene sequence of the two viruses. We thus named XC1 WTMV‐XC‐1 as a novel isolate of WTMV. The full sequence of WTMV‐XC‐1 may serve as a basis for future investigations on the gene diversity of WTMV.     

A Cocktail ELISA Semi‐nested RT‐PCR Assay to Detect Bean pod mottle virus in Soya bean Seeds

Bean pod mottle virus (BPMV) has been identified as an important pathogen for plant quarantine in China because large quantities of soya bean seeds (approximately 7 × 10⁷ tons) are imported annually. To develop a practical detection programme for BPMV, a cocktail enzyme‐linked immunosorbent assay (ELISA) nested RT‐PCR using a combination of serological and molecular methods was designed for soya bean seeds. The single‐vessel detection assay was performed in a 96‐well ELISA plate, which served as a carrier for the subsequent nested RT‐PCR assay. Assay specificity was demonstrated by the production of the expected 330‐ and 296‐bp bands using the external and internal primers, respectively. This method was 10⁴‐fold more sensitive than immunocapture‐RT‐PCR (IC‐RT‐PCR). In particular, it is important to note that this assay resulted in successful micro‐extraction from soya bean seeds and combined the advantages of each individual technique. The cocktail ELISA nested RT‐PCR is a specific, sensitive, rapid and economical procedure to rapidly identify and characterize BPMV and could be suitable for both primary‐level platforms and laboratories.     

Spectral feature differences between shrub and grass communities and shrub coverage retrieval in shrub-encroached grassland in Xianghuang Banner,Nei Mongol,China北大核心CSCD

Aims: Shrub-encroached grassland has become an important vegetation type in China's arid and semi-arid region. Our study objective is to explore the spectral features of shrub and grass communities, as well as their empirical relationships with shrub coverage. The quantitative estimation of shrub cover based on medium-resolution Landsat satellite imagery provides the practical basis for long term retrieval of large areas of shrub expansion in the grassland region. Methods: Linear models and Multiple Endmember Spectral Analysis Model (MESMA) based on medium resolution Landsat satellite imagery were developed to quantify the shrub coverage in a shrub-encroached grassland region in Xianghuang Banner, Nei Mongol using the spectral features and their seasonal differences between the shrub and grass communities. Important findings: Compared to Leymus chinensis and Stipa krylovii dominated grass communities, Caragana microphylla community had a higher normalized difference vegetation index (NDVI), modified red edge normalized difference vegetation index (mNDVI705), and red edge slope. The red edge position of C. microphylla community shifted to longer wavelengths. The average and the maximum shrub coverage was 13% and 25%, respectively, in the shrub-encroached grassland based on both models. The correlation coefficient of determination (R2) and root mean square error (RMSE) of the linear model was 0.31 and 0.05, respectively. We found that the linear model based on seasonal differences of shrub and grass community was more suitable for retrieving shrub coverage in the study area from medium resolution imagery than the MESMA model that is based on mid-summer images.     

Chalcone synthase homologous genes cloning and expression pattern in flowering <Emphasis Type="Italic">Fagopyrum tataricum</Emphasis> Gaertn

Tartary buckwheat (Fagopyrum tataricum Gaertn.) is highly nutritious and an excellent dietary source of flavonoid compounds. Chalcone synthase (CHS) is the first key enzyme involved in flavonoid biosynthesis. Here, three putative CHS genes (designated as FtCHS1 (GU172165), FtCHS2 (KT284884), and FtCHS3 (KT284885) were isolated from tartary buckwheat. Nucleotide sequence analysis indicated that FtCHS1 and FtCHS2 each contained one intron of 444 bp and 157 bp, respectively. FtCHS3 included two introns, one of 86 bp and another of 73 bp. The results of quantitative real-time PCR (qRT-PCR) showed the FtCHSs expression presented the same pattern in the stems and flowers, with FtCHS1>FtCHS3>FtCHS2. A different tendency was found in leaves, with FtCHS3>FtCHS2>FtCHS1. However, there was no direct correlation between the three CHS expression and total flavonoids. Furthermore, high-performance liquid chromatography (HPLC) performance reveals rutin is the most abundant flavonoid in all tissues, leaves should be the main location for quercetin storage in tartary buckwheat.