Cell lines from diamondback moth exhibiting differential susceptibility to baculovirus infection and expressing midgut genes

Abstract

Six new cell lines were established from embryonic tissues of the diamondback moth, Plutella xylostella (L.). The cell lines showed differential characteristics, including growth in attachment or in suspension, susceptibility to a baculovirus infection and expression of genes involved in the glucosinolate detoxification pathway in P. xylostella larvae. Five of the cell lines grew attached to the culture flask and one cell line grew unattached as a suspension cell line. The cell lines had population doubling times ranging from 18 to 23 h. Among five of the P. xylostella cell lines examined for infection of a nucleopolyhedrovirus from Autographa californica, AcMNPV, four cell lines were highly susceptible to AcMNPV infection, but one was only semi-permissive to AcMNPV infection. The production of two recombinant proteins, a β-galactosidase of bacterial origin and a secreted alkaline phosphatase of eukaryotic origin, in the P. xylostella cell lines was examined in comparison with that in the cell line Sf9 which is commonly used for recombinant protein production. In the P. xylostella cell lines, expression of three important midgut genes involved in the glucosinolate detoxification pathway, including the glucosinolate sulfatase genes GSS1 and GSS2 and the sulfatase modifying factor gene SUMF1, was detected. The P. xylostella cell lines developed in this study could be useful in in vitro research systems for studying insec-virus interactions and complex molecular mechanisms in glucosinolate detoxification and insect-plant interactions.

Large-scale genome-wide study reveals climate adaptive variability in a cosmopolitan pest

“…cDNA was synthesized with 1 μg of total RNA using GoScriptTM Reverse Transcription System (A5001, Promega Corporation, USA). RT-qPCR was carried out using the target gene-specific primer (forward: 5′-AACCCCCCCTTCATCCAAG-3′, reverse: 5′-CTGCTGAGGCTGTAGGTCATG-3′), which was designed by Oligo 7 and the reference gene primer for normalization (forward: 5′-CAATCAGGCCAATTTACCGC-3′, reverse: 5′-CTGGGTTTACGCCAGTTACG-3′) according to the previous reports 85 . qRT-PCR was conducted with 2 µL cDNA, 0.4 µL of each primer, 0.15 µL CXR Reference Dye, 7.05 µL DEPC water and 10 µL SYBR Green Supermix (A6001, Promega Corporation, USA) in a 20 µL total reaction mixture using QuantStudioTM 6 Flex real-time PCR system (ThermoFisher Scientific, USA).…”

Functional characterization of the cis‐regulatory region for the vitellogenin gene in Plutella xylostella

“…The mating adults were kept at 25 C and 80% RH, and fed with 10% honey solution. A previously established embryonic cell line of P. xylostella was maintained in Grace's medium supplemented with 10% foetal bovine serum (FBS; Invitrogen, Waltham, MA, USA) at 27 C (Ma et al, 2019).…”

“…no. AB180441) was selected as a reference gene (Ma et al, 2019). The cycle threshold (Ct) value was obtained for the target EGFP and reference PxRPL32 genes through qPCR.…”

Virulence and genetic characterization of six baculovirus strains isolated from different populations of Spodoptera frugiperda (Lepidoptera: Noctuidae)

“…Still, only four out of six isolates were able to replicate in vitro as SfNPV-Sin was unable to grow, which also showed very little virulence on larvae. Also, the SfGV-RV strain did not grow in cultured cells, although this result was expected, as very few cases of stale GV in vitro cultures have been achieved (Ma et al 2019).…”