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

Abstract

The vitellogenin gene promoter (VgP) is an essential cis‐regulatory element that plays a significant role in transcription of the vitellogenin (Vg) gene, leading to the production of yolk protein in insects, including lepidopterans. However, the function of VgP is still not clear in Plutella xylostella. Here, we cloned a 5.1 kb DNA fragment of the cis‐regulatory region adjacent to the 5′ end of the Vg gene of P. xylostella (PxVg). We identified two promoter sites in that 5′ upstream sequence of PxVg and performed in vitro analysis of two promoter sequences (PxVgP1, 4.9 kb, and PxVgP2, 2.9 kb) in the embryonic cell line of P. xylostella. PxVgP2 exhibited higher enhanced green fluorescent protein (EGFP) expression, so PxVgP2 was used for in vivo analysis. Strong EGFP fluorescence was observed in adult females and the fat body of females, with low expression in embryos. Our results suggest that PxVgP is an important stage‐, tissue‐ and sex‐specific endogenous cis‐regulatory element in P. xylostella.

Genome-Wide Identification, Expression Profiling, and Characterization of Cyclin-like Genes Reveal Their Role in the Fertility of the Diamondback Moth

“…The artificial diet was prepared by mixing different components such as agar, linoleic acid, canola oil, sucrose, radish seed powder, methyl paraben, vitamin, potassium sorbate ascorbic acid, and raw wheat material. The detailed procedure of the artificial diet preparation was described previously [ 26 , 27 ]. The newly hatched larvae of P. xylostella were maintained on an artificial diet until they developed into pupae at 25 °C, 35–50% relative humidity, and 16 h:8 h (light:dark) photoperiod.…”

Development of CRISPR/Cas9-Mediated Gene-Drive Construct Targeting the Phenotypic Gene in Plutella xylostella

“…The 250-bp Sv40 PolyA tail fragment was amplified from the GPXL-BacII-IE1-EGFP-SV40 vector ( Asad et al, 2020 ) by using primers of Sv40-F and Sv40-R ( Supplementary Table S1 ) with the overlapping region of Cas9 fragment and PJET1.2 vector. The PCR reaction was carried out with the Super-Fidelity DNA Polymerase (Vazyme, China) at the following conditions: 95 C for 3 min; 32 cycles of 95 C for 15 s, 58 C for 15 s, 72 C for 3 min; 72 C for 10 min; and then 4 C forever.…”

“…The Hr5IE1-EGFP-Sv40 fragment was amplified from the previously constructed vector GPXL-BacII-IE1-EGFP-SV40 ( Asad et al, 2020 ). Two oligonucleotides (IE1-F and IE1-R) were used to amplify the Hr5IE1-EGFP fragment ( Supplementary Table S1 ), which contains an overlapping region for insertion of this fragment at Abs Ⅰ cutting site of PJET-Cas9 vector ( Supplementary Figure S1 ).…”

“…The injection mixture was prepared by mixing Cas9-N-NLS Nuclease (GenScript, United States), gRNA targeting Pxyellow , dsRNA targeting Pxku70, LHA-Cas9-EGFP-gRNA-RHA vector with the injection buffer as previously described ( Asad et al, 2020 ). The parafilm sheets of 12 cm 2 coated with the cabbage leave extract were used to collect eggs, and the sheets were replaced every 30 min by new ones during the oviposition of female adults.…”

Corrigendum

“…The article “Functional characterization of the cis ‐regulatory region for the vitellogenin gene in Plutella xylostella ”, by M. Asad et al . [1], was incorrectly published as an “Invited Review”. The correct article category should have been “Original Article”.…”