Molecular Characterization of TRPA Subfamily Genes and Function in Temperature Preference in Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae)

Abstract

To reveal the mechanism of temperature preference in Tuta absoluta, one of the top 20 plant pests in the world, we cloned and identified TaTRPA1, TaPain, and TaPyx genes by RACE and bioinformatic analysis, and clarified their expression profiles during different development stages using real-time PCR, and revealed their function in preference temperature by RNAi. The full-length cDNA of TaPain was 3136 bp, with a 2865-bp open reading frame encoding a 259.89-kDa protein; and the partial length cDNA of TaPyx was 2326-bp, with a 2025-bp open reading frame encoding a 193.16-kDa protein. In addition, the expression of TaTRPA1 and TaPyx was significantly lower in larvae than other stages, and it was significantly higher in pupae and newly emerging males for TaPain. After feeding target double-stranded RNA (dsRNA), the preferred temperature decreased 2 °C more than the control group. In conclusion, the results firstly indicated the molecular characterization of TRPA subfamily genes and their key role in temperature perception in T. absoluta, and the study will help us to understand the temperature-sensing mechanism in the pest, and will provide some basis for study of other Lepidoptera insects’ temperature preference. Moreover, it is of great significance in enriching the research progress of “thermos TRP”.

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“…Quantitative real-time PCR (qPCR) was performed using an ABI 7500 real-time PCR system (Applied Biosystems, Waltham, MA, USA) with SYBR Green Master Mix according to the manufacturer guidelines. The large subunit 5 ribosomal protein ( RpL5 ) 52 and β-actin 53 were used as references for the analysis of larvae and tomatoes. Relative mRNA expression quantification used the Livak and Schmittgen 54 and Pfaffl 55 models, simplified to 2 −ΔΔCT , where ΔΔCT = (Ct target − Ct reference) treatment − (Ct target − Ct reference) control.…”

Chromatin-Remodelling ATPases ISWI and BRM Are Essential for Reproduction in the Destructive Pest Tuta absoluta

“…RT-qPCR was performed with a Hieff qPCR SYBR Green Master Mix (Yeasen, Shanghai, China) on an ABI 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The primers used in the present study are listed in Table 1 and include RpL5 (large subunit 5 ribosomal protein) as an internal control gene to normalise mRNA expression [ 54 , 55 ]. Each PCR reaction was performed in 20µL volumes consisting of 10.0 µL SYBR Green Master Mix, 1.0 µL cDNA template, 0.4 µL of each primer (10 µM), and 8.2 µL ddH 2 O.…”