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Identification And Functional Characterization Of Thermoreceptor Genes In Apolygus Lucorum(Meyer-dür)

Posted on:2016-02-21Degree:MasterType:Thesis
Country:ChinaCandidate:T FuFull Text:PDF
GTID:2283330461488216Subject:Agricultural Entomology and Pest Control
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The development, reproduction, migration, diapause and dormancy of insects are closely related to the change of environment temperature. During the long-term evolution, how to sense the change of temperature and respond accordingly is very important to the insects for their survival and reproduction. It has been reported that TRP(transient receptor potential) channels are considered as thermoreceptors in insects and mammals and play important roles in sensing temperature in insect. Apolygus lucorum(Meyer-Dür)(Hemiptera: Miridae, lygus Hahn) is a major pest to most crops and fruit trees in China. In this study, we obtained the sequence fragments of TRPA1 and Painless genes based on the transcriptome data of A. lucorum. RACE and RT-PCR techniques were applied subsequently to get the full-length of these two genes. Expression profiles of the genes were investigated by RT-PCR and q PCR in different adult tissues and across developmental stages. Functional characterization of A. lucorum TRPA1 and Painless were further conducted, followed by the behaviour studies of A. lucorum to temperature and chemicals. The primary results are as follows:1. With the bioinformatic analysis of the transcriptome data of A. lucorum, 4 sequence fragments of TRPA1 were isolated. Through sequences alignment and molecular cloning, 4 isoforms of TRPA1 were obtained, which were named as Aluc TRPA1-A, Aluc TRPA1-B, Aluc TRPA1-C and Aluc TRPA1-D respectively. Each isoform consisted of 6 conserved transmembrane domains and 16 ankyrin repeats. Aluc TRPA1 s appeared to express across all the life stages and also in all tissues of adults, with the highest gene expression level shown at the first-instar stage and in antennae, respectively. The gene expression level of Aluc TRPA1-C is higher in abdomen than other tissues, while Aluc TRPA1-B is higher in wing. Functional studies indicated that three of the isoforms could be activated by the increasing temperature from 20 ?C to 40 ?C except Aluc TRPA1-D. Moreover, Aluc TRPA1 s didn’t show significant adaptation to the repeated temperature stimulation. The activation temperature of Aluc TRPA1-A and Aluc TRPA1-B was <25 ?C, while Aluc TRPA1-C was in the range of 25-30 ?C. Only Aluc TRPA1-A and Aluc TRPA1-C can be activated by chemical compounds at high concentration(10-3 mol/L).2. Four c DNA fragments were identified in encoding a putative Painless based on the transcriptome data of A. lucorum. Full-length of the Painless gene was 2736 bp, encoding 911 amino acids. Aluc Painless consisted of 6 transmembrane domains and 8 ankyrin repeats. Expression profile of the Painless gene showed the highest express level in 1st instar nymphs and the adult legs, followed by antenna and wings. Functional characterization indicated that Aluc Painless could be activated by increasing temperature from 20 ?C to 45 ?C, but could not by chemicals. There was no significant difference of response values among the temperature ranges of 20-25 ?C, 20-30 ?C, 20-35 ?C, 20-40 ?C as control, which implied that the activation temperature of Aluc Painless should be >40 ?C.3. Behavior experiment results showed that the temperature preference of A. lucorum was the range of 25-28 ?C. There was no distribution of A. lucorum when the temperature was higher than 40 ?C. These indicated that A. lucorum could sense the environment temperature. According to two-choice assay, A. lucorum showed avoidance to 1% CA, 10% CA, 1% citronellal and 10% citronellal. We further demonstrated that A. lucorum could smell chemicals(AITC, CA, citronellal) by recording the kinetic adults to chemical stimulus through the Etho Vision XT 8.5.
Keywords/Search Tags:Apolygus lucorum, thermoreceptor, olfactory receptor, TRPA1, Painless, Xenopus oocytes
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