中国农业科学Issue(19)：3799-3809,11.DOI:10.3864/j.issn.0578-1752.2014.19.008
豌豆蚜温度受体基因Painless的克隆及时间和组织表达
Cloning and Spatio-Temporal Expression of the Thermoreceptor Gene Painless in Pea Aphids (Acyrthosiphon pisum)
摘要
Abstract
The objective of this study is to clone a candidate thermoreceptor gene Painless in the antenna of pea aphids, Acyrthosiphon pisum, and to illustrate the protein structure encoded by this gene, further to characterize the expression profiles of this gene across developmental stages and in different tissues. [Method] Based on the sequence of Painless from Drosophila melanogaster, full-length sequence of the ApisPainless from AphidBase was predicted by bioinformatic analysis, and the gene was cloned with cDNA template obtained from pea aphid antennae and specific primers were designed by Primer premier 5.0 software using RT-PCR. After determining the consistency of cloned sequence with predicted one by DNAMAN software, the protein structure of ApisPainless was depicted through the online tool SMART (simple modular architecture research tool) according to the analysis of ankyrin repeats at N-terminus and transmembrane domains. Subsequently, the Clustal Omega software was introduced to conduct alignment and analysis of ApisPainless with DmelPainless isforms (A, B and C). The phylogenetic tree was constructed for the analysis of insect TRP (transient receptor potential) channel superfamily, especially for the subfamily TRPA, which includes 4 members of TRPA1, Painless (ApisPainless), Pyrexia and Water witch, by comparing the length of their protein sequences and ankyrin repeats at N-terminus. Quantitative real-time PCR was applied to characterize the relative expression levels of ApisPainless at different developmental stages (1st-4th instar nymphs and adults) and in different tissues (antennae, heads, legs, and thoraxes and abdomens). [Result] The ApisPainless was successfully predicted and cloned, with a complete CDS of 2 832 bp, encoding 943 amino acids. Protein structure analysis indicated the ApisPainless had 8 ankyrin repeats and 6 transmembrane domains. The ApisPainless was more similar to the DmelPainless isoform A with the similarity of 42.3%, and clustered to the insect Painless cluster based on the results of multiple sequence alignment and phylogenetic analysis, respectively. TRPA1 was composed of about 1 200 amino acids, making it the longest among the TRPA subfamily, followed by Water witch, Pyrexia and Painless, with 920-1 000 amino acids. In addition, there were 8 ankyrin repeats observed at N-terminus in Painless, while 9 in both Water witch and Pyrexia, and 15-16 in TRPA1. ApisPainless was expressed at the highest level in the 1st instar nymphs, even though it was universally expressed across all the stages. Tissue-specific expression analysis elucidated that ApisPainless was expressed in a whole body-expressing manner, but mostly expressed in antennae, followed by legs.[Conclusion]ApisPainless obtained in this study was clustered to Painless, which is a member of insect TRPA subfamily. The ApisPainless is highly expressed in antennae and legs of pea aphids, which implies that it may be involved in noxious heat sensation, mechanosensation and gustatory detection as its homolog Painless in D. melanogaster.关键词
豌豆蚜/TRP通道/Painless/基因表达谱Key words
Acyrthosiphon pisum/TRP channel/Painless/gene expression profile引用本文复制引用
魏金金,曹德盼,杨婷,王桂荣..豌豆蚜温度受体基因Painless的克隆及时间和组织表达[J].中国农业科学,2014,(19):3799-3809,11.基金项目
国家重点基础研究发展计划（“973”计划）（2012CB114105）、国家自然科学基金重点项目 （）
