| After the Bt cotton had been large-area commercial implemented in China from 1997, the major cotton pest Helicoverpa armigera has been effective controlled, and the amount of insecticides used have decreased. Meanwhile, a series of the pest’s ecological niche in Bt cotton had changed. And Apolygus lucorum that has proved to be the secondary pest in cotton filed for a long time has become the main pest in Bt cotton and many other crops fields. Owing to the characteristics of the diversity of host plants and fast moving, it is difficult to control by the insecticides, A. lucorum has become a disaster factor. Because having strong tolerance to the environment temperature and pesticides stresses, A. lucorum has already distributed widely in China. A. lucorum now have happened from North to Heilongjiang, South to Guangdong, Guangxi and other places, West to Sichuan, Qinghai, Eastern to Gansu province etc. and the serious outbreak was focused on the eastern coastal area, and mainly in the Yangtze River and Yellow River basin area.Heat shock proteins (HSPs) are the important stress-resistant protein in insects, and could be enriched in cells quickly when insect under a variety of stress conditions. HSPs could help the insect cells and tissues pass through the poor living conditions. In order to understand how A. Lucorum regulated its AlHSP90 and AIHSC70 in response to temperature or pesticide stresses, the full-length cDNA of AIHSP90 and AIHSC70 were cloned and transcriptional and translational expression profiles of AIHSP90 and AlHSC70 under different temperature and 4 pesticide stresses were investigated. The main results of our study are as follows:1. Identificated the full-length of AIHSP90, AIHSC10 in A. Lucorum. The full-length cDNA of AlHSP90, AIHSC70 were cloned in A. Lucorum combined with the smart cDNA library and RACE approaches, and obtained the GenBank accession number of AIHSP90, AIHSC70 was KC109781, KC119044. Sequence analysis results indicated that the full-length cDNA of AIHSP90 is 2,548bp with the open reading frame (ORF) of 2,169 bp, encoding a 722-amino-acid peptide with the predicted molecular weight of 82.99 kDa and the theoretical isoelectric point of 4.93; the full-length cDNA of AlHSC70 is 2,333 bp with the open reading frame (ORF) of 1,971 bp, encoding a 656-amino-acid peptide with the predicted molecular weight of 71.57 kDa and the theoretical isoelectric point of 5.38. Protein signature analysis confirmed that AlHSP90, AlHSC70 have typical characteristics of HSP90 and HSC70 families respectively. Furthermore,15 characteristic residues (IVLVGGSTRIPKVQKL, AA 337-352), which only presented in constitutive HSC70 amino acid sequence but not in inducible HSP70 were also found in AlHSC70. In addition, multiple alignment results indicated that the amino acid sequence of AlHSP90, AlHSC70 deduced from its’ nucleotide sequence are in high homology with many other insects.2. The expression pattern of AIHSP90, AIHSC70 in A. lucorum adults and nymphs reared at different temperatures was identified by the real-time quantitative PCR (18℃、 21℃、24℃、27℃、30℃ and 33℃). Results indicated that the expression of AIHSP90, A1HSC70 in adults and nymphs were lowest at ～24℃ at the temperature range from 18℃ to 33℃. However, compared with 24℃ control, the expression of AlHSP90, AIHSC70 in adults and nymphs increased significantly at high temperature (30℃、33℃) or low temperature(18℃) (p<0.01). These results indicated that ～24℃ is the favorite suitable temperature range for A. lucorum development, and A. lucorum reared at these temperature range could be suffered the lowest temperature stress, whereas A. lucorum reared at temperature range of<18℃ or>30℃ should be suffered a higher temperature stress than that at any other temperatures.3. Transcriptional and translational expression profiles of AIHSP90, AIHSC70 in 2-day old A. lucorum adults treated by extreme high or low temperature (40℃,4℃) were identified by real-time quantitative PCR and Western blot technique. Results indicated that the translational expression profiles of AlHSP90, AlHSC70 in A. lucorum subjected to extreme high or low temperature (40℃,4℃) were in high agreement with that transcriptional expression profiles. After A. lucorum subjected to extreme high temperature (40℃) for 1 h, compared with the 24℃ control, the gene and protein expressions of AlHSP90, AlHSC70 in A. lucorum increased significantly (p< 0.01). After A. lucorum treated by extremely low temperature (4℃) for 1 h, compared with the 24℃ control, the gene and protein expressions of AlHSP90 increased insignificantly (p> 0.05), while the gene and protein expressions of AlHSC70 decreased significantly (p<0.01). These results indicated that AIHSP90 and AlHSC70 were important genes for A. lucorum in response to extreme high or low temperatures (40℃,4℃), especially for extreme high temperature (40℃), and different regulated mechanisms may be presented in A. lucorum facing to extreme high or low temperature (40℃,4℃).4. Transcriptional and translational expression profiles of AIHSP90, AlHSC10 in 2-day old A. lucorum adults treated with 4 different pesticide stresses [cyhalothrin, imidacloprid, emamectin benzoate and chlorpyrifos (induction dose= LD50)] were also identified by real-time quantitative PCR and Western blot technique. Results indicated that the gene and protein expressions of AIHSP90, A1HSC70 in male and female adults induced by cyhalothrin, imidacloprid and emamectin benzoate (induction dose= LD50) increased significantly than that in corresponding controls (p< 0.01). The gene and protein expressions of A1HSP90 in A. lucorum induced by chlorpyrifos (induction dose= LD50) increased significantly than that in corresponding controls (p< 0.01), while the gene and protein expressions of AlHSC70 in A. lucorum induced by chlorpyrifos (induction dose= LD50) decreased significantly than that in corresponding controls (p< 0.01). The translational expression profile of AlHSP90, AlHSC70 in A. lucorum subjected to each solo pesticide stress was in high agreement with that transcriptional expression profiles. In addition, these results also indicated that AlHSP90 and AlHSC70 may be involved in the resistance or tolerance to different pesticides, especially to cyhalothrin, imidacloprid, emamectin benzoate, and different regulated mechanisms of AlHSP90、AlHSC10 are also presented in A. lucorum facing to different pesticides5. The expression of AIHSP90 in A. lucorum treated by both temperature and pesticide stresses were also identified by real-time quantitative PCR. Results indicated that the expression of AIHSP90 in 2-day old adults subjected to both temperature (33 ℃) and the aforementioned 4 pesticide stresses increased significantly in A. lucorum than that treated with a single stress from heat shock or pesticide treatment (p<0.01). The results also confirmed that AIHSP90 is an important gene for A. lucorum defense against both temperature and pesticide stresses, and could provide a scientific research basis for cross-protection management in A. lucorum. |
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