| Heat shock proteins(HSPs)are important proteins which maintain the regular physiological activity and protect the organs against the damage at stress such as temperature,pesticides etc.HSPs could be divided into HSP110,HSP90,HSP70,HSP60 and small heat shock protein by molecular weight.The HSP70s are studied widely and play an important role on thermotolerance.Laodelphax striatella(small brown planthopper,SBPH)and Nilaparvata lugens(Stal)(brown planthopper,BPH)are the two main planthoppers affected rice in field in China,and faced the challenge of temperature stress and pesticides.In order to clarify the adaptation mechanism of rice planthopper to stresses of temperature and insecticide,the compositions,induced expression profiles and functions of HSP70s in Laodelphax striatella and Nilaparvata lugens(Stal)were studied,then the possible role of heat shock protein 70 at thermotolerance of chlorpyrifos resistant SBPH was analyzed.The main findings are as follows:1.Hsp70s clone and sequence analysisThe nucleotide sequences of heat shock protein 70 homologous genes of Laodelphax striatella and Nilaparvata lugens(Stal)was obtained from transcriptome database.12 HSP70 with a complete open reading frame were found in Laodelphax striatella,and 9 out of which expressed under normal or stress conditions.However,15 HSP70 were screened in Nilaparvata lugens(Stal),8 out of which were expressed under normal or temperature stress.ExPASy tool predicted that these genes contained three Hsp70 family signatures,with an isoelectric point of 5.05-6.15 and a molecular weight of 66801.97-75283.42.MEGA showed that KX695161,KX695162 and KX976472 were clustered together with endoplasmic reticulum HSP70s from human and Drosophila melanogaster,and KX976477,KX976478 and KX695164 were clustered together with mitochondrial HSP70s,others clustered with hsp70 localized to cytoplasm/nucleus,which suggested that these hsp70s would be localized to the cytoplasm/nucleus,endoplasmic reticulum and mitochondria,respectively.2.The induced expression profiles of hsp70s at temperature stressThe transcriptional expression of Hsp70s from SBPH and BPH was examined by qPCR under different temperature stress(0,6,10,14,18,22,30,33,36,39,42,44℃).For SBPH,the expression of KX695168 were significantly up-regulated,and the fold change was more than 1000 after 42℃ treatment;the expression of KX695160 was increased 4.4,5 and 4.5 times respectively after 30℃、33℃ and 35℃ heat treatment.Other genes did not obviously change their expression level at 30-44℃ treatment.However,the expression change of hsp70s at 0-22℃ was not in agreenment with 30-44℃.Only KX695160 increased its expression to 3.27 times after 14℃ temperature treatment,other genes showed similar or decreased expression.For BPH,the expression of KX976473 and KX976476 were increased significantly at 30-44℃,and the maximum fold change was 200 and 25 respectively.However,only KX976475 expression increased 2.38 times,the other seven genes expression were not changed or even significantly decreased after 0-22 ℃ treatment.These results suggested that these heat shock protein 70 genes in SBPH and BPH would play an important role on high temperature adaptation,but a limited role on cold adaptation.3.The induced expression profiles of HSP70 at chlorpyrifos stressThe relative expression of Hsp70s from SBPH and BPH were measured after exposure to LC25 and LC50 doses of chlorpyrifos for 0.5h,8h and 24h by qPCR.The results showed that the expression of hsp70s were affected by the concentration of chlorpyrifos and exposure time.In SBPH,the expression of KX695160 and KP994341 were inhibited after exposure to LC25 and LC50 dose of chlorpyrifos.the expression of KX695168 was increased significantly after exposure to LC25 dose of chlorpyrifos for 24h or LC50 dose for 0.5 or 8h.But the expression was inhibited when exposure to LC50 dose of chlorpyrifos for 24h.In BPH,KX976471,KX976473 and KX976475 expression were increased after treatment by LC25 dose of CPF for 0.5h,8h and 24h,and KX976473 expression increased 9.6 times after treatment for 8h.KX976473 expression increased significantly,and KX976471 and KX976475 did not change significantly after treatment by LC50 dose for 0.5h,8h and 24h.These results suggested that KX976473 and KX695168 would paly an important role on BPH and SBPH to chlorpyrifos stress,respectively.4.Study on the function of HSC70HSC70 is one of the most important members of the HSP70 family.It is often expressed under normal conditions and is slightly induced by temperature stress,and is important to thermotolerance in biology.In order to clear the role of HSC70 in thermotolerance of SBPH,RNAi was used to study the function of KP994340.The open reading frame(ORF)of KP994340 contained 1971 nucleotide and deduced a 656 amino acid sequence.Real-time quantitative PCR showed the expression level in female was significantly than that in 1-5 instars and male,and no difference was found between other stages except female.Downregulated expression of KP994340 due to RNAi significantly decreased the tolerance of the female to heat stress,while had a less influence on the oviposition.These results suggested that KP994340 would be related to constitutive resistance to heat but not to reproduction in SBPH.5.The study on the possible role of HSP70 on the thermotolerance of chlorpyrifos resistance SBPHTo determine the costs and benefits of resistance,the life tables of chlorpyrifos-resistant and-susceptible strains were constructed.The results showed the resistant SBPH had lower fitness at 24℃,but slightly higher fitness at 30℃.Transcriptomic analysis showed there are five heat shock protein genes changed their expression in the resistant SBPH,and the up-regulated genes are KX695160 and KX695161.KX695160 would be localized to the cytoplasmic/nucleus,whereas KX695161 would be localized to the endoplasmic reticulum.And the expression of KX695160 and KX695161 was slightly increased in the resistant strains after treatment by 30-44℃ heat.these results suggested the up-regulated expression of hsp70s may protect the chlorpyrifos-resistant pest against damage under high temperatures,increasing its relative fitness,but the lower relative fitness of this population under optimal temperature may be the trade-off. |
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