飞蝗羧酸酯酶基因LmCesF1的时空表达及与杀虫剂耐受性的关系

doi:10.3864/j.issn.0578-1752.2014.08.008

摘要/Abstract

摘要： 【目的】通过研究飞蝗（Locusta migratoria）羧酸酯酶基因LmCesF1的杀虫剂解毒功能，为害虫防治提供新的候选基因。【方法】从飞蝗转录组数据库搜索获得羧酸酯酶基因LmCesF1的cDNA序列，采用ExPASy在线软件推测LmCesF1的氨基酸序列，并预测该酶的分子量（MM）和等电点（pI）；在 NCBI保守域（CDD）数据库中预测该酶的催化三联体和底物结合位点；采用SignalP4.0在线软件预测该酶的信号肽序列；NetNGlyc在线软件预测该酶的N糖基化位点。将LmCesF1与其他昆虫物种羧酸酯酶进行NJ聚类分析；以飞蝗各龄期（卵期、5个若虫期和成虫期）整虫cDNA和5龄若虫不同组织部位cDNA为模板，运用reverse transcription quantitative PCR (RT-qPCR) 研究LmCesF1的时空表达特性；采用RT-qPCR 方法检测RNA干扰效率；LmCesF1基因沉默后24 h分别进行4种农药的生物学测定试验，每头若虫点滴3 μL的杀虫剂溶液（每个农药的剂量均为LD30，即240 ng马拉硫磷、15 ng毒死蜱、51 ng西维因和0.6 ng溴氰菊酯），点滴农药24 h后统计飞蝗的死亡率，进而分析该基因与农药解毒的关系。【结果】LmCesF1全长cDNA序列为3 121 bp，该基因开放阅读框（ORF）为2 490 bp，编码830个氨基酸，氨基酸序列N端存在信号肽序列，催化三联体为G-P-N，聚类分析表明该基因属于羧酸酯酶的F簇（属于非鳞翅目昆虫保幼激素酯酶）。LmCesF1在飞蝗卵期及若虫低龄期表达较低，在4龄若虫表达上调达到峰值，且一直持续到成虫第5天。5龄若虫不同组织部位表达结果显示，LmCesF1在前肠、后肠和脂肪体表达量最高，在胃盲囊和中肠的表达量最低。RNA干扰沉默效率检测结果显示，与对照组相比（注射等量的dsGFP），LmCesF1在12 h和24 h的沉默效率显著，mRNA表达量分别下降为22.6%和30%。RNA干扰结合农药生物学测定结果表明，在沉默LmCesF1后，与对照组比较（注射等量的dsGFP），西维因处理组飞蝗死亡率提高了32.9%，马拉硫磷、毒死蜱和溴氰菊酯处理组飞蝗的死亡率均没有显著性差异，推测LmCesF1可能参与西维因的解毒，而与其他3种农药的解毒没有相关性。【结论】从飞蝗中获得1条保幼激素酯酶（JHE）相似基因LmCesF1，该基因属于羧酸酯酶的F簇，推测的氨基酸序列催化三联体发生了改变；该基因在不同组织部位中均有表达，并可能参与西维因在飞蝗体内的解毒。

关键词: 飞蝗 , 羧酸酯酶 , reverse transcription quantitative PCR , RNA干扰

Abstract: 【Objective】 The objective of this study is to provide a new candidate gene for pest management through studying the function of pesticide detoxification of carboxylesterase gene LmCesF1 in Locusta migratoria. 【Method】The cDNA sequence of LmCesF1 was got from L. migratoria transcriptome database. Amino acid sequence was deduced by using the ExPASy proteomics website software. The molecular mass (MM) and isoelectric point (pI) were predicted by using ExPASy tools. To identify the catalytic triad and substrate binding pocket presented in deduced amino acid sequence, the BLASTp analyses were undertaken in NCBI conserved domain database (CDD). Signal peptide was predicted by SignalP4.0 web tools. The NetNGlyc1.0 Server was used to predict potential N-glycosylation sites of LmCesF1. Phylogenetic tree of LmCesF1 and carboxylesterases from other insect species was constructed with neighbor-joining method. Stage- (including egg, 1-5 instar nymphs and adult) and tissue-dependent expression patterns of LmCesF1 were conducted by reverse transcription quantitative PCR (RT-qPCR). The efficiency of RNAi was assessed by RT-qPCR. RNA interference followed by bioassay was applied to reveal the roles of LmCesF1 in insecticide detoxification. The insecticide was applied at 24 h after ds LmCesF1 injection, dsGFP was used as control. Each nymph was topically applied with 3 µL droplet of insecticide solution (240 ng for malathion, 15 ng for chlorpyrifos, 0.6 ng for deltamethrin and 51 ng for carbaryl) onto the abdomen between the 2nd and 3rd sterna. The doses of these insecticides were predetermined to be approximately LD30 by bioassay. Mortality was recorded at 24 h after topical application of the insecticides. 【Result】 The full-length cDNAs of LmCesF1 (3 121 bp) was obtained from L. migratoria transcriptome database. The open reading frame (ORF) of LmCesF1 was 2 490 bp, encoded 830 amino acid residues. LmCesF1 had a signal peptide at N-terminus. The deduced amino acid sequence of LmCesF1 had the catalytic triad G-P-N. Phylogenetic tree analysis showed that LmCesF1 was clustered to the F clade (nonlepidopteran juvenile hormone esterases). LmCesF1 showed lower expression at egg and early nymph stages, then reached a peak in the 4th-instar nymphs and kept high expression until the adult day 5. The results of tissue-dependent expression of LmCesF1 suggested that the highest expressions of LmCesF1 were found in the foregut, hindgut and fat bodies, but the lowest expressions were in the gastric caeca and midgut. The remaining transcript levels of LmCesF1 were 22.6% and 30% in the nymphs injected with dsLmCesF1 as compared with those in the control (injected with dsGFP) at 12 h and 24 h, respectively. These results indicated an effective silencing of LmCesF1 by RNAi at 12 h and 24 h. Insecticide bioassay showed that the nymph mortality in response to carbaryl treatment increased by 32.9% after the expression of LmCesF1 was suppressed. In contrast, similar exposures of the locusts to malathion, chlorpyrifos, and deltamethrin after the suppression of LmCesF1 did not show significant effects on the susceptibility of the locusts to these insecticides. These results indicated that LmCesF1 most likely to be involved in detoxification of carbaryl in L. migratoria. 【Conclusion】 A juvenile hormone esterase (JHE) like gene LmCesF1 was identified from L. migratoria. This gene was clustered in clade F. The catalytic triad of LmCesF1 has been changed to G-P-N. The transcripts of LmCesF1 were detectable in all tested tissues. LmCesF1 might play a significant role in detoxification of carbaryl in L. migratoria.

Key words: Locusta migratoria , carboxylesterases , reverse transcription quantitative PCR , RNA interference

张建琴, 葛娉婷, 李大琪, 王燕, 张建珍, 马恩波. 飞蝗羧酸酯酶基因LmCesF1的时空表达及与杀虫剂耐受性的关系[J]. 中国农业科学, 2014, 47(8): 1522-1530.

ZHANG Jian-Qin, GE Ping-Ting, LI Da-Qi, WANG Yan, ZHANG Jian-Zhen, MA EnBo. Spatio-Temporal Expression and Insecticide Tolerance Analysis of Carboxylesterase Gene LmCesF1 from Locusta migratoria[J]. Scientia Agricultura Sinica, 2014, 47(8): 1522-1530.

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