【摘要】：煙粉虱Bemisia tabaci (Gennadius)是一種世界性農(nóng)業(yè)害蟲,其中B型與Q型是危害程度最為嚴(yán)重的兩種生物型,相對于B型煙粉虱,Q型煙粉虱對殺蟲劑的耐受能力更強,且對包括有機磷類、擬除蟲菊酯類、新煙堿類和昆蟲生長調(diào)節(jié)劑等大部分殺蟲劑產(chǎn)生了不同程度的抗藥性,其中尤以對新煙堿類殺蟲劑的抗性問題最為突出。本文對我國華東地區(qū)8個Q型煙粉虱田間種群對新煙堿類殺蟲劑(吡蟲啉、烯啶蟲胺、噻蟲嗪和呋蟲胺)和氟啶蟲胺腈抗藥性水平進(jìn)行了監(jiān)測,通過RT-PCR克隆了Q型煙粉虱煙堿型乙酰膽堿受體p1亞基基因全長cDNA,并對Q型煙粉虱田間種群乙酰膽堿受體p1亞基是否存在抗性相關(guān)突變進(jìn)行了檢測。此外,運用基因組步移技術(shù)克隆了Q型煙粉虱抗性相關(guān)細(xì)胞色素P450CYP4C64基因側(cè)翼區(qū)序列,并對B型和Q型煙粉虱CYP4C64基因5’側(cè)翼區(qū)序列多態(tài)性進(jìn)行了比對分析。采用浸葉法對揚州、江陰、東臺、和縣、上海、合肥、杭州和南昌8個地區(qū)的Q型煙粉虱田間種群對新煙堿類殺蟲劑的抗性水平進(jìn)行了測定,結(jié)果表明,與敏感種群相比,8個地區(qū)Q型煙粉虱田間種群對吡蟲啉和烯啶蟲胺產(chǎn)生低至中等水平抗性,抗性倍數(shù)分別為4.07-21.75倍和3.37-16.14倍。除了揚州種群對噻蟲嗪產(chǎn)生高水平抗性(RF 40.38)外,其他種群對噻蟲嗪產(chǎn)生低水平抗性,抗性倍數(shù)為3.50-8.58倍。8個地區(qū)的煙粉虱田間種群對呋蟲胺與氟啶蟲胺腈較敏感,其中南昌種群對呋蟲胺和氟啶蟲胺腈的敏感性最高(RF分別為0.5和0.41),表明呋蟲胺和氟啶蟲胺腈與吡蟲啉、烯啶蟲胺和噻蟲嗪之間不存在交互抗性。通過RT-PCR克隆了煙粉虱乙酰膽堿受體β1亞基基因1597bp全長cDNA,編碼氨基酸序列與桃蚜Myzus persicae乙酰膽堿受體β1亞基的一致性為84.8%。對Q型煙粉虱田間種群乙酰膽堿受體β1亞基的序列分析發(fā)現(xiàn),在8個地區(qū)煙粉虱田間種群中存在23個核苷酸多態(tài)性位點,導(dǎo)致編碼19個氨基酸發(fā)生替換。雖然在D環(huán)上對應(yīng)桃蚜對吡蟲啉抗性相關(guān)T81R突變的R78氨基酸沒有發(fā)生突變,但在江陰種群中發(fā)現(xiàn)了位于D環(huán)的45個堿基的片段缺失,該缺失片段編碼的15個氨基酸包含了R78。利用基因組步移方法獲得煙粉虱抗性相關(guān)細(xì)胞色素P450基因CYP4C64的5’和3’側(cè)翼基因組序列。利用CONSITE軟件對CYP4C64 5'側(cè)翼基因組序列進(jìn)行分析,發(fā)現(xiàn)了多個轉(zhuǎn)錄因子結(jié)合位點,包括51個GATA-1、24個Ahr-ARNT、7個CREB、2個NRF-2和33個Broad-complex-4。對B型敏感種群和Q型田間種群煙粉虱CYP4C64基因5’側(cè)翼序列進(jìn)行的序列比對發(fā)現(xiàn),與B型敏感種群相比,Q型煙粉虱田間種群CYP4C64基因5’側(cè)翼區(qū)存在一個260bp的轉(zhuǎn)座子插入,該轉(zhuǎn)座子序列含有多個轉(zhuǎn)錄因子結(jié)合位點,包括3個Ahr-ARNT,7個Broad-complex-4和1個NRF-2。
[Abstract]:Bemisia tabaci (Gennadius) is a worldwide agricultural pest, among which type B and type Q are the two most serious biotypes. Compared with type B, type Q is more resistant to insecticides and is more resistant to organophosphorus. Most of the insecticides, such as pyrethroids, new nicotine and insect growth regulators, are resistant to different degrees of resistance, especially to new nicotine insecticides. The resistance levels of new nicotine insecticides (imidacloprid, dipyridamine, thiazide and furoximide) to new nicotine insecticides (imidacloprid, dipyridamine, thiazide and furoximide) and fluoridimidine in 8 field populations of Q-type whitefly in eastern China were monitored. The full-length cDNA, of nicotinic acetylcholine receptor subunit p1 gene was cloned by RT-PCR and the resistance-related mutation of acetylcholine receptor p1 subunit was detected in the field population of Q-type Tabaci (Bemisia Tabaci). In addition, the flanking region of the resistance-related cytochrome P450CYP4C64 gene was cloned by genomic walking technique, and the polymorphism of the 5 'flanking region of the CYP4C64 gene of type B and type Q were compared and analyzed. The resistance level of Q-type whitefly population to new nicotine insecticides in Yangzhou, Jiangyin, Dongtai, Hexian, Shanghai, Hefei, Hangzhou and Nanchang areas was determined by leaf soaking method. The results showed that compared with sensitive population, the resistance level of Q-type whitefly population to new nicotine insecticides was determined. The resistance to imidacloprid and enpyrimidine in the field populations of Q-type whitefly in 8 areas was low to medium, and the resistance ratios were 4.07 脳 21.75 times and 3.37% / 16.14 times, respectively. In addition to the high-level resistance of the Yangzhou population to thiazide (RF 40.38), other populations developed low-level resistance to thiothiazide. The resistance multiple was 3.50 脳 8.58. The field population of Bemisia Tabaci in 8 areas was more sensitive to furosemide and fludinamidonitrile, and Nanchang population was the most sensitive to furoximide and fludinamidonitrile (RF = 0.50 and 0.41, respectively), and the population of Nanchang had the highest sensitivity to furoximide and fluodacil (RF = 0.41, respectively). The results showed that there was no cross-resistance between furoximide and fludinamidonitrile with imidacloprid, dipyrimidine and thiazide. The full-length cDNA, coding amino acid sequence of acetylcholine receptor 尾 1 subunit (1597bp) gene of Lepidoptera Tabaci was cloned by RT-PCR, and the consistency of the amino acid sequence with Myzus persicae acetylcholine receptor 尾 1 subunit of Myzus persicae was 84.8%. The sequence analysis of acetylcholine receptor 尾 1 subunit in field population of Q type whitefly showed that there were 23 nucleotide polymorphism sites in the field population of B. Tabaci in 8 areas, which resulted in the substitution of 19 amino acids. Although there was no mutation of R78 amino acid in the D loop corresponding to the T81R mutation associated with imidacloprid resistance of the peach aphid, a deletion of 45 bases located in the D ring was found in Jiangyin population, and the 15 amino acids encoded by the deletion fragment contained R78. The 5 'and 3' flanking genomic sequences of resistance-related cytochrome P450 gene (CYP4C64) were obtained by genomic step-by-step method. The sequence of CYP4C64 5 'flanking genome was analyzed by CONSITE software. Several transcription factor binding sites were found, including 51 GATA-1,24 Ahr-ARNT,7, 33 CREB,2 NRF-2 and 33 Broad-complex-4.. The 5 'flanking sequences of CYP4C64 gene of B. Tabaci and B. Tabaci were compared with those of type B susceptible population. The results showed that there was a transposon insertion of 260bp in the 5' flanking region of CYP4C64 gene in the field population of B. Tabaci compared with B type susceptible population. The transposon sequence contains multiple transcription factor binding sites, including 3 Ahr-ARNT,7 Broad-complex-4 and 1 NRF-2..
【學(xué)位授予單位】：揚州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S433.39

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