华东地区Q型烟粉虱对新烟碱类杀虫剂的抗性监测及其靶标基因克隆

发布时间：2019-04-23 12:44

【摘要】：烟粉虱Bemisia tabaci (Gennadius)是一种世界性农业害虫,其中B型与Q型是危害程度最为严重的两种生物型,相对于B型烟粉虱,Q型烟粉虱对杀虫剂的耐受能力更强,且对包括有机磷类、拟除虫菊酯类、新烟碱类和昆虫生长调节剂等大部分杀虫剂产生了不同程度的抗药性,其中尤以对新烟碱类杀虫剂的抗性问题最为突出。本文对我国华东地区8个Q型烟粉虱田间种群对新烟碱类杀虫剂(吡虫啉、烯啶虫胺、噻虫嗪和呋虫胺)和氟啶虫胺腈抗药性水平进行了监测,通过RT-PCR克隆了Q型烟粉虱烟碱型乙酰胆碱受体p1亚基基因全长cDNA,并对Q型烟粉虱田间种群乙酰胆碱受体p1亚基是否存在抗性相关突变进行了检测。此外,运用基因组步移技术克隆了Q型烟粉虱抗性相关细胞色素P450CYP4C64基因侧翼区序列,并对B型和Q型烟粉虱CYP4C64基因5’侧翼区序列多态性进行了比对分析。采用浸叶法对扬州、江阴、东台、和县、上海、合肥、杭州和南昌8个地区的Q型烟粉虱田间种群对新烟碱类杀虫剂的抗性水平进行了测定,结果表明,与敏感种群相比,8个地区Q型烟粉虱田间种群对吡虫啉和烯啶虫胺产生低至中等水平抗性,抗性倍数分别为4.07-21.75倍和3.37-16.14倍。除了扬州种群对噻虫嗪产生高水平抗性(RF 40.38)外,其他种群对噻虫嗪产生低水平抗性,抗性倍数为3.50-8.58倍。8个地区的烟粉虱田间种群对呋虫胺与氟啶虫胺腈较敏感,其中南昌种群对呋虫胺和氟啶虫胺腈的敏感性最高(RF分别为0.5和0.41),表明呋虫胺和氟啶虫胺腈与吡虫啉、烯啶虫胺和噻虫嗪之间不存在交互抗性。通过RT-PCR克隆了烟粉虱乙酰胆碱受体β1亚基基因1597bp全长cDNA,编码氨基酸序列与桃蚜Myzus persicae乙酰胆碱受体β1亚基的一致性为84.8%。对Q型烟粉虱田间种群乙酰胆碱受体β1亚基的序列分析发现,在8个地区烟粉虱田间种群中存在23个核苷酸多态性位点,导致编码19个氨基酸发生替换。虽然在D环上对应桃蚜对吡虫啉抗性相关T81R突变的R78氨基酸没有发生突变,但在江阴种群中发现了位于D环的45个碱基的片段缺失,该缺失片段编码的15个氨基酸包含了R78。利用基因组步移方法获得烟粉虱抗性相关细胞色素P450基因CYP4C64的5’和3’侧翼基因组序列。利用CONSITE软件对CYP4C64 5'侧翼基因组序列进行分析,发现了多个转录因子结合位点,包括51个GATA-1、24个Ahr-ARNT、7个CREB、2个NRF-2和33个Broad-complex-4。对B型敏感种群和Q型田间种群烟粉虱CYP4C64基因5’侧翼序列进行的序列比对发现,与B型敏感种群相比,Q型烟粉虱田间种群CYP4C64基因5’侧翼区存在一个260bp的转座子插入,该转座子序列含有多个转录因子结合位点,包括3个Ahr-ARNT,7个Broad-complex-4和1个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..
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S433.39

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