本文選題：阿維菌素　切入點：朱砂葉螨　出處：《西南大學(xué)》2015年碩士論文

【摘要】：朱砂葉螨(Tetranychus cinnabarinus)是一種世界性害螨,因其世代周期短、繁殖能力強、受藥機會多、近親交配率高等特點而極易產(chǎn)生抗藥性。阿維菌素是一類具有殺蟲、殺螨和殺線蟲活性的大環(huán)內(nèi)酯化合物,隨著使用頻率的不斷增加,已有不少害蟲對其產(chǎn)生了抗藥性。P-糖蛋白(P-glycoprotein, Pgp)是ABC(ATP-binding cassette)轉(zhuǎn)運蛋白超家族成員之一,主要功能是利用其與ATP的結(jié)合和水解供能進行底物的跨膜轉(zhuǎn)運,保護組織免受內(nèi)源性代謝物和外源性有毒物質(zhì)的傷害。近年來已有研究表明許多寄生蟲和昆蟲對阿維菌素產(chǎn)生抗藥性與PgP有關(guān)。PgP引起的抗性也稱MDR (multidrug resistance)。本論文重點研究朱砂葉螨對阿維菌素的抗藥性與P-糖蛋白的相關(guān)關(guān)系,獲得了以下主要研究結(jié)果：1.室內(nèi)抗性篩選獲得了朱砂葉螨阿維菌素抗性品系(AbR,抗性倍數(shù)超過20倍),經(jīng)PgP特異性抑制劑維拉帕米處理12 h后,阿維菌素對朱砂葉螨敏感品系(SS)和AbR的毒力分別增大了19.91%和74.51%,初步說明PgP在阿維菌素抗性品系中作用更明顯,與阿維菌素抗性形成有關(guān)。2.朱砂葉螨P-糖蛋白ATP酶動力學(xué)測定結(jié)果為：Km分別為1.835 ± 0.142 mM(SS)和1.595 ± 0.353 mM (AbR), Vmax分別為0.097 ± 0.036μmol/mg/min (SS)和0.108 ± 0.054μmol/mg/min (AbR); Km和Vmax在敏感和抗性品系間無顯著差異。3.對朱砂葉螨P-糖蛋白ATP酶進行活性測定,結(jié)果發(fā)現(xiàn)該酶在AbR的活性顯著高于SS；用低劑量阿維菌素(LC30)分別對朱砂葉螨SS及AbR品系的雌成螨進行誘導(dǎo)后,ATP酶活性在SS中沒有顯著變化,但在AbR中表現(xiàn)為活性在誘導(dǎo)后顯著升高。4.克隆獲得兩條朱砂葉螨P-糖蛋白基因,分別命名為TcPgpl和TcPgp2。其中TcPgp1 cDNA序列全長3885 bp,編碼1295個氨基酸；TcPgp2 cDNA序列全長3879bp,編碼1293個氨基酸。兩條基因皆具有Walker-A、C-motif、Walker-B、D-loop和H-loop等特征序列。5.序列分析表明SS和AbR品系中TcPgp1和TcPgp2基因不存在氨基酸水平差異。RT-qPCR分析結(jié)果表明TcPgp1在各螨態(tài)的表達量差異不顯著,而TcPgp2在卵和幼螨的表達量顯著高于若螨和成螨; TcPgp1和TcPgp2的表達量在敏感和抗性品系之間均沒有顯著性差異。藥劑誘導(dǎo)實驗表明,SS品系TcPgp1和TcPgp2的表達量均沒有顯著變化,但AbR品系TcPgp1和TcPgp2的表達量在誘導(dǎo)后均顯著
[Abstract]:Tetranychus cinnabarinus (Tetranychus cinnabarinus) is a worldwide pest. Acaricidal and nematicidal macrolides, with the increasing frequency of use, many pests have developed resistance to them. P-glycoprotein (Pgp) is a member of the ABC(ATP-binding cassette-based transporter superfamily. Its main function is to utilize its binding with ATP and hydrolytic energy supply to carry out the transmembrane transport of the substrate. To protect tissues from endogenous metabolites and exogenous toxic substances. Recent studies have shown that resistance of many parasites and insects to avermectin is associated with resistance to PgP. PGP is also known as MDR multidrug resistance. To study the relationship between the resistance of Tetranychus cinnabarinus to abamectin and P- glycoprotein, The main results of this study were as follows: 1. The resistant strain of abamectin of Tetranychus cinnabinus was selected in laboratory, and the resistance ratio was more than 20 times. The strain was treated with verapamil, a specific inhibitor of PgP, for 12 h. The virulence of avermectin to AbR and AbR increased by 19.91% and 74.51%, respectively, which indicated that PgP was more effective in avermectin resistant strains. The results of kinetic analysis of P- glycoprotein ATP enzyme of Tetranychus cinnabinae were: 1.835 鹵0.142 mm ATP and 1.595 鹵0.353 mm ATP, Vmax 0.097 鹵0.036 渭 mol/mg/min and 0.108 鹵0.054 渭 mol/mg/min, respectively, but km and Vmax were not found in sensitive and resistant lines. The activity of P-glycoprotein ATP enzyme in Tetranychus cinnabarinus was determined. The results showed that the activity of this enzyme in AbR was significantly higher than that in SS.After induced by low dose avermectin (Avermectin), SS and female adult mites of AbR strain had no significant change in SS. But in AbR, the activity increased significantly after induction. 4. Two P- glycoprotein genes of Tetranychus cinnabarinus were cloned. They were named TcPgpl and TcPgp2 respectively. The TcPgp1 cDNA sequence was 3885 BP in length, encoding 1295 amino acids TcPgp2 cDNA sequence in 3879 BP, encoding 1293 amino acids. Both of the two genes had the characteristic sequences of Walker-Agna C-motifen Walker-BAND-loop and H-loop. The sequence analysis showed that TcPgp1 and H-loop in SS and AbR strains. There was no amino acid level difference in TcPgp2 gene. RT-qPCR analysis showed that there was no significant difference in the expression of TcPgp1 among mites. However, the expression of TcPgp2 in eggs and juvenile mites was significantly higher than that of mites and adults, and there was no significant difference in the expression of TcPgp1 and TcPgp2 between sensitive and resistant lines. The results of medicament induction showed that there was no significant change in the expression of TcPgp1 and TcPgp2 in SS strains. However, the expression of TcPgp1 and TcPgp2 in AbR lines were significant after induction.
【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S433.7

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