本文選題：朱砂葉螨　切入點(diǎn)：絲氨酸蛋白酶　出處：《西南大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：朱砂葉螨(Tetranychus cinnabarinus)是農(nóng)業(yè)生產(chǎn)中一種危害嚴(yán)重、分布廣泛的害螨,在世界各地均有分布。在我國華南、西北、西南、湖北等地方都有發(fā)生,其寄主植物多達(dá)1000多種,其中經(jīng)濟(jì)作物有100多種,因其具有繁殖能力強(qiáng)、世代周期短、活動(dòng)范圍小、近親交配率高等特點(diǎn),以及幾年來殺蟲(螨)劑的大量、持續(xù)和不科學(xué)使用,導(dǎo)致抗藥性問題尤為嚴(yán)重。絲氨酸蛋白酶作為昆蟲體內(nèi)一類重要的水解酶,在維持昆蟲機(jī)體正常生命活動(dòng)中扮演著重要的角色,它參與了昆蟲機(jī)體消化、胚胎發(fā)育、組織重建、細(xì)胞分化、血管形成、血液凝固、免疫反應(yīng)、激素活化等多種生理生化過程,它是否也可能與朱砂葉螨的抗性存在著相關(guān)關(guān)系,為探究?jī)烧咧g存在的相關(guān)關(guān)系,我們?cè)O(shè)計(jì)了相關(guān)研究?jī)?nèi)容:在室內(nèi)篩選出的甲氰菊酯抗性品系(FeR)、阿維菌素抗性品系(AbR)朱砂葉螨以及敏感品系(SS)朱砂葉螨的基礎(chǔ)上,研究絲氨酸蛋白酶與朱砂葉螨抗性之間存在的相關(guān)關(guān)系,并初步對(duì)其機(jī)制進(jìn)行初步探究,主要的研究?jī)?nèi)容和結(jié)果如下:1.對(duì)絲氨酸蛋白酶基因(SP-900)進(jìn)行核苷酸序列全長(zhǎng)克隆,獲得一條長(zhǎng)為897 bp堿基序列,并且順利翻譯成含有298個(gè)氨基酸的序列,序列分析確定其為一條絲氨酸蛋白酶基因,并且通過進(jìn)化樹構(gòu)建分析結(jié)果表明它亦是一條類胰蛋白酶基因。2.采用q-PCR技術(shù)檢測(cè)目的基因SP-900在SS、FeR、AbR三個(gè)品系中表達(dá)情況,利用數(shù)據(jù)分析軟件SPSS22.0采用獨(dú)立樣本T檢驗(yàn)的分析方法分析抗性品系與SS品系表達(dá)量的差異性,結(jié)果顯示FeR、AbR品系目的基因SP-900的表達(dá)量均高于SS品系,分別是SS品系的2.45、5.54倍。對(duì)三個(gè)品系朱砂葉螨經(jīng)甲氰菊酯或阿維菊素誘導(dǎo)后6h、12h、24h SP-900基因的表達(dá)量情況也進(jìn)行了檢測(cè),結(jié)果顯示,SS品系經(jīng)甲氰菊酯誘導(dǎo)后12h SP-900基因表達(dá)量上調(diào)至3.05倍,6h、24h表達(dá)量沒有上調(diào),FeR品系經(jīng)甲氰菊酯誘導(dǎo)后6h、12h SP-900基因表達(dá)量分別上調(diào)至2.77、2.65倍;SS品系經(jīng)阿維菌素誘導(dǎo)后12h SP-900基因表達(dá)量上調(diào)至2.42倍,6h、24h表達(dá)量沒有上調(diào),AbR品系經(jīng)阿維菌素誘導(dǎo)后12h SP-900基因表達(dá)量上調(diào)至2.25倍,6h、24h表達(dá)量沒有上調(diào)。上述研究結(jié)果表明在目前的抗性水平,朱砂葉螨抗性的增加可能與絲氨酸蛋白酶基因表達(dá)量的上調(diào)有關(guān),并且在藥劑誘導(dǎo)下,SP-900基因的表達(dá)量會(huì)上升,表明SP-900可能參與了朱砂葉螨在應(yīng)對(duì)藥劑帶來的不利影響時(shí)的某些生理生化反應(yīng)。3.SS、FeR、AbR三個(gè)品系朱砂葉螨絲氨酸蛋白酶粗酶活性測(cè)定結(jié)果為:SS、FeR、AbR三個(gè)品系朱砂葉螨絲氨酸蛋白酶粗酶活力分別為3.0024nmol/μg/min、5.2634 nmol/μg/min和6.2774 nmol/μg/min,FeR、AbR兩個(gè)抗性品系的粗酶活力分別是SS品系的1.75、2.09倍,具有顯著性差異。采用藥膜法對(duì)三個(gè)品系朱砂葉螨進(jìn)行絲氨酸蛋白酶抑制劑處理,這種處理方法能夠抑制朱砂葉螨體內(nèi)絲氨酸蛋白酶粗酶活性,處理后SS、FeR、AbR三個(gè)品系的粗酶活性分別下降了38.4%、24.8%和21.9%,三個(gè)品系朱砂葉螨絲氨酸蛋白酶活性被抑制后,其對(duì)甲氰菊酯和阿維菌素的敏感性上升。表明朱砂葉螨絲氨酸蛋白酶活性下降,對(duì)藥劑的敏感性上升。4.采用葉碟飼喂法對(duì)SS、FeR、AbR三個(gè)品系朱砂葉螨進(jìn)行RNAi處理,SP-900基因的表達(dá)量分別被抑制了49.19%、51.74%和53.69%;粗酶活性分別下降了28.6%、18.2%和23.5%;SS品系對(duì)甲氰菊酯、阿維菌素的LC50分別下降了174ppm、0.089ppm,FeR品系對(duì)甲氰菊酯的LC50下降了14234ppm,AbR品系對(duì)阿維菌素的LC50下降了0.211ppm。RNAi結(jié)果顯示,葉碟飼喂法能夠較好的抑制朱砂葉螨基因SP-900的表達(dá),基因SP-900表達(dá)量下降,酶活降低,藥劑敏感性上升,。5.絲氨酸蛋白酶與甲氰菊酯或阿維菌素結(jié)合實(shí)驗(yàn)的結(jié)果顯示,絲氨酸蛋白酶沒有與藥劑結(jié)合,表明其沒有直接參與朱砂葉螨對(duì)甲氰菊酯和阿維菌素的分解代謝活動(dòng);利用HPLC測(cè)定SS、FeR、AbR三個(gè)品系朱砂葉螨ATP含量,結(jié)果顯示三個(gè)品系之間ATP含量沒有表現(xiàn)出差異性�？赡苁且�?yàn)镕eR、AbR兩個(gè)抗性品系的解毒代謝活動(dòng)強(qiáng)于SS品系,ATP需求增加,但是兩個(gè)抗性產(chǎn)生了適合度代價(jià),導(dǎo)致其其它的生命活動(dòng)減弱(生殖力下降),ATP需求量下降,從而在ATP總量上沒有表現(xiàn)出差異性。
[Abstract]:Tetranychuscinnabarinus (Tetranychus cinnabarinus) is a kind of agricultural production in the serious, widespread pest mites, distributed throughout the world. In China, Southern China, northwest, southwest, Hubei and other places have occurred, the host plant as many as 1000 kinds, including 100 kinds of economic crops, because of its strong ability of reproduction the scope of activities, short generation cycle, small, high rate of inbreeding, and several years insect (mite) agent for large, continuous and scientific use, lead to drug resistance problem is especially serious. As a serine protease in insects is an important class of enzymes, play a critical role in maintaining the body's normal life activity in insects it is involved in the insect body, digestion, embryonic development, tissue remodeling, cell differentiation, angiogenesis, blood coagulation, immune response, hormone activation and other physiological and biochemical processes, it is impossible and tetranychuscinnabarinus resistant There is correlation between the relationship between the inquiry between the two, we designed the related research contents: fenpropathrin resistant strains were screened in laboratory (FeR), abamectin (AbR) tetranychuscinnabarinus and sensitive strains (SS) based on tetranychuscinnabarinus, correlation the research between serine protease and tetranychuscinnabarinus resistant, and the preliminary mechanism of the preliminary inquiry, the main research contents and results are as follows: 1. the serine protease gene (SP-900) full-length nucleotide sequence, have a length of 897 BP nucleotide sequence, and successfully translated into a sequence containing 298 amino acids. Sequence analysis identified it as a serine protease gene, and through the construction of phylogenetic tree analysis showed that it is also a kind of trypsin gene.2. q-PCR was used to detect the gene SP-900 in SS, FeR, AbR three The expression of strains, using data analysis software SPSS22.0 analysis using independent samples T test analysis of expression difference of resistant strains and SS strains, the results showed that FeR, expression of SP-900 gene of AbR strain was higher than that of the SS strain, which is 2.45,5.54 times of the SS strain of three. Strains of tetranychuscinnabarinus by fenpropathrin or acitretin pyrethrins after induction of 6h, 12h, 24h expression of SP-900 gene was detected, the results showed that the SS strain from fenpropathrin 12h after the induction of SP-900 gene expression was increased to 3.05 times, 6h, 24h expression did not increase, FeR the fenpropathrin after induction of 6h, 12h SP-900 expression were up to 2.77,2.65 times; SS strain by Avermectin 12h after the induction of SP-900 gene expression was increased to 2.42 times, 6h, 24h expression was not up-regulated AbR expression induced by strains of 12h SP-900 gene of avermectin quantity To 2.25 times, 6h, 24h expression was not up-regulated. These results demonstrated that the resistance level at present, tetranychuscinnabarinus resistance may be associated with increased serine protease gene expression amount increases, and in the chemical induction, the expression level of SP-900 gene will rise, suggest that SP-900 may be some physiological and biochemical reactions involved in.3.SS the adverse effects of tetranychuscinnabarinus agents to deal with the FeR, AbR of three strains of crude enzyme activity of tetranychuscinnabarinus serine protease determination results: SS, FeR, AbR in three strains of crude enzyme tetranychuscinnabarinus serine protease activity were 3.0024nmol/ g/min, 5.2634 nmol/ g/min and 6.2774 nmol/ g/min FeR, AbR, crude enzyme activity of two resistant strains were 1.75,2.09 times SS lines, with a significant difference. To tetranychuscinnabarinus three strains of serine protease inhibitor treatment by membrane method, this processing method Method can inhibit the crude enzyme activity in tetranychuscinnabarinus serine protease, SS after treatment, FeR, AbR crude enzyme activity of three strains were decreased by 38.4%, 24.8% and 21.9%, three strains of tetranychuscinnabarinus serine protease activity was inhibited, increased its sensitivity to fenpropathrin and avermectin showed a decrease. Tetranychuscinnabarinus serine protease activity and sensitivity to fungicides increased.4. by using leaf disc feeding method of SS, FeR, AbR of three strains of Tetranychus cinnabarinus were treated with RNAi, the expression level of SP-900 gene was inhibited by 49.19%, 51.74% and 53.69%; the crude enzyme activity decreased by 28.6%, 18.2% and 23.5%; SS Department of fenpropathrin, abamectin and LC50 were decreased by 174ppm, 0.089ppm, LC50 to fenpropathrin FeR strain decreased by 14234ppm and AbR strains of avermectin LC50 decreased by 0.211ppm.RNAi results show that the method can better suppress the Ye Diesi fed The expression of tetranychuscinnabarinus gene SP-900, SP-900 gene expression decreased, enzyme activity decreased, drug sensitivity increased,.5. serine protease and fenpropathrin and avermectin binding experiments showed that serine protease with no medicament combination, that is not directly involved in the catabolic activity of Tetranychus cinnabarinus to fenpropathrin and avermectin; determination SS, by HPLC FeR AbR, three strains of tetranychuscinnabarinus ATP content, showed that three strains of ATP content showed no difference. It may be because FeR, AbR of the two resistant strains of detoxification activity is strong in the SS strain, ATP demand increases, but the two resistance for the degree of price, the other life activities (reduced fertility decline), ATP demand dropped, resulting in the total ATP showed no difference.

【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S433.7

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 陳秋雙;趙舒;鄒晶;石力;何林;;朱砂葉螨抗藥性監(jiān)測(cè)[J];應(yīng)用昆蟲學(xué)報(bào);2012年02期

2 談善軍;余震;杜璐迪;陳必成;董千銅;王賢親;;HPLC法測(cè)定術(shù)后疲勞綜合征大鼠骨骼肌ATP,ADP,AMP的含量[J];藥物分析雜志;2011年11期

3 高坤;鄧祥元;郭錫杰;;免疫系統(tǒng)中絲氨酸蛋白酶的研究進(jìn)展[J];免疫學(xué)雜志;2010年11期

4 陸澄瀅;;昆蟲抗藥性分子機(jī)制的研究進(jìn)展[J];湖南農(nóng)機(jī);2010年09期

5 汪世華;王文勇;黃益洲;林琳;沙莉;;絲氨酸蛋白酶研究進(jìn)展[J];福建農(nóng)業(yè)學(xué)報(bào);2007年04期

6 亢春雨;趙春青;吳剛;;昆蟲抗藥性分子機(jī)制研究的新進(jìn)展[J];華東昆蟲學(xué)報(bào);2007年02期

7 王廣成;張忠明;高立明;陳丙坤;聶果;萬莉;吳春先;;阿維菌素的作用機(jī)理及其應(yīng)用現(xiàn)狀[J];植物醫(yī)生;2006年01期

8 繆宇,王承龍,殷惠軍,史大卓,陳可冀;高效液相色譜測(cè)定大鼠心肌組織腺苷酸含量[J];北京大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2005年02期

9 何林;楊羽;符建章;王進(jìn)軍;趙志模;;朱砂葉螨阿維菌素抗性品系選育及適合度研究[J];植物保護(hù)學(xué)報(bào);2004年04期

10 何林,譚仕祿,曹小芳,趙志模,鄧新平,王進(jìn)軍;朱砂葉螨的抗藥性選育及其解毒酶活性研究[J];農(nóng)藥學(xué)學(xué)報(bào);2003年04期

相關(guān)博士學(xué)位論文 前2條

1 申光茂;桔小實(shí)蠅消化和解毒代謝相關(guān)基因鑒定及其對(duì)藥劑脅迫的應(yīng)激反應(yīng)[D];西南大學(xué);2013年

2 何林;朱砂葉螨（Tetranychus Cinnabarinus）抗藥性機(jī)理及抗性適合度研究[D];西南農(nóng)業(yè)大學(xué);2003年

相關(guān)碩士學(xué)位論文 前3條

1 劉炎超;朱砂葉螨γ-氨基丁酸與阿維菌素殺螨活性關(guān)系研究[D];西南大學(xué);2016年

2 祝妮;小菜蛾中腸絲氨酸蛋白酶基因的克隆、轉(zhuǎn)錄及其酶活的研究[D];浙江大學(xué);2013年

3 張婷;甲氰菊酯對(duì)鯉鰓、肝、腎組織損傷的研究[D];華中農(nóng)業(yè)大學(xué);2013年

，

本文編號(hào)：1584583