本文選題：棉鈴蟲 + 中腸�。� 參考：《中國農(nóng)業(yè)科學(xué)院》2015年碩士論文

【摘要】：昆蟲中腸是蘇云金芽孢桿菌Bacillus thuringiensis(Bt)發(fā)揮毒性作用的主要部位,Bt殺蟲蛋白與昆蟲中腸刷狀緣膜囊泡brush border membrane vesicles(BBMV)上的受體蛋白結(jié)合是Bt作用機(jī)制中的關(guān)鍵步驟,因此中腸受體的改變被認(rèn)為是昆蟲對(duì)Bt毒蛋白產(chǎn)生抗性的主要機(jī)制。轉(zhuǎn)Bt基因棉花在有效控制棉鈴蟲(Helicoverpa armigera)危害的同時(shí)也使棉鈴蟲對(duì)其產(chǎn)生抗性演化的風(fēng)險(xiǎn),這將嚴(yán)重威脅到轉(zhuǎn)Bt棉花的長(zhǎng)期有效利用,因此了解Bt的抗性機(jī)制并制定相應(yīng)的抗性治理政策對(duì)于延長(zhǎng)Bt棉的使用壽命顯得至關(guān)重要。為了探究棉鈴蟲的抗性機(jī)制,我們利用iTRAQ實(shí)驗(yàn)比較敏感與抗性棉鈴蟲品系中腸BBMV之間的差異表達(dá)蛋白,發(fā)現(xiàn)了很多Vacuolar H+-ATP酶基因蛋白表達(dá)量存在顯著差異;而且已有研究報(bào)道V-ATP酶A亞基是一些昆蟲Bt-Cry1Ac的結(jié)合蛋白,因此,本研究克隆了棉鈴蟲V-ATP酶A亞基基因,并對(duì)其在Cry1Ac發(fā)揮殺蟲作用及抗性演化中的功能展開了初步研究。主要研究?jī)?nèi)容及結(jié)果如下:1、運(yùn)用PCR結(jié)合RACE(rapid-amplification of cDNA ends)cDNA末端快速擴(kuò)增技術(shù),克隆了棉鈴蟲中腸V-ATP酶A亞基基因的cDNA序列全長(zhǎng)。該cDNA序列全長(zhǎng)2 578 bp(GenBank登錄號(hào):KP090287),開放閱讀框1 863 bp,編碼621個(gè)氨基酸。預(yù)測(cè)其分子量和等電點(diǎn)分別為68 KD和5.13,且N端沒有信號(hào)肽,C端也沒有潛在的GPI錨定位點(diǎn),蛋白親疏水性預(yù)測(cè)顯示為親水性,亞細(xì)胞定位結(jié)果主要位于細(xì)胞質(zhì),與V-ATP酶結(jié)構(gòu)吻合。同源序列分析發(fā)現(xiàn),棉鈴蟲與其他物種的V-ATP酶A亞基氨基酸序列同源性非常高,其相似性達(dá)90%以上,其中與鱗翅目煙草天蛾序列一致性最高,為95.48%。不同種屬間的氨基酸序列差異主要在N端。說明V-ATP酶A亞基基因非常保守。2、利用實(shí)時(shí)熒光定量qRT-PCR對(duì)V-ATP酶A亞基的表達(dá)譜測(cè)定結(jié)果表明,棉鈴蟲不同發(fā)育歷期V-ATP酶A亞基的表達(dá)量不同,主要在幼蟲期表達(dá),其中4齡表達(dá)量最高,其次是2齡,卵、蛹和成蟲期的表達(dá)量較低。在腸道不同部位的表達(dá)量也有一定的差異,其中在中腸中表達(dá)量最高,其次是前腸,后腸的表達(dá)量顯著地低于中腸和前腸的表達(dá)量。棉鈴蟲4齡幼蟲取食含有Cry1Ac毒素蛋白的飼料后,中腸V-ATP酶A亞基表達(dá)量顯著降低。3、將V-ATP酶A亞基全長(zhǎng)進(jìn)行原核表達(dá),用western blot方法檢測(cè)發(fā)現(xiàn)棉鈴蟲中腸BBMV上V-ATP酶A亞基有較高的表達(dá)量,但將表達(dá)的蛋白與Cry1Ac進(jìn)行Ligand blot試驗(yàn),發(fā)現(xiàn)原核表達(dá)的蛋白與Cry1Ac毒素不結(jié)合。4、比較棉鈴蟲敏感品系和抗性品系之間的核苷酸序列和氨基酸序列差異,發(fā)現(xiàn)抗性品系核苷酸序列有75個(gè)堿基發(fā)生突變,但氨基酸序列一致。Cry1Ac抗性棉鈴蟲中腸V-ATP酶A亞基在mRNA水平和蛋白水平上表達(dá)量都顯著低于敏感品系棉鈴蟲的表達(dá)量。5、用RNAi方法將V-ATP酶A亞基基因沉默,檢測(cè)V-ATP酶A亞基基因沉默對(duì)Cry1Ac毒力的影響,結(jié)果發(fā)現(xiàn)siRNA-A1基因沉默效果較好,在各個(gè)時(shí)間段的沉默效率為42%~55%。敏感品系棉鈴蟲注射siRNA-A1后,體重抑制率增加,除第3天60μg/mL濃度處理外,其他處理的體重抑制率均顯著的大于對(duì)照。RNAi沉默基因表達(dá),使敏感品系對(duì)Cry1Ac的敏感性增強(qiáng)。以上結(jié)果表明,V-ATP酶A亞基在棉鈴蟲中腸高表達(dá),Cry1Ac處理敏感4齡幼蟲后基因表達(dá)量降低,而且通過RNAi試驗(yàn)證實(shí)V-ATP酶A亞基基因沉默后使敏感品系棉鈴蟲對(duì)Cry1Ac的敏感性增強(qiáng),但是棉鈴蟲對(duì)Cry1Ac產(chǎn)生抗性后,V-ATP酶A亞基基因和蛋白的表達(dá)量表現(xiàn)為下調(diào),推測(cè)V-ATP酶A亞基在棉鈴蟲生長(zhǎng)發(fā)育和能量代謝中起到重要作用,可能影響蛋白酶的合成和分泌,或影響B(tài)t信號(hào)傳導(dǎo)途徑中ATP的水解、激活蛋白激酶A的過程,參與抵御Cry1Ac的毒殺作用,并在在棉鈴蟲Cry1Ac產(chǎn)生抗性的過程中起到一定的作用。明確的作用機(jī)制有待于進(jìn)一步研究。
[Abstract]:The midgut of insect is the main part of the toxic effect of Bacillus thuringiensis Bacillus thuringiensis (Bt). The binding of Bt insecticidal protein with the receptor protein of brush border membrane vesicles (BBMV) in the midgut of the midgut is the key step in the mechanism of the action of Bt, because the change of the midgut receptor is considered to be the insect to Bt toxic protein. The main mechanism to produce resistance is that Bt transgenic cotton can effectively control the harm of cotton bollworm (Helicoverpa armigera) and also cause the risk of resistance evolution of cotton bollworm, which will seriously threaten the long-term effective utilization of Bt cotton. Therefore, understanding the resistance mechanism of Bt and formulating the corresponding resistance management policy for prolonging Bt cotton Life expectancy is very important. In order to explore the resistance mechanism of cotton bollworm, we use iTRAQ to compare the differential expression proteins between sensitive and resistant cotton bollworm strains in the midgut BBMV, and find that many Vacuolar H+-ATP enzyme gene protein expressions have significant differences, and the V-ATP enzyme A subunit has been reported to be some insect Bt-Cry1Ac Therefore, the V-ATP A subunit gene of Helicoverpa armigera was cloned, and the function of its insecticidal and resistance evolution in Cry1Ac was studied. The main contents and results were as follows: 1, the midgut of Helicoverpa armigera was cloned by PCR combined with RACE (rapid-amplification of cDNA ends) cDNA end rapid amplification. The cDNA sequence of the V-ATP A subunit gene was full length. The cDNA sequence was 2578 BP (GenBank login: KP090287), the open reading frame was 1863 BP, and 621 amino acids were encoded. The molecular weight and isoelectric points were predicted to be 68 KD and 5.13 respectively, and the N end had no signal peptide, the C end also had no potential anchor location, and the hydrophobicity prediction showed hydrophilic, The subcellular localization results were mainly located in the cytoplasm and anastomosed with the V-ATP enzyme structure. Homology analysis found that the homology of the V-ATP enzyme A subunit of Helicoverpa armigera and other species was very high and its similarity was more than 90%, which was the most consistent with the Lepidoptera tobacco sky moth sequence, which was the main difference of amino acid sequence among different 95.48%. species. At the N end, it is indicated that the V-ATP A subunit gene is very conservative.2, and the expression of the V-ATP enzyme A subunit by real-time fluorescent quantitative qRT-PCR shows that the expression of A subunit of the V-ATP enzyme A subunit of the cotton bollworm is different, mainly in the larval stage, the 4 age is the highest, the next is the 2 age, the egg, the pupa and the adult stage are low. The expression amount in the different parts of the intestinal tract was also different, in which the expression of the midgut was the highest, followed by the foregut, the expression of the back intestine was significantly lower than the expression of the midgut and the foregut. The expression of the V-ATP enzyme A subunit in the midgut of the 4 instar larvae of the Helicoverpa armigera was significantly reduced by.3, and the V-ATP enzyme A subunit was all long. The expression of V-ATP enzyme A subunit on the midgut BBMV of Helicoverpa armigera was detected by Western blot method, but the expressed protein and Cry1Ac were tested by Ligand blot, and the prokaryotic expression protein was not associated with Cry1Ac toxin.4, and the nucleotide sequence and amino acid were compared between the susceptible strain and the resistant strain of cotton bollworm. The sequence difference showed that the nucleotide sequence of the resistant strain was 75 base mutations, but the amino acid sequence consistent.Cry1Ac resistance of the midgut V-ATP enzyme A subunit of the cotton bollworm was significantly lower than that of the sensitive strain of cotton bollworm,.5. The V-ATP enzyme A subunit gene was silenced by RNAi method and the V-ATP enzyme A subunit was detected. The effect of silence on the virulence of Cry1Ac showed that the effect of siRNA-A1 gene silencing was better. The rate of weight inhibition increased after injection of siRNA-A1 in 42%~55%. sensitive strain of 42%~55%. sensitive strain of cotton bollworm, and the weight inhibition rate of other treatments was significantly greater than that of the control.RNAi silencing gene. The sensitivity of the sensitive strain to Cry1Ac was enhanced. The above results showed that the V-ATP enzyme A subunit was highly expressed in the midgut of Helicoverpa armigera, and the gene expression decreased after the Cry1Ac treatment sensitive 4 instar larvae, and the sensitivity of cotton bollworm to Cry1Ac was enhanced by the silencing of the A subunit gene of the V-ATP enzyme A, but the cotton bollworm was resistant to Cry1Ac. After sex, the expression of the V-ATP A subunit gene and protein expression is downregulated. It is suggested that the V-ATP enzyme A subunit plays an important role in the growth and energy metabolism of Helicoverpa armigera, may affect the synthesis and secretion of protease, or affects the hydrolysis of ATP in the Bt signal transduction pathway, activates the process of protein kinase A and participates in resisting the toxicity of Cry1Ac. It plays a role in the resistance of Helicoverpa armigera Cry1Ac to the production of Helicoverpa armigera.
【學(xué)位授予單位】：中國農(nóng)業(yè)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S433

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 周冬生,王學(xué)林,吳振廷,倪春耕,鄭厚今,夏靜;轉(zhuǎn)Bt基因抗蟲棉對(duì)棉鈴蟲拒食作用及其機(jī)理研究[J];昆蟲知識(shí);2001年06期

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本文編號(hào)：2035981