棉铃虫V-ATP酶A亚基在Cry1Ac杀虫及抗性演化中的作用研究

发布时间：2018-06-18 15:38

本文选题：棉铃虫 + 中肠　；参考：《中国农业科学院》2015年硕士论文

【摘要】：昆虫中肠是苏云金芽孢杆菌Bacillus thuringiensis(Bt)发挥毒性作用的主要部位,Bt杀虫蛋白与昆虫中肠刷状缘膜囊泡brush border membrane vesicles(BBMV)上的受体蛋白结合是Bt作用机制中的关键步骤,因此中肠受体的改变被认为是昆虫对Bt毒蛋白产生抗性的主要机制。转Bt基因棉花在有效控制棉铃虫(Helicoverpa armigera)危害的同时也使棉铃虫对其产生抗性演化的风险,这将严重威胁到转Bt棉花的长期有效利用,因此了解Bt的抗性机制并制定相应的抗性治理政策对于延长Bt棉的使用寿命显得至关重要。为了探究棉铃虫的抗性机制,我们利用iTRAQ实验比较敏感与抗性棉铃虫品系中肠BBMV之间的差异表达蛋白,发现了很多Vacuolar H+-ATP酶基因蛋白表达量存在显著差异;而且已有研究报道V-ATP酶A亚基是一些昆虫Bt-Cry1Ac的结合蛋白,因此,本研究克隆了棉铃虫V-ATP酶A亚基基因,并对其在Cry1Ac发挥杀虫作用及抗性演化中的功能展开了初步研究。主要研究内容及结果如下:1、运用PCR结合RACE(rapid-amplification of cDNA ends)cDNA末端快速扩增技术,克隆了棉铃虫中肠V-ATP酶A亚基基因的cDNA序列全长。该cDNA序列全长2 578 bp(GenBank登录号:KP090287),开放阅读框1 863 bp,编码621个氨基酸。预测其分子量和等电点分别为68 KD和5.13,且N端没有信号肽,C端也没有潜在的GPI锚定位点,蛋白亲疏水性预测显示为亲水性,亚细胞定位结果主要位于细胞质,与V-ATP酶结构吻合。同源序列分析发现,棉铃虫与其他物种的V-ATP酶A亚基氨基酸序列同源性非常高,其相似性达90%以上,其中与鳞翅目烟草天蛾序列一致性最高,为95.48%。不同种属间的氨基酸序列差异主要在N端。说明V-ATP酶A亚基基因非常保守。2、利用实时荧光定量qRT-PCR对V-ATP酶A亚基的表达谱测定结果表明,棉铃虫不同发育历期V-ATP酶A亚基的表达量不同,主要在幼虫期表达,其中4龄表达量最高,其次是2龄,卵、蛹和成虫期的表达量较低。在肠道不同部位的表达量也有一定的差异,其中在中肠中表达量最高,其次是前肠,后肠的表达量显著地低于中肠和前肠的表达量。棉铃虫4龄幼虫取食含有Cry1Ac毒素蛋白的饲料后,中肠V-ATP酶A亚基表达量显著降低。3、将V-ATP酶A亚基全长进行原核表达,用western blot方法检测发现棉铃虫中肠BBMV上V-ATP酶A亚基有较高的表达量,但将表达的蛋白与Cry1Ac进行Ligand blot试验,发现原核表达的蛋白与Cry1Ac毒素不结合。4、比较棉铃虫敏感品系和抗性品系之间的核苷酸序列和氨基酸序列差异,发现抗性品系核苷酸序列有75个碱基发生突变,但氨基酸序列一致。Cry1Ac抗性棉铃虫中肠V-ATP酶A亚基在mRNA水平和蛋白水平上表达量都显著低于敏感品系棉铃虫的表达量。5、用RNAi方法将V-ATP酶A亚基基因沉默,检测V-ATP酶A亚基基因沉默对Cry1Ac毒力的影响,结果发现siRNA-A1基因沉默效果较好,在各个时间段的沉默效率为42%~55%。敏感品系棉铃虫注射siRNA-A1后,体重抑制率增加,除第3天60μg/mL浓度处理外,其他处理的体重抑制率均显著的大于对照。RNAi沉默基因表达,使敏感品系对Cry1Ac的敏感性增强。以上结果表明,V-ATP酶A亚基在棉铃虫中肠高表达,Cry1Ac处理敏感4龄幼虫后基因表达量降低,而且通过RNAi试验证实V-ATP酶A亚基基因沉默后使敏感品系棉铃虫对Cry1Ac的敏感性增强,但是棉铃虫对Cry1Ac产生抗性后,V-ATP酶A亚基基因和蛋白的表达量表现为下调,推测V-ATP酶A亚基在棉铃虫生长发育和能量代谢中起到重要作用,可能影响蛋白酶的合成和分泌,或影响Bt信号传导途径中ATP的水解、激活蛋白激酶A的过程,参与抵御Cry1Ac的毒杀作用,并在在棉铃虫Cry1Ac产生抗性的过程中起到一定的作用。明确的作用机制有待于进一步研究。
[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.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S433

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