本文選題：桔小實(shí)蠅 + 蛋白酶��； 參考：《西南大學(xué)》2015年碩士論文

【摘要】：本學(xué)位論文基于桔小實(shí)蠅轉(zhuǎn)錄組數(shù)據(jù),篩選、鑒定出了了桔小實(shí)蠅5個(gè)胰蛋白酶基因(BdTryl、BdTry2、BdTry3、BdTry4 和 BdTry5)、2個(gè)天冬氨酸酶基因(BdAspl、BdAsp2)和2個(gè)羧肽酶基因(BdCPl、BdCP2)的全長(zhǎng)cDNA序列,在序列分析的基礎(chǔ)上,利用qPCR技術(shù)重點(diǎn)解析了這些在桔小實(shí)蠅不同發(fā)育階段、取食前后以及受胰蛋白酶抑制劑和高效氯氰菊酯脅迫處理后的應(yīng)激表達(dá)模式,同時(shí),在蛋白水平對(duì)相關(guān)酶活性進(jìn)行了檢測(cè)。研究結(jié)果將有助于明確桔小實(shí)蠅蛋白酶基因的功能及其調(diào)控機(jī)制,為桔小實(shí)蠅的持續(xù)防控提供新的思路和方法。主要研究結(jié)果如下：1.桔小實(shí)蠅9個(gè)蛋白酶基因的表達(dá)模式分析1.1.桔小實(shí)蠅9個(gè)蛋白酶基因在不同發(fā)育階段的表達(dá)模式分析利用qPCR技術(shù)分析桔小實(shí)蠅9個(gè)蛋白酶基因在幼蟲階段第一、三、五天,蛹和成蟲期的mRNA表達(dá)水平。結(jié)果顯示,這9個(gè)基因的表達(dá)高峰均出現(xiàn)在孵化后3-5 d的幼蟲期,化蛹后表達(dá)量迅速下降并維持在較低水平；羽化后這些蛋白酶基因的表達(dá)量逐漸上升�？傮w來(lái)看,9個(gè)蛋白酶基因的表達(dá)量在幼蟲期最高,成蟲期次之、卵期最低,推測(cè)這些基因的表達(dá)與桔小實(shí)蠅幼蟲、成蟲期的取食行為有密切的關(guān)系。1.2.桔小實(shí)蠅9個(gè)蛋白酶基因在胰蛋白酶抑制劑處理后的表達(dá)模式分析利用qPCR技術(shù)分析在胰蛋白酶抑制劑處理后桔小實(shí)蠅9個(gè)蛋白酶基因在成蟲中腸的表達(dá)情況。結(jié)果表明,經(jīng)抑制劑TLCK (100μg/mL)的處理,9個(gè)蛋白酶基因除ASP1外,均出現(xiàn)了2倍以上的上調(diào),而同濃度的SBTI對(duì)各基因的影響并不明顯,5個(gè)胰蛋白酶基因中只有Try3和Try4出現(xiàn)了顯著的上調(diào),而其余4個(gè)基因只有CP2和ASP2基因出現(xiàn)上調(diào)。表明桔小實(shí)蠅受到胰蛋白酶專性抑制劑脅迫時(shí),體內(nèi)蛋白酶基因的表達(dá)出現(xiàn)了上調(diào)的情況,以此抵御抑制劑對(duì)蟲體的影響。1.3.饑餓及飼喂條件下桔小實(shí)蠅9個(gè)蛋白酶基因的表達(dá)模式分析以桔小實(shí)蠅饑餓24 h后相關(guān)基因的表達(dá)量作為對(duì)照,分析飼喂后,胰蛋白酶基因24 h內(nèi)表達(dá)量的階段性變化,結(jié)果表明,除了Try3外,其他基因均在飼喂6h后出現(xiàn)顯著的上調(diào),而在12至18 h的時(shí)間段出現(xiàn)表達(dá)高峰,隨后在24 h以后出現(xiàn)顯著的回落。此外,羧肽酶基因也在飼喂18h后出現(xiàn)了2倍以上的上調(diào)現(xiàn)象。推測(cè)桔小實(shí)蠅對(duì)食物的消化高峰出現(xiàn)再取食后18h,并且由胰蛋白酶和羧肽酶等共同參與,是一個(gè)多種蛋白酶協(xié)同作用的過(guò)程。1.4.高效氯氰菊酯脅迫下桔小實(shí)蠅5個(gè)胰蛋白酶基因表達(dá)模式分析為明確逆境脅迫對(duì)桔小實(shí)蠅蛋白酶影響,對(duì)高效氯氰菊酯脅迫后桔小實(shí)蠅5個(gè)胰蛋白酶基因的表達(dá)模式進(jìn)行了檢測(cè)。結(jié)果表明,5個(gè)胰蛋白酶基因在脅迫后均出現(xiàn)不同程度的上調(diào),上調(diào)倍數(shù)介于1.5-2.3倍不等。說(shuō)明桔小實(shí)蠅會(huì)提高體內(nèi)的能量代謝來(lái)抵御逆境的脅迫。2.桔小實(shí)蠅胰蛋白酶活性及總蛋白酶活性的測(cè)定2.1.桔小實(shí)蠅胰蛋白酶活性的測(cè)定以BApNA為底物,測(cè)定桔小實(shí)蠅不同發(fā)育階段以及不同組織的胰蛋白酶活性。結(jié)果表明,在桔小實(shí)蠅幼蟲、蛹和成蟲3個(gè)發(fā)育階段中,幼蟲期的胰蛋白酶活性最高；在幼蟲的3個(gè)組織中(表皮、中腸和脂肪體),中腸胰蛋白酶活性最高。說(shuō)明桔小實(shí)蠅胰蛋白酶主要在幼蟲的中腸發(fā)揮作用。2.2.高效氯氰菊酯脅迫下桔小實(shí)蠅胰蛋白酶活性測(cè)定以含0.33μg/g高效氯氰菊酯的飼料飼喂桔小實(shí)蠅幼蟲5d后,分別以BApNA和TAME為底物檢測(cè)幼蟲中腸胰蛋白酶的酯水解活性和酰胺水解活性。結(jié)果顯示,在高效氯氰菊酯的脅迫下,幼蟲胰蛋白酶的酯水解活性和酰胺水解活性都顯著升高。說(shuō)明桔小實(shí)蠅受到藥劑脅迫后體內(nèi)消化酶活性出現(xiàn)了增強(qiáng)。2.3.蛋白抑制劑處理后桔小實(shí)蠅胰蛋白酶及總蛋白酶活性測(cè)定以含100μg/mL胰蛋白酶專性抑制劑TLCK和SBTI的飼料飼喂剛羽化的成蟲5d,測(cè)定成蟲中腸的胰蛋白酶及總蛋白酶活性。結(jié)果顯示,抑制劑處理后成蟲中腸的胰蛋白酶酶活被顯著抑制,且抑制率達(dá)到70%。但是,總蛋白酶活性卻保持恒定。說(shuō)明桔小實(shí)蠅在受到胰蛋白酶抑制劑脅迫時(shí),通過(guò)其它蛋白酶活性的提高來(lái)彌補(bǔ)胰蛋白酶活性的損失,從而使總的蛋白酶活性維持在一個(gè)相對(duì)恒定水平。
[Abstract]:Based on the data of the orange fly transcriptional group, the thesis screened out the full length cDNA sequence of 5 trypsin genes (BdTryl, BdTry2, BdTry3, BdTry4 and BdTry5), 2 aspartase gene (BdAspl, BdAsp2) and 2 carboxypeptidase genes (BdCPl, BdCP2). Based on the sequence analysis, it was analyzed by qPCR technology. The stress expression patterns in different developmental stages of the fruit fly, before and after feeding and under the stress of trypsin inhibitor and cypermethrin stress, were used to detect the activity of related enzymes at the protein level. The results will be helpful to clarify the function and regulation mechanism of the protein enzyme gene of the fruit fly. The main results are as follows: 1. the analysis of the expression pattern of 9 protease genes of 1. orange fly. Analysis of the expression pattern of 9 protease genes of the fly orange fly at different developmental stages. The analysis of the 9 protease genes of the orange fly with the 9 protease genes of the fly, first, third, five days, pupae and adult in the larval stage by the analysis of the 9 protease genes of the fruit fly The expression level of mRNA showed that the expression peak of these 9 genes appeared at the larval stage of 3-5 d after hatching, and the expression amount decreased rapidly and maintained at a lower level after the pupation. The expression of these protease genes increased gradually after the emergence. In general, the expression of the 9 protease genes was the highest in the larval stage, the adult stage, the eggs. The expression of these genes is closely related to the feeding behavior of the larva and the adult stage. The expression pattern of the 9 protease genes of the.1.2. citrus fruit fly after trypsin inhibitor treatment was analyzed by qPCR technique to analyze the table of the 9 protease genes in the adult after trypsin inhibitor treatment. The results showed that, with the treatment of inhibitor TLCK (100 mu g/mL), the 9 protease genes were up to up up to up to 2 times more than ASP1, while the same concentration of SBTI had no obvious effect on each gene. Only the 5 trypsin genes were only Try3 and Try4, but the remaining 4 genes only up to CP2 and ASP2 genes. The expression of protease gene in the body was up-regulated in vivo when the protease specific inhibitor was stressed. The effect of the inhibitor on the body was resisted. The expression pattern of 9 protease genes in the.1.3. starvation and the feeding condition of the fruit fly was analyzed. The expression of the related genes after the starvation of the fruit fly was 24 h as a pair. According to the analysis of the phased changes in the expression of trypsin gene 24 h after feeding, the results showed that except Try3, the other genes were significantly up-regulated after feeding 6h, while the peak expression appeared at the time of 12 to 18 h, followed by a significant decline after 24 h. In the process of food digestion, it is conjectured that it is 18h after feeding on the digestion peak of the food, and it is co involved by trypsin and carboxypeptidase. It is a synergistic process of a variety of proteases. The analysis of 5 trypsin gene expressions in the cypermethrin under the stress of.1.4. Effect of white enzyme, the expression pattern of 5 trypsin genes in citrus fruit fly after cypermethrin stress was detected. The results showed that the 5 trypsin genes were up regulated in varying degrees after stress, and the up-regulation multiplier was between 1.5-2.3 times. It indicated that the fruit fly could improve the energy metabolism in the body to resist the stress of.2. orange. Determination of trypsin activity and the activity of total protease in 2.1., the activity of trypsin was determined by BApNA as substrate. The trypsin activity of different developmental stages and different tissues of the fly was determined. The results showed that the trypsin activity in the larval stage of the larvae, pupae and adult were the highest in the 3 stages of the larvae, pupae and adult. In the 3 tissues of the larva (epidermis, midgut and fat body), the activity of trypsin in the midgut is the highest. It shows that the trypsin mainly plays a role in the midgut of the larvae. The activity of trypsin in the cypermethrin under the stress of high cypermethrin under the stress of.2.2. is fed with the feed of Cypermethrin containing 0.33 micron of Cypermethrin after feeding 5D, respectively. BApNA and TAME were used as substrates to detect the hydrolysis activity of trypsin in the midgut and the activity of amide hydrolysis. The results showed that the hydrolysis activity of trypsin and the hydrolysis activity of trypsin were significantly increased under the stress of Cypermethrin, indicating that the activity of internal digestive enzyme appeared to enhance the.2.3. eggs after the drug stress. Trypsin and total protease activity after treatment with white inhibitors were fed with 100 g/mL trypsin specific inhibitor TLCK and SBTI to feed the adult 5D, the trypsin and total protease activity in the midgut of the adult. The results showed that the trypsinase activity of the midgut of the adult was significantly inhibited, and the results showed that the trypsin activity of the midgut of the insect was significantly inhibited. The inhibitory rate reached 70%., but the activity of the total protease remained constant. It indicated that the total protease activity was maintained at a relatively constant level by the increase of the activity of other protease to compensate for the loss of trypsin activity when the trypsin inhibitor was stressed.
【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】：2015
【分類號(hào)】：S433

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本文編號(hào)：2017169