桔小实蝇胰蛋白酶酶学性质研究及蛋白酶基因表达模式分析

发布时间：2018-06-14 11:06

本文选题：桔小实蝇 + 蛋白酶　；参考：《西南大学》2015年硕士论文

【摘要】：本学位论文基于桔小实蝇转录组数据,筛选、鉴定出了了桔小实蝇5个胰蛋白酶基因(BdTryl、BdTry2、BdTry3、BdTry4 和 BdTry5)、2个天冬氨酸酶基因(BdAspl、BdAsp2)和2个羧肽酶基因(BdCPl、BdCP2)的全长cDNA序列,在序列分析的基础上,利用qPCR技术重点解析了这些在桔小实蝇不同发育阶段、取食前后以及受胰蛋白酶抑制剂和高效氯氰菊酯胁迫处理后的应激表达模式,同时,在蛋白水平对相关酶活性进行了检测。研究结果将有助于明确桔小实蝇蛋白酶基因的功能及其调控机制,为桔小实蝇的持续防控提供新的思路和方法。主要研究结果如下：1.桔小实蝇9个蛋白酶基因的表达模式分析1.1.桔小实蝇9个蛋白酶基因在不同发育阶段的表达模式分析利用qPCR技术分析桔小实蝇9个蛋白酶基因在幼虫阶段第一、三、五天,蛹和成虫期的mRNA表达水平。结果显示,这9个基因的表达高峰均出现在孵化后3-5 d的幼虫期,化蛹后表达量迅速下降并维持在较低水平；羽化后这些蛋白酶基因的表达量逐渐上升。总体来看,9个蛋白酶基因的表达量在幼虫期最高,成虫期次之、卵期最低,推测这些基因的表达与桔小实蝇幼虫、成虫期的取食行为有密切的关系。1.2.桔小实蝇9个蛋白酶基因在胰蛋白酶抑制剂处理后的表达模式分析利用qPCR技术分析在胰蛋白酶抑制剂处理后桔小实蝇9个蛋白酶基因在成虫中肠的表达情况。结果表明,经抑制剂TLCK (100μg/mL)的处理,9个蛋白酶基因除ASP1外,均出现了2倍以上的上调,而同浓度的SBTI对各基因的影响并不明显,5个胰蛋白酶基因中只有Try3和Try4出现了显著的上调,而其余4个基因只有CP2和ASP2基因出现上调。表明桔小实蝇受到胰蛋白酶专性抑制剂胁迫时,体内蛋白酶基因的表达出现了上调的情况,以此抵御抑制剂对虫体的影响。1.3.饥饿及饲喂条件下桔小实蝇9个蛋白酶基因的表达模式分析以桔小实蝇饥饿24 h后相关基因的表达量作为对照,分析饲喂后,胰蛋白酶基因24 h内表达量的阶段性变化,结果表明,除了Try3外,其他基因均在饲喂6h后出现显著的上调,而在12至18 h的时间段出现表达高峰,随后在24 h以后出现显著的回落。此外,羧肽酶基因也在饲喂18h后出现了2倍以上的上调现象。推测桔小实蝇对食物的消化高峰出现再取食后18h,并且由胰蛋白酶和羧肽酶等共同参与,是一个多种蛋白酶协同作用的过程。1.4.高效氯氰菊酯胁迫下桔小实蝇5个胰蛋白酶基因表达模式分析为明确逆境胁迫对桔小实蝇蛋白酶影响,对高效氯氰菊酯胁迫后桔小实蝇5个胰蛋白酶基因的表达模式进行了检测。结果表明,5个胰蛋白酶基因在胁迫后均出现不同程度的上调,上调倍数介于1.5-2.3倍不等。说明桔小实蝇会提高体内的能量代谢来抵御逆境的胁迫。2.桔小实蝇胰蛋白酶活性及总蛋白酶活性的测定2.1.桔小实蝇胰蛋白酶活性的测定以BApNA为底物,测定桔小实蝇不同发育阶段以及不同组织的胰蛋白酶活性。结果表明,在桔小实蝇幼虫、蛹和成虫3个发育阶段中,幼虫期的胰蛋白酶活性最高；在幼虫的3个组织中(表皮、中肠和脂肪体),中肠胰蛋白酶活性最高。说明桔小实蝇胰蛋白酶主要在幼虫的中肠发挥作用。2.2.高效氯氰菊酯胁迫下桔小实蝇胰蛋白酶活性测定以含0.33μg/g高效氯氰菊酯的饲料饲喂桔小实蝇幼虫5d后,分别以BApNA和TAME为底物检测幼虫中肠胰蛋白酶的酯水解活性和酰胺水解活性。结果显示,在高效氯氰菊酯的胁迫下,幼虫胰蛋白酶的酯水解活性和酰胺水解活性都显著升高。说明桔小实蝇受到药剂胁迫后体内消化酶活性出现了增强。2.3.蛋白抑制剂处理后桔小实蝇胰蛋白酶及总蛋白酶活性测定以含100μg/mL胰蛋白酶专性抑制剂TLCK和SBTI的饲料饲喂刚羽化的成虫5d,测定成虫中肠的胰蛋白酶及总蛋白酶活性。结果显示,抑制剂处理后成虫中肠的胰蛋白酶酶活被显著抑制,且抑制率达到70%。但是,总蛋白酶活性却保持恒定。说明桔小实蝇在受到胰蛋白酶抑制剂胁迫时,通过其它蛋白酶活性的提高来弥补胰蛋白酶活性的损失,从而使总的蛋白酶活性维持在一个相对恒定水平。
[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.
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S433

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