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摘要海藻糖合成酶（Trehalose-6-phosphate synthase，TPS）为海藻糖合成过程中的一个关键酶，其广泛存在于细菌、真菌和植物中，但不存在于哺乳动物中。为了更加深入研究昆虫TPS基因功能，评估其作为害虫控制靶标的潜力，本研究以赤拟谷盗 （Tribolium castaneum）作为研究对象，克隆获得编码828个氨基酸的TPS基因。合成TPS的dsRNA并注射到赤拟谷盗体内，48h和72h的检测结果表明TPS和TPP表达均被抑制，72h导致的死亡率和畸形率分别高达38%和31%，并出现蜕皮障碍。采用荧光定量PCR技术检测RNAi后几丁质合成通路中相关基因的表达，结果显示TcTreh1-3、TcTreh1-4、TcTreh2、TcHex1、TcHex2、TcGPI、TcGFAT、TcPFK和TcCHS2的表达在48h和72h都受到抑制。而当TPS表达被抑制后，TcTreh1-1的表达在48h下降，但在72h表达却上升。这些结果表明一旦TPS基因的表达被抑制，能够通过几丁质合成通路中的相关基因表达下降来控制昆虫的发育，并导致畸形和死亡，具有作为害虫控制靶标的潜力。46664
Abstract TPS (Trehalose-6-phosphate synthase) is a key enzyme for trehalose synthesis process, which are found in bacteria, fungi and plants, but it does not exist in mammals. In order to study the function of insect’s TPS gene, and to evaluate its potential as a pest control target, the Tribolium castaneum was a research object in this study. The results showed that the TPS gene encode 828 amino acids. The dsRNA of TPS and TPP domain were synthesised and then injected into T. castaneum. The results showed that the expression of TPS and TPP injected after 48 hours and 72 hours was inhibited, the mortality and malformation rate of 72h had up to 38% and 31% respectively, and exuvial obstacle happen to T. castaneum. Using Real-Time fluorescent quantitative PCR Technique to detect the expression of gene in chitin synthesis pathway after RNAi, the result showed that the expression of TcTreh1-3, TcTreh1-4, TcTreh2, TcHex1, TcHex2, TcGPI, TcGFAT, TcPFK, and TcCHS2 were inhibited after 48h and 72h. But when the expression of TPS was inhibited, the expression level of TcTreh1-1 after 48h was lower, but it increased after 72h. These results showed that once the TPS gene expression was inhibited, it could decrease the expression of related gene of chitin synthesis pathway and thus control insect’s development, and lead to deformity and death as it has the potential to be a pest control target.
毕业论文关键词：海藻糖；海藻糖合成酶；基因克隆；RNAi；实时荧光定量PCR
Key words: Trehalose; trehalose-6-phosphate synthase; gene cloning; RNAi; Real-time fluorogenic quantitative PCR
目 录
0. 引言 …5
1. 材料与方法5
1.1 试验材料…5
1.1.1 供试昆虫5
1.1.2 主要试剂5
1.2 试验方法…5
1.2.1 赤拟谷盗发育表达模式和组织材料选取…5
1.2.2 赤拟谷盗RNA 的抽提与一链cDNA合成 …6
1.2.3 赤拟谷盗海藻糖合成酶基因全长及TPS/TPP保守区域部分序列的克隆 6
1.2.4 赤拟谷盗TPS和TPP保守结构域dsRNA 的合成…6
1.2.5 dsTPS、dsTPP和dsGFP RNA注射到赤拟谷盗…7
1.2.6 赤拟谷盗RNAi 后表型的观察和取样 7
1.2.7 定量PCR检测RNAi后TPS及几丁质合成通路相关基因的表达及数据分析7
2 结果与分析7
2.1 TcTPS基因的序列全长 7
2.2 TPS和TPP RNAi后TPS和TPP表达…8
2.3 TPS和TPP表达抑制后畸形和死亡情况 …8
2.4 TPS和TPP RNAi后海藻糖酶表达9
2.5 TPS和TPP RNAi后几丁质合成通路中其它基因的表达…10
3 讨论…11