菜青虫中肠响应CoTI的转录组学分析

发布时间：2017-12-28 04:14

本文关键词：菜青虫中肠响应CoTI的转录组学分析　出处：《西南交通大学》2017年硕士论文　论文类型：学位论文

【摘要】：菜青虫(PierisrapaeL)属鳞翅目粉蝶科昆虫,是十字花科蔬菜的主要虫害之一。菜青虫利用消化道蛋白酶降解食物蛋白质以获得其生长发育必需的氨基酸,并利用细胞色素P450等解毒酶降解宿主植物来源的抗虫次生代谢物。本研究前期研究结果表明,来自菜青虫非宿主植物决明(Cassia obtusifolia)的胰蛋白酶抑制剂(CoTI)对菜青虫有较强的抗性。本文利用分子生物学、转录组测序等技术分析喂食CoTI前后,菜青虫中肠消化酶等的转录水平变化,为研究CoTI的抗虫机制打下基础。本文首先将CoTI基因克隆到原核表达载体pET28a中,成功构建了重组质粒pET28a/CoTI,经PCR、双酶切鉴定及测序分析,表明该重组质粒含有目的基因片段,且构建正确,表达纯化蛋白并检测浓度为920μg/ml。随后对菜青虫喂食含有CoTI的白菜叶,以喂食正常白菜叶的菜青虫为对照,分别提取中肠总RNA,利用Illumina 4000测序平台进行转录组测序。测序共获得了菜青虫转录组数据17.16Gb,分别获得28,918,396和28,678,331条高质量的reads。通过Trinity软件组装共获得51,544个Unigenes。将这些Unigenes序列与公共数据库Nr、Swiss-Prot、GO、KOG、COG和KEGG等进行比对注释,,共有22,233条Unigenes获得功能注释。通过比对Nr数据库发现了相关的消化酶与解毒酶家族,105个丝氨酸蛋白酶(79个胰蛋白酶和26个糜蛋白酶,PrSPs),74个脂肪酶(PrLPs),17个淀粉酶(PrALs),113个细胞色素P450(PrCYP450s),46个羧酸酯酶(PrCXs),54个UDP-糖基转移酶(PrUGTs)和41个谷胱甘肽S-转移酶(PrGSTs)。比较对照组与实验组菜青虫中肠的转录组数据,发现有3,066个差异表达基因,并对它们进行GO分析,COG分析与KEGG通路分析等。通过分析KEGG通路发现,参与生长和发育信号通路的几个关键基因wnt、Hippo和Notch在不同的处理条件下表达有所差异,这表明CoTI可能抑制了菜青虫获得正常的生长和发育的能力。对菜青虫进行qRT-PCR内参基因选择。先从转录组数据库中筛选8个比较稳定的 Unigene,包括 Sec61、PSMD9、eIF2g、UPF3、LRE、NADH、hnRNP、THOC2作为候选内参基因,然后选择RPL32、EF1、GADPH等3个常用的内参基因一起作为候选内参基因,分析它们在菜青虫不同组织,不同发育阶段与不同胁迫条件下的相对表达水平。然后利用GeNorm与NormFinder软件分析,发现UPF3的表达水平最为稳定,因此选择UPF3作为后续定量分析的内参基因。
[Abstract]:Pieris rapae (PierisrapaeL) belongs to Lepidoptera Pieridae insects, is one of the main pests of cruciferous vegetables. The use of food protein degradation Pieris rapae and digestive protease to obtain the growth and development of essential amino acids, and the use of insect resistant secondary metabolites such as cytochrome P450 enzyme degradation of host plant sources. The preliminary study results show that the non host plants from Pieris rapae (Cassia obtusifolia) Cassia trypsin inhibitor (CoTI) has strong resistance against Pieris rapae. This paper analysis by molecular biology, transcriptome sequencing technologies such as feeding before and after CoTI, the changes of transcription level of caterpillar midgut digestive enzymes and lay the foundation for the study of the mechanism of CoTI resistance. Firstly, the CoTI gene was cloned into prokaryotic expression vector pET28a, the recombinant plasmid pET28a/CoTI by PCR, double enzyme digestion and DNA sequencing analysis showed that the recombinant plasmid containing the target gene fragment, and constructed correctly, protein expression and purification and detection of the concentration of 920 g /ml. Then the feed containing CoTI rapae cabbage leaves, cabbage leaves to feed normal caterpillar as control, were extracted from the midgut total RNA of transcriptome sequencing using Illumina 4000 sequencing platform. Sequencing transcriptome data were obtained Pieris 17.16Gb, obtained 28918396 and 28678331 high quality reads respectively. A total of 51544 Unigenes were assembled through the Trinity software assembly. These Unigenes sequences are annotated with the common database Nr, Swiss-Prot, GO, KOG, COG, and KEGG, and there are 22233 Unigenes to get the functional annotations. By comparing the Nr database found digestive enzymes and detoxification enzyme family related, 105 serine proteases (79 trypsin and chymotrypsin 26, PrSPs), 74 (PrLPs), 17 lipase amylase (PrALs), 113 cytochrome P450 (PrCYP450s), 46 carboxylic acid ester enzyme (PrCXs) 54, UDP- glycosyltransferase (PrUGTs) and 41 glutathione S- transferase (PrGSTs). The transcriptome data comparing the experimental group and the control group of Pieris rapae midgut, found 3066 differentially expressed genes, and their GO analysis, COG analysis and KEGG pathway analysis. Through the analysis of the KEGG pathway that participates in several key genes Wnt, growth and development of the Hippo signaling pathway and Notch expression varied in different treatment conditions, suggesting that CoTI may inhibit the ability of Pieris rapae obtain normal growth and development. The qRT-PCR reference gene selection to pierisrapae. The first screening of 8 stable Unigene from the transcriptome database, including Sec61, PSMD9, eIF2g, UPF3, LRE, NADH, hnRNP, THOC2 as candidate reference genes, RPL32, EF1, GADPH and other 3 commonly used reference genes as candidate reference genes, analysis them in different tissues of Pieris rapae, different the development stage and different stress conditions, the relative expression level. Then the analysis using GeNorm and NormFinder software, found that the most stable reference gene expression level of UPF3, so UPF3 is selected as the subsequent quantitative analysis.
【学位授予单位】：西南交通大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S433.4

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