基于数字基因表达谱的甜菜夜蛾表皮蛋白基因CPR97和CPR63的研究

发布时间：2018-03-23 14:24

本文选题：甜菜夜蛾　切入点：突变体　出处：《华中农业大学》2015年硕士论文

【摘要】：昆虫表皮的主要成分是几丁质和表皮蛋白。一直以来,表皮蛋白种类和数量的变化被认为是影响昆虫表皮结构和功能的主要因素。甜菜夜蛾是一种世界性的杂食性害虫,在我国分布范围广,且寄主植物种类多样。甜菜夜蛾蛹黑突变体由本实验室正常体甜菜夜蛾于室内正常饲养条件下突变而来,蛹黑性状能稳定遗传且未发生性状分离。本研究以蛹黑突变和正常品系甜菜夜蛾为研究对象,第二代高通量测序为平台,对2个品系甜菜夜蛾的5龄幼虫、预蛹、0小时蛹的表皮组织进行数字基因表达谱升级版测序。主要研究结果如下:1通过高通量测序由正常体甜菜夜蛾5龄、预蛹、蛹的表皮组织样本中分别得到了350734013bp,346909171bp,345310840bp的核苷酸序列,reads数目分别为7157837,7079779,7079779。在蛹黑突变体甜菜夜蛾5龄、预蛹、蛹的表皮组织样本中分别得到了7397772,7188944,7305754个reads,核苷酸序列长度分别为362490828bp,352258256bp,357981946bp。以甜菜夜蛾转录组数据为参考基因进行注释,6个表达谱测序数据中的reads的比对率基本维持在75%,其中蛹黑突变品系的5龄幼虫的reads比对率偏低,为65.35%。2在差异基因筛选中,5龄幼虫蛹黑突变体较正常体甜菜夜蛾有7257个基因表达上调,664个基因表达下调。预蛹中460个基因表达上调,439个基因表达下调;0小时蛹中162个基因表达上调;293个基因表达下调。3在甜菜夜蛾3个时期的表皮组织中,均筛选到了多个表皮蛋白基因的表达有显著性差异。其中,在预蛹时期有9个表皮蛋白基因显著性上调,上调倍数最大为23.6,仅有一个表皮蛋白基因表达量下调,下调倍数为21.2;在蛹期5个表皮蛋白基因表达显著性上调,上调倍数最大为210.3倍。4通过荧光定量PCR分析目的基因CPR97在2个品系甜菜夜蛾预蛹晚期、化蛹初期中的表达量。在预蛹晚期和化蛹初期,蛹黑突变体中CPR97基因的表达量均高于正常体甜菜夜蛾。CPR63在化蛹初期蛹黑突变体中表达量明显较高。
[Abstract]:The main components of insect epidermis are chitin and epidermal protein. The variety and quantity of epidermal protein are considered to be the main factors that affect the structure and function of insect epidermis, and Spodoptera exigua is a worldwide omnivorous pest. The black mutant of Spodoptera exigua chrysalis was derived from a normal beet moth in our laboratory under normal rearing conditions. The pupa black traits can be inherited stably and no trait segregation occurred. In this study, the fifth instar larvae of two sugarbeet Spodoptera exigua were studied by using pupa black mutation and normal sugarbeet Spodoptera exigua on the platform of the second generation high-throughput sequencing. The epidermal tissues of prepupae were sequenced with the digital gene expression profile. The main results were as follows: 1. The results were as follows: 1: 1 by high-throughput sequencing from the 5th instar of normal beet exiguous moth, prepupae, prepupae, The number of nucleotide sequences of 35073404013bpnb, 346909171bptio 345310840bp was 7157837, 70797797079779 779 779 779 9. In epidermal tissue samples of pupae, 7397772n 7188944and 7305754 readswere obtained respectively, the nucleotide sequence length was 362490828bpn352258256bpn357981946bp. the reads alignment rate of the six expression profiling data was maintained at 755.The nucleotide sequence of the pupae was annotated using the transcriptome data of Spodoptera exigua as the reference gene. The reads ratio of the 5th instar larva of the medium pupa black mutant strain was lower than that of the middle pupa black mutant strain. 65.35.2 in differential gene screening, 7257 genes were up-regulated in black mutants of 5th instar larvae compared with normal beet Spodoptera exigua, while 460 genes were up-regulated in prepupae, 439 genes were down-regulated, 162 genes were down-regulated in 0-hour pupae. The expression of 293 genes was down-regulated in the epidermis of Spodoptera exigua in three stages. Among them, 9 epidermal protein genes were significantly upregulated in prepupae stage, the maximum up-regulation was 23.6, and only one epidermal protein gene was down-regulated. The expression of 5 epidermal protein genes was significantly up-regulated in pupal stage, and the maximum up-regulation was 210.3 times. The target gene CPR97 was analyzed by fluorescence quantitative PCR in the late stage of prepupae of two strains of Spodoptera exigua. The expression of CPR97 gene in the pupae black mutant was significantly higher than that in the normal beet Spodoptera exigua in the early pupae stage and in the early pupae stage.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S433.4

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