桑螟孵化酶基因特性分析与基于昆虫HE结构的进化研究

发布时间：2018-05-27 11:27

本文选题：桑螟 + 孵化酶基因　；参考：《江苏科技大学》2016年硕士论文

【摘要】：桑螟属鳞翅目螟蛾科,是桑树的主要害虫之一。本文以桑螟(Diaphania pyloalis)为研究对象,通过RACE技术克隆获得桑螟孵化酶基因(Diaphania pyloalis hatching enzyme,DpyHE)全长cDNA序列。DpyHEcDNA序列全长983bp,其中包含21bp的5’UTR、44bp的3’UTR和一段918bp的完整开放阅读框,编码305个氨基酸,其中N端有16个氨基酸为信号肽序列,C端成熟酶区由222个氨基酸组成。同源性分析显示DpyHE与鳞翅目其他昆虫孵化酶蛋白功能区序列相似性分别为70.9%至76%,与柞蚕同源性最高。DpyHE氨基酸序列Pro90至Ser240之间具有一个锌依赖性金属蛋白酶结构域。用同源建模的方法以虾红素蛋白酶Astacin为模板预测DpyHE蛋白的三维结构,结果显示DpyHE与Astacin三维结构相似,表明桑螟孵化酶具有与虾红素蛋白酶相似的蛋白酶功能区和活性中心。另外,我们将DpyHE的成熟酶序列、去信号肽序列、ORF序列对应的基因序列克隆到原核表达系统中进行蛋白表达,Western Blot验证其获得表达,这为进一步进行桑螟孵化酶活性分析提供基础。最近基于鱼类孵化酶基因及其内含子缺失角度探讨了进化分析,获得了有意义的结果。本课题组已发现小菜蛾孵化酶基因仅为单外显子,这与家蚕孵化酶基因的多外显子基因结构存在差异。因此本文调查了代表性昆虫的孵化酶同源序列的基因结构与其保守区对应的内含子,分析昆虫纲孵化酶基因内含子缺失的状态及相互间的亲缘关系。我们选择基因组数据已发表并具有完整孵化酶成熟酶同源序列的6种鳞翅目昆虫和2种双翅目昆虫进行调查,结果显示5种鳞翅目昆虫孵化酶成熟酶同源序列为6-外显子-5-内含子结构,2种双翅目昆虫均为3-外显子-2-内含子结构,而鳞翅目小菜蛾为单外显子,这可能与硬骨鱼孵化酶基因HCE类似,存在内含子丢失的现象。为进一步扩大调查样本,我们调查18种基因组已发表的昆虫孵化酶同源序列保守区,发现其各自对应基因组所包含内含子数分为0和1两类别;所调查昆虫物种进化途径中可能存在内含子缺失现象;推测存在内含子缺失的节点与根据分子进化系统分析获得的物种分类分支点相一致。上述初步结果表明昆虫孵化酶基因结构的内含子缺失可作为一个候选基因来研究昆虫进化关系。
[Abstract]:The family Lepidoptera is one of the main pests of mulberry. In this paper, Diaphania pyloalis was used as the research object. The full-length cDNA sequence of Diaphania pyloalis hatching enzyme DpyHE1 was cloned by RACE technique. The full-length cDNA sequence of DpyHEcDNA was 983bp. which contained the 3'UTR of 21bp's 5UTR44bp and a complete open reading frame of 918bp. It encodes 305 amino acids, of which 16 amino acids at the N-terminal are signal peptide sequences and the C-terminal mature enzyme region is composed of 222 amino acids. Homology analysis showed that the similarity between DpyHE and other lepidoptera insect hatching enzyme functional regions was 70.9% to 76%, respectively. There was a zinc dependent metalloproteinase domain between DpyHE and Antheraea pernyi with the highest homology. DpyHE amino acid sequence Pro90 to Ser240. The three-dimensional structure of DpyHE protein was predicted by using Astacin as template. The results showed that the three-dimensional structure of DpyHE was similar to that of Astacin, indicating that the hatching enzyme of mulberry borer had protease functional region and active center similar to that of shrimp protein. In addition, we cloned the mature enzyme sequence of DpyHE and the corresponding gene sequence of designaled peptide sequence into prokaryotic expression system. The protein expression was verified by Western Blot, which provided the basis for further analysis of incubation enzyme activity of Mulberry borer. The evolutionary analysis of fish hatching enzyme gene and its intron deletion has been discussed recently, and some significant results have been obtained. Our group has found that the hatching enzyme gene of Plutella xylostella is only a single exon, which is different from the structure of multiple exons of the hatching enzyme gene of silkworm, Bombyx mori. Therefore, the genetic structure of the homologous sequence of the incubation enzyme and the corresponding intron of the conserved region of the representative insect were investigated, and the status of the deletion of the intron of the incubator gene of the class Insecta and the relationship between them were analyzed. We selected 6 species of Lepidoptera and 2 species of Diptera that had published genomic data and had complete mature enzyme sequence. The results showed that the homologous sequence of hatching enzyme maturation enzymes of five Lepidoptera insects was 6-exon-5- intron structure. Both species of Diptera insects had 3-exon-2-intron structure, while Lepidoptera Plutella xylostella had a single exon. This may be similar to the HCE gene of hatching enzyme in bony fish, and the loss of intron exists. In order to further expand the sample, we investigated the homologous regions of insect hatching enzymes published in 18 genomes, and found that the number of introns contained in their respective genomes could be divided into two categories: 0 and 1; The loss of intron may exist in the evolutionary pathway of insect species, and the node with the deletion of intron is consistent with the taxonomic branch point obtained by molecular phylogenetic analysis. These preliminary results suggest that the deletion of intron in the structure of insect hatching enzyme gene can be used as a candidate gene to study the evolutionary relationship of insects.
【学位授予单位】：江苏科技大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S888.722

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