發(fā)布時(shí)間：2018-05-27 11:27

  本文選題：桑螟 + 孵化酶基因　； 參考：《江蘇科技大學(xué)》2016年碩士論文

【摘要】：桑螟屬鱗翅目螟蛾科,是桑樹的主要害蟲之一。本文以桑螟(Diaphania pyloalis)為研究對象,通過RACE技術(shù)克隆獲得桑螟孵化酶基因(Diaphania pyloalis hatching enzyme,DpyHE)全長cDNA序列。DpyHEcDNA序列全長983bp,其中包含21bp的5’UTR、44bp的3’UTR和一段918bp的完整開放閱讀框,編碼305個(gè)氨基酸,其中N端有16個(gè)氨基酸為信號(hào)肽序列,C端成熟酶區(qū)由222個(gè)氨基酸組成。同源性分析顯示DpyHE與鱗翅目其他昆蟲孵化酶蛋白功能區(qū)序列相似性分別為70.9%至76%,與柞蠶同源性最高。DpyHE氨基酸序列Pro90至Ser240之間具有一個(gè)鋅依賴性金屬蛋白酶結(jié)構(gòu)域。用同源建模的方法以蝦紅素蛋白酶Astacin為模板預(yù)測DpyHE蛋白的三維結(jié)構(gòu),結(jié)果顯示DpyHE與Astacin三維結(jié)構(gòu)相似,表明桑螟孵化酶具有與蝦紅素蛋白酶相似的蛋白酶功能區(qū)和活性中心。另外,我們將DpyHE的成熟酶序列、去信號(hào)肽序列、ORF序列對應(yīng)的基因序列克隆到原核表達(dá)系統(tǒng)中進(jìn)行蛋白表達(dá),Western Blot驗(yàn)證其獲得表達(dá),這為進(jìn)一步進(jìn)行桑螟孵化酶活性分析提供基礎(chǔ)。最近基于魚類孵化酶基因及其內(nèi)含子缺失角度探討了進(jìn)化分析,獲得了有意義的結(jié)果。本課題組已發(fā)現(xiàn)小菜蛾孵化酶基因僅為單外顯子,這與家蠶孵化酶基因的多外顯子基因結(jié)構(gòu)存在差異。因此本文調(diào)查了代表性昆蟲的孵化酶同源序列的基因結(jié)構(gòu)與其保守區(qū)對應(yīng)的內(nèi)含子,分析昆蟲綱孵化酶基因內(nèi)含子缺失的狀態(tài)及相互間的親緣關(guān)系。我們選擇基因組數(shù)據(jù)已發(fā)表并具有完整孵化酶成熟酶同源序列的6種鱗翅目昆蟲和2種雙翅目昆蟲進(jìn)行調(diào)查,結(jié)果顯示5種鱗翅目昆蟲孵化酶成熟酶同源序列為6-外顯子-5-內(nèi)含子結(jié)構(gòu),2種雙翅目昆蟲均為3-外顯子-2-內(nèi)含子結(jié)構(gòu),而鱗翅目小菜蛾為單外顯子,這可能與硬骨魚孵化酶基因HCE類似,存在內(nèi)含子丟失的現(xiàn)象。為進(jìn)一步擴(kuò)大調(diào)查樣本,我們調(diào)查18種基因組已發(fā)表的昆蟲孵化酶同源序列保守區(qū),發(fā)現(xiàn)其各自對應(yīng)基因組所包含內(nèi)含子數(shù)分為0和1兩類別;所調(diào)查昆蟲物種進(jìn)化途徑中可能存在內(nèi)含子缺失現(xiàn)象;推測存在內(nèi)含子缺失的節(jié)點(diǎn)與根據(jù)分子進(jìn)化系統(tǒng)分析獲得的物種分類分支點(diǎn)相一致。上述初步結(jié)果表明昆蟲孵化酶基因結(jié)構(gòu)的內(nèi)含子缺失可作為一個(gè)候選基因來研究昆蟲進(jìn)化關(guān)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.
【學(xué)位授予單位】：江蘇科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：S888.722

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