本文選題：甜菜夜蛾 + 幾丁質(zhì)脫乙酰酶　； 參考：《河北農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：甜菜夜蛾(Spodoptera exigua Hübner),屬鱗翅目夜蛾科,是一種世界性分布的害蟲。其取食范圍非常廣泛,對(duì)多種糧食作物、蔬菜、經(jīng)濟(jì)作物和油料作物等造成嚴(yán)重危害。昆蟲幾丁質(zhì)脫乙酰酶(Chitin deacetylase,CDA)是幾丁質(zhì)的修飾酶之一,可水解幾丁質(zhì)中的乙酰胺基,將幾丁質(zhì)降解成脫乙酰幾丁質(zhì),是昆蟲幾丁質(zhì)代謝中的一種關(guān)鍵酶,在昆蟲的多種生理活動(dòng)中發(fā)揮重要作用。本研究以甜菜夜蛾S.exigua 5齡幼蟲中腸為材料提取總RNA,利用RT-PCR及RACE技術(shù),擴(kuò)增得到甜菜夜蛾幾丁質(zhì)脫乙酰酶1(chitin deacetylase 1)基因的c DNA全長(zhǎng)序列,Gen Bank登錄號(hào)為KJ621414。該基因全長(zhǎng)1659bp,包括一個(gè)1554bp的開放閱讀框,編碼518個(gè)氨基酸,N-端具有23個(gè)氨基酸的信號(hào)肽序列,預(yù)測(cè)蛋白(Se CDA1)分子量為61.5k Da,等電點(diǎn)為4.83;NCBI BLAST分析結(jié)果表明,Se CDA1含有低密度脂蛋白結(jié)合區(qū)(LDLa)、幾丁質(zhì)結(jié)合區(qū)(Ch BD)和脫乙酰基酶催化區(qū)(CDA),屬于GroupⅠ類CDA蛋白。氨基酸序列分析發(fā)現(xiàn),該序列分別含有3個(gè)N-聯(lián)糖基化位點(diǎn)和5個(gè)O-聯(lián)糖基化位點(diǎn)。secda1基因能夠在原核細(xì)胞中表達(dá)61.5k Da目的蛋白,免疫家兔獲得Se CDA1蛋白的特異性抗體。secda1基因在畢赤酵母細(xì)胞中表達(dá)80k Da的目的蛋白。構(gòu)建重組桿狀病毒表達(dá)載體p Fast Bac-secda1,Western blot分析表明secda1基因在昆蟲細(xì)胞BTI-Tn-5B1-4(High Five)中成功表達(dá)80k Da蛋白。脫乙�；富钚詼y(cè)定結(jié)果顯示昆蟲細(xì)胞表達(dá)的Se CDA1蛋白及畢赤酵母表達(dá)的Se CDA1蛋白均具有脫乙酰基酶活性,分別為1.88U/m L及1.35U/m L。利用Se CDA1特異性抗體對(duì)Se CDA1進(jìn)行免疫組織定位顯示,Se CDA1蛋白在卵、幼蟲和蛹期中均有表達(dá)。q PCR結(jié)果顯示secda1基因在卵、幼蟲和蛹期中均有表達(dá),而在卵中表達(dá)量最高,在5齡幼蟲頭、中腸、馬氏管、脂肪體中也有表達(dá),且在體壁中表達(dá)水平最高,推測(cè)Se CDA1可能參與卵的孵化及幼蟲蛻皮過(guò)程。為進(jìn)一步驗(yàn)證Se CDA1蛋白功能,利用RNA干擾(RNAi)技術(shù)將secda1 536bp保守序列克隆至p GEM-T載體,成功構(gòu)建了RNAi重組質(zhì)粒,大量提取純化重組質(zhì)粒,經(jīng)線性化后合成ds RNA-secda1,為下一步功能研究提供了材料。
[Abstract]:Spodoptera exigua H 眉 bneridae, belonging to the family Lepidoptera, is a worldwide pest. Its feeding range is very extensive, which causes serious harm to many kinds of food crops, vegetables, cash crops and oil crops. Chitin deacetylase (Chitin deacetylase CDA) is one of the modification enzymes of chitin, which can hydrolyze acetamide group in chitin and deacetylate into deacetylated chitin, which is a key enzyme in the metabolism of insect chitin. It plays an important role in many physiological activities of insects. In this study, total RNAs were extracted from the midgut of 5th instar larvae of Spodoptera exigua (S.exigua). By using RT-PCR and RACE techniques, the full-length sequence of c DNA 1(chitin deacetylase 1) gene of Spodoptera exigua was amplified and its accession number was KJ621414. The gene is 1659bp in length and contains an open reading frame of 1554bp encoding 518 amino acids with a signal peptide sequence of 23 amino acids. The molecular weight of predicted protein se CDA1 was 61.5kDa.The isoelectric point was 4.83NCBI BLAST. The results showed that se CDA1 contained low density lipoprotein binding region (LDLaN), chitin binding region (CHBDD) and deacetylase catalyzed region (CDAN), which belonged to Group class I CDA protein. Amino acid sequence analysis showed that the sequence contained three N-glycosylation sites and five O-glycosylation sites. Secda1 gene was able to express 61.5 kDa target protein in prokaryotic cells. The specific antibody of se CDA1 protein. Secda1 gene was expressed in Pichia pastoris cells by immunizing rabbits with 80 kDa target protein. Western blot analysis of recombinant baculovirus expression vector p Fast Bac-secda1 showed that the secda1 gene was successfully expressed in insect cell line BTI-Tn-5B1-4(High. The results of deacetylase activity test showed that se CDA1 protein expressed by insect cells and se CDA1 protein expressed by Pichia pastoris had deacetylase activity, which were 1.88U/m L and 1.35U/m L, respectively. Immunohistochemical localization of se CDA1 with se CDA1 specific antibody showed that se CDA1 protein was expressed in eggs, and in larval and pupa stage. Q PCR showed that secda1 gene was expressed in eggs, larvae and pupal stages, but the highest expression level was found in eggs. The expression of se CDA1 was also found in the head, midgut, Markov tube and adipose body of the 5th instar larva, and the highest expression level was found in the body wall. It was suggested that se CDA1 might be involved in the hatching of eggs and the molting process of larvae. In order to further verify the function of se CDA1 protein, the conserved sequence of secda1 536bp was cloned into p GEM-T vector by RNA interference RNAi technique. The recombinant plasmid of RNAi was successfully constructed, and a large number of purified recombinant plasmids were extracted and purified. After linearization, DS RNA-secda1 was synthesized, which provided materials for further functional research.
【學(xué)位授予單位】：河北農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S433.4

【共引文獻(xiàn)】

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1 劉海南;蔡文兵;陳建平;徐文華;劉標(biāo);;射陽(yáng)棉區(qū)甜菜夜蛾的發(fā)生規(guī)律與防治對(duì)策[J];江西農(nóng)業(yè)學(xué)報(bào);2010年01期

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本文編號(hào)：1939318