【摘要】：翅是昆蟲(chóng)重要的附屬結(jié)構(gòu),在昆蟲(chóng)覓食、求偶、躲避敵害、遷飛等方面發(fā)揮著重要的作用。飛蝗是一種不完全變態(tài)發(fā)育的世界性作物害蟲(chóng),因其具有功能強(qiáng)大的前后翅能夠遠(yuǎn)距離、大規(guī)模遷移,導(dǎo)致大面積蝗災(zāi)的發(fā)生,致災(zāi)嚴(yán)重。因此,對(duì)飛蝗翅的生長(zhǎng)、發(fā)育及變態(tài)的研究有利于掌握飛蝗生長(zhǎng)發(fā)育規(guī)律,從而為蝗災(zāi)防治提供基礎(chǔ)資料。本文以飛蝗為研究對(duì)象,利用轉(zhuǎn)錄組數(shù)據(jù)庫(kù)鑒定其所含表皮蛋白基因,并以翅表皮蛋白基因LmACP7為研究靶標(biāo),利用RNAi結(jié)合透射電鏡技術(shù)探討其在飛蝗翅發(fā)育過(guò)程中的生物學(xué)功能,得到如下結(jié)果:1)利用課題組飛蝗轉(zhuǎn)錄組數(shù)據(jù)庫(kù)鑒定出81個(gè)表皮蛋白基因,分為5個(gè)家族,其中含51個(gè)CPR家族表皮蛋白基因、2個(gè)Tweedle基因、9個(gè)含CPF/CPFL家族基因、9個(gè)CPAPs家族的表皮蛋白基因以及10個(gè)不屬于上述任何家族的表皮蛋白基因。在CPR家族表皮蛋白基因中發(fā)現(xiàn)3個(gè)含有幾丁質(zhì)結(jié)合域的翅表皮蛋白基因,其中LmACP7在四齡蛻皮前高表達(dá)。2)以翅表皮蛋白基因LmACP7為研究靶標(biāo),對(duì)該基因進(jìn)行分析,發(fā)現(xiàn)其具有3個(gè)外顯子和2個(gè)內(nèi)含子,編碼的蛋白含有一個(gè)RR-2基序,屬于RR-2亞家族成員;同源性分析顯示該蛋白與歐洲玉米螟cuticle protein 7具有較高的同源性,可能在飛蝗翅發(fā)育過(guò)程中具有重要作用。五齡飛蝗不同組織RT-qPCR結(jié)果分析顯示LmACP7在飛蝗翅組織中特異性高表達(dá);不同發(fā)育時(shí)期RT-qPCR表達(dá)分析顯示LmACP7在不同齡期若蟲(chóng)的翅芽中均有表達(dá),而且均在齡期末期表達(dá)量最高,前期及中期表達(dá)較低,在成蟲(chóng)1、2天的表達(dá)也較低,具有時(shí)期特異性,是蛻皮前表皮蛋白基因,可能參與翅外表皮的形成。免疫組化結(jié)果發(fā)現(xiàn)LmACP7在若蟲(chóng)前期主要定位在細(xì)胞層,在蛻皮前轉(zhuǎn)運(yùn)到新形成的表皮層中(外表皮);利用膠體金免疫技術(shù)精細(xì)定位發(fā)現(xiàn),LmACP7主要存在于成蟲(chóng)翅外表皮層中。同時(shí),體外結(jié)合實(shí)驗(yàn)證實(shí)LmACP7能夠與幾丁質(zhì)特異性結(jié)合。3)利用飛蝗對(duì)RNAi的敏感性,在四齡若蟲(chóng)期對(duì)該基因進(jìn)行沉默后發(fā)現(xiàn),飛蝗蛻皮后翅芽出現(xiàn)發(fā)育異常,HE染色發(fā)現(xiàn)細(xì)胞排列發(fā)生紊亂;在五齡若蟲(chóng)期對(duì)該基因進(jìn)行沉默后發(fā)現(xiàn),飛蝗蛻皮后成蟲(chóng)翅發(fā)育異常、皺縮畸形、蛻皮困難,最終死亡。對(duì)其翅進(jìn)行超微結(jié)構(gòu)觀察發(fā)現(xiàn),與對(duì)照組相比干擾掉該基因后翅細(xì)胞層,微絨毛以及細(xì)胞連接等被破壞,影響翅外表皮的形成,由于外表皮形成受阻導(dǎo)致內(nèi)表皮結(jié)構(gòu)也受到影響。同時(shí),研究發(fā)現(xiàn)缺失LmACP7后引起翅表皮細(xì)胞排列發(fā)生紊亂、細(xì)胞連接被破壞,從而引發(fā)線粒體途徑介導(dǎo)的細(xì)胞凋亡。飛蝗是典型的漸變態(tài)昆蟲(chóng),具有功能強(qiáng)大的前后翅,極具遷飛能力,其翅的結(jié)構(gòu)特點(diǎn)與發(fā)育過(guò)程與家蠶、果蠅和赤擬谷盜等全變態(tài)模式昆蟲(chóng)不同。本文探討了翅特異表皮蛋白基因LmACP7在飛蝗翅生長(zhǎng)發(fā)育中的表達(dá)規(guī)律,并闡明了其在飛蝗翅形成中的作用,豐富了漸變態(tài)昆蟲(chóng)的研究?jī)?nèi)容,為以翅表皮蛋白基因?qū)w飛害蟲(chóng)(如粘蟲(chóng)、玉米螟等)防治的靶標(biāo)提供重要的理論依據(jù)。
[Abstract]:Wing is an important accessory structure of insects, which plays an important role in foraging, courting, avoiding enemies and migrating. Locusta migratoria is an incomplete metamorphosis of the world crop pests, because of its powerful forewings and hind wings can be long-distance, large-scale migration, leading to large-scale locust disasters, causing serious disasters. The study of wing growth, development and metamorphosis is helpful to grasp the law of locust growth and development, so as to provide basic data for locust control. In this paper, locusts were selected as the research object, and the epidermal protein gene LmACP7 was identified by transcriptome database, and the target gene LmACP7 was studied by using RNA I and transmission electron microscopy. The results are as follows: 1) 81 epidermal protein genes were identified and classified into 5 families, including 51 CPR family epidermal protein genes, 2 Tweedle genes, 9 CPF / CPFL family genes, 9 CPAPs family epidermal protein genes and 10 non-genus genes. Three wing epidermis protein genes containing chitin binding domain were found in the CPR family epidermis protein genes, of which LmACP7 was highly expressed before molting at the fourth instar. Homology analysis showed that LmACP7 had high homology with cuticle protein 7 of the European corn borer and might play an important role in the development of locust wings. RT-q-PCR analysis showed that LmACP7 was expressed in the wing buds of different instar nymphs, and the highest expression was found at the end of instar. The expression was low at the early and middle stages of instar. The expression of LmACP7 was also low at the first and second day of adult. It was a period-specific gene of preecdysis protein, which might be involved in the formation of the wing outer epidermis. It was found that LmACP7 was mainly localized in the cell layer at the early stage of nymph and transported to the newly formed epidermis (outer epidermis) before molting, and that LmACP7 was mainly located in the outer epidermis of adult wing by colloidal gold immunoassay. After the gene was silenced in the 4th instar nymph stage, it was found that the wing buds of locusts developed abnormally and the cells arranged disorderly by HE staining. After the gene was silenced in the 5th instar nymph stage, the wings of locusts developed abnormally, shrunk abnormally, molted difficultly and died eventually. It was found that the formation of the outer epidermis was affected by the disruption of the outer epidermis and the formation of the inner epidermis was also affected by the disruption of the outer epidermis. At the same time, it was found that the deletion of LmACP7 caused the disorder of the arrangement of the epidermal cells and the destruction of the cell junction, which led to the formation of the outer epidermis. Mitochondrial pathway mediates cell apoptosis. Locusta migratoria is a typical metamorphic insect with powerful forewings and hind wings. The structural characteristics and developmental process of its wings are different from those of silkworms, Drosophila melanogaster and Rhododendron oryzae. The expression pattern and its role in the wing formation of migratory locusts were elucidated, which enriched the research contents of metamorphic insects and provided important theoretical basis for the control of migratory insects (such as armyworm, corn borer, etc.) with wing epidermal protein gene.
【學(xué)位授予單位】：山西大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：S433.2

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本文編號(hào)：2200244