飞蝗翅特异表皮蛋白基因LmACP7的功能研究

发布时间：2018-08-24 08:42

【摘要】：翅是昆虫重要的附属结构,在昆虫觅食、求偶、躲避敌害、迁飞等方面发挥着重要的作用。飞蝗是一种不完全变态发育的世界性作物害虫,因其具有功能强大的前后翅能够远距离、大规模迁移,导致大面积蝗灾的发生,致灾严重。因此,对飞蝗翅的生长、发育及变态的研究有利于掌握飞蝗生长发育规律,从而为蝗灾防治提供基础资料。本文以飞蝗为研究对象,利用转录组数据库鉴定其所含表皮蛋白基因,并以翅表皮蛋白基因LmACP7为研究靶标,利用RNAi结合透射电镜技术探讨其在飞蝗翅发育过程中的生物学功能,得到如下结果:1)利用课题组飞蝗转录组数据库鉴定出81个表皮蛋白基因,分为5个家族,其中含51个CPR家族表皮蛋白基因、2个Tweedle基因、9个含CPF/CPFL家族基因、9个CPAPs家族的表皮蛋白基因以及10个不属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发现3个含有几丁质结合域的翅表皮蛋白基因,其中LmACP7在四龄蜕皮前高表达。2)以翅表皮蛋白基因LmACP7为研究靶标,对该基因进行分析,发现其具有3个外显子和2个内含子,编码的蛋白含有一个RR-2基序,属于RR-2亚家族成员;同源性分析显示该蛋白与欧洲玉米螟cuticle protein 7具有较高的同源性,可能在飞蝗翅发育过程中具有重要作用。五龄飞蝗不同组织RT-qPCR结果分析显示LmACP7在飞蝗翅组织中特异性高表达;不同发育时期RT-qPCR表达分析显示LmACP7在不同龄期若虫的翅芽中均有表达,而且均在龄期末期表达量最高,前期及中期表达较低,在成虫1、2天的表达也较低,具有时期特异性,是蜕皮前表皮蛋白基因,可能参与翅外表皮的形成。免疫组化结果发现LmACP7在若虫前期主要定位在细胞层,在蜕皮前转运到新形成的表皮层中(外表皮);利用胶体金免疫技术精细定位发现,LmACP7主要存在于成虫翅外表皮层中。同时,体外结合实验证实LmACP7能够与几丁质特异性结合。3)利用飞蝗对RNAi的敏感性,在四龄若虫期对该基因进行沉默后发现,飞蝗蜕皮后翅芽出现发育异常,HE染色发现细胞排列发生紊乱;在五龄若虫期对该基因进行沉默后发现,飞蝗蜕皮后成虫翅发育异常、皱缩畸形、蜕皮困难,最终死亡。对其翅进行超微结构观察发现,与对照组相比干扰掉该基因后翅细胞层,微绒毛以及细胞连接等被破坏,影响翅外表皮的形成,由于外表皮形成受阻导致内表皮结构也受到影响。同时,研究发现缺失LmACP7后引起翅表皮细胞排列发生紊乱、细胞连接被破坏,从而引发线粒体途径介导的细胞凋亡。飞蝗是典型的渐变态昆虫,具有功能强大的前后翅,极具迁飞能力,其翅的结构特点与发育过程与家蚕、果蝇和赤拟谷盗等全变态模式昆虫不同。本文探讨了翅特异表皮蛋白基因LmACP7在飞蝗翅生长发育中的表达规律,并阐明了其在飞蝗翅形成中的作用,丰富了渐变态昆虫的研究内容,为以翅表皮蛋白基因对迁飞害虫(如粘虫、玉米螟等)防治的靶标提供重要的理论依据。
[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.
【学位授予单位】：山西大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S433.2

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