飞蝗内表皮蛋白基因LmAbd-5的表达与功能分析

发布时间：2018-06-05 23:44

  本文选题：飞蝗 + 表皮蛋白　； 参考：《中国农业科学》2017年10期

【摘要】：【目的】基于飞蝗(Locusta migratoria)转录组数据库获得内表皮蛋白(endocuticle structural glycoprotein)基因Lm Abd-5的c DNA序列,分析该基因的序列特征和m RNA表达特性,采用RNAi方法分析其生物学功能,探讨其在飞蝗表皮形成中的作用,为害虫防治提供新的分子靶标。【方法】采用生物信息学方法搜索飞蝗转录组数据库,获得Lm Abd-5 c DNA全长序列并克隆验证;采用Signal P在线软件分析蛋白的信号肽,利用SMART网站预测其功能域,使用MEGA 7.0软件中neighbor-joining(NJ)方法,与其他昆虫同源序列进行聚类分析;采用reverse-transcription quantitative PCR(RT-q PCR)方法检测Lm Abd-5在飞蝗5龄第2天若虫不同组织部位和5龄不同发育时期体壁组织中的表达情况,揭示其组织和时期表达模式;采用RNA干扰(RNAi)技术及透射电镜技术(TEM)观察沉默Lm Abd-5后对飞蝗生长发育和表皮结构的影响。【结果】通过搜索得到Lm Abd-5 c DNA全长序列并进行了克隆和测序验证,获得520 bp全长c DNA序列,其中ORF为303 bp;基因结构分析显示该基因含有3个外显子;功能域分析发现其含有1个信号肽和1个几丁质结合域(chitin binding domain 4,Cht BD4),与沙漠蝗、白蚁、赤拟谷盗等的Abd-5结构类似;BLAST分析结果表明Abd-5在昆虫中高度保守,飞蝗与沙漠蝗Abd-5序列一致度高达81%;对其保守基序进行Web Logo分析发现Lm Abd-5属于表皮蛋白CPR家族中的RR-1亚类;聚类分析结果显示,Lm Abd-5与沙漠蝗和白蚁的Abd-5显示出较近的亲缘关系;RT-q PCR结果显示Lm Abd-5在前肠、后肠、气管和体壁等由外胚层形成的组织中高表达,而在胃盲囊、中肠、马氏管、脂肪体和翅芽中低表达或不表达;不同时期表达分析发现,Lm Abd-5在4龄若虫蜕皮后0—72 h(5龄早期)具有高表达,其中蜕皮后72 h时达到最大表达量,随后表达量急剧降低(96—168 h),其表达时期与内表皮形成时间一致;采用RNAi技术分析该基因的生物学功能,对5龄第2天若虫分别注射等量的ds Lm Abd-5和ds GFP(对照),发现注射ds Lm Abd-5的5龄若虫和对照组相同,均可正常蜕皮,发育至成虫第2天目的基因表达量显著降低,但未出现肉眼可见的异常表型。分别取成虫第2天处理组和对照组成虫表皮进行超微结构观察,发现与对照组相比,处理组成虫内表皮片层结构较为疏松,导致内表皮片层变厚,最终表现为整个内表皮变厚。【结论】根据转录组数据库分析获得1个CPR家族表皮蛋白Lm Abd-5,该蛋白含有1个信号肽和1个几丁质结合域Cht BD4,属于RR-1亚类;Lm Abd-5主要在外胚层起源的组织中高表达,沉默Lm Abd-5后飞蝗没有肉眼可见的表型,但超微结构分析发现其参与飞蝗内表皮片层结构的形成。
[Abstract]:[objective] to obtain the c DNA sequence of endocuticle structural glycoprotein gene LM Abd-5 based on Locusta migratoria transcriptional database, analyze the sequence characteristics and m RNA expression characteristics of the gene, and analyze its biological function by RNAi method. To explore its role in epidermis formation of migratory locust, and to provide a new molecular target for pest control. [methods] A bioinformatics method was used to search the transcriptional database of migratory locusts, and the full-length sequence of LM Abd-5 c DNA was obtained and cloned. The signal peptide of protein was analyzed by Signal P online software, the functional domain was predicted by SMART website, and the homologous sequence of other insects was analyzed by using the method of neighbor-joiningnj in MEGA 7.0 software. The expression of Lm Abd-5 in different tissues of nymphs at the 2nd day of 5th instar and in different development stages of fifth instar were detected by reverse-transcription quantitative PCR(RT-q Abd-5 method, and the expression patterns of Lm Abd-5 in different tissues and periods were revealed. The effects of silencing Lm Abd-5 on the growth and epidermal structure of locust migratoria were observed by RNA interference RNAi technique and transmission electron microscopy (TEM). [results] the full-length sequence of Lm Abd-5 c DNA was obtained by searching and was cloned and sequenced. The full-length c DNA sequence of 520bp was obtained, in which ORF was 303bp.The gene structure analysis showed that the gene contained three exons, and the functional domain analysis showed that the gene contained a signal peptide and a chitin binding domain 4 Cht BD4N, which was associated with desert locust and termites. The results of Abd-5 analysis showed that Abd-5 was highly conserved in insects, the consistency of Abd-5 sequence between migratory locust and desert locust was as high as 81.Lm Abd-5 was found to belong to RR-1 subclass of epidermal protein CPR family by Web Logo analysis of its conserved motif. Cluster analysis showed that Lm Abd-5 was closely related to Abd-5 of grasshopper and termites. RT-q PCR showed that LM Abd-5 was highly expressed in tissues formed by ectoderm, such as foregut, hindgut, trachea and body wall, while in gastric blind sac, midgut and Markov tube. The low or no expression of Lm Abd-5 was found in adipose body and wing bud at different stages, and the expression of Lm Abd-5 was found to be high in the early 5th instar after molting at 0-72 h after molting, and the maximum expression was reached at 72 h after molting. Subsequently, the expression level decreased sharply from 96 to 168 h, and the expression period was the same as that of the inner epidermis. The biological function of the gene was analyzed by RNAi technique. On the second day of the 5th instar, the nymphs were injected with the same amount of DS Lm Abd-5 and DS GFP respectively. It was found that the 5th instar nymph injected with DS LM Abd-5 could molt normally, and the gene expression of the second day of adult growth decreased significantly. But there was no abnormal phenotype visible to the naked eye. The epidermis of adult and control group were observed respectively on the second day. The results showed that the structure of the inner epidermal lamellae was looser than that of the control group, which resulted in the thickening of the inner epidermal lamellae. [conclusion] according to transcriptional database analysis, a CPR family epidermal protein LmAbd-5 was obtained, which contains a signal peptide and a chitin binding domain Cht BD4, which belongs to the RR-1 subclass Lm Abd-5. Highly expressed in the tissues of ectodermal origin, There was no visible phenotype of migratory locust after silencing Lm Abd-5, but ultrastructural analysis showed that it was involved in the formation of epidermal lamellae in migratory locust.
【作者单位】： 山西大学应用生物学研究所;山西大学生命科学学院;
【基金】：国家自然科学基金(31640075,31672364) 山西省青年科学基金(201601D021102) 山西省高校科技创新基金(2016113)
【分类号】：S433.2
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本文编号：1983946