本文選題：飛蝗 + 表皮蛋白　； 參考：《中國農業(yè)科學》2017年10期

【摘要】：【目的】基于飛蝗(Locusta migratoria)轉錄組數(shù)據(jù)庫獲得內表皮蛋白(endocuticle structural glycoprotein)基因Lm Abd-5的c DNA序列,分析該基因的序列特征和m RNA表達特性,采用RNAi方法分析其生物學功能,探討其在飛蝗表皮形成中的作用,為害蟲防治提供新的分子靶標�！痉椒ā坎捎蒙镄畔W方法搜索飛蝗轉錄組數(shù)據(jù)庫,獲得Lm Abd-5 c DNA全長序列并克隆驗證;采用Signal P在線軟件分析蛋白的信號肽,利用SMART網站預測其功能域,使用MEGA 7.0軟件中neighbor-joining(NJ)方法,與其他昆蟲同源序列進行聚類分析;采用reverse-transcription quantitative PCR(RT-q PCR)方法檢測Lm Abd-5在飛蝗5齡第2天若蟲不同組織部位和5齡不同發(fā)育時期體壁組織中的表達情況,揭示其組織和時期表達模式;采用RNA干擾(RNAi)技術及透射電鏡技術(TEM)觀察沉默Lm Abd-5后對飛蝗生長發(fā)育和表皮結構的影響。【結果】通過搜索得到Lm Abd-5 c DNA全長序列并進行了克隆和測序驗證,獲得520 bp全長c DNA序列,其中ORF為303 bp;基因結構分析顯示該基因含有3個外顯子;功能域分析發(fā)現(xiàn)其含有1個信號肽和1個幾丁質結合域(chitin binding domain 4,Cht BD4),與沙漠蝗、白蟻、赤擬谷盜等的Abd-5結構類似;BLAST分析結果表明Abd-5在昆蟲中高度保守,飛蝗與沙漠蝗Abd-5序列一致度高達81%;對其保守基序進行Web Logo分析發(fā)現(xiàn)Lm Abd-5屬于表皮蛋白CPR家族中的RR-1亞類;聚類分析結果顯示,Lm Abd-5與沙漠蝗和白蟻的Abd-5顯示出較近的親緣關系;RT-q PCR結果顯示Lm Abd-5在前腸、后腸、氣管和體壁等由外胚層形成的組織中高表達,而在胃盲囊、中腸、馬氏管、脂肪體和翅芽中低表達或不表達;不同時期表達分析發(fā)現(xiàn),Lm Abd-5在4齡若蟲蛻皮后0—72 h(5齡早期)具有高表達,其中蛻皮后72 h時達到最大表達量,隨后表達量急劇降低(96—168 h),其表達時期與內表皮形成時間一致;采用RNAi技術分析該基因的生物學功能,對5齡第2天若蟲分別注射等量的ds Lm Abd-5和ds GFP(對照),發(fā)現(xiàn)注射ds Lm Abd-5的5齡若蟲和對照組相同,均可正常蛻皮,發(fā)育至成蟲第2天目的基因表達量顯著降低,但未出現(xiàn)肉眼可見的異常表型。分別取成蟲第2天處理組和對照組成蟲表皮進行超微結構觀察,發(fā)現(xiàn)與對照組相比,處理組成蟲內表皮片層結構較為疏松,導致內表皮片層變厚,最終表現(xiàn)為整個內表皮變厚�！窘Y論】根據(jù)轉錄組數(shù)據(jù)庫分析獲得1個CPR家族表皮蛋白Lm Abd-5,該蛋白含有1個信號肽和1個幾丁質結合域Cht BD4,屬于RR-1亞類;Lm Abd-5主要在外胚層起源的組織中高表達,沉默Lm Abd-5后飛蝗沒有肉眼可見的表型,但超微結構分析發(fā)現(xiàn)其參與飛蝗內表皮片層結構的形成。
[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.
【作者單位】： 山西大學應用生物學研究所;山西大學生命科學學院;
【基金】：國家自然科學基金(31640075,31672364) 山西省青年科學基金(201601D021102) 山西省高�？萍紕�(chuàng)新基金(2016113)
【分類號】：S433.2
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本文編號：1983946