本文選題：飛蝗 + 表皮蛋白　； 參考：《中國(guó)農(nóng)業(yè)科學(xué)》2017年10期

【摘要】：【目的】基于飛蝗(Locusta migratoria)轉(zhuǎn)錄組數(shù)據(jù)庫(kù)獲得內(nèi)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(dá)特性,采用RNAi方法分析其生物學(xué)功能,探討其在飛蝗表皮形成中的作用,為害蟲(chóng)防治提供新的分子靶標(biāo)�！痉椒ā坎捎蒙镄畔W(xué)方法搜索飛蝗轉(zhuǎn)錄組數(shù)據(jù)庫(kù),獲得Lm Abd-5 c DNA全長(zhǎng)序列并克隆驗(yàn)證;采用Signal P在線軟件分析蛋白的信號(hào)肽,利用SMART網(wǎng)站預(yù)測(cè)其功能域,使用MEGA 7.0軟件中neighbor-joining(NJ)方法,與其他昆蟲(chóng)同源序列進(jìn)行聚類(lèi)分析;采用reverse-transcription quantitative PCR(RT-q PCR)方法檢測(cè)Lm Abd-5在飛蝗5齡第2天若蟲(chóng)不同組織部位和5齡不同發(fā)育時(shí)期體壁組織中的表達(dá)情況,揭示其組織和時(shí)期表達(dá)模式;采用RNA干擾(RNAi)技術(shù)及透射電鏡技術(shù)(TEM)觀察沉默Lm Abd-5后對(duì)飛蝗生長(zhǎng)發(fā)育和表皮結(jié)構(gòu)的影響。【結(jié)果】通過(guò)搜索得到Lm Abd-5 c DNA全長(zhǎng)序列并進(jìn)行了克隆和測(cè)序驗(yàn)證,獲得520 bp全長(zhǎng)c DNA序列,其中ORF為303 bp;基因結(jié)構(gòu)分析顯示該基因含有3個(gè)外顯子;功能域分析發(fā)現(xiàn)其含有1個(gè)信號(hào)肽和1個(gè)幾丁質(zhì)結(jié)合域(chitin binding domain 4,Cht BD4),與沙漠蝗、白蟻、赤擬谷盜等的Abd-5結(jié)構(gòu)類(lèi)似;BLAST分析結(jié)果表明Abd-5在昆蟲(chóng)中高度保守,飛蝗與沙漠蝗Abd-5序列一致度高達(dá)81%;對(duì)其保守基序進(jìn)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(lèi);聚類(lèi)分析結(jié)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(guān)系;RT-q PCR結(jié)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(dá),而在胃盲囊、中腸、馬氏管、脂肪體和翅芽中低表達(dá)或不表達(dá);不同時(shí)期表達(dá)分析發(fā)現(xiàn),Lm Abd-5在4齡若蟲(chóng)蛻皮后0—72 h(5齡早期)具有高表達(dá),其中蛻皮后72 h時(shí)達(dá)到最大表達(dá)量,隨后表達(dá)量急劇降低(96—168 h),其表達(dá)時(shí)期與內(nèi)表皮形成時(shí)間一致;采用RNAi技術(shù)分析該基因的生物學(xué)功能,對(duì)5齡第2天若蟲(chóng)分別注射等量的ds Lm Abd-5和ds GFP(對(duì)照),發(fā)現(xiàn)注射ds Lm Abd-5的5齡若蟲(chóng)和對(duì)照組相同,均可正常蛻皮,發(fā)育至成蟲(chóng)第2天目的基因表達(dá)量顯著降低,但未出現(xiàn)肉眼可見(jiàn)的異常表型。分別取成蟲(chóng)第2天處理組和對(duì)照組成蟲(chóng)表皮進(jìn)行超微結(jié)構(gòu)觀察,發(fā)現(xiàn)與對(duì)照組相比,處理組成蟲(chóng)內(nèi)表皮片層結(jié)構(gòu)較為疏松,導(dǎo)致內(nèi)表皮片層變厚,最終表現(xiàn)為整個(gè)內(nèi)表皮變厚。【結(jié)論】根據(jù)轉(zhuǎn)錄組數(shù)據(jù)庫(kù)分析獲得1個(gè)CPR家族表皮蛋白Lm Abd-5,該蛋白含有1個(gè)信號(hào)肽和1個(gè)幾丁質(zhì)結(jié)合域Cht BD4,屬于RR-1亞類(lèi);Lm Abd-5主要在外胚層起源的組織中高表達(dá),沉默Lm Abd-5后飛蝗沒(méi)有肉眼可見(jiàn)的表型,但超微結(jié)構(gòu)分析發(fā)現(xiàn)其參與飛蝗內(nèi)表皮片層結(jié)構(gòu)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(xué)應(yīng)用生物學(xué)研究所;山西大學(xué)生命科學(xué)學(xué)院;
【基金】：國(guó)家自然科學(xué)基金(31640075,31672364) 山西省青年科學(xué)基金(201601D021102) 山西省高�？萍紕�(chuàng)新基金(2016113)
【分類(lèi)號(hào)】：S433.2
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本文編號(hào)：1983945