本文關(guān)鍵詞：家蠶細(xì)胞及絲腺退化和凋亡的程序性細(xì)胞信號(hào)通路中BmDredd基因的功能　出處：《浙江大學(xué)》2017年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 家蠶 絲腺 凋亡 BmDredd lncRNA miRNA

【摘要】：家蠶是一種重要的經(jīng)濟(jì)動(dòng)物和模式昆蟲,絲腺是家蠶非常重要的吐絲器官,直接決定著家蠶產(chǎn)絲量的多少。在家蠶由幼蟲到蛹的變型期,絲腺器官發(fā)生了劇烈的退化,在短時(shí)間內(nèi)消亡,這一過程涉及到了細(xì)胞凋亡。目前,細(xì)胞凋亡分子調(diào)控機(jī)制在哺乳動(dòng)物中研究的較為清楚,但我們對(duì)于家蠶中的凋亡分子機(jī)制知之甚少。Dredd為caspase家族同源蛋白,本研究克隆到了家蠶BmDredd基因,并分別在細(xì)胞水平和絲腺組織水平揭示了其功能,調(diào)查了其與相關(guān)凋亡蛋白BmFadd的互作關(guān)系。同時(shí),我們利用高通量測(cè)序技術(shù),進(jìn)行了家蠶五齡第3天和吐絲36 h后部絲腺mRNA、lncRNA與miRNA的差異表達(dá)分析,并對(duì)差異表達(dá)lncRNAs與miRNAs的靶基因做了 GO功能預(yù)測(cè)。分析了BmDredd與lncRNA、miRNA的互作網(wǎng)絡(luò)。對(duì)這些內(nèi)容的探索使得家蠶細(xì)胞及絲腺凋亡的分子調(diào)控機(jī)制研究更為完善,主要研究?jī)?nèi)容和結(jié)果如下:1)克隆到了BmDredd基因,并且對(duì)其生物學(xué)信息進(jìn)行了分析,其ORF全長(zhǎng)1632 bp,編碼543個(gè)氨基酸,分子量大小約為63 KDa,含有N端的long prodomain和C端的 CASc domain。2)在細(xì)胞水平進(jìn)行BmDredd的功能研究:對(duì)BmN細(xì)胞進(jìn)行了誘導(dǎo)凋亡、dsRNA干擾處理。我們發(fā)現(xiàn)凋亡的細(xì)胞中BmDredd表達(dá)量升高,而RNA干擾BmDredd的表達(dá)則可以減小細(xì)胞凋亡率,提高正常細(xì)胞數(shù)量。另外,我們構(gòu)建了 BmDredd的N端、C端融合表達(dá)載體以及ORF序列過表達(dá)載體,結(jié)果發(fā)現(xiàn)過表達(dá)BmDredd可以引起細(xì)胞凋亡,細(xì)胞凋亡時(shí),BmDredd由細(xì)胞質(zhì)遷移到細(xì)胞核中,其核定位片段既不是獨(dú)立的N端longprodomain序列,也不是C端CASc序列,推測(cè)定位序列位于376-987 bp之間。dsRNA干擾凋亡相關(guān)基因后實(shí)時(shí)定量檢測(cè)其他基因表達(dá)量結(jié)果顯示,BmDredd與BmDaxx,BmCide-b,BmFadd,BmCreb之間存在著復(fù)雜的調(diào)控關(guān)系,他們一起參與調(diào)控細(xì)胞凋亡的執(zhí)行。3)絲腺水平的BmDredd功能研究:我們檢測(cè)了不同時(shí)期家蠶絲腺中BmDredd表達(dá)量以及家蠶不同組織在吐絲18 h時(shí)BmDredd表達(dá)量,發(fā)現(xiàn)絲腺凋亡期間,BmDredd表達(dá)量升高,表明BmDredd與絲腺凋亡有著極重要的聯(lián)系。并且,在蛻皮激素飼喂家蠶后,BmDredd表達(dá)量升高,說明BmDredd屬于蛻皮激素下游靶基因。Caspase抑制劑處理絲腺可以降低BmDredd表達(dá)量,注射dsRNA-BmDredd到家蠶絲腺可以延遲絲腺凋亡,在絲腺中過表達(dá)BmDredd引起過表達(dá)部位caspase-3活性的升高,所有結(jié)果都顯示BmDredd參與和誘導(dǎo)絲腺凋亡。4)Fadd屬于Fas結(jié)合蛋白,在哺乳動(dòng)物中可以與procaspase-8結(jié)合形成DISC(Death-inducing signalling complex),引起細(xì)胞死亡。本實(shí)驗(yàn)我們純化到了含GST標(biāo)簽的BmFadd蛋白,通過與凋亡細(xì)胞胞質(zhì)總蛋白孵育,清洗和洗脫后質(zhì)譜檢測(cè),發(fā)現(xiàn)凋亡時(shí)BmFadd并沒有與BmDredd蛋白發(fā)生互作。5)使用二代測(cè)序技術(shù)我們發(fā)現(xiàn)被歸到Death相關(guān)功能下的mRNAs在表達(dá)量上都沒有顯示出顯著性變化,這從側(cè)面反應(yīng)了 lncRNA和miRNA在絲腺凋亡發(fā)生期間對(duì)mRNA的調(diào)節(jié)可能十分重要。我們共鑒定到家蠶中10947個(gè)lncRNAs,預(yù)測(cè)到索引號(hào)為 TCONS_00023629、TCONS_35829、TCONS_28940、TCONS_28943、TCONS_30941的lncRNAs參與了凋亡過程。另外有344個(gè)miRNAs靶向調(diào)節(jié)著285個(gè)mRNAs都與GO條目下Death process有關(guān)。這說明,相比于lncRNA,miRNA在家蠶絲腺凋亡的分子調(diào)控中起到了更為廣泛和重要的作用。最后,我們篩選出了可能與BmDredd互作的746個(gè)lncRNAs和20個(gè)miRNAs,并做了三者間的網(wǎng)絡(luò)互作圖。
[Abstract]:Silkworm is an important economic animal and model insect. The silk gland is a very important silk thread organ of the silkworm, which directly determines the amount of silk production in the silkworm. In the stage of the silkworm from the larva to the pupa, the silk gland organs degenerate sharply and die out in a short time. This process involves apoptosis. At present, the mechanism of apoptosis molecular regulation is more clear in mammals, but we know little about the mechanism of apoptosis in silkworm. Dredd is a homologous protein of caspase family. The BmDredd gene of Bombyx Mori was cloned, and its function was revealed at cell level and silk gland tissue level. The interaction between BmFadd and its related apoptosis protein was investigated. Meanwhile, we used high-throughput sequencing technology to analyze the differential expression of mRNA, lncRNA and miRNA in the posterior silkgland of silkworm, Bombyx mori, five days, third days and 36 h, and predicted GO function of differentially expressed target genes of lncRNAs and miRNAs. The interwork network of BmDredd and lncRNA and miRNA is analyzed. Study on the molecular mechanism of the apoptosis of cells and silkworm silk gland to explore the content of which is more perfect, the main research contents and results are as follows: 1) BmDredd gene was cloned, and its biological information is analyzed, its ORF was 1632 BP, encoding 543 amino acids, the molecular weight of approximately 63 KDa in size. The long prodomain contains N end and C end CASc domain. 2) the functional study of BmDredd at the cell level: induced apoptosis and dsRNA interference treatment for BmN cells. We found that the expression of BmDredd in the apoptotic cells increased and the expression of RNA interfered with the expression of BmDredd could reduce the rate of apoptosis and increase the number of normal cells. In addition, we constructed the BmDredd N terminal and the C terminal fusion protein expression vector and ORF vector sequence, the results found that overexpression of BmDredd can induce cell apoptosis, apoptosis, BmDredd from cytoplasm to migrate to the nucleus, the nuclear localization is not independent of the N terminal fragment of longprodomain sequence, nor the C end CASc sequences. That is located in the 376-987 BP localization sequence. DsRNA interferes with apoptosis related genes, and real-time quantitative detection of other gene expression shows that there is a complex regulatory relationship between BmDredd and BmDaxx, BmCide-b, BmFadd and BmCreb. They are involved in regulating the execution of apoptosis. 3) BmDredd function of silk gland level: we detected the amount and amount of BmDredd expression in different tissues of Bombyx mori silk 18 h expression of BmDredd in different period in silk gland and silk gland found during apoptosis, BmDredd expression increased, indicating that BmDredd and silk gland apoptosis has a very important connection. And, in the silkworm feeding ecdysone, BmDredd expression increased, indicating that BmDredd is a downstream target gene of ecdysone. Caspase inhibitor can reduce the expression of BmDredd in silk gland. DsRNA-BmDredd injection to silkgland can delay the apoptosis of silk gland, and overexpression of BmDredd in silk gland can increase the activity of Caspase-3 in overexpression area, all results show that BmDredd participates in and induces apoptosis of silk gland. 4) Fadd belongs to Fas binding protein, which can combine with procaspase-8 to form DISC (Death-inducing signalling complex) in mammals, causing cell death. In this experiment, we purified the BmFadd protein containing GST tag and incubated with the total protein of apoptotic cells. After cleaning and elution, mass spectrometry revealed that BmFadd did not interact with BmDredd protein during apoptosis. 5) using the two generation sequencing technology, we found that the expression of mRNAs under Death related function did not show significant change in expression level. This reacted side by side to lncRNA and miRNA, which might be important in regulating mRNA during the apoptosis of silk gland. We identified 10947 lncRNAs in silkworm, and predicted that lncRNAs with TCONS_00023629, TCONS_35829, TCONS_28940, TCONS_28943 and TCONS_30941 participated in the apoptosis process. In addition, 344 miRNAs targets and 285 mRNAs are all related to the Death process under the GO entry. This suggests that miRNA plays a more extensive and important role in the molecular regulation of the apoptosis of the silk gland, compared with lncRNA. Finally, we screened 746 lncRNAs and 20 miRNAs that might interact with BmDredd, and did the network interaction between the three.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：S881.2

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