家蚕细胞及丝腺退化和凋亡的程序性细胞信号通路中BmDredd基因的功能

发布时间：2017-12-27 11:14

  本文关键词：家蚕细胞及丝腺退化和凋亡的程序性细胞信号通路中BmDredd基因的功能　出处：《浙江大学》2017年博士论文　论文类型：学位论文

  更多相关文章： 家蚕 丝腺 凋亡 BmDredd lncRNA miRNA

【摘要】：家蚕是一种重要的经济动物和模式昆虫,丝腺是家蚕非常重要的吐丝器官,直接决定着家蚕产丝量的多少。在家蚕由幼虫到蛹的变型期,丝腺器官发生了剧烈的退化,在短时间内消亡,这一过程涉及到了细胞凋亡。目前,细胞凋亡分子调控机制在哺乳动物中研究的较为清楚,但我们对于家蚕中的凋亡分子机制知之甚少。Dredd为caspase家族同源蛋白,本研究克隆到了家蚕BmDredd基因,并分别在细胞水平和丝腺组织水平揭示了其功能,调查了其与相关凋亡蛋白BmFadd的互作关系。同时,我们利用高通量测序技术,进行了家蚕五龄第3天和吐丝36 h后部丝腺mRNA、lncRNA与miRNA的差异表达分析,并对差异表达lncRNAs与miRNAs的靶基因做了 GO功能预测。分析了BmDredd与lncRNA、miRNA的互作网络。对这些内容的探索使得家蚕细胞及丝腺凋亡的分子调控机制研究更为完善,主要研究内容和结果如下:1)克隆到了BmDredd基因,并且对其生物学信息进行了分析,其ORF全长1632 bp,编码543个氨基酸,分子量大小约为63 KDa,含有N端的long prodomain和C端的 CASc domain。2)在细胞水平进行BmDredd的功能研究:对BmN细胞进行了诱导凋亡、dsRNA干扰处理。我们发现凋亡的细胞中BmDredd表达量升高,而RNA干扰BmDredd的表达则可以减小细胞凋亡率,提高正常细胞数量。另外,我们构建了 BmDredd的N端、C端融合表达载体以及ORF序列过表达载体,结果发现过表达BmDredd可以引起细胞凋亡,细胞凋亡时,BmDredd由细胞质迁移到细胞核中,其核定位片段既不是独立的N端longprodomain序列,也不是C端CASc序列,推测定位序列位于376-987 bp之间。dsRNA干扰凋亡相关基因后实时定量检测其他基因表达量结果显示,BmDredd与BmDaxx,BmCide-b,BmFadd,BmCreb之间存在着复杂的调控关系,他们一起参与调控细胞凋亡的执行。3)丝腺水平的BmDredd功能研究:我们检测了不同时期家蚕丝腺中BmDredd表达量以及家蚕不同组织在吐丝18 h时BmDredd表达量,发现丝腺凋亡期间,BmDredd表达量升高,表明BmDredd与丝腺凋亡有着极重要的联系。并且,在蜕皮激素饲喂家蚕后,BmDredd表达量升高,说明BmDredd属于蜕皮激素下游靶基因。Caspase抑制剂处理丝腺可以降低BmDredd表达量,注射dsRNA-BmDredd到家蚕丝腺可以延迟丝腺凋亡,在丝腺中过表达BmDredd引起过表达部位caspase-3活性的升高,所有结果都显示BmDredd参与和诱导丝腺凋亡。4)Fadd属于Fas结合蛋白,在哺乳动物中可以与procaspase-8结合形成DISC(Death-inducing signalling complex),引起细胞死亡。本实验我们纯化到了含GST标签的BmFadd蛋白,通过与凋亡细胞胞质总蛋白孵育,清洗和洗脱后质谱检测,发现凋亡时BmFadd并没有与BmDredd蛋白发生互作。5)使用二代测序技术我们发现被归到Death相关功能下的mRNAs在表达量上都没有显示出显著性变化,这从侧面反应了 lncRNA和miRNA在丝腺凋亡发生期间对mRNA的调节可能十分重要。我们共鉴定到家蚕中10947个lncRNAs,预测到索引号为 TCONS_00023629、TCONS_35829、TCONS_28940、TCONS_28943、TCONS_30941的lncRNAs参与了凋亡过程。另外有344个miRNAs靶向调节着285个mRNAs都与GO条目下Death process有关。这说明,相比于lncRNA,miRNA在家蚕丝腺凋亡的分子调控中起到了更为广泛和重要的作用。最后,我们筛选出了可能与BmDredd互作的746个lncRNAs和20个miRNAs,并做了三者间的网络互作图。
[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.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S881.2

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