DNA甲基化及其对植物发育的调控

DNA甲基化属于一种表观遗传修饰,主要发生在CpG双核苷酸序列中的胞嘧啶上,是在DNA甲基转移酶催化下,以S-腺苷甲硫氨酸为甲基供体,将甲基转移到胞嘧啶上,生成5-甲基胞嘧啶的一种反应。DNA甲基化在植物生长过程中具有极其重要的作用。综述了植物DNA甲基化的特征、调控机制,及其对植物基因表达影响的研究进展。     

DNA甲基化--浅谈发育生物学中的几个问题

甲基化和非甲基化是一种碱基修饰 ,现已表明DNA甲基化程度与基因表达的调控有关。在哺乳动物的 DNA中常常有 2 %～ 7%的胞嘧啶被酶所修饰 ,形成5 -甲基胞嘧啶。这种现象在卫星 DNA中很明显 (赵寿元等 ,1996 )。动物染色质中平均有 70 %的 CCGG甲基化(童克中 ,1996 )。甲基化现象发生在 5′- CG- 3′二核苷酸上。那么甲基化在基因的表达和调控中有着什么作用 ,又是如何得到这样的结论呢 ?人们假设了这样一种情况 :基因无论在激活状态或是在钝化状态都包含相同的核苷酸。由此 ,可以说在红血细胞前体中的 β珠蛋白基因与同一个动物的成纤…     

DNA甲基化与发育调节

甲基化修饰是脊椎动物ＤＮＡ唯一的自然修饰方式,动物基因组甲基化与基因表达密切相关,ＤＮＡ甲基化通过与反式作用因子相互作用或通过改变染色质结构而影响表达,在胚胎发育、Ｘ染色体失活、基因组印记及肿瘤发生发展中起重要作用。     

活性氧调控植物细胞自噬的研究进展

真核生物通过双层膜结构包裹细胞内受损的蛋白、细胞器或外源物质, 经溶酶体(或液泡)将内含物降解并进行循环利用, 这种高度保守的生物学过程称为自噬。活性氧是细胞有氧代谢的副产物, 作为一种信号分子广泛参与不同生物学过程的调控。研究表明, 真核生物中自噬与活性氧之间存在密切联系。该文结合近年的研究进展, 对植物细胞中活性氧的种类及作用和自噬的分子机制等进行概述, 旨在探讨活性氧对自噬的调控作用。     

Bmttv在家蚕翅发育过程中的功能分析

【目的】探析家蚕Bombyx mori糖基转移酶基因Bmttv在家蚕翅发育过程中的功能。【方法】通过RACE克隆家蚕Bmttv的CDS全长序列并进行生物信息学分析;使用qRT-PCR检测Bmttv在家蚕不同发育阶段(5龄幼虫、游走期幼虫、预蛹、蛹和成虫)和5龄幼虫不同组织(表皮、翅原基、精巢、马氏管、气管、丝腺、头、血淋巴、脂肪体和中肠)中的表达量;构建过表达载体,瞬时转染BmE细胞,通过免疫荧光对BmTTV进行亚细胞定位,利用qRT-PCR检测家蚕翅发育关键基因BmHippo,Bmwg,BmDpp,Bmsotv和BmHh的表达量。【结果】成功克隆到家蚕Bmttv的CDS全长序列,长2 228 bp; BmTTV蛋白具有两个特殊结构域,均与硫酸肝素聚糖(heparan sulfate proteoglycans, HSPGs)的合成相关,与黑腹果蝇Drosophila melanogaster和智人Homo sapiens TTV的氨基酸序列一致性达45%左右,与鳞翅目昆虫TTV的亲缘关系更近。Bmttv主要在家蚕蛹期开始高量表达,在5龄幼虫中肠、精巢和翅原基中高量表达。BmTTV主要...     

植物细胞自噬研究进展

细胞自噬是一类依赖于溶酶体和液泡的蛋白质降解途径。在动物细胞中, 靶物质通过自噬体包裹被运送到溶酶体中,由特定的水解酶降解; 而植物和酵母细胞中该过程在液泡内进行。近年来, 在模式植物拟南芥(Arabidopsis thaliana)中鉴定到多个关键ATG基因, 它们对植物细胞自噬体的形成及自噬调控起到关键作用。该文全面综述了植物细胞自噬的调控及其在植物逆境胁迫中的生理功能。     

细胞自噬调控的分子机制研究进展

细胞自噬是一种进化上保守的分解代谢过程,涉及细胞内长寿命蛋白和受损伤细胞器的降解,其在细胞内稳态、肿瘤、心力衰竭、衰老相关性疾病、神经退行性疾病以及传染病等多种生命进程中发挥着重要作用。泛素样蛋白系统、m TOR信号通路、micro RNA、caspase等均参与了细胞自噬调控过程。该文综述了细胞自噬过程、功能和分子调控机制的研究进展,以期有助于研究细胞自噬机理,为治疗心脏疾病(如动脉粥样硬化)、癌症(如乳腺癌)等提供理论基础。     

DNA甲基化在中枢神经系统发育中的研究进展

DNA甲基化是最早被发现的表观遗传修饰之一。近年来,大量的研究显示DNA甲基化在中枢神经系统(CNS)发育中发挥了重要作用。不同种类的DNA甲基转移酶(Dnmt)和DNA甲基结合蛋白(MBD)在CNS发育的不同阶段发挥不同的作用。DNA甲基化促进神经干细胞向神经元方向分化,抑制其向胶质细胞分化。Dnmt和MBD主要在神经元中表达,而在胶质细胞不表达或表达较少。DNA甲基化调节神经发生和突触的形成,参与学习记忆。星型胶质细胞的标志物GFAP去甲基化促进早期神经上皮分化为星型胶质细胞。少突胶质细胞相关基因MAG和Sox10等也受甲基化的调节。本文主要从以上方面综述了DNA甲基化在中枢神经系统发育中的作用。     

细胞自噬与肿瘤

自噬是广泛存在于真核细胞内的一种细胞分解自身构成成分的生命现象.细胞内的双层膜结构与溶酶体结合后其内包裹的受损、变形或衰老细胞器蛋白质等被水解酶类降解.细胞自噬具有多种生理功能,生命体借此维持蛋白质代谢平衡及细胞环境稳定,这一过程在细胞清除废物、结构重建、生长发育调节中发挥重要作用. 细胞自噬也与肿瘤的存活和死亡等过程密切相关. 近年来对细胞自噬的研究有了较大的深入,本文主要对自噬体的形态和发生过程及其分子机制、信号调节通路、自噬研究的检测方法,以及自噬与细胞凋亡和肿瘤发生的关系等方面进行概述,以期较全面地了解细胞自噬作用和最新研究动态.     

细胞自噬研究进展

细胞自噬是真核生物在进化过程中高度保守、基于溶酶体的一种胞内降解途径,对维持细胞和生物体的稳态平衡有重要作用。研究表明,自噬参与生物体发育、免疫反应、代谢调节、细胞凋亡和衰老等多种过程。自噬功能异常与神经退行性疾病、肿瘤等的发生发展密切相关。近30年,我们对细胞自噬的认识无论是在分子机制上还是生理功能方面都有了长足的发展。为进一步加深对细胞自噬的认识,该文主要对细胞自噬的概念、自噬核心机器的组成及调控机制、自噬类型、生理功能及与疾病的关系作一简单综述。     

Comparative transcriptome analysis of wing discs from Bombyx mori and Bombyx mandarina

The insect wing is developed from the wing imaginal disc which is designed from the embryonic ectoderm. To get insight into gene expression profiles in wing discs of Bombyx mori during metamorphosis, we compared the gene expression in the wing between B. mori and B. mandarina moth through RNA-seq. Out of total valid reads identified from the 5th day of 5th instar larvae of silkworm (L5), 7th day of pupae (P7), 1st day of moth (M1) and 1st day of wild silkworm moth (WM1), 20,092,004, 29,251,647, 24,654,695 and 19,753,089 reads were mapped to the mRNA reference sequences of silkworm, respectively. 9229, 7048, 9268 and 6701 differentially expressed genes (DEGs) were respectively recorded in P7 vs L5, M1 vs P7, M1 vs L5 and WM1 vs M1. Further, the peroxisome, ribosome, endocytosis and oxidative phosphorylation pathways were significantly regulated in the metamorphosis of the silkworm. Our study identified 16 orthologous genes with a positive selection from M1, which might be subjected to artificial selection in the domestication of B. mori and would play vital roles in the flight of B. mandarina.     

Functional identification of Bombyx mori Atg13 in autophagy

The autophagy process involves a series of autophagy-related (Atg) proteins, which are conserved in eukaryotes. ULK1/Atg1-ATG13/Atg13 is the core protein complex for autophagy initiation in response to nutrient and hormone signaling. However, how Atg13 is regulated to participate in autophagy is unclear in insects. Here in Bombyx mori, the variation of BmAtg13 was correlated with autophagy induced by steroid hormone 20-hydroxyecdysone (20E) or starvation. Developmental profiles from feeding to prepupal stage revealed that there were two bands of BmAtg13 protein detected by western blot analysis, therein the upper band was intensively decreased, while the lower band was significantly increased which was in accordance with its mRNA variation; and immunofluorescent staining indicated that BmAtg13 was nucleocytoplasmic translocated during larval-pupal metamorphosis when autophagy was dramatically induced. BmAtg13 knockdown and overexpression both inhibits autophagy. Besides, 20E treatment-induced BmAtg13 gene expression, while blocking 20E signaling transduction by knockdown of BmUsp reduced both gene expression and protein level of BmAtg13. These results reveal that BmAtg13 is required for 20E- and starvation-induced autophagy in B. mori, which provides the foundation for further related studies.     

A palmitoyltransferase Approximated gene Bm-app regulates wing development in Bombyx mori

The silkworm Bombyx mori is an important lepidopteran model insect in which many kinds of natural mutants have been identified.However,molecular mechanisms of most of these mutants remain to be explored.Here we report the identification of a gene Bm-app is responsible for the silkworm minute wing(mw)mutation which exhibits exceedingly small wings during pupal and adult stages.Compared with the wild type silkworm,relative messenger RNA expression of Bm-app is significantly decreased in the ul 1 mutant strain which shows mw phenotype.A 10 bp insertion in the putative promoter region of the Bm-app gene in mw mutant strain was identified and the dual luciferase assay revealed that this insertion decreased Bm-app promoter activity.Furthermore,clustered regularly interspaced short palindromic repeats/RNA-guided Cas9 nucleases-mediated depletion of the Bm-app induced similar wing defects which appeared in the mw mutant,demonstrating that Bm-app controls wing development in B.mori.Bm-app encodes a palmitoyltransferase and is responsible for the palmitoylation of selected cytoplasmic proteins,indicating that it is required for cell mitosis and growth during wing development.We also discuss the possibility that Bm-app regulates wing development through the Hippo signaling pathway in B.mori.     

家蚕鞣化激素基因对翅展及繁殖力的调控作用

[目的]昆虫鞣化激素(Bursicon)是由神经系统分泌的一种异源二聚体神经肽,对昆虫表皮鞣化、翅展等功能具有调控作用.本研究旨在探究鞣化激素基因与家蚕Bombyx mori翅发育及对繁殖力相关基因的关系,明确其对翅展和繁殖力的调控作用.[方法]采用RNAi技术,分别注射Bursicon基因的dsRNA(dsBmBur...     

家蚕BmRpt4基因在翅膀发育中的功能研究

【目的】研究分析编码蛋白酶体调控复合物亚基基因BmRpt4(Regulatoryparticletriple-A ATPase4,BGIBMGA010794)在家蚕翅膀发育过程中的功能。【方法】利用CRISPR/Cas9基因组编辑技术,将BmRpt4的gRNAs和Cas9mRNA直接注射到家蚕胚胎中,观察G0代家蚕个体雏翅率,并分析雏翅个体中BmRpt4基因突变情况。【结果】经统计,对照组G0代家蚕个体均表现为正常翅,而实验组注射BmRpt4的gRNAs和Cas9 mRNA后,G0代约66.7%家蚕个体表现出小翅表型。在分子水平上对小翅表型个体进行检测,发现BmRpt4基因在两个gRNA及其之间位置均有不同长度片段的缺失或者插入,说明在这些突变个体中BmRpt4基因被不同程度的敲除,从而其功能缺失,导致出现小翅表型。【结论】该研究结果表明编码蛋白酶体调控亚基的BmRpt4基因在家蚕翅膀发育中具有重要作用,为蛋白酶体在生物组织器官发育中调控作用研究提供重要实验依据。     

Ecdysteroid-induced programmed cell death and cell proliferation during pupal wing development of the silkworm, Bombyx mori

The wing margin of adult wings of Lepidoptera is defined by the position of a "bordering lacuna"(BL). During adult wing development, cell proliferation and scale formation proximal to this lacuna and programmed cell death distal to the lacuna are generally observed. To determine the effect of 20-hydroxyecdysone (20E) on these events, we cultured the silkworm pupal wings with or without 20E and analyzed regional specificity for cell death by the TUNEL method and cell proliferation by 5-bromodeoxyuridine labeling. Programmed cell death was induced by 20E after 5 days of culture and was detected only in the region distal to BL. Cell proliferation after 1 day of culture and scale formation after 5 days of culture were also inducible by 20E and detected in the region proximal to BL. These results suggest that two types of pupal wing cells, which are divided by the position of the BL, respond to ecdysteroid in different manners. Higher concentrations of 20E (more than 1,000 ng/ml) repressed the scale formation, while such repression could not be observed in the peripheral cell death even with 5,000 ng/ml 20E. The ecdysteroid may work both as a trigger to make the wing margin and scales and as a developmental timer to arrange these cellular responses.     

The programming of silk-gland development in Bombyx mori

Summary The cytological development of the silk gland has been studied by light and electron microscopy in silkworms experimentally starved at different periods of the natural feeding stage during the fifth instar. When newly molted animals are not provided with food, no sign of growth is observed. Starvation initiated early during the obligatory feeding period, stops cell growth and development of the organelles involved in protein synthesis and secretion, whereas it induces the appearance of organelles concerned with autolysis. These effects are reversible if starvation is not prolonged beyond two days. Starvation during the facultative feeding period, at the time of massive fibroin production, results in quantitative and qualitative modifications of organelles related to the decrease of fibroin production and the onset of autolysis.Rough endoplasmic reticulum, responsible for fibroin synthesis, forms transitory whorls. Fibroin transport via the Golgi apparatus and secretion of the protein into the gland lumen decrease parallel to fibroin synthesis, so that no fibroin storage can be detected in any organelle. After food deprivation, autophagosomes and secondary lysosomes rapidly develop in the cytoplasm, and if starvation continues portions of the cytoplasm are sequestered and completely destroyed. If animals are refed, fibroin production is resumed and autolysis declines.These ultrastructural alterations of the silk gland during experimental starvation are very similar to those observed during the periods of physiological starvation (molt and cocoon spinning) and generally considered to be under hormonal control. Our results raise the question of the nature of interactions between alimentary and hormonal factors which control silk-gland development.This study was supported by a grant from the Centre National de la Recherche Scientifique (A.T.P., Contract n 1472) and was partly carried out in the Centre de Microscopie Electronique, CMEABG, Université Claude Bernard, Lyon and the Laboratoire d'Histologie, Service de Quantimétrie, Faculté de Médicine, Université Claude Bernard, Lyon     

DNA replication events during larval silk gland development in the silkworm, Bombyx mori

The silk gland is an important organ in silkworm as it synthesizes silk proteins and is critical to spinning. The genomic DNA content of silk gland cells dramatically increases 200-400 thousand times for the larval life span through the process of endomitosis. Using in vitro culture, DNA synthesis was measured using BrdU labeling during the larval molt and intermolt periods. We found that the cell cycle of endomitosis was activated during the intermolt and was inhibited during the molt phase. The anterior silk gland, middle silk gland, and posterior silk gland cells asynchronously exit the endomitotic cycle after day 6 in 5th instar larvae, which correlated with the reduced expression of the cell cycle-related cdt1, pcna, cyclin E, cdk2 and cdk1 mRNAs in the wandering phase. Additional starvation had no effect on the initiation of silk gland DNA synthesis of the freshly ecdysed larvae.