冠突散囊菌转录因子stf1基因的克隆及其表达分析

利用RACE技术获得了冠突散囊菌stf1基因的全长DNA序列。该序列全长3 029bp,开放阅读框长度为2 664bp,从114–2 908bp,在253bp处含有一个131bp的内含子,预测编码887个氨基酸。同源分析结果表明该基因与Snd1/p100转录因子同源。应用相对定量SYBR Green I荧光定量PCR技术对stf1基因在不同发育时期的表达量的差异进行了检测。结果表明,这个基因在子囊孢子时期的表达量最高,在分生孢子时期的表达量最低,相比子囊孢子时期下降了1倍。为深入研究冠突散囊菌的产孢调控机制奠定了一定的基础。     

Editor's choice: A comprehensive,genome-wide analysis of autophagy-related genes identified in tobacco suggests a central role of autophagy in plant response to various environmental cues

Autophagy is an evolutionarily conserved mechanism in both animals and plants, which has been shown to be involved in various essential developmental processes in plants. Nicotiana tabacum is considered to be an ideal model plant and has been widely used for the study of the roles of autophagy in the processes of plant development and in the response to various stresses. However, only a few autophagy-related genes (ATGs) have been identified in tobacco up to now. Here, we identified 30 ATGs belonging to 16 different groups in tobacco through a genome-wide survey. Comprehensive expression profile analysis reveals an abroad expression pattern of these ATGs, which could be detected in all tissues tested under normal growth conditions. Our series tests further reveal that majority of ATGs are sensitive and responsive to different stresses including nutrient starvation, plant hormones, heavy metal and other abiotic stresses, suggesting a central role of autophagy, likely as an effector, in plant response to various environmental cues. This work offers a detailed survey of all ATGs in tobacco and also suggests manifold functions of autophagy in both normal plant growth and plant response to environmental stresses.     

Expression pattern and functions of autophagy-related gene atg5 in zebrafish organogenesis

The implications of autophagy-related genes in serious neural degenerative diseases have been well documented. However, the functions and regulation of the family genes in embryonic development remain to be rigorously studied. Here, we report on for the first time the important role of atg5 gene in zebrafish neurogenesis and organogenesis as evidenced by the spatiotemporal expression pattern and functional analysis. Using morpholino oligo knockdown and mRNA overexpression, we demonstrated that zebrafish atg5 is required for normal morphogenesis of brain regionalization and body plan as well as for expression regulation of neural gene markers: gli1, huC, nkx2.2, pink1, β-synuclein, xb51 and zic1. We further demonstrated that ATG5 protein is involved in autophagy by LC3-II/LC3I ratio and rapamycin-induction experiments, and that ATG5 is capable of regulating expression of itself gene in the manner of a feedback inhibition loop. In addition, we found that expression of another autophagy-related gene, atg12, is maintained at a higher constant level like a housekeeping gene. This indicates that the formation of the ATG12–ATG5 conjugate may be dependent on ATG5 protein generation and its splicing, rather than on ATG12 protein in zebrafish. Importantly, in the present study, we provide a mechanistic insight into the regulation and functional roles of atg5 in development of zebrafish nervous system.     

光敏色素与转录因子结合直接调控植物基因表达和发育

植物的光控发育一直是植物学中一个非常活跃的研究领域,是近该领域又取得重大突破性进展,人们通过酵母双杂交技术克隆到在体内与光敏色素相互作用的转录因子,并证实被光活化的光敏色素直接进入细胞核与转录因子结构合启动基因表达,本文就此研究进展作简要介绍。     

Historical landmarks of autophagy research

The year of 2013 marked the 50th anniversary of C de Duve''s coining of the term “autophagy” for the degradation process of cytoplasmic constituents in the lysosome/vacuole. This year we regretfully lost this great scientist, who contributed much during the early years of research to the field of autophagy. Soon after the discovery of lysosomes by de Duve, electron microscopy revealed autophagy as a means of delivering intracellular components to the lysosome. For a long time after the discovery of autophagy, studies failed to yield any significant advances at a molecular level in our understanding of this fundamental pathway of degradation. The first breakthrough was made in the early 1990s, as autophagy was discovered in yeast subjected to starvation by microscopic observation. Next, a genetic effort to address the poorly understood problem of autophagy led to the discovery of many autophagy-defective mutants. Subsequent identification of autophagy-related genes in yeast revealed unique sets of molecules involved in membrane dynamics during autophagy. ATG homologs were subsequently found in various organisms, indicating that the fundamental mechanism of autophagy is well conserved among eukaryotes. These findings brought revolutionary changes to research in this field. For instance, the last 10 years have seen remarkable progress in our understanding of autophagy, not only in terms of the molecular mechanisms of autophagy, but also with regard to its broad physiological roles and relevance to health and disease. Now our knowledge of autophagy is dramatically expanding day by day. Here, the historical landmarks underpinning the explosion of autophagy research are described with a particular focus on the contribution of yeast as a model organism.     

STIM proteins,Orai1, and gene expression

Cytoplasmic Ca²⁺ is an universal intracellular messenger that activates cellular responses over a broad temporal range, from neurotransmitter release to cell growth and proliferation.^1,2 Inherent to the use of the multifarious Ca²⁺ signal is the question of specificity: how can some Ca²⁺-dependent responses be activated in a cell and not others? A rise in cytoplasmic Ca²⁺ can evoke a response either by binding directly to the target (as occurs with certain Ca²⁺-activated K⁺ and Cl⁻ channels, for example) or through recruitment of intermediary proteins, such as calmodulin and troponin C. A substantial body of evidence has now established that Ca²⁺-binding proteins differ both in their affinities for Ca²⁺ and in their on- and off-rates for Ca²⁺ binding/unbinding. Furthermore, different Ca²⁺-binding proteins often occupy distinct locations within the cell. Therefore, the size, kinetics and spatial profile of a cytoplasmic Ca²⁺ signal are all important in determining which Ca²⁺-dependent response will be activated, when and for how long.³     

Morphometric analysis of autophagy-related structures in Saccharomyces cerevisiae

When macroautophagy (autophagy) is induced by nutrient starvation or rapamycin treatment, Atg (autophagy-related) proteins are assembled at a restricted region close to the vacuole. Subsequently, the phagophore expands to form a closed autophagosome. In Saccharomyces cerevisiae cells overexpressing precursor Ape1 (prApe1), a specific autophagosome cargo protein, the phagophore can be visualized as a cup-shaped structure labeled with green fluorescent protein (GFP)-tagged Atg8. Previously, our group has shown that the maximum length of GFP-Atg8-labeled structures reflects the magnitude of bulk autophagy. In that study, the morphological parameters of the autophagy-related structures were extracted manually, requiring a great deal of time. Moreover, only well-expanded phagophores were subjected to further analysis. Here we report Qautas (Quantitative autophagy-related structure analysis system), a high-throughput and comprehensive system for morphological analysis of autophagy-related structures using a combination of image processing and machine learning. We describe both the manual method and Qautas in detail.     

Lys05: A new lysosomal autophagy inhibitor

Lys05 is a previously undescribed dimeric chloroquine which more potently accumulates in the lysosome and blocks autophagy compared with HCQ. Lys05 produced more potent antitumor activity as a single agent both in vitro and in vivo in multiple human cancer cell lines and xenograft models compared with HCQ. Initial structure-activity relationship studies demonstrated that the increased activity associated with Lys05 was due to the bivalent aminoquinoline rings, C7-Chlorine, and a short triamine linker. While lower doses of Lys05 were well tolerated and associated with antitumor activity, at the highest dose tested, Lys05 produced Paneth cell dysfunction and intestinal toxicity, similar to what can be observed in mice and humans with genetic defects in the autophagy gene ATG16L1. Lys05 is therefore a new lysosomal autophagy inhibitor that has potential to be developed further into a drug for cancer and other medical applications.     

构树转录因子BpbZIP1的鉴定及镉胁迫响应分析

重金属污染不仅影响土壤有效面积,限制植被分布,也会对食物链和人体健康造成危害,其中镉（Cd）污染尤为突出。选择重金属耐受性强的植物应用于尾矿区的土壤修复亟待进行。构树（Broussonetia papyrifera）是重金属污染土壤的先锋树种,为探明构树响应重金属胁迫的分子机制,本研究从构树中克隆获得1个碱性亮氨酸拉链（basic leucine zipper,bZIP）转录因子（命名为BpbZIP1）,对其基本生物信息、Cd胁迫响应及转化酵母的功能进行分析,预测BpbZIP1响应Cd的功能。结果显示,BpbZIP1基因开放阅读框长1 713 bp,编码的蛋白含570个氨基酸,分子量为62 902.38 Da,等电点为4.62。与拟南芥（Arabidopsis thaliana）AtbZIP1具有较近的进化关系。在150 μmol·L^-1 CdCl₂处理下,BpbZIP1基因能被不同程度的诱导表达,在3 h时BpbZIP1在根中的表达为对照的17.4倍。将BpbZIP1转入酵母能显著提高转基因酵母的抗Cd能力,当浓度高于0.6%时,转基因酵母的生长活力是对照的1.54~1.71倍。以上结果表明BpbZIP1基因能积极响应Cd胁迫,其表达可改善Cd胁迫耐受力,是构树Cd胁迫响应的重要基因。     

Expression pattern and functions of autophagy-related gene atg5 in zebrafish organogenesis

The implications of autophagy-related genes in serious neural degenerative diseases have been well documented. However, the functions and regulation of the family genes in embryonic development remain to be rigorously studied. Here, we report on for the first time the important role of atg5 gene in zebrafish neurogenesis and organogenesis as evidenced by the spatiotemporal expression pattern and functional analysis. Using morpholino oligo knockdown and mRNA overexpression, we demonstrated that zebrafish atg5 is required for normal morphogenesis of brain regionalization and body plan as well as for expression regulation of neural gene markers: gli1, huC, nkx2.2, pink1, β-synuclein, xb51 and zic1. We further demonstrated that ATG5 protein is involved in autophagy by LC3-II/LC3I ratio and rapamycin-induction experiments, and that ATG5 is capable of regulating expression of itself gene in the manner of a feedback inhibition loop. In addition, we found that expression of another autophagy-related gene, atg12, is maintained at a higher constant level like a housekeeping gene. This indicates that the formation of the ATG12–ATG5 conjugate may be dependent on ATG5 protein generation and its splicing, rather than on ATG12 protein in zebrafish. Importantly, in the present study, we provide a mechanistic insight into the regulation and functional roles of atg5 in development of zebrafish nervous system.