基于黏虫转录组的几丁质酶基因筛选和表达分析

昆虫的几丁质酶对昆虫的生长发育致关重要,是生物农药的重要靶标。本研究使用高通量测序技术对迁飞性害虫黏虫Mythimna separata的中肠和表皮组织进行了转录组测序、序列组装、功能注释及差异表达基因分析。对转录组数据库中鉴定出的几丁质酶基因进行了理化性质的预测,包括cDNA长度、蛋白质分子量、氨基酸序列、等电点、不稳定系数、跨膜结构和蛋白结构域等。使用MEGA软件构建了黏虫和其他昆虫几丁质酶的系统进化树,并通过q-PCR验证了黏虫基因在不同组织和发育阶段的表达模式。通过中肠与表皮的转录组测序,获得了19.42 Gb的数据,在COG、GO、KEGG、KOG、Pfam、Swissprot、eggNOG、nr数据库注释到了25 236个Unigene;基因表达分析结果表明,中肠和表皮的差异表达基因共有3 137个,其中中肠高表达基因有1 872个,表皮高表达基因有1 265个。从转录组数据中鉴定出9个几丁质酶基因,其中7个是新的几丁质酶基因,这些基因的cDNA长度在1 362~9 816 bp,SMART结构预测表明几丁质酶含有1个或多个催化结构域。构建的系统进化树将昆虫几丁质酶基因分为9个亚家族。q-PCR结果表明〖STBX〗MsCht2、MsCht5、MsCht6、MsCht7、MsIDGF1在表皮中表达量较高,MsCht4、MsCht11和MsChi-H在中肠中表达量较高,与转录组数据一致;多数几丁质酶基因在蛹期或预蛹期表达量最高,而MsCht4〖STBZ〗在5龄期表达量最高,在蛹期表达量很低。黏虫几丁质酶基因表达上存在不同的差异,不同的几丁质酶基因可能具有不同的功能。本研究筛选出了7个新的几丁质酶基因,为黏虫的生物防治提供了新的靶标。研究结果为进一步研究黏虫几丁质酶的功能奠定了基础。     

Developmental expression patterns of connexin26 and ‐30 in the rat cochlea

Connexin proteins form transmembranous gap junction channels that connect adjacent cells. Connexin26 and connexin30 have been previously shown to be strongly expressed in the inner ear of adult rats and to be mainly colocalized. Because intercellular connections by gap junction proteins are crucial for maturation of different tissues, we investigated the developmental expression of connexin26 and connexin30 in pre‐ and postnatal rats using immunocytochemistry. In the rat otocyst, staining for connexin26 as well as for connexin30 appeared at the 17th day of gestation. However, at this stage, expression of connexin30 was low and restricted to the neurosensory epithelium. Beginning from the 3rd postnatal day connexin26 and ‐30 were expressed with highest immunoreaction in the spiral limbus, the neurosensory epithelium, and between the stria vascularis and the spiral ligament. Beginning from postnatal day 12 the staining pattern resembled that of adult animals, with additional strong staining between all fibrocytes of the spiral ligament. Double labeling experiments demonstrated strongest colocalization of both connexins between the stria vascularis and the spiral ligament. These results demonstrate that development of the cochlear gap junction system precedes the functional maturation of the rat inner ear, which takes place between the 2nd and 3rd postnatal week. In the cochlea of a 22‐week‐old human embryo, connexin26 and connexin30 could be detected in the lateral wall, suggesting that both connexins also play a crucial role in function of the human inner ear. Dev. Genet. 25:306–311, 1999. © 1999 Wiley‐Liss, Inc.     

Differential expression of mRNAs for sialyltransferase isoenzymes induced in the hippocampus of mouse following kindled seizures

Sialic acids play important roles in various biological functions. In the brain, evidence suggests that sialylation of glycoproteins and glycolipids affects neural plasticity. While the 18 sialyltransferase isoenzymes (STs) identified to date synthesize individual sialyl-oligosaccharide structures, they each exhibit activity toward more than one substrate and can overlap in their specificity. Therefore, the distribution of STs is a secondary factor in the study of specific sialylation. Here, seven STs; ST3Gal I-IV, ST8Sia IV, ST6Gal I and ST6GalNAc II, the expressions of which were identified in the adult hippocampus by RT-PCR, showed diverse localization patterns in the hippocampus on in situ hybridization, suggesting that the individual cells expressed relevant STS: Furthermore, to assay activity-related changes in ST expression, we used amygdaloid-kindling among models of neural plasticity. Differential expression of the STs participating in the kindling, notably, up-regulation of ST3Gal IV and ST6GalNAc II mRNAs, and down-regulation of ST3Gal I and ST8Sia IV mRNAs, were observed in the hippocampus following kindled seizures. These results indicate that ST expressions are regulated by physiological activity and may play a role in neural plasticity.     

Synopsis of the International Gap Junction Conference in Elsinore, Denmark August 5-9, 2007

This synopsis covers the main results and conclusions from the platform presentations during the International Gap Junction Conference. More detailed information is provided in the mini reviews on controversial scientific issues, short reports of research results and conference abstracts published in this issue of Cell Communication and Adhesion.     

Functional expression of connexin30 and connexin31 in the polarized human airway epithelium

Gap junctions are documented in the human airway epithelium but the functional expression and molecular identity of their protein constituents (connexins, Cx) in the polarized epithelium is not known. To address this question, we documented the expression of a family of epithelial Cx (Cx26, Cx30, Cx30.3, Cx31, Cx31.1, Cx32, Cx37, Cx40, and Cx43) in primary human airway epithelial cells (AEC) grown on porous supports. Under submerged conditions, AEC formed a monolayer of airway cells whereas the air-liquid interface induced within 30-60 days AEC differentiation into a polarized epithelium for up to 6-9 months. Maturation of AEC was associated with the down-regulation of Cx26 and Cx43. The well-differentiated airway epithelium exhibited gap junctional communication between ciliated and between ciliated and basal cells. Interestingly, Cx30 was mostly present between ciliated cells whereas Cx31 was found between basal cells. These results are supportive of the establishment of signal-selective gap junctions with maturation of AEC, likely contributing to support airway epithelium function. These results lay the ground for studying the role of Cx-mediated cell-cell communication during repair following AEC injury and exploring Cx-targeted interventions to modulate the healing process.     

启动以细胞为基本功能单元的系统人类基因转录组研究

基因组学、分子生物学和细胞生物学等基础生命科学领域前沿的概念和技术都在高速形成和发展,这些领域和新概念、新技术的不断融合推动生命科学研究从一个生长点到又一个新的生长点。人类基因组研究将在未来的五到十年里,从以一个基因组为对象的研究进入到以每个体基因组为对象的研究。基因功能和功能相关性的研究也将进入到以细胞为单元的研究,转录组研究必将成为这一研究的基础和出发点。转录组研究包括转录组的构成、调控和与蛋白质组的关联等基本部分,以及在临床诊断和药物研发中的应用。在生理状态下,每一种细胞中基因的共有和特异功能最终构成物种的生长、发育和演化。在病理状态下,每一种细胞中基因的变异和失控最终导致器官、组织、乃至个体的衰亡。人类转录组研究是基因功能研究的最基本层次之一,作为基因产物的RNA和蛋白质在这个层次上的存在、变化、关联和在细胞间的差异构成转录组研究的基本内容。     

Regulation of regulators of G protein signaling mRNA expression in rat brain by acute and chronic electroconvulsive seizures

G protein-coupled receptor (GPCR) signaling cascades may be key substrates for the antidepressant effects of chronic electroconvulsive seizures (ECS). To better understand changes in these signaling pathways, alterations in levels of mRNA's encoding regulators of G protein signaling (RGS) protein subtypes-2, -4, -7, -8 and -10 were evaluated in rat brain using northern blotting and in situ hybridization. In prefrontal cortex, RGS2 mRNA levels were increased several-fold 2 h following an acute ECS. Increases in RGS8 mRNA were of lesser magnitude (30%), and no changes were evident for the other RGS subtypes. At 24 h following a chronic ECS regimen, RGS4, -7, and -10 mRNA levels were reduced by 20-30%; only RGS10 was significantly reduced 24 h after acute ECS. Levels of RGS2 mRNA were unchanged 24 h following either acute or chronic ECS. In hippocampus, RGS2 mRNA levels were markedly increased 2 h following acute ECS. More modest increases were seen for RGS4 mRNA expression, whereas levels of the other RGS subtypes were unaltered. At 24 h following chronic ECS, RGS7, -8 and -10 mRNA levels were decreased in the granule cell layer, and RGS7 and -8 mRNA levels were decreased in the pyramidal cell layers. Only RGS8 and -10 mRNA levels were significantly reduced in hippocampus 24 h following an acute ECS. Paralleling neocortex, RGS2 mRNA content was unchanged in hippocampus 24 h following either acute or chronic ECS. In ventromedial hypothalamus, RGS4 mRNA content was increased 24 h following chronic ECS, whereas RGS7 mRNA levels were only increased 24 h following an acute ECS. The increased RGS4 mRNA levels in hypothalamus were significant by 2 h following an acute ECS. These studies demonstrate subtype-, time-, and region-specific regulation of RGS proteins by ECS, adaptations that may contribute to the antidepressant effects of this treatment.     

慢性强直电刺激右侧尾壳核诱导大鼠癫痫样电图和行为发作特征

目的 :慢性强直电刺激右侧尾壳核 (CPu)诱导大鼠电图和行为癫痫点燃样现象 ,观察CPu或海马 (HPC)网络异常的靶向癫痫样行为表达特征。方法 :共用雄性SD大鼠 58只。强直电刺激 ( 60Hz ,0 .4～ 0 .6mA ,2s)大鼠右侧CPu或右侧前背HPC ,1time/d ,连续刺激 7～ 12d。结果 :①CPu电图节律性尖波样发放或HPC电图阵发性高幅失律。②CPu或HPC刺激组大鼠均可以出现原发性、继发性或点燃样湿狗样抖动 (wetdogshakes ,WEDS)、直立、洗面、好静、咀嚼和节律性点头等行为发作。③CPu刺激组大鼠原发性WEDS频率明显低于HPC刺激组大鼠( 2 .10± 0 .12和 2 .89± 0 .2 0times/min ,P <0 .0 1) ,继发性WEDS频率明显高于HPC刺激组大鼠 ( 1.2 3± 0 .11和0 .78± 0 .0 6times/min ,P <0 .0 1)。④CPu刺激组大鼠点燃样效应出现之前的行为静止天数较长。结论 :如同刺激HPC一样 ,慢性电刺激大鼠CPu可以出现类似的癫痫样行为发作。结果提示 :CPu功能异常有可能成为癫痫发作的起源病灶 ,与HPC类似 ,参与了颞叶癫痫电网络的重建 ,具有特征性的癫痫样靶行为表达