Construction and characterization of genetically modified synechocystis sp. PCC 6803 photosystem II core complexes containing carotenoids with shorter pi-conjugation than beta-carotene

Beta-carotene has been identified as an intermediate in a secondary electron transfer pathway that oxidizes Chl(Z) and cytochrome b(559) in Photosystem II (PS II) when normal tyrosine oxidation is blocked. To test the redox function of carotenoids in this pathway, we replaced the zeta-carotene desaturase gene (zds) or both the zds and phytoene desaturase (pds) genes of Synechocystis sp. PCC 6803 with the phytoene desaturase gene (crtI) of Rhodobacter capsulatus, producing carotenoids with shorter conjugated pi-electron systems and higher reduction potentials than beta-carotene. The PS II core complexes of both mutant strains contain approximately the same number of chlorophylls and carotenoids as the wild type but have replaced beta-carotene (11 double bonds), with neurosporene (9 conjugated double bonds) and beta-zeacarotene (9 conjugated double bonds and 1 beta-ionylidene ring). The presence of the ring appears necessary for PS II assembly. Visible and near-infrared spectroscopy were used to examine the light-induced formation of chlorophyll and carotenoid radical cations in the mutant PS II core complexes at temperatures from 20 to 160 K. At 20 K, a carotenoid cation radical is formed having an absorption maximum at 898 nm, an 85 nm blue shift relative to the beta-carotene radical cation peak in the WT, and consistent with the formation of the cation radical of a carotenoid with 9 conjugated double bonds. The ratio of Chl(+)/Car(+) is higher in the mutant core complexes, consistent with the higher reduction potential for Car(+). As the temperature increases, other carotenoids become accessible to oxidation by P(680)(+).     

质膜NAD(P)H氧化酶参予金瓜炭疽(Colletotrichum lagerarium)细胞壁激发子诱导人参细胞产生激发反应(英文)

在人参(Panax ginseng C.A.Meyer)悬浮细胞质膜上测出了NAD(P)H氧化酶活性。这类NAD(P)H氧化酶活性可以被金瓜炭疽细胞壁激发子(Cle)诱导。Cle处理还能诱导人参悬浮细胞的氧进发、促进人参悬浮细胞的皂苷合成、提高苯丙氨酸解氨酶(PAL)的活力、以及诱导查尔式酮酶(CHS)的累积和细胞壁上抗性相关蛋白基因脯氨酸富裕蛋白基因hrgp(Hydroxyprolin-rich glycoproleins)的表达。当用哺乳动物白细胞质膜NADPH氧化酶的特异性抑制剂二亚苯基碘(Diphenylene iodonium,DPI)与奎吖因(quinacrine)预处理人参悬浮细胞30 min 后,Cle诱导的H2O2释放与Cle激活的质膜NAD(P)H氧化酶活性被抑制,同时Cle诱导的PAL活性及CHS的积累下降,皂苷合成与hrgp的表达被抑制。由此推测:人参细胞质膜NAD(P)H氧化酶与哺乳动物白细胞质膜NADPH氧化酶有很大的相似性。在Cle激发人参悬浮细胞产生氧进发的过程中,NAD(P)H氧化酶活性被诱导从而导致H2O2的产生,H2O2作为第二信使,激活苯丙氨酸途径,诱发人参皂苷的合成及hrgp防御基因的表达。这一过程中还涉及到Ca2+内流,胞内Ca2+浓度的升高,蛋白磷酸化与去磷酸化。人参细胞质膜NAD(P)H氧化酶在人参细胞对Cle的反应过程中起一种介导作用。因此可能存在由Cle刺激,NAD(P)H氧化酶被诱导,H2O2释放,到人     

人肺腺癌细胞分化相关基因cDNAs的克隆

在用10^-5 mol/L全反式维甲酸(RA)诱导人肺腺癌细胞系GLC-82分化的基础上,以M13噬菌粒pSPORT1为载体,应用定向克隆技术,分别构建了未经RA诱导和RA诱导1d及4d细胞的3个cDNA文库.以含重组子的诱导文库单链DNA为靶标(Target)同未诱导文库的cDNA驱除子(Driver)进行消减杂交,富集RA特异性单链DNA,将富集的单链DNA回复为双链后转化感受态菌,建立细胞诱导分化过程中活化表达基因的cDNA消减文库,得到124个cDNA消减克隆.经同源性分析和与文库总cDNA作Southern印迹杂交,进而与RA诱导前后细胞的RNA作Northern印迹杂交,筛选出2个(RA5,RA28)诱导后呈早期瞬时表达和1个(RA42)呈早期并持续表达的cDNA克隆,cDNA全长分别为1.8,1.5和0.7kb.序列测定及初步功能分析结果表明,RA5,RA28和RA42这3个首次报道的序列,可能是人肺腺癌细胞分化相关基因的cDNA克隆.     

东亚飞蝗hunchback基因在卵子形成和胚胎发育过程中的原位表达

hb（hunchback）基因是昆虫胚胎前后轴模式形成的关键基因．对东亚飞蝗（Locusta migratoria manilensis,Meyen）hb基因的功能已有报道,但其表达模式还不清楚．为了研究胁基因在东亚飞蝗卵子形成和胚胎发育过程中的时空表达情况,本研究采用免疫组化方法在蛋白质水平上检测了hb基因的时空表达模式．在卵子形成过程中,hb基因局限在卵细胞核区中表达,随着卵子的发育逐渐移至卵细胞的后端;卵受精后,核区里的Hb蛋白向外扩散,在卵后端形成浓度梯度;胚盘期,hb基因在胚盘中央呈带状表达;胚盘分化为原头和原躯干后,表达条带变宽,并呈现出梯度表达,该表达区域将形成颌、胸部的部分体节;随着腹节开始形成,hb基因在颌胸部的表达逐渐减弱,而在腹部后端的“生长区”表达,并呈现出不连续性．经比较,hb易基因在昆虫颌胸部的表达较为保守,而在卵子形成过程中和腹部的表达具有较大的变异性．与黑腹果蝇等长胚带昆虫相比,东亚飞蝗hb基因在体节形成的基因级联调控中具有更重要、更直接的调控作用．     

心脏特异表达LMNA^E82K转基因小鼠的建立

目的建立心脏特异表达LMNAE82K转基因小鼠,为研究LMNAE82K与心肌病发病机制的关系提供工具动物。方法把LMNAE82K基因插入α-MHC启动子下游,构建转基因表达载体,显微注射法建立C57BL/6JLMNAE82K转基因小鼠,PCR鉴定转基因小鼠的基因型,采用Western Blot鉴定LMNAE82K在心脏组织中的表达,H＆E染色和超声检测转基因小鼠心脏的病理改变。结果建立了2个心脏组织特异表达LMNAE82K的转基因小鼠品系。超声检查显示转基因小鼠心室壁变薄,收缩期容积和舒张期容积增加,射血分数及短轴缩短率降低。结论LMNAE82K转基因小鼠具有LMNAE82K引起的家族性扩心病有类似的病理变化,为研究LMNAE82K与心肌病发病机制的关系的研究提供了有价值的疾病动物模型。     

Global transcriptional response of pig brain and lung to natural infection by Pseudorabies virus

Background  

Pseudorabies virus (PRV) is an alphaherpesviruses whose native host is pig. PRV infection mainly causes signs of central nervous system disorder in young pigs, and respiratory system diseases in the adult.     

The draft genome of the pest tephritid fruit fly Bactrocera tryoni: resources for the genomic analysis of hybridising species

Background

The tephritid fruit flies include a number of economically important pests of horticulture, with a large accumulated body of research on their biology and control. Amongst the Tephritidae, the genus Bactrocera, containing over 400 species, presents various species groups of potential utility for genetic studies of speciation, behaviour or pest control. In Australia, there exists a triad of closely-related, sympatric Bactrocera species which do not mate in the wild but which, despite distinct morphologies and behaviours, can be force-mated in the laboratory to produce fertile hybrid offspring. To exploit the opportunities offered by genomics, such as the efficient identification of genetic loci central to pest behaviour and to the earliest stages of speciation, investigators require genomic resources for future investigations.

Results

We produced a draft de novo genome assembly of Australia’s major tephritid pest species, Bactrocera tryoni. The male genome (650 -700 Mbp) includes approximately 150Mb of interspersed repetitive DNA sequences and 60Mb of satellite DNA. Assessment using conserved core eukaryotic sequences indicated 98% completeness. Over 16,000 MAKER-derived gene models showed a large degree of overlap with other Dipteran reference genomes. The sequence of the ribosomal RNA transcribed unit was also determined. Unscaffolded assemblies of B. neohumeralis and B. jarvisi were then produced; comparison with B. tryoni showed that the species are more closely related than any Drosophila species pair. The similarity of the genomes was exploited to identify 4924 potentially diagnostic indels between the species, all of which occur in non-coding regions.

Conclusions

This first draft B. tryoni genome resembles other dipteran genomes in terms of size and putative coding sequences. For all three species included in this study, we have identified a comprehensive set of non-redundant repetitive sequences, including the ribosomal RNA unit, and have quantified the major satellite DNA families. These genetic resources will facilitate the further investigations of genetic mechanisms responsible for the behavioural and morphological differences between these three species and other tephritids. We have also shown how whole genome sequence data can be used to generate simple diagnostic tests between very closely-related species where only one of the species is scaffolded.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1153) contains supplementary material, which is available to authorized users.