集胞藻PCC6803基因sll0853的克隆、优化表达及其结构功能预测

从集胞藻(Synechocystis sp.)PCC6803提取总DNA,利用PCR扩增目的基因sll0853,构建重组T-0853克隆载体和pET-0853原核表达载体.为了提高sll0853在大肠杆菌E.coli BL21(DE3)中的表达量,通过改变诱导温度、诱导时间及诱导剂浓度等条件对表达量产生影响,以SDS-PAGE电泳分析证明sll0853基因蛋白表达的最佳条件.结果表明:目的蛋白在28℃、0.2mmol/L IPTG、诱导6h表达量分别达高峰.通过生物信息学软件预测基因sll0853可能具有裂合酶的功能.     

Bifunctional enzyme FBPase/SBPase is essential for photoautotrophic growth in cyanobacterium Synechocystis sp. PCC 6803

From a random insertion mutant library of Synechocystis sp. PCC 6803, a mutant defective in photoautotrophic growth was obtained. The interrupted gene was identified to be slr2094 （fbpl）, which encodes the fructose-1,6-biphosphatase （FBPase）/sedoheptulose-1,7-biphosphatase （SBPase） bifunctional enzyme （F-I）. Two other independently constructed slr2094 mutants showed an identical phenotype. The FBPase activity was found to be virtually lacking in an slr2094 mutant, which was sensitive to light under mixotrophic growth conditions. These results indicate that slr2094 is the only active FBPase-encoding gene in this cyanobacterium. Inactivation of photosystem II by interrupting psbB in slr2094 mutant alleviated the sensitiveness to light. This report provides the direct genetic evidence for the essential role of F-I in the photosynthesis of Synechocystis sp. PCC 6803.     

Role of two phosphohexomutase genes in glycogen synthesis in Synechocystis sp. PCC6803

Phosphohexomutases catalyze the interconversion between hexose-6-phosphate and hexose-l-phosphate and play important roles in polysaccharide synthesis. In Synechocystis sp. PCC 6803, sl10726 is predicted to encode PGM （phosphoglucomutase）, slr1334 is predicted to encode a PGM/PMM （phosphomannomutase） bifunction enzyme. In comparison to the wild type, a sllO726-null mutant showed 3.4% PGM activity but 45%-69% glycogen content. Down-regulation of slr1334, an essential gene, by using a copper regulated promoter further decreased the PGM activity in the sllO726：：Kmr PpetE-slr1334 double mutant to 0.3% of the wild type level. However, the glycogen content was not further decreased in parallel. In vitro, recombinant Sl10726 or S1r1334 showed predicted enzyme activities. Our results indicate that a relatively high level of glycogen can be maintained in Synechocystis mutants with low levels of PGM activity. The high PGM activity in the cyanobacterium may be required for turnover of glycogen or synthesis of other polysaccharides or oligosaccharides.     

拟南芥BLI基因调控植物衰老的研究

BLISTER(BLI)蛋白是多种应激反应的调节因子,可以促进植物对环境的适应性.BLI蛋白通过抑制IRE 1蛋白以维持植物的正常生长,缺失BLI蛋白的拟南芥植株出现多种发育缺陷表型.文章以拟南芥bli突变体为实验材料,通过细胞生物学及遗传学方法检测其衰老相关表征,发现bli突变体植株早期出现叶片黄化、叶绿素含量下降、...     

Effects of Chlorophyll Availability on Fluorescence Components of Photosystems in the ORF469-Deletion Mutant of Cyanobacterium

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Characterization of Arabidopsis calreticulin mutants in response to calcium and salinity stresses

As an important calcium-binding protein, calreticulin plays an important role in regulating calcium homeostasis in endoplasmic reticulum （ER） of plants. Here, we identified three loss-of-function mutants of calreticulin genes in Arabidopsis to demonstrate the function of calreticulin in response to calcium and salinity stresses. There are three genes encoding calreticulin in Arabidopsis, and they are named AtCRT1, 2, and 3, respectively. We found that both single mutant of crt3 and double mutant of crtl crt2 were more sensitive to low calcium environment than wild-type Arabidopsis. Moreover, crt3 mutant showed more sensitivity to salt treatment at germination stage, but tolerance to salt stress at later stage compared with wild-type plant. However, there was no obvious growth difference in the mutant crtl and crt2 compared with wild-type Arabidopsis under calcium and salt stresses. These results suggest that calreticulin functions in plant responses to calcium and salt stresses.     

Sequence of a Drosophila segmentation gene: protein structure homology with DNA-binding proteins

Mutations in the fushi tarazu (ftz) locus of Drosophila result in embryos with half the usual number of body segments. The sequences of the wild-type gene, a temperature-sensitive allele and a dominant mutant allele are presented. A portion of the conserved protein domain present in ftz and several homoeotic genes resembles the DNA-binding region of prokaryotic DNA-binding proteins, and is also similar to products of the yeast mating-type locus.     

酿酒酵母YBR019C基因缺失突变的分析

【目的】研究目的基因YBR019C缺失对酿酒酵母(Saccharomyces cerevisiae)菌株糖代谢和乙醇发酵的影响。【方法】以酿酒酵母野生菌NF1002为出发菌株,选择2号染色体上的基因YBR019C为目的基因,以质粒pUG6和pUG66为模板进行PCR,构建带有Cre/loxP系统的酿酒酵母YBR019C基因敲除组件,并转化酿酒酵母NF1002,利用筛选标记Kan r和Ble r与YBR019C基因进行同源重组,筛选YBR019C双倍体缺陷型菌株。利用蔗糖和甘蔗糖蜜为碳源,对突变菌进行发酵特性的研究。【结果】成功获得YBR019C双倍体缺陷型菌株NFybr。碳源同化实验表明,突变株和野生菌均能利用葡萄糖和蔗糖,不能利用乳糖和木糖;但相比野生菌,突变株利用棉子糖和麦芽糖的能力有所下降,而且完全不能利用半乳糖。蔗糖发酵实验表明:突变株NF-ybr与野生菌株相比,在发酵终点乙醇浓度提高10.7%,发酵周期有所延长。按目前甘蔗糖蜜乙醇生产的发酵工艺,突变株在30℃发酵72h的醪液乙醇含量为12.52%,低于野生菌的13.89%。【结论】YBR019C基因的缺失影响了菌株对糖份的利用,导致乙醇发酵能力不及野生菌。本研究为菌株高效快捷的基因改造提供了参考。     

利用转录组测序分析大豆矮小突变体中差异表达基因

植株高度是影响农作物产量的重要性状,本实验以大豆矮小突变体为研究材料,发现GA3处理可以部分恢复其矮化表型。为了探究其至矮的分子机制,运用RNA-Seq技术测序分析了野生型和突变体的转录组差异,结果表明有多个与植物激素相关的基因差异表达,其中Glyma19g01590与植物激素GA调节有关,生物信息学分析表明差异表达基因Glyma19g01590的开放阅读框有594 bp,推测编码198个氨基酸,该蛋白具有GASA(Gibberellic Acid-Stimulated in Arabidopsis)保守区域,GASA保守区域与GA调节有关。半定量RT-PCR实验表明Glyma19g01590在突变体中的表达水平明显高于野生型,qRT-PCR实验表明Glyma19g01590在突变体的表达量大约是是野生型的24倍。根据以上结果推测Glyma19g01590基因是导致大豆矮小表型的因素之一。     

一个水稻白化致死突变体abl25鉴定及其基因定位

经Co60辐照的粳稻嘉花1号得到一个新的致死白化突变体albino lethal 25(abl25),该突变体从发芽至4叶期表现为白化苗,之后逐渐死亡.与野生型嘉花1号相比,abl25突变体的叶绿素含量和类胡萝卜素的含量大大降低,叶绿体结构不正常,说明其叶绿体发育受到严重阻碍,导致植物死亡.遗传分析表明:该突变体受一对隐性核基因(abl25)控制,进一步利用abl25与广占63S杂交的F2分离群体,将该突变体基因(abl25)定位于第2染色体上SSR标记RM424与Indel分子标记ID7330之间,随后利用新开发的分子标记和扩大群体将其定位在Indel分子标记ID9111和ID9261之间的150 kb内,发现abl25是一个新的水稻苗期白化致死基因.     

拟南芥中一个新的constans等位基因及特性

植物开花是由内外信号途径共同调控的,CONSTANS是长日照途径上控制开花的基因.在筛选拟南芥滞绿突变体的过程中筛选到一个晚花突变体,工作名称为fnc25.后来证实为一个新的constans突变体co-9.在长日照条件下该突变体植株高大,叶片呈深绿色,莲座叶数目增多,开花延迟,寿限显著延长;春化处理和外源施加赤霉素对其开花时间几乎没有响应,在短日照条件下开花时间几乎不变.测序发现co-9中CO基因编码区中有10个碱基的缺失导致了CO蛋白C末端92个氨基酸没有被合成,这其中包含一个CCT结构域.CO基因的功能缺失很可能是导致co-9晚花的原因.RT-PCR实验表明co-9中CO直接调控的基因FT的mRNA水平显著下调,而另一个调控的基因SOC1的mRNA水平和野生型相比没有改变.说明CO通过不同的结构域作用于下游目的基因,一个结构域的改变只影响下游一个目的基因的表达,导致co-9的晚花表型.     

Molecular basis of transmembrane signalling by sensory rhodopsin II-transducer complex

Microbial rhodopsins, which constitute a family of seven-helix membrane proteins with retinal as a prosthetic group, are distributed throughout the Bacteria, Archaea and Eukaryota. This family of photoactive proteins uses a common structural design for two distinct functions: light-driven ion transport and phototaxis. The sensors activate a signal transduction chain similar to that of the two-component system of eubacterial chemotaxis. The link between the photoreceptor and the following cytoplasmic signal cascade is formed by a transducer molecule that binds tightly and specifically to its cognate receptor by means of two transmembrane helices (TM1 and TM2). It is thought that light excitation of sensory rhodopsin II from Natronobacterium pharaonis (SRII) in complex with its transducer (HtrII) induces an outward movement of its helix F (ref. 6), which in turn triggers a rotation of TM2 (ref. 7). It is unclear how this TM2 transition is converted into a cellular signal. Here we present the X-ray structure of the complex between N. pharaonis SRII and the receptor-binding domain of HtrII at 1.94 A resolution, which provides an atomic picture of the first signal transduction step. Our results provide evidence for a common mechanism for this process in phototaxis and chemotaxis.     

高致病性猪链球菌双组分系统Ihk/Irr双基因缺失突变株的构建

目的构建Ⅱ型猪链球菌05ZYH33菌株双组分系统Ihk/Irr的双基因缺失突变株.方法首先对双组分系统Ihk/Irr基因进行序列分析;选取Ihk/Irr基因的相关片段克隆至p ET30a表达载体,进行可溶表达并注射家兔制备抗体;分别将ihk基因上游irr基因下游各1 kb的片段扩增,将两个片段连接起来;克隆至温敏型p JRS233的穿梭质粒中;利用两步同源重组法获得突变体;分别提取突变株的基因组DNA,RNA及菌体总蛋白,利用PCR,RT-PCR,Western-blot方法验证突变体.结果成功制备了双组分系统Ihk/Irr可溶性表达的蛋白,成功构建了重组的温敏型ikr-p JRS233重组质粒,成功将重组质粒转入猪链球菌05ZYH33中并获得突变株,基因组PCR,RTPCR及Western-blot分别证实了双组分系统Ihk/Irr双基因被成功敲除.结论成功构建重组温敏型的穿梭质粒,导入到猪链球菌05ZYH33菌株中经两步同源重组获得突变株,在基因组DNA,RNA及蛋白水平上验证了双组分系统Ihk/Irr双基因缺失株的成功构建.     

Combinational biosynthesis and characterization of a fluorescent 82β-phycocyanin of Spirulina platensis

To biosynthesize fluorescent Spirulina platensis (Sp)β -phycocyanin (PC) in Escherichia coli, a BLASTP search for homologs of the cpeS gene, a chromophore lyase, was performed against the Synechocystis sp. PCC 6803 (S6) proteome. A highly homologous gene, slr2049, was obtained from the S6 genome. Sites 82 and 153 in -phycocyanin of Sp were modified by site-directed muta- genesis. Two recombinant expression vectors were constructed and transformed into E. coli BL21: (i) pCDF-cpcB (C153A)- slr2049-sll0583-ho1-pcyA; and (ii) pCDF-cpcB (C82I)-slr2049-sll0583-ho1-pcyA. Lyases encoded by the genes slr2049 and sll0583 catalyzed the linking of Sp 82β -PC to phycocyanobilin (PCB), and fluorescent CpcB (C153A)-PCB was generated. We present a strategy for the co-expression of multiple genes in a single expression vector to identify the function of an unknown gene. Recombinant phycobiliproteins produced on a large scale are promising fluorescent tags for diagnostics and pharmacology.     

Uncoupling of hypomyelination and glial cell death by a mutation in the proteolipid protein gene.

Proteolipid protein (PLP; M(r) 30,000) is a highly conserved major polytopic membrane protein in myelin but its cellular function remains obscure. Neurological mutant mice can often provide model systems for human genetic disorders. Mutations of the X-chromosome-linked PLP gene are lethal, identified first in the jimpy mouse and subsequently in patients with Pelizaeus-Merzbacher disease. The unexplained phenotype of these mutations includes degeneration and premature cell death of oligodendrocytes with associated hypomyelination. Here we show that a new mouse mutant rumpshaker is defined by the amino-acid substitution Ile-to-Thr at residue 186 in a membrane-embedded domain of PLP. Surprisingly, rumpshaker mice, although myelin-deficient, have normal longevity and a full complement of morphologically normal oligodendrocytes. Hypomyelination can thus be genetically separated from the PLP-dependent oligodendrocyte degeneration. We suggest that PLP has a vital function in glial cell development, distinct from its later role in myelin assembly, and that this dichotomy of action may explain the clinical spectrum of Pelizaeus-Merzbacher disease.     

Involvement of the 'leucine zipper' region in the oligomerization and transforming activity of human c-myc protein

c-Myc plays a part in the regulation of important cellular processes such as growth, differentiation and neoplastic transformation. Although c-myc gene structure and expression are well characterized, the function and biochemical properties of the protein are less well understood. Human c-myc is a 439-amino acid phosphoprotein which binds DNA in vitro and belongs to a discrete subset of nuclear proteins. Using the human c-myc mutants generated by linker-insertion and deletion mutagenesis, we have defined regions of the protein that are important for its transforming activities and its nuclear localization. Here, we show that human c-myc exists as an oligomer in vitro and use mutant proteins to localize the oligomerization domain to a carboxyl-terminal peptide containing the 'leucine zipper' motif. The 'leucine zipper' describes a structure found in a number of DNA-binding proteins that contains leucines occurring at intervals of every seventh amino acid in a region predicted to be alpha-helical. The 'leucine zipper' might mediate dimerization by intermolecular interdigitation of the leucine side-chains. We show that a c-myc mutant, which is inactive but can oligomerize, dominantly inhibits the cotransforming activity with wild-type c-myc of rat embryo cells, whereas inactive mutants which cannot oligomerize properly because of deletions in the oligomerization domain are recessive.     

Identification of a novel tillering dwarf mutant and fine mapping of the TDDL（T） gene in rice （Oryza sativa L.）

Rice plant architecture is an important agronomic trait that affects the grain yield. To understand the molecular mechanism that controls plant architecture, a tillering dwarf mutant with darker-green leaves derived from an indica cultivar IR64 treated with EMS is characterized. The mutant, designated as tddl（t）, is nonallelic to the known tiilering dwarf mutants. It is controlled by one recessive nuclear gene, TDDL（T）, and grouped into the dn-type dwarfism according to Takeda＇s definition. The dwarfism of the mutant is independent of gibberellic acid based on the analyses of two GA-mediated processes. The independence of brassinosteroid （BR） and naphthal-3-acetic acid （NAA） of the tddl（t） mutant, together with the decreased size of parenchyma cells in the vascular bundle, indicates that the TDDL（7） gene might participate in another hormone pathway. TDDL（T） is fine mapped within an 85.51 kb region on the long arm of rice chromosome 4, where 20 ORFs are predicted by RiceGAAS （http：//ricegaas.dna.affrc. go.jp/rgadb/）. Further cloning of TDDL（T） will benefit both marker assisted selection （MAS） of plant architecture and dissection of the molecular mechanism underlying tillering dwarf in rice.     

转录因子TDF1对拟南芥雄性不育突变体atms1的温敏调控机制研究

TDF1作为MYB家族的转录因子,参与了胼胝质的降解过程,并控制绒毡层的分化从而影响着花粉粒的正常发育.以一株通过甲基磺酸乙酯(EMS)诱变Col-0野生型拟南芥获得的温敏雄性不育突变体atms1(ambient temperature-sensory male sterility1)对温度敏感机制进行了研究.通过构建TDF1反义RNA表达载体,在atms1突变体背景下抑制TDF1的表达来观察突变体花粉育性的变化,发现在27℃时的atms1突变体花粉的育性得以恢复.这一结果表明在花粉发育的过程中TDF1对ATMS1m具有一定的调控作用.