Cloning and functional analysis of chloroplast division gene NtFtsZ2-1 in Nicotiana tabacum

FtsZ protein plays an important role in the division of chloroplasts. With the finding and functional analysis of higher plant FtsZ proteins, people have deepened the understanding in the molecular mechanism of chloroplast division. Multiple ftsZ genes are diversified into two families in higher plants, ftsZ1 and ftsZ2. On the basis of the research on ftsZ1 family, we analyzed the function of NtFtsZ2-1 gene in Nicotiana tabacum. Microscopic analysis of the sense and antisense NtFtsZ2-1 transgenic tobacco plants revealed that the chloroplasts were abnormal in size and also in number when compared with wild-type tobacco chloroplasts. Our investigations confirmed that the NtFtsZ2-1 gene is involved in plant chloroplast division.     

Cloning and functional analysis of chloroplast division gene NtFtsZ2-l in Nicotiana tabacum

FtsZ protein plays an important role in the division of chloroplasts. With the finding and functional analysis of higher plant FtsZ proteins, people have deepened the understanding in the molecular mechanism of chloroplast division. Multiple ftsZ genes are diversified into two families in higher plants, ftsZ1 and ftsZ2 . On the basis of the research on ftsZl family, we analyzed the function of NtFtsZ2-l gene in Nicotiana tabacum . Microscopic analysis of the sense and antisense NtFtsZ2-l transgenic tobacco plants revealed that the chloroplasts were abnormal in size and also in number when compared with wild-type tobacco chloroplasts. Our investigations confirmed that the NtFtsZ2-l gene is involved in plant chloroplast division.     

Cloning of plastid division gene GlFtsZ from Gentiana lutea and its expression during petal development

A full-length cDNA of GlFtsZ was isolated by screening the cDNA library of Gentiana lutea. Analysis of the deduced amino acid sequence encoded by GlFtsZ indicated that GlFtsZ protein possesses the typical conservative motifs existed in all FtsZ proteins. The existence of putative plastid transit peptide in its N-terminus suggested that GlFtsZ might function inside of plastids. With the deve- lopmental process of petals of Gentiana lutea, the expression of plastid division gene GlFtsZ declined gradually, whereas the expression of carotenoids biosynthesis gene Zds increased obviously; meanwhile, in contrast to the increment of carotenoids, the content of chlorophyll in petals decreased sharply. The chloroplasts turned into chromoplasts, and the color of petals also turned from green to golden. All of these results suggested that the expression of GlFtsZ is accompanied with the development and differentiation of plastids.     

<Emphasis Type="Italic">ftsZ</Emphasis> gene and plastid division

As the important cellular organelles in plants, plas-tids comprise one of the primary features that distinguish plant cells from those of other eukaryotes. Seen from the origin, plastids derive from endosymbiotic photosynthetic bacteria. Subsequently, plastids have evolved to become essential components for plant cell function. Besides the important role of chloroplasts in photosynthesis, some water-soluble proteins that involved in biosynthesis of starch, fatty acids, amino acids, nucleic aci…     

菠菜性别差异NUPTs序列的克隆及分析

质体DNA向核基因组转移能够形成核质体DNA,核质体DNA序列的插入是推动动植物基因组及染色体组演化的重要动力.但是核质体DNA的插入和植物性染色体起源及演化之间的关系仍不清楚.以雌雄异株植物菠菜为材料,利用基因组消减杂交技术筛选分离菠菜雌雄基因组差异的NUPTs序列,并进行验证和分析.结果表明,从构建的菠菜消减杂交文库中共得到39条长度为75~308 bp的雌雄差异序列,其平均长度约为154 bp.对获得的序列进行Blastn同源比对发现了12条序列为叶绿体基因组来源序列,这些序列与菠菜叶绿体基因组相似度在98%以上,说明所获得的差异片段为核质体DNA序列.将筛选出的核质体DNA序列进行进一步验证后获得了两个稳定的长度分别为146 bp和199 bp的雄性偏好核质体DNA序列,说明所获得的两个NUPTs序列在菠菜雄性基因组中有更多的累积.     

Transfer of a eubacteria-type cell division site-determining factor CrMinD gene to the nucleus from the chloroplast genome in Chlamydomonas reinhardtii

MinD is a ubiquitous ATPase that plays a crucial role in selection of the division site in eubacteria, chloroplasts, and probably Archaea. In four green algae, Mesostigma viride, Nephroselmis olivacea, Chlorella vulgaris and Prototheca wickerhamii, MinD homologues are encoded in the plastid genome. However, in Arabidopsis, MinD is a nucleus-encoded, chloroplast-targeted protein involved in chloro- plast division, which suggests that MinD has been transferred to the nucleus in higher land plants. Yet the lateral gene transfer (LGT) of MinD from plastid to nucleus during plastid evolution remains poorly understood. Here, we identified a nucleus-encoded MinD homologue from unicellular green alga Chlamydomonas reinhardtii, a basal species in the green plant lineage. Overexpression of CrMinD in wild type E. coli inhibited cell division and resulted in the filamentous cell formation, clearly demon- strated the conservation of the MinD protein during the evolution of photosynthetic eukaryotes. The transient expression of CrMinD-egfp confirmed the role of CrMinD protein in the regulation of plastid division. Searching all the published plastid genomic sequences of land plants, no MinD homologues were found, which suggests that the transfer of MinD from plastid to nucleus might have occurred be- fore the evolution of land plants.     

Transfer of a eubacteria-type cell division site-determining factor CrldfnD 8ene to the nucleus from the chloroplast genome in Chlamydomonas reinhardtii

MinD is a ubiquitous ATPase that plays a crucial role in selection of the division site in eubacteria, chloroplasts, and probably Archaea. In four green algae, MesosUgma viride, Nephroselmis olivacea, Chlorella vulgaris and Prototheca wickerhamii, MinD homologues are encoded in the plastid genome. However, in Arabidopsis, MinD is a nucleus-encoded, chloroplast-targeted protein involved in chloroplast division, which suggests that MinD has been transferred to the nucleus in higher land plants. Yet the lateral gene transfer （LGT） of MinD from plastid to nucleus during plastid evolution remains poorly understood. Here, we identified a nucleus-encoded MinD homologue from unicellular green alga Chlamydomonas reinhardtii, a basal species in the green plant lineage. Overexpression of CrMinD in wild type E. coil inhibited cell division and resulted in the filamentous cell formation, clearly demonstrated the conservation of the MinD protein during the evolution of photosynthetic eukaryotes. The transient expression of CrMinD-egfp confirmed the role of CrMinD protein in the regulation of plastid division. Searching all the published plastid genomic sequences of land plants, no MinD homologues were found, which suggests that the transfer of MinD from plastid to nucleus might have occurred before the evolution of land plants.     

甜菊钙调蛋白基因的克隆及结构分析

钙调蛋白（Calmodulin）是生物细胞内一种重要的调控蛋白，通过其与靶酶的相互作用控制细胞正常的生长发育及细胞对外界环境变化的反应，我们从甜菊顶芽和花芽中提取总RNA，逆转录合成cDNA第一链，以此为模板，参考GenBank上已发表植物的钙调蛋白基因序列合成5′端和3′端引物，利用多聚酶链式反应（PCR）扩增并克隆得到了甜菊钙调蛋白基因的两个异型基因，序列分析表明，它们均由450个核苷酸组成，编码148个氨基酸，在核苷酸序列上与迄今已知的多种植物钙调蛋白均有很高的同源性，同源率在83％以上，编码的氨基酸序列同源性更同，同源率高达95％以上，这两个基因之间存在差异，其核苷酸序列同源率为85％，编码区的氨基酸序列的同源率为99％，仅在第122个氨基酸由ALA代替了VAL。     

Isolation of cDNAs Enconding Two Distinct Transcription Factors Binding to DRE cis-acting Element Involved in Cold- and drought-induced Expression of Arabidopsis rd29A Gene

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The essential bacterial cell-division protein FtsZ is a GTPase.

Cytokinesis defines the last stage in the division cycle, in which cell constriction leads to the formation of daughter cells. The biochemical mechanisms responsible for this process are poorly understood. In bacteria, the ftsZ gene product, FtsZ, is required for cell division, playing a prominent role in cytokinesis. The cellular concentration of FtsZ regulates the frequency of division and genetic studies have indicated that it is the target of several endogenous division inhibitors. At the time of onset of septal invagination, the FtsZ protein is recruited from the cytoplasm to the division site, where it assembles into a ring that remains associated with the leading edge of the invaginating septum until septation is completed. Here we report that FtsZ specifically binds and hydrolyses GTP. The reaction can be dissociated into a GTP-dependent activation stage that is markedly affected by the concentration of FtsZ, and a hydrolysis stage in which GTP is hydrolysed to GDP. The results indicate that GTP binding and hydrolysis are important in enabling FtsZ to support bacterial cytokinesis, either by facilitating the assembly of the FtsZ ring and/or by catalysing an essential step in the cytokinetic process itself.     

Escherichia coli cell-division gene ftsZ encodes a novel GTP-binding protein.

Escherichia coli divides by forming a septum across the middle of the cell. The biochemical mechanism underlying this process is unknown. Genetic evidence suggests that of all the fts (filamentation temperature sensitive) genes involved in E. coli cell division, ftsZ plays a central role at the earliest known step of septation. Here we show that FtsZ protein binds GTP in vitro using unusual sequence elements. In contrast, such binding to the product of the conditional-lethal ftsZ84 allele is impaired. Purified FtsZ displays a Mg(2+)-dependent GTPase activity which is markedly reduced in the FtsZ84 protein. FtsZ copurifies with near stoichiometric amounts of noncovalently-bound GDP, implying the presence of a GTPase cycle in vivo, similar to that known for signal-transducing GTP-binding proteins. We also show that a small fraction of FtsZ exists as a distinct membrane-associated species that binds GTP. The membrane association of FtsZ and the known ability of GTPases to act as molecular switches implicate FtsZ in a GTP-activated signal transduction pathway that may regulate the start of septation in E. coli.     

不同折叠类型蛋白中基于密码子的二级结构偏向性分析

在传统的Chou-Fasman蛋白质二级结构预测方法的基础上引入同义密码子使用的信息,计算了200个蛋白(49种全α结构蛋白,69种全β结构蛋白,38种仅α β结构蛋白,44种α/β结构蛋白)中不同密码子对应的氨基酸形成不同二级结构(α：螺旋,β：折叠,C：卷曲)的偏向性参数．通过对这些密码子对应氨基酸二级结构偏向性的分析,得到了氨基酸二级结构偏向性分析中所忽略的同义密码子的蛋白结构信息．这些新的信息量对于指导蛋白质设计以及提高蛋白质二级结构预测的准确率有着一定的作用．     

西瓜八氢番茄红素合成酶基因全长的克隆与表达分析

通过RT-PCR和RACE技术从西瓜果实中克隆八氢番茄红素合成酶（PSY）基因的cDNA全长,用生物信息学方法对其cDNA序列及推测氨基酸序列进行分析,并用实时荧光定量PCR技术研究PSY在不同瓤色西瓜果实发育过程中的表达情况.结果表明,PSY基因cDNA全长1 561 bp（GenBank登录号为KC166870）,其开放阅读框为1 266 bp,编码421个氨基酸.该基因及其推导的氨基酸序列与同为葫芦科的其他植物的PSY及氨基酸序列的同源性分别为87％和91％以上;序列分析显示,PSY氨基酸序列N末端存在转运肽信号序列.不同瓤色西瓜果实发育过程中PSY的表达存在明显差异,红瓤果实中的表达量最高,这表明PSY可能与红瓤果实中积累较多的类胡萝卜素有关;PSY的表达量均高于PSY-A,推测PSY基因主要负责果实中类胡萝卜素的合成.     

叶绿体CrFtsZ2蛋白对E.coli形态的影响

FtsZ蛋白在细菌的分裂中担任着重要作用,能够在分裂位点形成一个环状结构而控制细菌的分裂过程。胞内FtsZ蛋白浓度的异常升高或降低均可阻断正常的细胞分裂过程进而形成分裂异常的丝状菌体。我们为了进一步研究衣藻FtsZ蛋白的生物学活性,建立了CrFtsZ2-EGFP融合蛋白原核表达的对照体系。结果表明：低浓度IPTG促进转化有表达质粒pLGZ2的大肠杆菌JM109伸长,高浓度IPTG则抑制其伸长;融合表达质粒的过量表达导致宿主菌形成了丝状菌体,不但菌体长度随浓度梯度而有规律性的变化,而且CrFtsZ2-EGFP融合蛋白沿着宿主菌体的纵轴方向有规律地聚集成荧光点或荧光带。更进一步验证了衣藻CrFtsZ2蛋白的功能。     

Genome organization and phylogenetic tree analysis of Garlic virus E, a new member of genus Allexivirus

The complete sequence of an Allexivirus isolated from garlic plants in Yuhang City, Zhejiang Province, China had been determined. The single-strand, positive RNA genome was 8451 nucleotides in length excluding poly(A) tail. The genome organization of this virus was similar to that of the other Allexiviruses but only with 62.8%-64.8% nucleotide acid identities. The amino acid sequences of proteins encoded by ORF1-6 shared 67.6%-78.5%, 55.4%-66.2%, 56.7%- 66.4%, 40.3%-55.6%, 66.3%-79.7% and 52.2%- 68.8% identities with those of the others respectively. The homology range between it and the other Allexiviruses was similar to that between the other distinct species in this genus. A more comprehensive comparison using all available CP amino acid sequences showed that it shared only 63.9%- 79.8% amino acids identical with the others. Therefore, it had been considered as a new member of the genus, named as garlic virus E (GarV-E). Phylogenetic analysis confirmed GarV-E as a distinct member and the correct names and classification of some members of genus Allexivirus were also discussed.     

Yeast and mammalian ras proteins have conserved biochemical properties

Mammalian ras oncogenes encode polypeptides of relative molecular mass (Mr) 21,000 (p21) which bind GTP and GDP. Oncogenic ras-encoded proteins differ from their normal homologues by an amino acid substitution for Gly 12, Ala 59 or Gln 61. Recently, we and others have observed that normal p21, encoded by the Ha-ras gene, has a GTP hydrolytic activity that is reduced by the oncogenic substitutions Val 12 or Thr 59. The yeast Saccharomyces cerevisiae contains two ras-related genes, RASsc1 and RASsc2, the expression of either of which is sufficient for viability. RASsc1 and RASsc2 encode proteins of 309 (SC1) and 322 (SC2) residues which are 62% homologous to mammalian p21 in their 172-amino acid N-terminal sequences. We report here that the N-terminal domain of SC1 binds GTP and GDP and has a GTP hydrolytic activity that is reduced in the variants SC1[Thr 66] and SC1[Leu 68] which are analogous to oncogenic Ha[Thr 59] and Ha[Leu 61], respectively. These results suggest that yeast and mammalian ras proteins have similar biochemical and possibly biological functions.     

FtsZ ring structure associated with division in Escherichia coli

Genes for cell division have been identified in Escherichia coli by the isolation of conditional lethal mutations that block cell division, but do not affect DNA replication or segregation. Of these genes, ftsZ is of great interest as it acts earliest in the division pathway, is essential, its level dictates the frequency of division, and it is thought to be the target of two cell-division inhibitors, SulA, produced in response to DNA damage, and MinCD, which prevents division at old sites. Here we have used immunoelectronmicroscopy to localize the FtsZ protein to the division site. The results suggest that FtsZ self-assembles into a ring structure at the future division site and may function as a cytoskeletal element. The formation of this ring may be the point at which division is regulated.     

桑树MaACS和MaACO基因的鉴定和表达模式研究

研究目的：分离和鉴定桑树中参与乙烯生物合成的酶的编码基因MaACS 和MaACO,研究其表达模式。创新要点：基于最新公布的桑树基因组数据库数据,获得5个MaACS 基因和2个MaACO 基因,对其进行了生物信息分析,同时鉴定了其在不同桑树组织中、不同发育时期桑椹中和不同激素作用下的表达模式。研究方法：通过生物信息学方法筛选和鉴定基因,利用荧光定量逆转录聚合酶链式反应（qRT-PCR）分析基因的表达量。重要结论：MaACS 和MaACO 基因在根、茎、叶等不同组织中呈现出不同的表达模式,在桑椹发育过程中呈现出两种表达模式,其表达量被脱落酸和乙烯利上调。