小立碗藓扩展蛋白基因家族的鉴定与生物信息学分析

扩展蛋白(Expansins,EXP)是一类基因家族,几乎参与了植物发育的全过程,从种子萌发到果实成熟都有扩展蛋白的参与。该研究利用生物信息学的方法对小立碗藓(Physcomitrella patens) Expansin基因家族成员进行鉴定,分析了其基因结构、染色体定位以及系统发生关系。结果表明:小立碗藓基因组中含有Expansin A(EXPA) 32个、Expansin-like A(EXLA) 6个,并未发现Expansin-like B(EXLB)及Expansin B(EXPB)。扩展蛋白氨基酸序列长度在228～290 aa之间,编码蛋白质具有两个保守的结构域Pollen_allerg_1和DPBB_1。蛋白质亚细胞定位预测结果表明:运用CELLO在线工具预测发现小立碗藓中约4/5的EXP家族基因定位于细胞外;而Euk-mPLoc预测结果则显示小立碗藓EXP基因家族成员全定位于细胞外。基因结构分析表明,小立碗藓中约68%Expansin基因有含有1～3个内含子。以上结果可为深入研究小立碗藓扩展蛋白基因的分子进化与生物学功能奠定基础。     

紫杉醇组合生物合成的研究进展

紫杉醇是植物次级代谢产物中一种具有抗癌作用的萜类物质。但由于在自然界中含量低、难获得使其成本昂贵,临床应用受限。近年来,利用组合生物合成技术制备紫杉醇成为研究热点,研究用的体系以大肠杆菌、酵母为主,还涉及到小立碗藓、拟南芥、番茄、人参等植物体系。文中综述了不同体系中紫杉醇的组合生物合成研究进展,分析了目前存在的问题,并对不同体系的发展方向提出建议。最后,结合本实验室对紫杉醇组合生物合成的小立碗藓体系的研究,阐述了其发展前景和优势。     

小立碗藓LysM型类受体激酶基因家族生物信息学分析

植物LysM型类受体激酶(lysin motif receptor-like kinase,LYKs)是植物中发现的一类重要的RLK,在植物生长发育、抵御逆境胁迫等方面具有不可忽视的作用,是植物中基因功能的研究热点。为更好地了解小立碗藓中的LYK基因,该文利用生物信息学的方法对小立碗藓(Physcomitrella patens) LysM型类受体激酶基因家族成员进行鉴定及分析。通过分析小立碗藓LYK家族成员的基本物理信息、基因结构、染色体定位及系统发生关系,初步探讨了其LYK基因结构、进化与功能间的联系。结果表明:(1)小立碗藓中共有21个LYK基因,其氨基酸序列大小在625～755 aa之间,分子量为69.54～82.02 kDa,等电点在5.98～7.78之间。(2)将小立碗藓所有LYK蛋白与3种典型模式植物(水稻、拟南芥和蒺藜苜蓿)的LYK蛋白共同构建系统进化树,所有LYK蛋白被分为4个亚组(LYK-I、LYK-Ⅱ、LYR-I和LYR-Ⅱ)。小立碗藓各亚组内成员的基因结构、保守域特征显示出较为相似的特征,由此推测其可能具有相同或相似的功能。(3)染色体定位发现21个LYK基因集中分布于4条染色体上并出现小型基因簇,这可能与基因功能相联系。该文分析了小立碗藓LysM型类受体蛋白激酶基因家族的基本信息,可为后续深入研究其LYK基因家族成员的生理生化功能奠定基础。     

植物分子生物学研究极具前景的模式系统——小立碗藓

小立碗藓作为植物分子生物学研究极具前景的模式系统已日益受到人们的重视,它的生活史周期短,易于培养,转基因植株易于分析,核基因组容易和外源DNA 发生同源重组,这些特点使它成为研究基因功能的良好材料。一些成功的基因敲除和基因破坏已经在小立碗藓中实现,这些基因的功能也通过小立碗藓转化植株的特点得以证实。小立碗藓标签突变文库已经建立,其应用为小立碗藓基因的进一步研究打下了基础。关于小立碗藓的ESTs 数据库已经建立,已有67 000 条ESTs 信息。     

植物分子生物学研究极具前景的模式系统--小立碗藓

小立碗藓作为植物分子生物学研究极具前景的模式系统已日益受到人们的重视,它的生活史周期短,易于培养,转基因植株易于分析,核基因组容易和外源DNA发生同源重组,这些特点使它成为研究基因功能的良好材料.一些成功的基因敲除和基因破坏已经在小立碗藓中实现,这些基因的功能也通过小立碗藓转化植株的特点得以证实.小立碗藓标签突变文库已经建立,其应用为小立碗藓基因的进一步研究打下了基础.关于小立碗藓的ESTs数据库已经建立,已有67 000条ESTs信息.     

小立碗藓PpAGO7基因的克隆及表达分析

该研究采用同源克隆的方法获得了小立碗藓AGO7蛋白编码基因PpAGO7,对PpAGO7基因序列特征进行生物信息学分析,并利用Real-time PCR方法分析PpAGO7基因的时空表达模式,为揭示小立碗藓中PpAGO7基因的生物学功能提供依据。结果表明:PpAGO7基因的全长cNDA为3 583bp,其中开放阅读框3 363bp,编码1 120个氨基酸,分子量123.38kD,等电点9.26,含有AGO蛋白典型的PAZ和PIWI结构域;PpAGO7基因在小立碗藓整个生活周期都有表达,且在茎叶体生长时期表达水平较高,但在原丝体时期表达水平较低。研究结果推测PpAGO7基因可能在茎叶体拟叶的生长发育过程中起作用。     

小立碗藓（Physcomitrella patens）植物microRNA结合靶位预测

利用857条植物miRNA序列对27546条小立碗藓mRNA序列进行搜索,预测出162个植物miRNA家族在小立碗藓中存在结合靶位。miRNA结合靶位数目和miRNA协同作用网络分析结果同时显示,miR482和miR1168在小立碗藓中结合靶位多、协同作用广,提示它们对于小立碗藓可能具有重要生物学功能。52个菜茵衣藻特有的miRNA被预测在小立碗藓中存在结合靶位,显示小立碗藓在从藻类向种子植物进化过程中处在独特演化位置。     

RNA编辑修复藻苔(Takakia lepidozioides)叶绿体accD基因中的突变

藻苔(Takakia lepidozioides)叶绿体基因组中存在着非常高的RNA编辑频率,这种现象至今没有得到很好的解释。本研究采用比较基因组学方法,对小立碗藓(Physcomitrella patens)、青苔(Syntrichia ruralis)和藻苔3种苔藓植物中accD基因的DNA序列和基因编码的蛋白氨基酸序列进行比较分析。研究发现,经过RNA编辑,藻苔的基因产物获得了与其同源基因相同的氨基酸序列。藻苔中高频发生的RNA编辑是对基因组中DNA突变的一种修复,提高了藻苔对强辐射环境的生存适应能力。     

基于HMM的齿肋赤藓VOZ转录因子的预测与分析

VOZ(Vascular plant One Zinc finger protein)作为与植物的进化与发育密切相关的基因,在极端耐旱荒漠苔藓植物齿肋赤藓(Syntrichia caninervis)中对VOZ基因进行挖掘和分析有利于更好的揭示VOZ基因的进化关系,且可作为抗逆基因进行更为深入的分子生物学研究。在VOZ转录因子蛋白中VOZ-domain是一个保守的DNA结合结构功能域,利用VOZ-domain多序列联配构建隐马尔可夫模型序列谱能够很好的进行家族成员的识别和预测。利用拟南芥、小立碗藓和水稻等植物已知的转录因子序列信息构建HMM序列谱模型,对荒漠苔藓齿肋赤藓转录组进行比对搜索。最终得到一条新的齿肋赤藓VOZ转录因子ScVOZ1(NCBI/EBI检索号:HG764415),序列长度为1 495 bp,具有完整的VOZ-domain结构域。生物信息学分析表明其具有转录调控功能和核定位潜能。多序列比对、进化和保守基序分析表明,ScVOZ1蛋白序列与小立碗藓VOZ家族和拟南芥AtVOZ1相似度较高。本研究为进一步研究ScVOZ1基因的功能以及其进化起源奠定了基础。     

小立碗藓PpAGO7基因启动子分离及其启动活性分析

AGO7蛋白在多种植物中被发现并预测在叶片生长发育过程中起作用,但是在高等植物中最古老且唯一没有维管束的苔藓植物中却未有报道。该文通过BLAST比对水稻OsAGO7基因编码的氨基酸序列得到小立碗藓AGO7蛋白编码基因PpAGO7,扩增PpAGO7基因起始密码子上游的启动子序列,利用在线软件PlantCARE和PLACE分析该启动子的结构特征;并构建启动子分析载体pPpAGO7-GUS,转化拟南芥,通过转基因拟南芥GUS染色结果分析推测PpAGO7启动子的启动特性。结果显示:(1)PpAGO7基因启动子序列中含有大量的光反应有关的顺式作用元件以及数个分生组织相关和防御与胁迫相关作用元件。(2)T2代转基因拟南芥的GUS染色结果表明,PpAGO7基因启动子会启动GUS在拟南芥的不同部位和不同生长时期表达,而且在根尖、叶片顶端、雄蕊的花药、雌蕊的柱头和种子等部位的染色较其他部位更深。(3)生长在光照强度1 000lx和4 000lx下转基因拟南芥的GUS酶活性比光照强度7 000lx和10 000lx下的低。研究表明所克隆的PpAGO7基因启动子具有组成性启动活性,且在生长旺盛的部位启动活性较强,此外其启动活性还受到光照因素的影响,为进一步研究小立碗藓的PpAGO7基因功能提供了重要依据。     

PpASCL, a moss ortholog of anther-specific chalcone synthase-like enzymes, is a hydroxyalkylpyrone synthase involved in an evolutionarily conserved sporopollenin biosynthesis pathway

Sporopollenin is the main constituent of the exine layer of spore and pollen walls. Recently, several Arabidopsis genes, including polyketide synthase A (PKSA), which encodes an anther-specific chalcone synthase-like enzyme (ASCL), have been shown to be involved in sporopollenin biosynthesis. The genome of the moss Physcomitrella patens contains putative orthologs of the Arabidopsis sporopollenin biosynthesis genes. We analyzed available P.patens expressed sequence tag (EST) data for putative moss orthologs of the Arabidopsis genes of sporopollenin biosynthesis and studied the enzymatic properties and reaction mechanism of recombinant PpASCL, the P.patens ortholog of Arabidopsis PKSA. We also generated structure models of PpASCL and Arabidopsis PKSA to study their substrate specificity. Physcomitrella patens orthologs of Arabidopsis genes for sporopollenin biosynthesis were found to be expressed in the sporophyte generation. Similarly to Arabidopsis PKSA, PpASCL condenses hydroxy fatty acyl-CoA esters with malonyl-CoA and produces hydroxyalkyl α-pyrones that probably serve as building blocks of sporopollenin. The ASCL-specific set of Gly-Gly-Ala residues predicted by the models to be located at the floor of the putative active site is proposed to serve as the opening of an acyl-binding tunnel in ASCL. These results suggest that ASCL functions together with other sporophyte-specific enzymes to provide polyhydroxylated precursors of sporopollenin in a pathway common to land plants.     

Cloning of the PpMSH-2 cDNA of Physcomitrella patens, a moss in which gene targeting by homologous recombination occurs at high frequency

In the moss Physcomitrella patens integrative transformants from homologous recombination are obtained at an efficiency comparable to that found for yeast. This property, unique in the plant kingdom, allows the knockout of specific genes. It also makes the moss a convenient model to study the regulation of homologous recombination in plants. We used degenerate oligonucleotides designed from AtMSH2 from Arabidopsis thaliana and other known MutS homologues to isolate the P. patens MSH2 (PpMSH2) cDNA. The deduced sequence of the PpMSH2 protein is respectively 60.8% and 59.6% identical to the maize and A. thaliana MSH2. Phylogenic studies show that PpMSH2 is closely related to the group of plant MSH2 proteins. Southern analysis reveals that the gene exists as a single copy in the P. patens genome.     

Classification of the genes involved in the two-component system of the moss Physcomitrella patens

Physcomitrella patens, belonging to bryopsida, is a basal lineage of land plants. To gain insight into the diversification of the two-component system (TCS), which is widely conserved from prokaryotes to eukaryotes, we compiled TCS-associated genes by employing P. patens genome databases. The moss has a set of His-kinases (HKs), including homologs of the cytokinin- and ethylene-receptors in seed plants. In addition, it has a number of coding-sequences specifying unique HKs. We found evidence that a putative cytokinin-receptor HK in P. patans serves as a sensor for this hormone, and that the HK activity of a putative ethylene-receptor homolog is regulated by ethylene, as observed for Arabidopsis thaliana.     

Genomic organization of plant terpene synthases and molecular evolutionary implications

Terpenoids are the largest, most diverse class of plant natural products and they play numerous functional roles in primary metabolism and in ecological interactions. The first committed step in the formation of the various terpenoid classes is the transformation of the prenyl diphosphate precursors, geranyl diphosphate, farnesyl diphosphate, and geranylgeranyl diphosphate, to the parent structures of each type catalyzed by the respective monoterpene (C(10)), sesquiterpene (C(15)), and diterpene synthases (C(20)). Over 30 cDNAs encoding plant terpenoid synthases involved in primary and secondary metabolism have been cloned and characterized. Here we describe the isolation and analysis of six genomic clones encoding terpene synthases of conifers, [(-)-pinene (C(10)), (-)-limonene (C(10)), (E)-alpha-bisabolene (C(15)), delta-selinene (C(15)), and abietadiene synthase (C(20)) from Abies grandis and taxadiene synthase (C(20)) from Taxus brevifolia], all of which are involved in natural products biosynthesis. Genome organization (intron number, size, placement and phase, and exon size) of these gymnosperm terpene synthases was compared to eight previously characterized angiosperm terpene synthase genes and to six putative terpene synthase genomic sequences from Arabidopsis thaliana. Three distinct classes of terpene synthase genes were discerned, from which assumed patterns of sequential intron loss and the loss of an unusual internal sequence element suggest that the ancestral terpenoid synthase gene resembled a contemporary conifer diterpene synthase gene in containing at least 12 introns and 13 exons of conserved size. A model presented for the evolutionary history of plant terpene synthases suggests that this superfamily of genes responsible for natural products biosynthesis derived from terpene synthase genes involved in primary metabolism by duplication and divergence in structural and functional specialization. This novel molecular evolutionary approach focused on genes of secondary metabolism may have broad implications for the origins of natural products and for plant phylogenetics in general.     

Gene targeting in Physcomitrella patens

Gene-targeting efficiency in the land plant Physcomitrella patens (Bryophyta) can only be compared with that observed in Saccharomyces cerevisiae. Sequencing programs and microbiological molecular genetic approaches are now being developed to unravel the precise function of plant genes. Physcomitrella patens, as the new 'green yeast', might well become a major tool for functional genomic studies of multicellular eukaryotes.     

An Arabidopsis homolog of the bacterial peptidoglycan synthesis enzyme MurE has an essential role in chloroplast development

Enzymes encoded by bacterial MurE genes catalyze the ATP-dependent formation of uridine diphosphate- N -acetylmuramic acid-tripeptide in bacterial peptidoglycan biosynthesis. The Arabidopsis thaliana genome contains one gene with homology to the bacterial MurE : AtMurE . Under normal conditions AtMurE is expressed in leaves and flowers, but not in roots or stems. Sequence-based predictions and analyses of GFP fusions of the N terminus of AtMurE, as well as the full-length protein, suggest that AtMurE localizes to plastids. We identified three T-DNA-tagged and one Ds -tagged mutant alleles of AtMurE in A. thaliana . All four alleles show a white phenotype, and A. thaliana antisense AtMurE lines showed a pale-green phenotype. These results suggest that AtMurE is involved in chloroplast biogenesis. Cells of the mutants were inhibited in thylakoid membrane development. RT-PCR analysis of the mutant lines suggested that the expression of genes that depend on a multisubunit plastid-encoded RNA polymerase was decreased. To analyze the functional relationships between the MurE genes of cyanobacteria, the moss Physcomitrella patens and higher plants, a complementation assay was carried out with a P. patens ( Pp ) MurE knock-out line, which exhibits a small number of macrochloroplasts per cell. Although the Anabaena MurE, fused with the N-terminal region of PpMurE, complemented the macrochloroplast phenotype in P. patens , transformation with AtMurE did not complement this phenotype. These results suggest that AtMurE is functionally divergent from the bacterial and moss MurE proteins.