拟南芥AtVQ29基因的克隆与植物表达载体构建

目的:VQ模序蛋白是植物中所特有的一类具有高度保守序列的蛋白质,广泛参与植物的生长发育与逆境反应,本研究拟克隆拟南芥的AtVQ29基因并进一步构建由组成型启动子CaMV 35S驱动的植物表达载体pSN1301-AtVQ29。方法:采用CTAB法提取拟南芥基因组DNA,根据已报道的AtVQ29基因序列设计并合成引物,通过PCR技术扩增获得拟南芥AtVQ29基因,经T载体克隆后测序。利用生物信息学软件对序列进行初步分析,同时基于基因重组技术构建植物表达载体。结果:序列分析表明已成功克隆AtVQ29基因,该基因编码区全长为372bp,共编码123个氨基酸残基,具有保守的VQ模序。并进一步构建了由组成型启动子CaMV 35S驱动的AtVQ29基因植物表达载体pSN1301-AtVQ29。结论:本研究所构建的AtVQ29基因植物表达载体能够在转基因植株中过量表达AtVQ29基因,为后期开展基因功能研究与植物基因改良奠定了基础。     

小立碗藓叶绿体分裂相关基因PpMinE的克隆及其功能与进化分析

用RT-PCR技术从小立碗藓中(Physcomitrella patens)克隆了核编码的MinE基因,命名为PpMinE,并克隆了该基因的基因组DNA。序列比对显示该基因编码的蛋白质与真细菌和绿藻叶绿体编码的MinE蛋白具有较高的相似性。pMinE-EGFP融合蛋白在烟草中的瞬时表达证明该蛋白定位于叶绿体内。在大肠杆菌中过量表达PpMinE导致细胞不正常分裂,产生无染色体的小细胞,这表明MinE的功能在进化上是保守的。在系统发育树中,PpMinE和高等陆生植物有较近的亲缘关系。在已知的陆生植物的叶绿体基因组中没有找到MinE的同源蛋白,这暗示在进化过程中MinE从叶绿体到细胞核的水平转移可能发生在陆生植物发生以前。     

玉米大斑病抗性基因Ht1定位区域内候选序列的生物信息学分析

为获得玉米大斑病抗性基因Ht1候选序列, 文章采用生物信息学方法对与玉米大斑病抗性基因Ht1紧密连锁的分子标记umc22和umc122定位区域内候选序列进行了分析, 其中得到的63条ORF序列中有14条序列可编码蛋白质结构域。将14条核苷酸酸序列预测出的氨基酸序列与已克隆的24条抗性基因编码氨基酸序列进行Blast比对及进化树构建。结果发现, 候选序列gpm565a具有植物抗性基因编码产物的高度保守结构域, 而且与抗性基因Xal相似性高、亲缘关系近, 推测可能与抗性基因Ht1有关。其他候选序列由于不具有植物抗性基因编码产物的高度保守结构域或者相似性低、亲缘关系远等原因, 不能确定与抗性基因Ht1有关。通过对候选序列gpm565a进行二级结构及三维结构分析, 发现有大量构成蛋白质特异功能结构组件的无规则卷曲存在, 推测gpm565a可能是Ht1功能域的一部分。     

小麦泛素融合降解蛋白基因的克隆及特征分析

酵母UFD1基因编码的泛素融合降解蛋白是泛素依赖性降解系统或泛素融合降解途径中的一个关键因子。利用RT-PCR技术在小麦(Triticum aestivum L.)中分离到一个UFD1类似基因。该基因的编码区长948 bp,编码长315个氨基酸的多肽,其氨基酸序列与GenBank中登录的一个拟南芥UFD1类似蛋白有74％的同源性。在多肽链的N-端具有在真核生物中高度保守的UFD1结构域。我们将该基因定位在小麦的第六染色体群并将其命名为了UFD1。Southern杂交和数据库搜索表明植物的UFD1基因是单拷贝或低拷贝的。无论是在单子叶中还是在双子叶植物中,UFD1蛋白都高度同源。除了N端UFD1结构域外,该类蛋白还有3个高度保守的C端结构域。TUFD1基因在小麦幼苗的根、茎、胚芽鞘、叶片以及幼穗和腊熟期子粒中呈组成性表达。     

一组高度保守的水稻trs-like基因的鉴定与分析

基于对水稻基因组序列的注解和同源搜索的结果,用RT-PCR结合测序的方法证明了水稻中至少有10个具有转录活性的trs-like基因。这10个基因的编码产物与酵母TRAPP蛋白复合体已知10个亚基中的6个分别同源。其中4对基因是双拷贝的,另2个则是单拷贝的(基于已知的水稻基因组序列)。所有这10个基因均在不同时期的水稻组织中广泛表达,并与其他真核生物的trs-like基因在基因结构及编码蛋白质序列水平上高度保守。     

新基因水稻OsLSD1的克隆及拟南芥和水稻类 LSD1基因家族的生物信息学分析

类LSD1 (LSD1-like)基因家族是一类特殊的C2C2型锌指蛋白基因,编码植物特有的转录因子.目前已经研究的2个成员拟南芥LSD1(1esions stimulating disease resistance 1)和LOL1(LSD-One-Like 1)基因均参与植物细胞程序化死亡(programmed cell death,PCD)的调控.从水稻cDNA文库中克隆到1个类LSD1基因,命名为OsLSD1.该基因长988 bp,包含一个432bp的开放阅读框,推导的氨基酸序列(143个氨基酸)含有3个内部保守的锌指结构域.DNA印迹结果表明OsLSD1基因在水稻基因组中为单拷贝,且在根、茎和叶中表达.借助于生物信息学分析技术,从拟南芥和水稻数据库中各识别出5个和7个(包括OsLSD1)类LSD1基因.分析了这些类LSD1基因的结构,蛋白质结构域组成.系统进化分析表明,无论基于编码区的核苷酸或氨基酸序列都可以将这些类LSD1基因分为2类.虽然不存在拟南芥或水稻特有的类LSD1蛋白,但有些结构域是水稻所特有的,也有些基因是来源于复制事件.     

新基因水稻 OsLSD1 的克隆及拟南芥和水稻类 LSD1 基因家族的生物信息学分析

类 LSD1 (LSD1-like) 基因家族是一类特殊的 C2C2 型锌指蛋白基因,编码植物特有的转录因子 . 目前已经研究的 2 个成员拟南芥 LSD1 (lesions stimulating disease resistance 1) 和 LOL1 (LSD-One-Like 1) 基因均参与植物细胞程序化死亡 (programmed cell death, PCD) 的调控 . 从水稻 cDNA 文库中克隆到 1 个类 LSD1 基因,命名为 OsLSD1. 该基因长 988 bp ,包含一个 432 bp 的开放阅读框,推导的氨基酸序列 (143 个氨基酸 ) 含有 3 个内部保守的锌指结构域 . DNA 印迹结果表明 OsLSD1 基因在水稻基因组中为单拷贝,且在根、茎和叶中表达 . 借助于生物信息学分析技术,从拟南芥和水稻数据库中各识别出 5 个和 7 个 ( 包括 OsLSD1) 类 LSD1 基因 . 分析了这些类 LSD1 基因的结构,蛋白质结构域组成 . 系统进化分析表明,无论基于编码区的核苷酸或氨基酸序列都可以将这些类 LSD1 基因分为 2 类 . 虽然不存在拟南芥或水稻特有的类 LSD1 蛋白,但有些结构域是水稻所特有的,也有些基因是来源于复制事件 .     

小麦泛素融合降解蛋白基因的克隆及特征分析

酵母UFD1基因编码的泛素融合降解蛋白是泛素依赖性降解系统或泛素融合降解途径中的一个关键因子.利用RT-PCR技术在小麦(Triticum aestivum L.)中分离到一个UFD1类似基因.该基因的编码区长948 bp,编码长315个氨基酸的多肽,其氨基酸序列与GenBank中登录的一个拟南芥UFD1类似蛋白有74%的同源性.在多肽链的N-端具有在真核生物中高度保守的UFD1结构域.我们将该基因定位在小麦的第六染色体群并将其命名为TUFD1.South-ern杂交和数据库搜索表明植物的UFD1基因是单拷贝或低拷贝的.无论是在单子叶中还是在双子叶植物中,UFD1蛋白都高度同源.除了N端UFD1结构域外,该类蛋白还有3个高度保守的C端结构域.TUFD1基因在小麦幼苗的根、茎、胚芽鞘、叶片以及幼穗和腊熟期子粒中呈组成性表达.     

商陆高亲和性K＋转运体基因PaHAK1的克隆与表达分析

为了研究高钾植物商陆高亲和性K＋吸收机制,本试验选用野生商陆为材料,以拟南芥、冰叶日中花、小麦、大麦和水稻等多种植物的HAK家族基因的保守序列,设计简并引物,获得了981bp的PaHAK1的基因片段。然后应用RACE技术克隆到PaHAK1基因序列,其cDNA全长为2337bp,编码771个氨基酸。该编码蛋白质分子量为86．66kDa,等电点为7．54。蛋白质疏水性和拓扑结构分析显示该氨基酸序列共有12-13个跨膜区。该cDNA序列与冰叶日中花HAK1基因序列相似性高达88％。Real-TimePCR分析表明PaHAK1主要在商陆根中表达,低钾状态可以诱导PaHAK1的高表达。     

拟南芥和水稻CPP转录因子家族的生物信息学分析

CPP(cystein-rich polycomb-like protein or Tesmin/TOS1-like)家族是含有保守的富含Cystein的CRC结构域的蛋白质的统称,参与花器官的发育,在控制生殖组织发育和细胞分裂过程中起着极其重要的作用。本研究系统鉴定了8个拟南芥(Arabidopsis thaliana)和11个水稻(Orysa sativa japonicacv.Nipponbare)的CPP基因,并对这些基因编码的蛋白质序列进行了序列保守性和系统进化分析,最后对相关基因的表达进行了分析。结果表明,所有的CPP转录因子都具有高度保守的CRC结构域;单子叶和双子叶植物分化之前,植物CPP基因发生过大幅度的扩张;尽管C1域和C2域氨基酸序列一致性较高,但在该蛋白质的生物学功能中可能起着不同的作用;CPP基因主要在花组织中表达,在拟南芥中,AtCPP5、AtCPP6和AtCPP7极有可能存在功能互补作用。     

Intron loss mediated structural dynamics and functional differentiation of the polygalacturonase gene family in land plants

The polygalacturonase (PG) gene family is one of the largest gene families in plants. PGs are involved in various plant development steps. The evolutionary processes accounting for the functional divergence and the specialized functions of PGs in land plants are unclear. Whole sets of PG genes were retrieved from the genome web sites of model organisms in algae and land plants. The number of PG genes was expanded by lineage-specific manner with the biological complexity of the organism. Differentiation of PGs was related with phylogenetic hierarchy such as presence of rhamno-PGs from algae to plants, endo- and exo-PGs in land plants, exo-PGs in flowering plants. Gene structure analysis revealed that land plant PG genes resulted from differential intron gain and loss, with the latter event predominating. Differential intron losses partitioned the PGs into separate clades to be expressed differentially during plant development. Intron position and phase were not conserved between PGs of algae and land plants but conserved among PG genes of land plants from moss to vascular plants, indicating that the current introns in the PGs in land plants appeared after the split between unicellular algae and multicelluar land plants. The results demonstrate that the functional divergence and differentiation of PGs in land plants is attributable to intron losses.     

The lineage and diversity of putative amino acid sensor ACR proteins in plants

Amino acid metabolic enzymes often contain a regulatory ACT domain, named for aspartate kinase, chorismate mutase, and TyrA (prephenate dehydrogenase). Arabidopsis encodes 12 putative amino acid sensor ACT repeat (ACR) proteins, all containing ACT repeats but no identifiable catalytic domain. Arabidopsis ACRs comprise three groups based on domain composition and sequence: group I and II ACRs contain four ACTs each, and group III ACRs contain two ACTs. Previously, all three groups had been documented only in Arabidopsis. Here, we extended this to algae and land plants, showing that all three groups of ACRs are present in most, if not all, land plants, whereas among algal ACRs, although quite diverse, only group III is conserved. The appearance of canonical group I and II ACRs thus accompanied the evolution of plants from living in water to living on land. Alignment of ACTs from plant ACRs revealed a conserved motif, DRPGLL, at the putative ligand-binding site. Notably, the unique features of the DRPGLL motifs in each ACT domain are conserved in ACRs from algae to land plants. The conservation of plant ACRs is reminiscent of that of human cellular arginine sensor for mTORC1 (CASTOR1), a member of a small protein family highly conserved in animals. CASTOR proteins also have four ACT domains, although the sequence identities between ACRs and CASTORs are very low. Thus, plant ACRs and animal CASTORs may have adapted the regulatory ACT domains from a more ancient metabolic enzyme, and then evolved independently.     

Genome-wide analysis of a land plant-specific acyl:coenzyme A synthetase (ACS) gene family in Arabidopsis, poplar, rice and Physcomitrella

The plant enzyme 4-coumarate:coenzyme A ligase (4CL) is part of a family of adenylate-forming enzymes present in all organisms. Analysis of genome sequences shows the presence of '4CL-like' enzymes in plants and other organisms, but their evolutionary relationships and functions remain largely unknown. 4CL and 4CL-like genes were identified by BLAST searches in Arabidopsis, Populus, rice, Physcomitrella, Chlamydomonas and microbial genomes. Evolutionary relationships were inferred by phylogenetic analysis of aligned amino acid sequences. Expression patterns of a conserved set of Arabidopsis and poplar 4CL-like acyl-CoA synthetase (ACS) genes were assayed. The conserved ACS genes form a land plant-specific class. Angiosperm ACS genes grouped into five clades, each of which contained representatives in three fully sequenced genomes. Expression analysis revealed conserved developmental and stress-induced expression patterns of Arabidopsis and poplar genes in some clades. Evolution of plant ACS enzymes occurred early in land plants. Differential gene expansion of angiosperm ACS clades has occurred in some lineages. Evolutionary and gene expression data, combined with in vitro and limited in vivo protein function data, suggest that angiosperm ACS enzymes play conserved roles in octadecanoid and fatty acid metabolism, and play roles in organ development, for example in anthers.     

Convergent evolution of disease resistance gene specificity in two flowering plant families

Plant disease resistance (R) genes that mediate recognition of the same pathogen determinant sometimes can be found in distantly related plant families. This observation implies that some R gene alleles may have been conserved throughout the diversification of land plants. To address this question, we have compared R genes from Glycine max (soybean), Rpg1-b, and Arabidopsis thaliana, RPM1, that mediate recognition of the same type III effector protein from Pseudomonas syringae, AvrB. RPM1 has been cloned previously, and here, we describe the isolation of Rpg1-b. Although RPM1 and Rpg1-b both belong to the coiled-coil nucleotide binding site (NBS) Leu-rich repeat (LRR) class of R genes, they share only limited sequence similarity outside the conserved domains characteristic of this class. Phylogenetic analyses of A. thaliana and legume NBS-LRR sequences demonstrate that Rpg1-b and RPM1 are not orthologous. We conclude that convergent evolution, rather than the conservation of an ancient specificity, is responsible for the generation of these AvrB-specific genes.     

Genomic organization and evolutionary conservation of plant D-type cyclins

Plants contain more genes encoding core cell cycle regulators than other organisms but it is unclear whether these represent distinct functions. D-type cyclins (CYCD) play key roles in the G1-to-S-phase transition, and Arabidopsis (Arabidopsis thaliana) contains 10 CYCD genes in seven defined subgroups, six of which are conserved in rice (Oryza sativa). Here, we identify 22 CYCD genes in the poplar (Populus trichocarpa) genome and confirm that these six CYCD subgroups are conserved across higher plants, suggesting subgroup-specific functions. Different subgroups show gene number increases, with CYCD3 having three members in Arabidopsis, six in poplar, and a single representative in rice. All three species contain a single CYCD7 gene. Despite low overall sequence homology, we find remarkable conservation of intron/exon boundaries, because in most CYCD genes of plants and mammals, the first exon ends in the conserved cyclin signature. Only CYCD3 genes contain the complete cyclin box in a single exon, and this structure is conserved across angiosperms, again suggesting an early origin for the subgroup. The single CYCD gene of moss has a gene structure closely related to those of higher plants, sharing an identical exon/intron structure with several higher plant subgroups. However, green algae have CYCD genes structurally unrelated to higher plants. Conservation is also observed in the location of potential cyclin-dependent kinase phosphorylation sites within CYCD proteins. Subgroup structure is supported by conserved regulatory elements, particularly in the eudicot species, including conserved E2F regulatory sites within CYCD3 promoters. Global expression correlation analysis further supports distinct expression patterns for CYCD subgroups.     

Putative subunits of the maize origin of replication recognition complex ZmORC1-ZmORC5

The finding in animal species of complexes homologous to the products of six Saccharomyces cerevisiae genes, origin of replication recognition complex (ORC), has suggested that ORC-related mechanisms have been conserved in all eukaryotes. In plants, however, the only cloned putative homologs of ORC subunits are the Arabidopsis ORC2 and the rice ORC1. Homologs of other subunits of plant origin have not been cloned and characterized. A striking observation was the absence from the Arabidopsis genome of an obvious candidate gene-homolog of ORC4. This fact raised compelling questions of whether plants, in general, and Arabidopsis, in particular, may have lost the ORC4 gene, whether ORC-homologous subunits function within a complex in plants, whether an ORC complex may form and function without an ORC4 subunit, whether a functional (but not sequence) protein homolog may have taken up the role of ORC4 in Arabidopsis, and whether lack of ORC4 is a plant feature, in general. Here, we report the first cloned and molecularly characterized five genes coding for the maize putative homologs of ORC subunits ZmORC1, ZmORC2, ZmORC3, ZmORC4 and ZmORC5. Their expression profiles in tissues with different cell-dividing activities are compatible with a role in DNA replication. Based on the potential of ORC-homologous maize proteins to bind each other in yeast, we propose a model for their possible assembly within a maize ORC. The isolation and molecular characterization of an ORC4-homologous gene from maize argues that, in its evolution, Arabidopsis may have lost the homologous ORC4 gene.     

Characterization of a tomato protein that inhibits a xyloglucan-specific endoglucanase

A basic, 51 kDa protein was purified from suspension-cultured tomato and shown to inhibit the hydrolytic activity of a xyloglucan-specific endoglucanase (XEG) from the fungus Aspergillus aculeatus. The tomato (Lycopersicon esculentum) protein, termed XEG inhibitor protein (XEGIP), inhibits XEG activity by forming a 1 : 1 protein:protein complex with a Ki approximately 0.5 nm. To our knowledge, XEGIP is the first reported proteinaceous inhibitor of any endo-beta-1,4-glucanase, including the cellulases. The cDNA encoding XEGIP was cloned and sequenced. Database analysis revealed homology with carrot extracellular dermal glycoprotein (EDGP), which has a putative role in plant defense. XEGIP also has sequence similarity to ESTs from a broad range of plant species, suggesting that XEGIP-like genes are widely distributed in the plant kingdom. Although Southern analysis detected only a single XEGIP gene in tomato, at least five other XEGIP-like tomato sequences have been identified. Similar small families of XEGIP-like sequences are present in other plants, including Arabidopsis. XEGIP also has some sequence similarity to two previously characterized proteins, basic globulin 7S protein from soybean and conglutin gamma from lupin. Several amino acids in the XEGIP sequence, notably 8 of the 12 cysteines, are generally conserved in all the XEGIP-like proteins we have encountered, suggesting a fundamental structural similarity. Northern analysis revealed that XEGIP is widely expressed in tomato vegetative tissues and is present in expanding and maturing fruit, but is downregulated during ripening.