葡萄SBP基因家族生物信息学分析

SBP(squamosa promoter binding protein,SBP)基因家族是植物所特有的一类重要转录因子,广泛参与植物生长、发育以及多种生理生化反应的信号传导。葡萄是继拟南芥、水稻和杨树之后完成全基因组测序的第四种开花植物,因此葡萄逐渐成为分子生物学研究的重点对象,进行葡萄基因组信息挖掘与分析对于葡萄功能基因组学的发展具有重要意义。本文利用生物信息学方法对葡萄家族45条SBP蛋白序列的系统发生和SBP基因组定位进行分析,然后对其氨基酸组成成分、理化性质以及二级和三级结构进行预测和分析,同时还分析了葡萄与拟南芥的SBP基因家族之间的联系。结果显示这45条蛋白序列与拟南芥16个SBP基因蛋白序列一起分成了3个亚族,说明拟南芥与葡萄SBP基因间具有较高的保守性;进一步的基因组定位结果发现其分布在14条染色体上,较拟南芥在染色体上的分布更为分散。研究还发现不同亚家族间氨基酸数目、氨基酸序列间的疏水性存在一定的差异;而二级结构预测结果发现,41条氨基酸序列以随机卷曲为主要组成部分,这与拟南芥相似,且45条氨基酸序列三维结构十分相似。本文实验结果均为葡萄SBP基因家族的进一步功能分析提供了重要研究基础。     

Evolutionary Analysis for Functional Divergence of the Toll-Like Receptor Gene Family and Altered Functional Constraints

The Toll-like receptor (TLR) gene family consists of type 1 transmembrane receptors, which play essential roles in both innate immunity and adaptive immune response by ligand recognition and signal transduction. Using all available vertebrate TLR protein sequences, we inferred the phylogenetic tree and then characterized critical amino acid residues for functional divergence by detecting altered functional constraints after gene duplications. We found that the extracellular domain of TLR genes showed higher functional divergence than that of the cytoplasmic domain, particularly in the region between leucine-rich repeat (LRR) 10 and LRR 15 of TLR 4. Our finding supports the concept that sequence evolution in the extracellular domain may be responsible for the broad diversity of TLR ligand-binding affinity, providing a testable hypothesis for potential targets that could be verified by further experimentation.     

芪合酶基因的分子生物学研究

植物中存在芪类次生代谢产物(stilbenes)作为一种重要的植保素,不仅能够使植物体本身的抗逆性提高,在人类健康医疗领域也有很好的应用前景.由于其合成途径具有专一性,需要芪合酶(Stilbene synthase,STS)的存在,近年来芪合酶基因工程日益引起人们的研究和重视.介绍了芪合酶基因的结构功能及其诱导表达的调控机理,并对其转基因工程的研究进展进行了综述,以期为进一步开展芪类次生代谢物在作物品质改良及人类健康营养中的应用提供参考.     

Functional Analysis of the Brassica napus L. Phytoene Synthase (PSY) Gene Family

Phytoene synthase (PSY) has been shown to catalyze the first committed and rate-limiting step of carotenogenesis in several crop species, including Brassica napus L. Due to its pivotal role, PSY has been a prime target for breeding and metabolic engineering the carotenoid content of seeds, tubers, fruits and flowers. In Arabidopsis thaliana, PSY is encoded by a single copy gene but small PSY gene families have been described in monocot and dicotyledonous species. We have recently shown that PSY genes have been retained in a triplicated state in the A- and C-Brassica genomes, with each paralogue mapping to syntenic locations in each of the three “Arabidopsis-like” subgenomes. Most importantly, we have shown that in B. napus all six members are expressed, exhibiting overlapping redundancy and signs of subfunctionalization among photosynthetic and non photosynthetic tissues. The question of whether this large PSY family actually encodes six functional enzymes remained to be answered. Therefore, the objectives of this study were to: (i) isolate, characterize and compare the complete protein coding sequences (CDS) of the six B. napus PSY genes; (ii) model their predicted tridimensional enzyme structures; (iii) test their phytoene synthase activity in a heterologous complementation system and (iv) evaluate their individual expression patterns during seed development. This study further confirmed that the six B. napus PSY genes encode proteins with high sequence identity, which have evolved under functional constraint. Structural modeling demonstrated that they share similar tridimensional protein structures with a putative PSY active site. Significantly, all six B. napus PSY enzymes were found to be functional. Taking into account the specific patterns of expression exhibited by these PSY genes during seed development and recent knowledge of PSY suborganellar localization, the selection of transgene candidates for metabolic engineering the carotenoid content of oilseeds is discussed.     

Evolutionary Relationship and Structural Characterization of the EPF/EPFL Gene Family

EPF1-EPF2 and EPFL9/Stomagen act antagonistically in regulating leaf stomatal density. The aim of this study was to elucidate the evolutionary functional divergence of EPF/EPFL family genes. Phylogenetic analyses showed that AtEPFL9/Stomagen-like genes are conserved only in vascular plants and are closely related to AtEPF1/EPF2-like genes. Modeling showed that EPF/EPFL peptides share a common 3D structure that is constituted of a scaffold and loop. Molecular dynamics simulation suggested that AtEPF1/EPF2-like peptides form an additional disulfide bond in their loop regions and show greater flexibility in these regions than AtEPFL9/Stomagen-like peptides. This study uncovered the evolutionary relationship and the conformational divergence of proteins encoded by the EPF/EPFL family genes.     

Expression of the Stilbene Synthase (StSy) Gene from Grapevine in Transgenic White Poplar Results in High Accumulation of the Antioxidant Resveratrol Glucosides

When present, stilbene synthase leads to the production of resveratrol compounds, which are major components of the phytoalexin response against fungal pathogens of the plant and are highly bioactive substances of pharmaceutical interest. White poplar (Populus alba L.) was transformed with a construct containing a cDNA insert encoding stilbene synthase from grapevine (Vitis vinifera L.), under the control of the cauliflower mosaic virus (CaMV) 35S promoter, and a chimeric kanamycin resistance gene. Southern blot hybridization analysis demonstrated the presence and integration of exogenous DNA sequences in the poplar genome. Expression of the stilbene synthase-encoding gene in different transgenic lines was confirmed by Western blot and Northern analyses. Compared to the controls, in the transgenic plants two new compounds were detected and were identified as the trans- and cis-isomers of resveratrol-3-glucoside (piceid) by high-pressure liquid chromatography (HPLC), UV spectrophotometry, electrospray mass spectrometry (HPLC-ESI-MS) and enzymatic hydrolysis. Since poplar is a good biomass producer and piceids are accumulated in substantial amounts (up to 615.2 microg/g leaf fresh weight), the transgenic plants represent a potential alternative source for the production of these compounds with high pharmacological value. Despite the presence of piceid, in our experimental conditions no increased resistance against the pathogen Melampsora pulcherrima, which causes rust disease, was observed when in vitro bioassays were performed.     

Functional Divergence and Evolutionary Dynamics of the Putative AAAP Gene Family in Brassica rapa

The amino acid/auxin permease (AAAP) protein family is ubiquitously present in almost all eukaryotic species and functions in various aspects of growth and development. To investigate the evolution of AAAP proteins, here 83 AAAP genes in Brassica rapa were identified, and their sequence features, and evolutionary relationships were analyzed using in silico methods. According to the phylogenetic analysis, the AAAP genes of B. rapa are divided into six clades, and these clades share relatively similar sequence features, including gene structures, conserved motifs, and domain organizations. Synteny mapping strongly suggested that segmental duplications could be responsible for the expansion of this family. Adaptive evolution analysis demonstrated that most of AAAP proteins were subject to purifying selection. However, the site Tyr²⁵⁷ on eight AAAP proteins from clade 2b underwent significant positive selection. Functional divergent analysis showed that type I functional divergence coefficients (θ _I ) were significantly greater than zero in six pair-wise comparisons. However, functional divergence sites (Q _k ?>?0.95) found only in the AAAP I/II and AAAP I/III comparisons were localized mainly to the trans-membrane (TM) regions, suggesting highly divergent TM structures between these groups might be associated with group-specific functions. Our results could provide a valuable clue for further investigations of the evolutionary history and biological functions of the AAAP genes in B. rapa.     

中国野生毛葡萄芪合成酶基因克隆及表达分析

采用RT-PCR技术克隆中国野生毛葡萄‘丹凤-2’芪合成酶基因,命名为VqDSTS1,并进行序列及表达模式分析.结果表明:VqDSTS1基因cDNA编码区全长为1 179bp,GenBank登录号为JQ342086,编码392个氨基酸;氨基酸序列分析表明,VqDSTS1含有芪合成酶基因家簇的特征识别序列‘IPNSAGAIAGN’和‘GVLFGFG-PGLT’;序列比对显示,VqDSTS1与其他葡萄种质的芪合成酶氨基酸序列一致性在95.2%～98.7%之间;半定量RT-PCR分析表明,VqDSTS1受白粉病诱导表达,呈双峰模式.为进一步研究中国野生毛葡萄‘丹凤-2’芪合成酶基因家族的表达及功能分析提供了基础.     

Functional and Evolutionary Characterization of the CONSTANS Gene Family in Short-Day Photoperiodic Flowering in Soybean

CONSTANS (CO) plays a central role in photoperiodic flowering control of plants. However, much remains unknown about the function of the CO gene family in soybean and the molecular mechanisms underlying short-day photoperiodic flowering of soybean. We identified 26 CO homologs (GmCOLs) in the soybean genome, many of them previously unreported. Phylogenic analysis classified GmCOLs into three clades conserved among flowering plants. Two homeologous pairs in Clade I, GmCOL1a/GmCOL1b and GmCOL2a/GmCOL2b, showed the highest sequence similarity to Arabidopsis CO. The mRNA abundance of GmCOL1a and GmCOL1b exhibited a strong diurnal rhythm under flowering-inductive short days and peaked at dawn, which coincided with the rise of GmFT5a expression. In contrast, the mRNA abundance of GmCOL2a and GmCOL2b was extremely low. Our transgenic study demonstrated that GmCOL1a, GmCOL1b, GmCOL2a and GmCOL2b fully complemented the late flowering effect of the co-1 mutant in Arabidopsis. Together, these results indicate that GmCOL1a and GmCOL1b are potential inducers of flowering in soybean. Our data also indicate rapid regulatory divergence between GmCOL1a/GmCOL1b and GmCOL2a/GmCOL2b but conservation of their protein function. Dynamic evolution of GmCOL regulatory mechanisms may underlie the evolution of photoperiodic signaling in soybean.     

Evolutionary Aspects of Functional and Pseudogene Members of the Phytochrome Gene Family in Scots Pine

According to the neutral theory of evolution, mutation and genetic drift are the only forces that shape unconstrained, neutral, gene evolution. Thus, pseudogenes (which often evolve neutrally) provide opportunities to obtain direct estimates of mutation rates that are not biased by selection, and gene families comprising functional and pseudogene members provide useful material for both estimating neutral mutation rates and identifying sites that appear to be under positive or negative selection pressures. Conifers could be very useful for such analyses since they have large and complex genomes. There is evidence that pseudogenes make significant contributions to the size and complexity of gene families in pines, although few studies have examined the composition and evolution of gene families in conifers. In this work, I examine the complexity and rates of mutation of the phytochrome gene family in Pinus sylvestris and show that it includes not only functional genes but also pseudogenes. As expected, the functional PHYO does not appear to have evolved neutrally, while phytochrome pseudogenes show signs of unconstrained evolution.     

芪合酶基因Fm-STS在何首乌毛状根中的过量表达分析

目的:通过过量表达探究在何首乌中得到的芪合酶基因Fm-STS的功能.方法:由含CaMV 35S启动子驱动以及荧光标记蛋白(Green fluorescent protein,GFP)基因的植物转基因基础表达质粒pBIN-35S-GFP构建过量表达质粒pBIN-35S-STS-GFP(阳性),并同空白表达质粒pBIN-35S-GFP(空白)均导入野生型发根农杆菌ATCC15834中,转化何首乌外植体(无菌苗叶片),诱导生成毛状根并培养,对毛状根进行高效液相色谱分析芪合物二苯乙烯苷含量变化以及实时荧光定量检测基因Fm-STS表达差异.结果:在过表达组和空白组中毛状根中发根农杆菌Ri质粒中的rolB基因和外源基因GFP均有表达,芪合物二苯乙烯苷含量依次为4.67 mg/g和2.18 mg/g(干重),在mRNA水平上检测基因Fm-STS表达量:过表达组是空白组的2.41倍.结论:基因FM-STS是何首乌中芪合物二苯乙烯苷生物合成过程中的酶基因,过量表达在基因功能研究中有良好的应用.     

芪合酶基因转化番茄产生白藜芦醇的研究

为了获得含有白藜芦醇的转基因番茄,从葡萄雷司令中克隆到芪合酶基因,以之构建了含有组成型启动子的植物表达载体pBS2,用于农杆菌介导对番茄品种Tx00l4的遗传转化。通过对诱导愈伤、出芽、生根、再生植株的筛选,得到5株再生小苗,经PCR、Southem检测证实,有3株为真正的转基因植株。用HPLC对3株转基因植株叶片进行白藜芦醇含量鲜重分析,它们中白藜芦醇的含量分别为12．45μg/g,5.35μg/g,4.55μg/g。     

Evolutionary Dynamics of the Cellulose Synthase Gene Superfamily in Grasses

Phylogenetic analyses of cellulose synthase (CesA) and cellulose synthase-like (Csl) families from the cellulose synthase gene superfamily were used to reconstruct their evolutionary origins and selection histories. Counterintuitively, genes encoding primary cell wall CesAs have undergone extensive expansion and diversification following an ancestral duplication from a secondary cell wall-associated CesA. Selection pressure across entire CesA and Csl clades appears to be low, but this conceals considerable variation within individual clades. Genes in the CslF clade are of particular interest because some mediate the synthesis of (1,3;1,4)-β-glucan, a polysaccharide characteristic of the evolutionarily successful grasses that is not widely distributed elsewhere in the plant kingdom. The phylogeny suggests that duplication of either CslF6 and/or CslF7 produced the ancestor of a highly conserved cluster of CslF genes that remain located in syntenic regions of all the grass genomes examined. A CslF6-specific insert encoding approximately 55 amino acid residues has subsequently been incorporated into the gene, or possibly lost from other CslFs, and the CslF7 clade has undergone a significant long-term shift in selection pressure. Homology modeling and molecular dynamics of the CslF6 protein were used to define the three-dimensional dispositions of individual amino acids that are subject to strong ongoing selection, together with the position of the conserved 55-amino acid insert that is known to influence the amounts and fine structures of (1,3;1,4)-β-glucans synthesized. These wall polysaccharides are attracting renewed interest because of their central roles as sources of dietary fiber in human health and for the generation of renewable liquid biofuels.Recent attempts to better understand the chemistry and biology of plant cell walls have been driven by the importance of these walls as biomass sources for biofuel production systems, as sources of dietary fiber that is increasingly recognized as being highly beneficial for human health, and as key components of livestock forage and fodder. Plant cell walls consist predominantly of polysaccharides and lignin. In addition to cellulose, walls contain a wide array of complex noncellulosic polysaccharides that vary across the plant kingdom (Carpita, 1996; Popper and Fry, 2003; Niklas, 2004; Popper and Tuohy, 2010). In the dicotyledons, pectic polysaccharides and xyloglucans predominate, although smaller amounts of heteroxylans and heteromannans are also found. In evolutionary terms, a major change in noncellulosic wall composition is observed with the emergence of the Poaceae family, which contains the grasses and important cereal species. In contrast to dicots, walls of the Poaceae have relatively low levels of pectic polysaccharides and xyloglucans and correspondingly higher levels of heteroxylans, which appear to constitute the core noncellulosic wall polysaccharides in this family. In addition, walls of the Poaceae often contain (1,3;1,4)-β-glucans, which are not widely distributed in dicotyledons or other monocotyledons (Carpita, 1996; Fincher, 2009).Following the identification of the genes that encode cellulose synthases, which were designated CesA genes (Pear et al., 1996; Arioli et al., 1998), analyses of EST databases quickly revealed that the CesA group of cellulose synthase genes was in fact just one clade of a much larger superfamily that contained up to about 50 genes in most land plants (Richmond and Somerville, 2000; Hazen et al., 2002). The other members of the large gene family were designated cellulose synthase-like genes (Csl), which represent several clades in the overall phylogeny of the superfamily (Supplemental Fig. S1). The plant CesA genes were shown to have both conserved and hypervariable regions (Delmer, 1999; Doblin et al., 2002) and, together with the related Csl genes, were predicted to be integral membrane proteins and to have conserved, active-site D,D,D,QxxRW amino acid sequences. The CesA and Csl genes are members of the GT2 family of glycosyltransferases (Cantarel et al., 2009; http://www.cazy.org/).Several of the Csl genes have now been implicated in the biosynthesis of noncellulosic wall polysaccharides. Certain CslA genes mediate mannan and glucomannan synthesis (Dhugga et al., 2004; Liepman et al., 2005). Genes in the CslC clade are believed to be involved in xyloglucan biosynthesis (Cocuron et al., 2007), while genes from both the CslF and CslH clades mediate (1,3;1,4)-β-glucan synthesis in the Poaceae (Burton et al., 2006; Doblin et al., 2009). The CslJ group of enzymes is also believed to be involved in (1,3;1,4)-β-glucan synthesis (Farrokhi et al., 2006; Fincher, 2009), but the phylogeny of this group of genes remains unresolved (Yin et al., 2009). The fact that the CslF group does not form a clade with the CslH and CslJ groups on the phylogenetic tree (Supplemental Fig. S1) led to the suggestion that the genes mediating (1,3;1,4)-β-glucan synthesis have evolved independently on more than one occasion (Doblin et al., 2009; Fincher, 2009).Against this background and considering the sequence similarities between genes in the cellulose synthase gene superfamily, we have used Bayesian phylogenetic analyses of these genes from seven fully sequenced taxa to reconstruct the evolutionary origins of the CesA and Csl families in the grasses and, in particular, to investigate the evolution of the CslF, CslH, and CslJ genes. The distributions of the genes across genomes were compared, CslF gene clusters were analyzed, and the rates of synonymous and nonsynonymous nucleotide substitution were estimated to assess and compare selection histories of individual members of clades within the gene superfamily. Finally, we have constructed a refined model of the barley CslF6 enzyme to observe how selection on specific residues and regions of the enzyme has operated in a structural and functional context.     

哺乳动物BMP4蛋白功能位点的进化踪迹分析

哺乳动物骨形态发生蛋白(BMPs)具有促进动物软骨形成,调节细胞增殖、分化及迁移的多种功能。此外,该蛋白在动物个体发育及人(Homo sapiens)肿瘤的发生、发展过程中也扮演着重要角色。本文以人源骨形态发生蛋白4(BMP4)为种子序列,利用多种生物信息学工具进行哺乳动物BMP4蛋白的序列查找及其同源蛋白的搜索,共得到具有完整结构域的同源蛋白序列72条,并以此为基础对哺乳动物BMP4的进化踪迹位点及相关的功能位点进行比较研究。结果表明,BMP4蛋白家族的TGFβ-propeptide结构域具22个全家族保守残基,而TGF-β结构域仅具84个亚家族特异性残基。人BMP4蛋白TGF-β结构域的配基结合位点主要分布于结合口袋的边缘区域。本研究可为BMP4蛋白重要功能区残基的确定及未知功能位点的预测提供信息。     

Functional and Evolutionary Analysis of the CASPARIAN STRIP MEMBRANE DOMAIN PROTEIN Family

CASPARIAN STRIP MEMBRANE DOMAIN PROTEINS (CASPs) are four-membrane-span proteins that mediate the deposition of Casparian strips in the endodermis by recruiting the lignin polymerization machinery. CASPs show high stability in their membrane domain, which presents all the hallmarks of a membrane scaffold. Here, we characterized the large family of CASP-like (CASPL) proteins. CASPLs were found in all major divisions of land plants as well as in green algae; homologs outside of the plant kingdom were identified as members of the MARVEL protein family. When ectopically expressed in the endodermis, most CASPLs were able to integrate the CASP membrane domain, which suggests that CASPLs share with CASPs the propensity to form transmembrane scaffolds. Extracellular loops are not necessary for generating the scaffold, since CASP1 was still able to localize correctly when either one of the extracellular loops was deleted. The CASP first extracellular loop was found conserved in euphyllophytes but absent in plants lacking Casparian strips, an observation that may contribute to the study of Casparian strip and root evolution. In Arabidopsis (Arabidopsis thaliana), CASPL showed specific expression in a variety of cell types, such as trichomes, abscission zone cells, peripheral root cap cells, and xylem pole pericycle cells.Biological membranes are conceptually simple structures that may be generated in vitro according to simple physicochemical principles. In vivo, however, membranes are highly complex and host a plethora of proteins that mediate the transfer of molecules and communication across the membrane. Proteins may be trapped in membrane by their transmembrane domains, anchored by lipid tails, or attach to membrane-integral proteins. A further level of complexity is seen when membrane proteins are not equally distributed but occupy only a limited fraction of the available surface (i.e. when they are polarly localized or when they form small membrane subdomains in the micrometer range). The question of how membrane proteins are retained locally and prevented from diffusing freely is of high importance to cell biology. Polarly localized proteins may be retained in their respective domains by membrane fences; in such a situation, polarly localized proteins are mobile in their domains but cannot diffuse through tightly packed scaffold proteins forming a molecular fence within the membrane. Membrane fences delimiting polar domains have been described in different organisms. For example, diffusion between membrane compartments is prevented in budding yeast (Saccharomyces cerevisiae) at the level of the bud neck (Barral et al., 2000; Takizawa et al., 2000); in ciliated vertebrate cells, between ciliary and periciliary membranes (Hu et al., 2010); in epithelial cells, between apical and basolateral membranes (van Meer and Simons, 1986); in neurons, between axon and soma (Kobayashi et al., 1992; Winckler et al., 1999; Nakada et al., 2003); and in spermatozoa, at the level of the annulus (Myles et al., 1984; Nehme et al., 1993). The existence of membrane scaffolds that prevent free protein diffusion has also been described in bacteria (Baldi and Barral, 2012; Schlimpert et al., 2012). In plants, we have shown the existence of a strict membrane fence in the root endodermis, where a median domain splits the cell in two lateral halves occupied by different sets of proteins (Alassimone et al., 2010). The situation in the plant endodermis is analogous to the separation of animal epithelia into apical and basolateral domains; indeed, a parallel between epithelia and endodermal cells has been drawn, despite the different origin of multicellularity in plants and animals (Grebe, 2011).The protein complexes responsible for the formation of membrane fences have been identified. Septins are a family of proteins able to oligomerize and form filaments (Saarikangas and Barral, 2011); their role in the formation of membrane fences has been demonstrated in several organisms and cellular situations, including the yeast bud neck (Barral et al., 2000; Takizawa et al., 2000), animal cilia (Hu et al., 2010), and mammalian spermatozoa (Ihara et al., 2005; Kissel et al., 2005; Kwitny et al., 2010). At the axonal initial segment of neurons, AnkyrinG is necessary to establish and maintain a membrane scaffold where different membrane proteins are immobilized and stabilized (Hedstrom et al., 2008; Sobotzik et al., 2009). In Caulobacter crescentus, the stalk protein Stp forms a complex that prevents diffusion between the cell body and stalk and between stalk compartments. Claudins and occludin are the main components of epithelial tight junctions (Furuse et al., 1993, 1998). Occludins are four-membrane-span proteins and belong to the MARVEL protein family (Sánchez-Pulido et al., 2002), as do Tricellulin and MARVELD3, which are also tight junction-associated proteins (Furuse et al., 1993; Ikenouchi et al., 2005; Steed et al., 2009).In Arabidopsis (Arabidopsis thaliana), our group identified a family of proteins that form a membrane fence in the endodermis (Roppolo et al., 2011). These CASPARIAN STRIP MEMBRANE DOMAIN PROTEINS (CASP1 to CASP5) are four-transmembrane proteins that form a median domain referred to as the Casparian strip membrane domain (CSD). CASPs are initially targeted to the whole plasma membrane, then they are quickly removed from lateral plasma membranes and remain localized exclusively at the CSD; there, they show an extremely low turnover, although they are eventually removed (Roppolo et al., 2011). The membrane proteins NOD26-LIKE INTRINSIC PROTEIN5;1 and BORON TRANSPORTER1 are restricted from diffusing through the CSD and remain polarly localized in the outer and inner lateral membranes, respectively; a fluorescent lipophilic molecule, when integrated in the outer endodermal membrane, was blocked at the level of the CSD and could not diffuse into the inner membrane (Roppolo et al., 2011). Besides making a plasma membrane diffusion barrier, CASPs have an important role in directing the modification of the cell wall juxtaposing their membrane domain: by interacting with secreted peroxidases, they mediate the deposition of lignin and the building up of the Casparian strips (Roppolo et al., 2011; Naseer et al., 2012; Lee et al., 2013). The two CASP activities, making membrane scaffolds and directing a modification of the cell wall, can be uncoupled: indeed, (1) formation of the CASP domain is independent from the deposition of lignin, and (2) interaction between CASPs and peroxidases can take place outside the CSD when CASPs are ectopically expressed (Lee et al., 2013).As CASPs are currently the only known proteins forming membrane fences in plants and because of their essential role in directing a local cell wall modification, we were interested in characterizing the repertoire of a large number of CASP-like (CASPL) proteins in the plant kingdom. Our aim was to provide the molecular basis for the discovery of additional membrane domains in plants and for the identification of proteins involved in local cell wall modifications. We extended our phylogenetic analysis outside of the plant kingdom and found conservation between CASPLs and the MARVEL protein family. Conserved residues are located in transmembrane domains, and we provide evidence suggesting that these domains are involved in CASP localization. We explored the potential use of the CASPL module in plants by investigating CASPL expression patterns and their ability to form membrane domains in the endodermis. Moreover, we related the appearance of the Casparian strips in the plant kingdom to the emergence of a CASP-specific signature that was not found in the genomes of plants lacking Casparian strips.     

小桐子肌醇半乳糖苷合成酶3的基因克隆及其生物信息学分析和功能初步验证

旨在研究可溶性糖在小桐子抗冷性形成中的作用机制及其相关抗冷基因工程的应用。克隆了小桐子肌醇半乳糖苷合成酶家族成员3基因(Jc GS3)的全长c DNA,序列长1 053 bp,包含完整的开放阅读框1 008 bp,编码由335个氨基酸组成的酶蛋白(理论分子量为38 k D、等电点为5.44,含有典型的Dx D基序)。对其相应基因组序列中启动子区域进行分析,鉴定到了TATA框、CAAT框、CRT/DRE低温响应元件以及ABA应答元件等。进一步将其在酵母中表达,发现该基因的表达能够显著提高其重组酵母菌的低温抵抗能力。     

伴矿景天植物螯合肽合酶基因的克隆及功能分析

重金属超积累植物由于长期生长在高浓度的重金属环境中,使得经由植物螯合肽（phytochelatins, PCs）解毒途径来应对重金属毒害代价高昂。我们从Zn/Cd超积累植物伴矿景天（Sedum plumbizincicola）中克隆了植物螯合肽合酶（phytochelatin synthase, PCS）基因SepPCS。该基因在裂殖酵母和拟南芥中表达后都具有PCS活性,而且能够互补它们的PCs缺失突变体的Cd敏感表型。SepPCS在伴矿景天中的表达受到高浓度Cd处理的诱导。与其近亲非超积累生态型东南景天（S. alfredii）相比,虽然伴矿景天地上部PCs与Cd的摩尔比远低于东南景天,但是在高浓度Cd处理时PCs含量以及PCs与Cd的摩尔比急剧增加。我们推测在伴矿景天应对Cd毒害的过程中, PCs起到一定的作用,并且在高浓度Cd胁迫时地上部PCs依赖的解毒作用有所加强。