人工microRNAs对拟南芥At1g13770和At2g23470基因的特异沉默

DUF647(Domain of unknown function 647)蛋白家族是在真核生物中广泛存在的、高度保守的蛋白家族。拟南芥中该基因家族共有6个成员,迄今为止拟南芥DUF647家族中4个成员的功能尚不清楚。文章以拟南芥内源MIR319a前体为骨架,构建了敲减DUF647家族中2个基因At1g13770和At2g23470表达的人工microRNAs(Artifical microRNAs,amiRNAs)。利用WMD(Web microRNA designer)平台设计分别靶向At1g13770和At2g23470基因的amiRNAs序列,通过重叠PCR置换拟南芥MIR319a前体序列。构建融合amiRNAs前体的植物表达载体pCHF3-amiRNAs,在农杆菌介导下转化拟南芥。RT-PCR分析表明,amiRNAs能够显著抑制At1g13770和At2g23470基因的表达,获得了抑制效果明显的转基因株系。At2g23470-amiRNA转基因植株At2g23470转录水平的下调导致育性严重下降。文章为进一步研究这两个基因的功能奠定了良好的基础。     

拟南芥At5g01510和At5g49820基因人工microRNAs的构建与鉴定

以拟南芥内源MIR319a前体为骨架,构建沉默DUF647家族基因At5t01510和At5g49820表达的人工microRNAs,研究其对目的基因表达的抑制效果。利用WMD平台设计分别靶向At5g01510和At5g49820的amiRNAs序列,通过重叠PCR改造拟南芥MIR319a骨架序列,使其包含我们设计的特异amiRNAs序列。构建35S：：amiR-At5g0150和35S：：amiR-At5g49820融合基因,以农杆菌介导的花苞浸染法转化获得转基因拟南芥。RT-PCR分析表明,人工microRNAs能够显著抑制靶基因的表达,获得了抑制效果明显的转基因植株。本工作为进一步研究这两个基因的功能奠定了良好的基础。     

拟南芥F-box基因At3g16740的表达分析

拟南芥At3g16740基因为F-box基因家族成员,其功能尚不清楚.通过连续和瞬时光照处理分析,发现蓝光、红光和远红光都诱导At3g16740基因的表达,其中远红光的诱导作用最明显.蓝光受体cry1、cry2,红光受体phyB或远红光受体phyA突变导致At3g16740基因表达的光诱导作用减弱或者消失,表明该基因为光信号通路相关基因.通过实时荧光定量PCR分析At3g16740基因在拟南芥不同组织器官中的表达,发现其在拟南芥根、茎、叶、花和果荚中都有表达,花和果荚中的表达量最高,推测该基因可能参与植物花和/或果荚的发育.酵母双杂交分析发现,At3g16740蛋白通过F-box结构域与拟南芥ASK(arabidopsis-SKP1-like)家族成员ASK1、ASK2和ASK11相互作用,表明At3g16740是SCF(Skp、Cullin、F-box)复合物的成员.     

拟南芥砷诱导基因At4g13180的表达模式研究

拟南芥(Arabidopsis thaliana)砷诱导基因At4g13180编码蛋白是短链脱氢酶(Short-Chain Dehydrogenase/Reductase Superfamily,SDR)家族的成员之一,其过表达可以增强植物对过氧化氢的耐受性。该实验通过半定量RT-PCR,构建ProAt4g13180:GUS、At4g13180-EGFP和At4g13180-OE表达载体,获得At4g13180基因过表达转基因株系,并研究了At4g13180基因的表达模式及其编码蛋白的亚细胞定位。结果显示,At4g13180基因在根尖、叶脉、萼片和花丝等组织都强烈表达,该基因编码蛋白主要定位于胞质和核中。该研究结果为深入探究拟南芥砷诱导基因At4g13180的功能奠定了一定的基础。     

拟南芥F-box基因At5g22700的功能初步分析

F-box蛋白作为SCF（Skpl,Cullin and anF-boxprotein）复合体的成员,参与调节植物的生长发育过程。At5g22700为功能未知的F-box基因家族成员。本研究通过酵母双杂交分析At5g22700蛋白与ASK（Arabidop-sis-SKP1-1ike）家族蛋白的相互作用,发现At5g22700蛋白的F-box结构域与ASK4蛋白相互作用。实时定量PCR分析该基因在不同组织器官中的表达,发现该基因在根和花中的表达量最高,说明At5g2700可能在根和花的发育中具有重要作用。以At5g22700基因的T—DNA插入突变体和过量表达转基因株系为材料,分析不同光照条件下幼苗的表型,发现蓝光下At5g22700过量表达转基因幼苗的主根比野生型长。这些研究结果表明,At5g22700在植物体内可能形成SCF复合体,并在植物幼苗主根伸长生长中起促进作用。     

拟南芥醛还原酶编码基因At3g04000表达模式分析及过量表达转基因植株获得

植物体内的α,β-不饱和活性醛类化合物对植物细胞具有毒害作用,清除这些α,β-不饱和活性醛类化合物对于植物细胞维持正常的生命活动至关重要。前人研究报道通过体外酶活测定和异源瞬时表达鉴定拟南芥 At3g04000基因编码的蛋白为 NADPH 依赖的叶绿体醛还原酶(Arabidopsis NADPH-dependent chloroplastic aldehyde reductases, AtChlADRs),推测其在清除叶绿体中长链(≥5)α,β-不饱和醛类物质中具有重要的功能。该研究主要构建了拟南芥 At3g04000基因的表达模式分析载体 ProAt3g04000：GUS、亚细胞定位分析载体At3g04000-EGFP 和过量表达载体 At3g04000-OE,并获得了转基因拟南芥,并通过实时定量 PCR 分析了At3g04000基因在拟南芥不同组织中的转录水平。结果表明：拟南芥 At3g04000基因在幼苗中的转录水平最高,在莲座叶、茎生叶、花序和角果中均有较高的转录水平;而在根部和茎秆中的转录水平较低。通过对ProAt3g04000：GUS 转基因植株的 GUS 染色分析可知,At3g04000基因在子叶、莲座叶和萼片的维管组织和保卫细胞中均有较强的表达,在根的维管组织中有较弱的表达。通过共聚焦显微镜对 At3g04000-EGFP 转基因植株的观察和分析发现,At3g04000不是定位于叶绿体中,而是定位在细胞质和细胞核中。该研究结果为深入研究拟南芥醛还原酶编码基因 At3g04000的功能奠定了基础。     

拟南芥未知功能基因At3g61870编码蛋白的叶绿体定位研究

利用反向遗传学研究方法对1个预测的拟南芥叶绿体未知功能基因At3g61870编码蛋白进行了亚细胞定位研究.通过克隆At3g61870基因5′端长229 bp的DNA片段,与绿色荧光蛋白(GFP)基因构建重组表达载体pMON530-CP-TP-GFP,经农杆菌介导转化拟南芥.转基因植株的叶肉细胞经激光共聚焦显微镜观察,叶绿素自发荧光与GFP荧光共定位于叶绿体中.结果表明,未知功能基因At3g61870编码的蛋白质为叶绿体蛋白质.     

拟南芥未知功能基因At4g22890 编码蛋白的叶绿体定位

蛋白质的亚细胞定位信息对于深入了解该蛋白质的功能具有重要意义。本文对一个预测的拟南芥叶绿体未知功能基因At4g22890 编码蛋白进行了叶绿体定位研究。我们克隆了该基因5′端长208 bp 的DNA 片段, 与绿色荧光蛋白(GFP) 基因构建重组表达载体pMON530-cTP-GFP, 经农杆菌介导转化拟南芥。转基因植株经激光共聚焦显微镜观察, GFP 荧光仅在叶绿体中观察到, 表明所克隆的DNA 序列编码的多肽能够将At4g22890 编码蛋白质引导进入叶绿体, 由此推测该蛋白质为叶绿体蛋白质。     

拟南芥未知功能基因At4g22890编码蛋白的叶绿体定位

蛋白质的亚细胞定位信息对于深入了解该蛋白质的功能具有重要意义。本文对一个预测的拟南芥叶绿体未知功能基因At4g22890编码蛋白进行了叶绿体定位研究。我们克隆了该基因5′端长208bp的DNA片段,与绿色荧光蛋白(GFP)基因构建重组表达载体pMON530-cTP-GFP,经农杆菌介导转化拟南芥。转基因植株经激光共聚焦显微镜观察,GFP荧光仅在叶绿体中观察到,表明所克隆的DNA序列编码的多肽能够将At4g22890编码蛋白质引导进入叶绿体,由此推测该蛋白质为叶绿体蛋白质。     

人工microRNA干扰拟南芥AtCDKC;1和AtCDKC;2基因表达的初步研究

以来源于拟南芥的microRNA序列为骨架,构建抑制AtCDKC;1和AtCDKC;2基因的人工microRNAs载体,研究其对目的基因表达的抑制效果。选择AtCDKCs基因的特异性序列,通过重叠PCR的方法改造拟南芥microRNA164a骨架序列,连接到双元载体pPZPY122,在农杆菌介导下转化拟南芥。RT-PCR分析表明,人工microRNA能够显著抑制目的基因的表达,获得了抑制效果明显的转基因植株,并且对AtCDKCs在拟南芥生长发育中的作用进行了初步的研究。     

Analysis of two L-Galactono-1,4-lactone-responsive genes with complementary expression during the development of Arabidopsis thaliana

Unraveling the role of genes annotated as protein of unknown function is of importance in progression of plant science. l-Galactono-1,4-lactone (l-GalL) is the terminal precursor for ascorbic acid (AsA) biosynthesis in Arabidopsis thaliana, and a previous study showed two DUF (domains of unknown function) 642 family genes (At1g80240 and At5g25460, designated as DGR1 and DGR2, respectively) to be sensitive to it. In this work, leaves from wild-type Arabidopsis were fed with d-glucose, l-galactose, l-GalL and AsA, and the expression level of the At1g80240 and At5g25460 genes showed a specific response to l-GalL, but not to the other supplements despite the increases of the tissue AsA contents. Analysis of promoter-β-glucuronidase (GUS) transgenic plants showed the two genes to be complementarily expressed at the root apex and in the rest of the root excluding the apex, respectively, in both young and old seedlings, and to be expressed at the leaf primordia. The GUS activity under the control of the At5g25460 promoter was high in the cotyledon and leaf veins of young seedlings. These findings were consistent with the results of quantitative real-time PCR. Interestingly, the T-DNA insertion mutant of At5g25460 (SALK_125079) displayed shorter roots and smaller rosettes than Col-0; however, no phenotypic difference was observed between the T-DNA insertion mutant of At1g80240 and the wild type. This is the first report on the expression and functional analysis of these two DUF642 family genes, with the results revealing the contribution of DGR genes to the development of Arabidopsis.     

Computational identification of novel family members of microRNA genes in Arabidopsis thaliana and Oryza sativa

MicroRNAs (miRNAs) are a class of endogenous small RNAs that play important regulatory roles in both animals and plants, miRNA genes have been intensively studied in animals, but not in plants. In this study, we adopted a homology search approach to identify homologs of previously validated plant miRNAs in Arabidopsis thaliana and Oryza sativa. We identified 20 potential miRNA genes in Arabidopsis and 40 in O. sativa, providing a relatively complete enumeration of family members for these miRNAs in plants. In addition, a greater number of Arabidopsis miRNAs (MIR168, MIR159 and MIR172) were found to be conserved in rice. With the novel homologs, most of the miRNAs have closely related fellow miRNAs and the number of paralogs varies in the different miRNA families. Moreover, a probable functional segment highly conserved on the elongated stem of pre-miRNA fold-backs of MIR319 and MIR159 family was identified. These results support a model of variegated miRNA regulation in plants, in which miRNAs with different functional elements on their pre-miRNA fold-backs can differ in their function or regulation, and closely related miRNAs can be diverse in their specificity or competence to downregulate target genes. It appears that the sophisticated regulation of miRNAs can achieve complex biological effects through qualitative and quantitative modulation of gene expression profiles in plants.     

Microarray expression profiling and functional characterization of AtTPS genes: duplicated Arabidopsis thaliana sesquiterpene synthase genes At4g13280 and At4g13300 encode root-specific and wound-inducible (Z)-gamma-bisabolene synthases

The Arabidopsis thaliana genome contains at least 32 terpenoid synthase (AtTPS) genes [Aubourg et al., Mol. Genet. Genom. 267 (2002) 730] a few of which have recently been characterized. Based on hierarchical cluster analysis of AtTPS gene expression, measured by microarray profiling and validated with published expression data, we identified two groups of predominantly root expressed AtTPS genes containing five members with previously unknown biochemical functions (At4g13280, At4g13300, At5g48110, At1g33750, and At3g29410). Among the root expressed AtTPS genes, a pair of tandem-organized genes, At4g13280 (AtTPS12) and At4g13300 (AtTPS13), shares 91% predicted amino acid identity indicating recent gene duplication. Bacterial expression of cDNAs and enzyme assays showed that both At4g13280 and At4g13300 encode sesquiterpene synthases catalyzing the conversion of farnesyl diphosphate to (Z)-gamma-bisabolene and the additional minor products E-nerolidol and alpha-bisabolol. Expression of beta-glucuronidase (GUS) reporter gene fused to upstream genomic regions of At4g13280 or At4g13300 showed constitutive promoter activities in the cortex and sub-epidermal layers of Arabidopsis roots. In addition, highly localized promoter activities were found in leaf hydathodes and flower stigmata. Mechanical wounding of Arabidopsis leaves induced local expression of At4g13280 and At4g13300. The functional characterization of At4g13280 gene product AtTPS12 and At4g13230 gene product AtTPS13 as (Z)-gamma-bisabolene synthases, together with the recent characterization of two flower-specific AtTPS [At5g23960 and At5g44630; Tholl et al., Plant J. 42 (2005) 757], concludes the biochemical functional annotation of all four predicted Arabidopsis sesquiterpene synthase genes. Our data suggest biological functions for At4g13280 and At4g13300 in the rhizosphere with additional roles in aerial plant tissues.     

The Arabidopsis Domain of Unknown Function 1218 (DUF1218) Containing Proteins,MODIFYING WALL LIGNIN-1 and 2 (At1g31720/MWL-1 and At4g19370/MWL-2) Function Redundantly to Alter Secondary Cell Wall Lignin Content

DUF1218 is a land plant-specific innovation and has previously been shown to be associated with cell wall biology, vasculature patterning and abiotic/biotic stress response. The Arabidopsis genome encodes 15 members, two of which (At1g31720 and At4g27435) are preferentially expressed in the secondary cell wall depositing inflorescence stems. To further our understanding of the roles of DUF1218-containing proteins in secondary cell wall biology, we functionally characterized At1g31720 (herein referred to as MODIFYING WALL LIGNIN-1 or MWL-1). Since related gene family members may contribute to functional redundancy, we also characterized At4g19370 (MWL-2), the most closely related gene to MWL-1 in the protein family. Subcellular localization revealed that both Arabidopsis proteins are targeted to the cell periphery. The single T-DNA knockout lines, mwl-1 and mwl-2, and independent overexpression lines showed no significant differences in plant growth or changes in total lignin content relative to wild-type (WT) control plants. However, the double homozygous mutant, mwl-1/mwl-2, had smaller rosettes with a significant decrease in rosette fresh weight and stem height relative to the WT control at four weeks and six weeks, respectively. Moreover, mwl-1/mwl-2 showed a significant reduction in total lignin content (by ca. 11% relative to WT) and an increase in syringyl/guaiacyl (S/G) monomer ratio relative to the control plants. Our study has identified two additional members of the DUF1218 family in Arabidopsis as novel contributors to secondary cell wall biology, specifically lignin biosynthesis, and these proteins appear to function redundantly.