棉花叶绿体基因RNA编辑位点的测定及分析

陆生植物叶绿体RNA编辑是转录后基因表达调控的一种重要方式。该文在预测棉花（Gossypium hirsutum）叶绿体基因RNA编辑位点的基础上,选取中棉10（CRRI 10）为实验材料,采用PCR、RT-PCR及测序等方法,确定CRRI 10的27个叶绿体蛋白编码基因共有55个编辑位点,均是C→U的转换。与棉种柯字310（C310）的编辑位点比对后发现,CRRI 10多出accD-468和rpoC1-163两个编辑位点,同时缺失psbN-10。利用生物信息学分析这3个位点,rpoC1-163和psbN-10的编辑可能会改变各自蛋白的二级结构。对CRRI 10中55个编辑位点上游的顺式作用元件（？30–？1）分析显示,共有8组顺式作用元件的相似性达到60%或以上,推测各组中的编辑位点可能由相同的反式作用因子来识别。     

小麦叶绿体蛋白质编码基因RNA编辑位点的测定及与返白现象的关系

RNA编辑是一种转录后基因加工修饰现象,广泛存在于高等植物细胞器中。已有研究表明,RNA编辑与植物发生白化或者黄化有关。通过PCR、RT-PCR及测序的方法,对具有阶段性白化特性的小麦（Triticum aestivum）返白系FA85及其野生型矮变一号（Aibian 1）的叶绿体蛋白质编码基因RNA编辑位点进行了测定,在14个基因上发现了26个编辑位点。有5个编辑位点在2个株系之间存在编辑效率的差异,且这些差异的位点均位于编码叶绿体RNA聚合酶的基因上,其中3个位点编辑前后对应的蛋白质二级结构可能有差异。对2个株系叶绿体中PEP、NEP及PEP、NEP共同依赖基因转录水平的检测显示,除psbA和clpP外,其它基因在小麦返白系中的转录水平均有不同程度的下降。这种转录水平的显著下降及叶绿体RNA聚合酶基因上RNA编辑位点编辑效率的改变,可能与小麦返白系叶片的返白有关。     

Predictable Alteration of Sequence Recognition by RNA Editing Factors from Arabidopsis

RNA editing factors of the pentatricopeptide repeat (PPR) family show a very high degree of sequence specificity in the recognition of their target sites. A molecular basis for target recognition by editing factors has been proposed based on statistical correlations but has not been tested experimentally. To achieve this, we systematically mutated the pentatricopeptide motifs in the Arabidopsis thaliana RNA editing factor CLB19 to investigate their individual contribution to RNA recognition. We find that the motifs contributing significantly to the specificity of binding follow the previously proposed recognition rules, distinguishing primarily between purines and pyrimidines. Our results are consistent with proposals that each motif recognizes one nucleotide in the RNA target with the protein aligned parallel to the RNA and contiguous motifs aligned with contiguous nucleotides such that the final PPR motif aligns four nucleotides upstream of the edited cytidine. By altering S motifs in CLB19 and another editing factor, OTP82, and using the modified proteins to attempt to complement the respective mutants, we demonstrate that we can predictably alter the specificity of these factors in vivo.     

A Suite of Lotus japonicus Starch Mutants Reveals Both Conserved and Novel Features of Starch Metabolism

The metabolism of starch is of central importance for many aspects of plant growth and development. Information on leaf starch metabolism other than in Arabidopsis (Arabidopsis thaliana) is scarce. Furthermore, its importance in several agronomically important traits exemplified by legumes remains to be investigated. To address this issue, we have provided detailed information on the genes involved in starch metabolism in Lotus japonicus and have characterized a comprehensive collection of forward and TILLING (for Targeting Induced Local Lesions IN Genomes) reverse genetics mutants affecting five enzymes of starch synthesis and two enzymes of starch degradation. The mutants provide new insights into the structure-function relationships of ADP-glucose pyrophosphorylase and glucan, water dikinase1 in particular. Analyses of the mutant phenotypes indicate that the pathways of leaf starch metabolism in L. japonicus and Arabidopsis are largely conserved. However, the importance of these pathways for plant growth and development differs substantially between the two species. Whereas essentially starchless Arabidopsis plants lacking plastidial phosphoglucomutase grow slowly relative to wild-type plants, the equivalent mutant of L. japonicus grows normally even in a 12-h photoperiod. In contrast, the loss of GLUCAN, WATER DIKINASE1, required for starch degradation, has a far greater effect on plant growth and fertility in L. japonicus than in Arabidopsis. Moreover, we have also identified several mutants likely to be affected in new components or regulators of the pathways of starch metabolism. This suite of mutants provides a substantial new resource for further investigations of the partitioning of carbon and its importance for symbiotic nitrogen fixation, legume seed development, and perenniality and vegetative regrowth.Recent studies in Arabidopsis (Arabidopsis thaliana) have greatly enhanced our knowledge about pathways of transitory starch metabolism (Zeeman et al., 2007; Keeling and Myers, 2010; Kötting et al., 2010; Zeeman et al., 2010). The pathway of synthesis is well established for several species, but the degradative pathway is understood only in Arabidopsis. During synthesis, the plastidial isoforms of phosphoglucoisomerase (PGI1) and phosphoglucomutase (PGM1), together with ADP-Glc pyrophosphorylase (AGPase), catalyze the conversion of the Calvin cycle intermediate Fru 6-P to ADPGlc, the substrate for starch synthases (Supplemental Fig. S1). Leaves of mutants lacking any of these three enzymes either have strongly reduced starch contents or lack starch almost completely (Caspar et al., 1985; Hanson and McHale, 1988; Lin et al., 1988a, 1988b; Kruckeberg et al., 1989; Harrison et al., 1998; Yu et al., 2000; Streb et al., 2009). In contrast, the phenotypes of mutants lacking individual enzymes that convert ADPGlc into starch vary between species and are often much less pronounced (starch synthases [Delvallé et al., 2005; Zhang et al., 2005] and starch-branching enzymes [Tomlinson et al., 1997; Blauth et al., 2001; Dumez et al., 2006]).The degradation of the starch granule in Arabidopsis leaves is catalyzed primarily by β-amylases and isoamylase 3 (Wattebled et al., 2005; Delatte et al., 2006; Fulton et al., 2008). Normal rates of degradation require phosphorylation of the starch polymers by two glucan, water dikinases, GWD1 (Ritte et al., 2002) and GWD3 (or PWD, for phosphoglucan water, dikinase; Baunsgaard et al., 2005; Kötting et al., 2005), followed by dephosphorylation by a phosphoglucan phosphatase, STARCH EXCESS4 (SEX4; Kötting et al., 2009). Maltose produced by starch degradation is exported from the chloroplast by a maltose transporter and further metabolized to hexose phosphates in the cytosol (Zeeman et al., 2007; Supplemental Fig. S1). Mutations in numerous components of this pathway result in a starch-excess phenotype, in which the starch content of leaves at the end of the night is higher than that of wild-type plants.These studies have also revealed the importance of starch turnover for the productivity of the plant. Mutants of Arabidopsis that are essentially unable to synthesize transitory starch, or with reduced rates of starch degradation at night, have a reduced rate of growth and delayed flowering time relative to wild-type plants under most conditions (Caspar et al., 1985, 1991; Eimert et al., 1995; Corbesier et al., 1998; Smith and Stitt, 2007). However, it is not known whether information about the nature and importance of starch turnover in Arabidopsis is widely applicable. Plant species differ considerably in the extent to which starch is stored in leaves at night as well as in diurnal patterns of growth and metabolic demand. The function and regulation of starch metabolism in heterotrophic organs and its importance in major physiological and developmental processes such as perenniality, vegetative regrowth, symbiotic nitrogen fixation, and the accumulation of seed storage reserves cannot be studied easily in Arabidopsis and remain largely unknown. These processes represent traits of agronomic value in legumes (Fabaceae), a family that includes some of the most agriculturally important forage (e.g. alfalfa [Medicago sativa] and clover [Trifolium spp.]), grain (e.g. pea [Pisum sativum] and common bean [Phaseolus vulgaris]), and oilseed (e.g. soybean [Glycine max]) crops.Some information is already available about starch metabolism in pea and other legume crops (Martin and Smith, 1995; Wang et al., 1998b, and refs. therein). However, characteristics including large genome sizes and recalcitrant transformation and regeneration have limited progress on these species. There is insufficient information to allow either an overview of the nature and importance of starch metabolism in legumes or a meaningful comparison with the detailed picture emerging for Arabidopsis. The development of both Lotus japonicus and Medicago truncatula as legume model systems, and the wide range of genetic and genomic resources generated for them, offer the opportunity for a systematic analysis.To elucidate the pathway of starch synthesis and degradation in legumes and provide resources for future experimentation, we screened an ethyl methanesulfonate (EMS)-mutagenized population of L. japonicus (Perry et al., 2003) for mutants altered in transitory starch metabolism and carried out genetic mapping to identify the mutation responsible for their phenotype. We also used TILLING (for Targeting Induced Local Lesions IN Genomes; McCallum et al., 2000) to confirm that the mutations identified were indeed responsible for the mutant phenotype and to obtain additional mutations in genes known to affect leaf starch content in other species. We present the results of molecular and phenotypic analyses on the mutants that provide novel insights into the structure-function relationship of the AGPase and GWD1 enzymes. In addition, our analyses reveal new information on the nature and importance of starch metabolism for plant growth and development in L. japonicus. The importance of starch accumulation and degradation and a comparison with pathways in other plant species are also discussed.     

Comparative Analysis of Asteraceae Chloroplast Genomes: Structural Organization,RNA Editing and Evolution

Plant Molecular Biology Reporter - Comparative chloroplast genome analysis presents new opportunities for performing molecular phylogeny studies and revealing the significant evolutionary features...     

银杏叶绿体ndhF RNA编辑现象分析及C290位编辑对胁迫处理的响应

RNA编辑是一种转录后修饰加工过程, 通过碱基的插入、缺失或替换可改变氨基酸的种类, 增加蛋白质的疏水性和同源蛋白在不同物种间的保守性。该文通过DNA与cDNA序列的比对, 分析了裸子植物银杏(Ginkgo biloba)叶绿体功能基因ndhF的编辑现象, 该基因共含有21个编辑位点, 且这21个位点均为部分编辑。生物信息学分析及与其它物种比对结果表明, ndhF C290位编辑可能会影响该蛋白的正确折叠。进一步使用单克隆酶切方法测定了不同胁迫处理对ndhF C290位编辑效率的影响, 结果表明该位点的编辑效率对温度和黑暗敏感。     

银杏叶绿体ndhF RNA编辑现象分析及C290位编辑对胁迫处理的响应

RNA编辑是一种转录后修饰加工过程,通过碱基的插入、缺失或替换可改变氨基酸的种类,增加蛋白质的疏水性和同源蛋白在不同物种间的保守性。该文通过DNA与cDNA序列的比对,分析了裸子植物银杏（Ginkgobiloba）叶绿体功能基因ndhF的编辑现象,该基因共含有21个编辑位点,且这21个位点均为部分编辑。生物信息学分析及与其它物种比对结果表明,ndhFC290位编辑可能会影响该蛋白的正确折叠。进一步使用单克隆酶切方法测定了不同胁迫处理对ndhFC290位编辑效率的影响,结果表明该位点的编辑效率对温度和黑暗敏感。     

植物细胞器RNA编辑因子的功能及其作用机制 *

在植物线粒体和叶绿体转录本上,数百个胞嘧啶(C)位点经脱氨基反应变为尿嘧啶(U),这是一种在转录本水平上对遗传信息进行修饰或调控的机制.在植物细胞器中,RNA编辑过程需要不同家族的RNA编辑因子相互作用组装成复杂的编辑复合体,特异地识别编辑位点进行编辑.最初的研究发现,植物RNA编辑受到高特异性五环肽重复(pentatricopeptide repeat, PPR)蛋白的调控,目前在植物中发现400多种PPR家族蛋白,编辑作用复杂.之后对RNA编辑因子互作蛋白/多细胞器RNA编辑因子(RNA editing factor interacting proteins /multiple organellar RNA editing factors,RIP/MORF),细胞器RNA识别基序(organelle RNA recognition motif,ORRM),细胞器锌指蛋白(organelle zinc-finger,OZ)等的研究表明,这些非PPR蛋白组分可以与PPR蛋白形成编辑复合体,共同参与编辑,且RNA编辑复合体具有多样性.RNA编辑因子的缺失会引起植物的生长发育受阻,果实成熟延迟等,对RNA编辑因子的研究显得尤为重要.对植物中RNA编辑因子的功能及其作用机制研究进展进行综述,旨在为后续RNA编辑的研究提供一定的参考.     

High-Throughput Genotyping of Green Algal Mutants Reveals Random Distribution of Mutagenic Insertion Sites and Endonucleolytic Cleavage of Transforming DNA

A high-throughput genetic screening platform in a single-celled photosynthetic eukaryote would be a transformative addition to the plant biology toolbox. Here, we present ChlaMmeSeq (Chlamydomonas MmeI-based insertion site Sequencing), a tool for simultaneous mapping of tens of thousands of mutagenic insertion sites in the eukaryotic unicellular green alga Chlamydomonas reinhardtii. We first validated ChlaMmeSeq by in-depth characterization of individual insertion sites. We then applied ChlaMmeSeq to a mutant pool and mapped 11,478 insertions, covering 39% of annotated protein coding genes. We observe that insertions are distributed in a manner largely indistinguishable from random, indicating that mutants in nearly all genes can be obtained efficiently. The data reveal that sequence-specific endonucleolytic activities cleave the transforming DNA and allow us to propose a simple model to explain the origin of the poorly understood exogenous sequences that sometimes surround insertion sites. ChlaMmeSeq is quantitatively reproducible, enabling its use for pooled enrichment screens and for the generation of indexed mutant libraries. Additionally, ChlaMmeSeq allows genotyping of hits from Chlamydomonas screens on an unprecedented scale, opening the door to comprehensive identification of genes with roles in photosynthesis, algal lipid metabolism, the algal carbon-concentrating mechanism, phototaxis, the biogenesis and function of cilia, and other processes for which C. reinhardtii is a leading model system.     

Arabidopsis Mutants Lacking the 43- and 54-Kilodalton Subunits of the Chloroplast Signal Recognition Particle Have Distinct Phenotypes

The chloroplast signal recognition particle (cpSRP) is a protein complex consisting of 54- and 43-kD subunits encoded by the fifty-four chloroplast, which encodes cpSRP54 (ffc), and chaos (cao) loci, respectively. Two new null alleles in the ffc locus have been identified. ffc1-1 is caused by a stop codon in exon 10, while ffc1-2 has a large DNA insertion in intron 8. ffc mutants have yellow first true leaves that subsequently become green. The reaction center proteins D1, D2, and psaA/B, as well as seven different light-harvesting chlorophyll proteins (LHCPs), were found at reduced levels in the young ffc leaves but at wild-type levels in the older leaves. The abundance of the two types of LHCP was unaffected by the mutation, while two others were increased in the absence of cpSRP54. Null mutants in the cao locus contain reduced levels of the same subset of LHCP proteins as ffc mutants, but are distinguishable in four ways: young leaves are greener, the chlorophyll a/b ratio is elevated, levels of reaction center proteins are normal, and there is no recovery in the level of LHCPs in the adult plant. The data suggest that cpSRP54 and cpSRP43 have some nonoverlapping roles and that alternative transport pathways can compensate for the absence of a functional cpSRP.