植物功能基因组研究进展

随着植物基因组计划的深入,植物基因组学研究的重点已经转变为基因组功能的研究,即利用基因组序列的信息和高通量的系统分析技术,在基因组水平研究植物结构和组织与植物功能在细胞、有机体和进化上的关系.对功能基因组学研究的内容、方法以及最新研究进展进行了综述.     

植物转座子及其在番茄功能基因组的应用

转座子作为插入突变原或分子标签被广泛应用于基因的分离和克隆,已成为发现新基因和基因功能分析的有效工具。该文综述了植物转座子及其在基因分离中的研究进展,并讨论其在番茄功能基因遗传资源、功能基因组分离等研究中的应用。     

Stable Plastid Transformation in <Emphasis Type="Italic">Nicotiana benthamiana</Emphasis>

Plastids from Nicotiana benthamiana were transformed with the vector for dicistronic expression of two genes—aminoglycoside 3'-adenyltransferase (aadA) and green fluorescent protein (gfp)—in the plastids of Nicotiana tabacum. Transplastomic shoots exhibited green fluorescence under UV light. Transformation efficiencies were similar between species. Although the border sequence (trnI and trnA) for homologous recombination to transform the plastid genome of N. benthamiana was identical to that sequence of N. tabacum, the exception was a 9-bp addition in the intron of trnI. This indicated that the N. tabacum sequence used as a border region for recombination was sufficient to insert the foreign gene into the target site between the trnI and trnA of N. benthamiana with similar efficiency. Southern blot analysis detected the presence of aadA and gfp between trnI and trnA in the plastid genome of N. benthamiana. Northern and western blot analyses revealed high expression of gfp in the plastids from petals and leaves. Our results suggest that the plastid transformation system established here is applicable to investigations of the interactions between plastid and nucleus in N. benthamiana.     

植物功能基因组学的发展现状与发展趋势

本文将评述植物功能基因组学研究领域最新的发展状况及其趋势。     

基于植物重组蛋白产量提高的研究进展

重组蛋白为疾病治疗提供了新手段,同时创造了可观的经济效益。利用经济作物(主要是烟草)、谷类作物、豆科作物和蔬菜作物生产具有药用价值的重组蛋白是“分子农业”最热门的研究内容。尽管许多重组蛋白已在植物中表达,但只有一小部分已成功投入使用。为了极大地克服限制植物生产重组蛋白发展的问题,研究人员改进表达系统以增加重组蛋白的产量。本文从分析植物产生重组蛋白产量低和/或生物活性低等问题入手,综述了近些年来解决这些问题的优化策略,同时提出了提高植物生产重组蛋白产量的研究方向。     

Development of a quantitative tool for measuring changes in the coefficient of conductivity of plasmodesmata induced by developmental, biotic, and abiotic signals

Summary. The regulation of intercellular and interorgan communication is pivotal for cell fate decisions in plant development and probably plays a significant role in the systemic regulation of gene expression and in defense reactions against pathogens or other biotic and abiotic environmental factors. In plants, symplasmic cell-to-cell communication is provided by plasmodesmata (Pd), coaxial membranous tunnels that span cell walls interconnecting adjacent cytoplasms. Macromolecules, proteins, and RNA may be transported through Pd by passive diffusion or by a facilitated mechanism. A quantitative tool was developed to measure the coefficient of conductivity, C(Pd), for diffusion-driven transport via Pd and to assess changes in the coefficient induced by developmental, biotic and abiotic signals. ^GFPC(Pd), the coefficient of conductivity for cell-to-cell spread of green-fluorescent protein (GFP), a protein with a Stokes radius of 2.82 nm, was determined in epidermal cells of sink and source leaves of wild-type and transgenic Nicotiana benthamiana plants expressing the movement protein of tobacco mosaic virus (MP^TMV) incubated both in dark and light and at 16 and 25°C. Under all conditions, Pd in source leaves conducted macromolecules, with ^GFPC(Pd)_sink > ^GFPC(Pd)_source. Light down-regulated ^GFPC(Pd) (all conditions); down-regulation was stronger for sink cells. The effect of MP^TMV on ^GFPC(Pd) between epidermal cells was dependent on temperature and leaf development; at 16°C, MP^TMV down-regulated ^GFPC(Pd) only in source leaves, while at 25°C, MP^TMV had no significant effect. This quantitative tool should be useful for investigating differences in Pd conductivity that are induced by mutations or silencing. Correspondence and reprints: Department of Plant Sciences, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel. Present address: Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel.     

Reverse Genetic Approaches for Functional Genomics of Rice

T-DNA and transposable elements e.g., Ds and Tos17, are used to generate a large number of insertional mutant lines in rice. Some carry the GUS or GFP reporter for gene trap or enhancer trap. These reporter systems are valuable for identifying tissue- or organ-preferential genes. Activation tagging lines have also been generated for screening mutants and isolating mutagenized genes. To utilize these resources more efficiently, tagged lines have been produced for reverse genetic approaches. DNA pools of the T-DNA tagged lines and Tos17 lines have been prepared for PCR screening of insertional mutants in a given gene. Tag end sequences (TES) of the inserts have also been produced. TES databases are beneficial for analyzing the function of a large number of rice genes.     

Virus-induced silencing of WIPK and SIPK genes reduces resistance to a bacterial pathogen,but has no effect on the INF1-induced hypersensitive response (HR) in Nicotiana benthamiana

Activation of two mitogen-activated protein kinases (MAPKs), wound-induced protein kinase (WIPK) and salicylic acid-induced protein kinase (SIPK), is one of the earliest responses that occur in tobacco plants that have been wounded, treated with pathogen-derived elicitors or challenged with avirulent pathogens. We isolated cDNAs for these MAPKs ( NbWIPK and NbSIPK) from Nicotiana benthamiana. The function of NbWIPK and NbSIPK in mediating the hypersensitive response (HR) triggered by infiltration with INF1 protein (the major elicitin secreted by Phytophthora infestans), and the defense response to an incompatible bacterial pathogen ( Pseudomonas cichorii), was investigated by employing virus-induced gene silencing (VIGS) to inhibit expression of the WIPK and SIPK genes in N. benthamiana. Silencing of WIPK or SIPK, or both genes simultaneously, resulted in reduced resistance to P. cichorii, but no change was observed in the timing or extent of HR development after treatment with INF1.Communicated by R. G. Herrmann     

Recent Progress in Development of Tnt1 Functional Genomics Platform for Medicago truncatula and Lotus japonicus in Bulgaria

Legumes, as protein-rich crops, are widely used for human food, animal feed and vegetable oil production. Over the past decade, two legume species, Medicago truncatula and Lotus japonicus, have been adopted as model legumes for genomics and physiological studies. The tobacco transposable element, Tnt1, is a powerful tool for insertional mutagenesis and gene inactivation in plants. A large collection of Tnt1-tagged lines of M. truncatula cv. Jemalong was generated during the course of the project 'GLIP': Grain Legumes Integrated Project, funded by the European Union (www.eugrainlegumes.org). In the project 'IFCOSMO': Integrated Functional and COmparative genomics Studies on the MOdel Legumes Medicago truncatula and Lotus japonicus, supported by a grant from the Ministry of Education, Youth and Science, Bulgaria, these lines are used for development of functional genomics platform of legumes in Bulgaria. This review presents recent advances in the evaluation of the M. truncatula Tnt1 mutant collection and outlines the steps that are taken in using the Tnt1-tagging for generation of a mutant collection of the second model legume L. japonicus. Both collections will provide a number of legume-specific mutants and serve as a resource for functional and comparative genomics research on legumes. Genomics technologies are expected to advance genetics and breeding of important legume crops (pea, faba bean, alfalfa and clover) in Bulgaria and worldwide.     

本氏烟草悬浮细胞NBS-1的创建及底盘特征分析

植物合成生物学在植物天然产物的发掘制造方面具有明显的理论优势,但因缺乏高效的底盘系统及相关使能技术,其对生物合成领域的贡献有限。最常用的植物底盘——烟草由于操作周期长、改造难度大、代谢及纯化背景复杂、烟碱毒性和农业生产难精准控制等问题,而烟草悬浮细胞底盘体系可有效解决上述问题。本研究旨在开发出生长快速、生物量高、分散度好、转化效率高、烟碱含量极低并具备较高科研价值及工业化潜力的烟草悬浮细胞底盘。选取分子技术高适用性的本氏烟草(Nicotiana benthamiana),诱导获得的悬浮细胞NBS-1生长迅速、分散性好,最高生物量可达到476.39 g/L (鲜重),各项参数均远超常用的烟草BY-2 (Nicotiana tabacum L. cv Bright Yellow 2)细胞系。常用的pEAQ-HT高效瞬时表达系统在NBS-1中的转化表达效率可达81%,远高于BY-2。利用转录组数据对BY-2和NBS-1的代谢特征及偏好性进行分析,发现NBS-1的黄酮类等化合物合成通路基因的表达显著高于BY-2,且NBS-1的生物碱合成通路基因的表达显著低于BY-2,并通过代谢物含量测定实验验证了该分析结果,表明NBS-1极低烟碱含量的同时,为该底盘对应产品的选择提供参考。综上,本研究开发出的生长转化优异、黄酮类含量较高且烟碱含量极低的本氏烟草悬浮细胞底盘NBS-1,对于开发烟草悬浮细胞底盘有重要指导意义。     

3种烟草基因组SSR位点信息分析和标记开发

利用生物信息学方法对绒毛状烟草、林烟草和本塞姆氏烟草3份烟草野生种基因组数据中的SSR位点信息进行了分析。结果表明,在全长分别为2.14Gb、2.44Gb和2.59Gb的绒毛状烟草、林烟草和本塞姆氏烟草基因组中分别获得153 357个、196 493个和278 784个SSR位点,平均相隔13.94kb、12.42kb和9.31kb出现一个SSR。在SSR位点分布区域上,绝大部分的SSR位点分布在内含子和UTR(尤其是5′-UTR)区域;在SSR基序类型上,主要集中在二、三碱基基序且二者占总SSR位点数目的80%以上,并以二碱基基序类型丰度最高;在SSR基序结构上,基因组中出现频率及数量最高的是含有A(T)n的基序结构;在SSR基序的重复次数上,除单碱基基序类型外,重复次数多在3~10次之间。利用分别属于5个不同烟草组的8份烟草材料验证所合成的300对引物,所有合成的引物均能扩增获得目标片段,其中有80对引物存在扩增多态性。表明来源于绒毛状烟草、林烟草和本塞姆氏烟草基因组的SSR标记在亲缘关系相对较近的烟草种间具有高度保守性和通用性,基于此3份烟草野生种基因组数据开发SSR引物用于后续的相关遗传研究具有可行性。     

模式植物短柄草研究新进展

短柄草(Brachypodium distachyon)株型矮小,易于种植栽培,生长周期短,自花授粉,容易繁殖。另外,短柄草基因组比较小,易于转化,与小麦具有比较近的亲缘关系,是理想的草类特别是禾本科模式植物。近年来,短柄草的研究工作在细胞遗传学、基因组学、比较基因组学、植物-病原菌相互作用、功能基因组学等研究领域取得了许多进展,包括完成了Bd-21全基因组的测序工作、构建了T-DNA插入突变体库、用遗传学的方法首次研究短柄草基因的生物学功能等。本文综述了近年来特别是2009年以来短柄草的研究进展,并对未来的研究工作做了展望。     

Mass-Spectrometry-Linked Screening of Protein Fractions for Enzymatic Activities—A Tool for Functional Genomics

A simple and rapid strategy is described to screen protein fractions for defined enzymatic activity. A protein fraction from a porcine kidney extract was immobilized by covalent coupling to activated affinity beads. The immobilized proteins were incubated with probes specific for different enzyme activities. The reaction products were analyzed by matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry. The MALDI spectra indicate the presence of 5′-nucleotidase, phosphatase, kinase, glutathione reductase, and renin activities in the kidney protein extract. Furthermore, the method can be used to screen for inhibitors of enzymatic reactions. The method is adaptable to high-throughput sample handling and automated mass spectrometric analysis and therefore suited for functional genomics.     

Expression of the rabies virus nucleoprotein in plants at high-levels and evaluation of immune responses in mice

Transgenic plants have been employed successfully as a low-cost system for the production of therapeutically valuable proteins including antibodies, antigens and hormones. Here, we report expression of a full-length nucleoprotein gene of rabies virus in transgenic tomato plants. The nucleoprotein was also transiently expressed in Nicotiana benthamiana plants by agroinfiltration. In both cases, the nucleoprotein was expressed at high levels, 1–5% of total soluble protein in tomato and 45% in N. benthamiana. Previously, only epitopes of the nucleoprotein had been expressed in plants. The presence and expression of the transgene was verified by PCR, Southern, northern and western blots. Mice were immunized both intraperitoneally (i.p.) and orally with tomato protein extracts containing the N protein induced the production of antibodies. The antibody titer of mice immunized i.p., was at least four times higher than that of mice immunized orally. These results were reflected in the challenge experiments where i.p.-immunized mice were partially protected against a peripheral virus challenge whereas orally immunized mice were not. This protection was comparable to that obtained in previous experiments employing different expression systems. Work is in progress to express both G and N proteins in transgenic plants and evaluate protection in mice.