培育具有安全选择标记或无选择标记的转基因植物

转基因植物中选择标记的安全性已成为植物基因工程研究的热点之一。从两个方面可以解决转基因植物中的选择标记问题。一是选用安全的正向选择标记,主要是与糖代谢和激素代谢相关的基因。二是构建能去除选择标记基因的转化系统,主要有共转化系统、双T-DNA边界载体系统、位点特异性重组系统和转座子系统等。这些植物基因工程的方法将有助于培育安全的转基因植物。 Abstract:The bio-safety of selective markers in transgenic plants has been a hot spot in the field of plant genetic engineering.To solve the problem of selective markers in the transgenic plants,two means of producing transgenic plants have been developed.One is the utilization of bio-safe positive selective markers which are genes mainly related to metabolism of auxins and carbohydrates.The other is the establishment of transformation systems allowing marker genes to be eliminated from the transgenic plants,which include co-ransformation,double T-DNA border vectors,site-specific recombination and transposition.All these approaches of plant genetic engineering will benefit breeding transgenic plants with bio-safety.     

Identification and genetic mapping of four novel genes that regulate leaf development in Arabidopsis

Molecular and genetic characterizations of mutants have led to a better understanding of many developmental processes in the model system Arabidopsis thaliana.However,the leaf development that is specific to plants has been little studies.With the aim of contributing to the genetic dissection of leaf development,we have performed a large-scare screening for mutants with abnormal leaves.Among a great number of leaf mutants we have generated by T-DNA and transposon tagging and ethylmethae sulfonate (EMS) mutagenesis,four independent mutant lines have been identified and studied genetically.Phenotypes of these mutant lines represent the defects of four novel muclear genes designated LL1(LOTUS LEAF 1),LL2(LOTUS LEAF2),URO(UPRIGHT ROSETTE),and EIL(ENVIRONMENT CONDITION INDUCED LESION).The phenotypic analysis indicates that these genes play important roles during leaf development.For the further genetic analysis of these genes and the map-based cloning of LL1 and LL2,we have mapped these genes to chromosome regions with an efficient and rapid mapping method.     

Molecular strategies of plant defense and insect counter-defense

The prediction of human population growth worldwide indicates there will be a need to substantially increase food production in order to meet the demand on food supply.This can be achieved in part by the effective management of insect pests. Since plants have co-evolved with herbivorous insects for millions of years, they have developed an array of defense genes to protect themselves against a wide variety of chewing and sucking insects.Using these naturally-occurring genes via genetic engineering represents an environmentally friendly insect pest-control measure. Insects, however, have been actively evolving adaptive mechanisms to evade natural plant defenses. Such evolved adaptability undoubtedly has helped insects during the last century to rapidly overcome a great many humanimposed management practices and agents, including chemical insecticides and genetically engineered plants. Thus, better understanding of the molecular and genetic basis of plant defense and insect counter-defense mechanisms is imperative, not only from a basic science perspective, but also for biotechnology-based pest control practice. In this review, we emphasize the recent advance and understanding of molecular strategies of attack-counterattack and defense-counter-defense between plants and their herbivores.     

金属结合蛋白基因及其在清除重金属污染中的应用

一些微生物和植物由于对毒性金属具有独特的抗性机制,使得利用它们来清除日益严重的环境污染已发展成为一种十分有效的技术——生物修复。研究表明,不同的金属结合蛋白（如MT 和PC）,在生物忍耐和降解过量重金属毒性机制中起重要作用。愈来愈多的MT 和PC基因被克隆,并已成功地应用于生物遗传转化,这些转基因生物在清除重金属污染方面已显示出潜在的应用价值。 Abstract:Heavy metal pollution has become a global environmental hazard.The use of microorganisms and plants for the decontamination of heavy metals is recognized as a low lost and high efficiency method for cleaning up metal contamination.It shows that various metal-binding proteins such as metallothioneins (MTs) or phytochelatines (PCs) play an important role in defense systems and detoxification to heavy metals in organisms.Many genes of MTs and PCs have been cloned and utilized successfully in genetically modified bacteria and plants for increasing remediation capacity.These transgenic organisms have been displayed a great potential in bioremediation and phytoremediation of heavy metals.     

Progress of chromosome engineering mediated by asymmetric somatic hybridization

Plant somatic hybridization has progressed steadily over the past 35 years. Many hybrid plants have been generated from fusion combinations of different phylogenetic species, some of which have been utilized in crop breeding programs. Among them, asymmetric hybrid, which usually contains a fraction of alien genome, has received more attention because of its importance in crop improvement. However, few studies have dealt with the heredity of the genome of somatic hybrid for a long time, which has limited the progress of this approach. Over recent ten years, along with the development of an effective cytogenetical tool ＂in situ hybridization （ISH）＂, asymmetric fusion of common wheat （Triticum aestivum L.） with different grasses or cereals has been greatly developed. Genetics, genomes, functional genes and agricultt, ral traits of wheat asymmetric hybrids have been subject to systematic investigations using gene cloning, genomic in situ hybridization （GISH） and molecular makers. The future goal is to fully elucidate the functional relationships among improved agronomic traits, the genes and underlying molecular mechanisms, and the genome dynamics of somatic introgression lines. This will accelerate the development of elite germplasms via somatic hybridization and the application of these materials in the molecular improvement of crop plants.     

Abiotic and Biotic Stresses and Changes in the Lignin Content and Composition in Plants

Lignin is a polymer of phenylpropanoid compounds formed through a complex biosynthesis route,represented by a metabolic grid for which most of the genes involved have been sequenced in several plants,mainly in the model-plants Arabidopsis thaliana and Populus.Plants are exposed to different stresses,which may change lignin content and composition.In many cases,particularly for plant-microbe interactions,this has been suggested as defence responses of plants to the stress.Thus,understanding how a stressor modulates expression of the genes related with lignin biosynthesis may allow us to develop study-models to increase our knowledge on the metabolic control of lignin deposition in the cell wall.This review focuses on recent literature reporting on the main types of abiotic and biotic stresses that alter the biosynthesis of lignin in plants.     

TRANSGENIC PLANTS EXPRESSING BACILLUS THURINGIENSIS DELTA-ENDOTOXINS

Commercial varieties of transgenic Bacillus thuringiensis (Bt) plants have been developed in many countries to control target pests. Initially, the expression of native Bt genes in plants was low due to mRNA instability, improper splicing, and post-translation modifications. Subsequently, modifications of the native Bt genes greatly enhanced expression levels. This is a review of the developments that made modem high-expression transgenic Bt plants possible, with an emphasis on the reasons for the low-level expression of native Bt genes in plant systems, and the techniques that have been used to improve plant expression of Bt toxin genes.     

Melatonin: A master regulator of plant development and stress responses

Melatonin is a pleiotropic molecule with multiple functions in plants. Since the discovery of melatonin in plants, numerous studies have provided insight into the biosynthesis, catabolism, and physiological and biochemical functions of this important molecule. Here, we describe the biosynthesis of melatonin from tryptophan, as well as its various degradation pathways in plants. The identification of a putative melatonin receptor in plants has led to the hypothesis that melatonin is a hormone involved in regulating plant growth,aerial organ development, root morphology, and the floral transition. The universal antioxidant activity of melatonin and its role in preserving chlorophyll might explain its anti-senescence capacity in aging leaves. An impressive amount of research has focused on the role of melatonin in modulating postharvest fruit ripening by regulating the expression of ethylene-related genes.Recent evidence also indicated that melatonin functions in the plant's response to biotic stress,cooperating with other phytohormones and wellknown molecules such as reactive oxygen species and nitric oxide. Finally, great progress has been made towards understanding how melatonin alleviates the effects of various abiotic stresses, including salt, drought, extreme temperature, and heavy metal stress. Given its diverse roles, we propose that melatonin is a master regulator in plants.     

The Genetic and Molecular Basis of Plant Resistance to Pathogens

Plant pathogens have evolved numerous strategies to obtain nutritive materials from their host,and plants in turn have evolved the preformed physical and chemical barriers as well as sophisticated two-tiered immune system to combat pathogen attacks.Genetically, plant resistance to pathogens can be divided into qualitative and quantitative disease resistance,conditioned by major gene(s) and multiple genes with minor effects,respectively.Qualitative disease resistance has been mostly detected in plant defense against biotrophic pathogens,whereas quantitative disease resistance is involved in defense response to all plant pathogens,from biotrophs,hemibiotrophs to necrotrophs.Plant resistance is achieved through interception of pathogen-derived effectors and elicitation of defense response.In recent years,great progress has been made related to the molecular basis underlying host-pathogen interactions.In this review,we would like to provide an update on genetic and molecular aspects of plant resistance to pathogens.     

Expression and signal regulation of the alternative oxidase genes under abiotic stresses

Plants in their natural environment frequently face various abiotic stresses, such as drought, high salinity, and chilling. Plant mitochondria contain an alternative oxidase （AOX）, which is encoded by a small family of nuclear genes. AOX genes have been shown to be highly responsive to abiotic stresses. Using transgenic plants with varying levels of AOX expression, it has been confirmed that AOX genes are im- portant for abiotic stress tolerance. Although the roles of AOX under abiotic stresses have been extensively studied and there are several excellent reviews on this topic, the differential expression patterns of the AOX gene family members and the signal regulation of AOX gene（s） under abiotic stresses have not been extensively summarized. Here, we review and discuss the current progress of these two important issues.     

Comparative genomics of rice and Arabidopsis. Analysis of 727 cytochrome P450 genes and pseudogenes from a monocot and a dicot

Data mining methods have been used to identify 356 Cyt P450 genes and 99 related pseudogenes in the rice (Oryza sativa) genome using sequence information available from both the indica and japonica strains. Because neither of these genomes is completely available, some genes have been identified in only one strain, and 28 genes remain incomplete. Comparison of these rice genes with the 246 P450 genes and 26 pseudogenes in the Arabidopsis genome has indicated that most of the known plant P450 families existed before the monocot-dicot divergence that occurred approximately 200 million years ago. Comparative analysis of P450s in the Pinus expressed sequence tag collections has identified P450 families that predated the separation of gymnosperms and flowering plants. Complete mapping of all available plant P450s onto the Deep Green consensus plant phylogeny highlights certain lineage-specific families maintained (CYP80 in Ranunculales) and lineage-specific families lost (CYP92 in Arabidopsis) in the course of evolution.     

Genome-wide identification and characterization of putative cytochrome P450 genes in the model legume <Emphasis Type="Italic">Medicago truncatula</Emphasis>

In plants, cytochrome P450 is a group of monooxygenases existing as a gene superfamily and plays important roles in metabolizing physiologically important compounds. However, to date only a limited number of P450s have been identified and characterized in legumes. In this study, data mining methods were used, and 151 putative P450 genes in the model legume Medicago truncatula were identified, including 135 novel sequences. These genes were classified into 9 clans and 44 families by sequence similarity, and among those 4 new clans and 21 new families not reported previously in legumes. By comparison of these genes with P450 genes in Arabidopsis and rice, it was found that most of the known P450 families in dicot species exist in M. truncatula. The representative protein sequences of putative P450s were aligned, and the secondary elements were assigned based on the known structure P450BM3. Putative substrate recognition sites (SRSs) and substrate binding sites were also identified in these sequences. In addition, the ESTs-derived expression profiles (digital Northern) of the putative P450 genes were analyzed, which was confirmed by semi-quantitative RT-PCR analyses of several selected P450 genes. These results will provide a base for catalogue information on P450 genes in M. truncatula and for further functional analysis of P450 superfamily genes in legumes.     

Cytochromes P450 for engineering herbicide tolerance

In recent years, genome sequencing has revealed that cytochromes P450 (P450s) constitute the largest family of enzymatic proteins in higher plants. P450s are mono-oxygenases that insert one atom of oxygen into inert hydrophobic molecules to make them more reactive and hydrosoluble. Besides their physiological functions in the biosynthesis of hormones, lipids and secondary metabolites, P450s help plants to cope with harmful exogenous chemicals including pesticides and industrial pollutants, making them less phytotoxic. The recovery of an increasing number of plant P450 genes in recombinant form has enabled their use in experimentation, which has revealed their extraordinary potential for engineering herbicide tolerance, biosafening, bioremediation and green chemistry.     

A general cloning strategy for divergent plant cytochrome P450 genes and its application in Lolium rigidum and Ocimum basilicum

The investigation of plant cytochrome P450 genes and enzymes is a field of growing interest. Apparently, an even greater diversity of cytochrome P450 genes exists in plants in comparison to other eukaryotes. This may be due to their role in the biosynthesis of secondary metabolites that are present in plants in an enormous variety. Most cloning approaches are hampered by the large sequence diversity of plant cytochrome P450 genes. We present a method to clone divergent cytochrome P450 ESTs by a nested RT-PCR-strategy. These ESTs were used for the subsequent cloning of the corresponding full-size cDNAs of divergent families via cDNA-library screening. Sixteen cytochrome P450 genes belonging to different cytochrome P450-families have been identified in this way, proving the efficacy of the strategy. Received: 1 December 2000 / Accepted: 26 February 2001     

The cytochrome P450 gene superfamily in Drosophila melanogaster: annotation, intron-exon organization and phylogeny

The cytochrome P450 gene superfamily is represented by 90 sequences in the Drosophila melanogaster genome. Of these 90 P450 sequences, 83 code for apparently functional genes whereas seven are apparent pseudogenes. More than half of the genes belong to only two families, CYP4 and CYP6. The CYP6 family is insect specific whereas the CYP4 family includes sequences from vertebrates. There are eight genes coding for mitochondrial P450s as deduced from their homology to CYP12A1 from the house fly. The genetic map of the distribution of D. melanogaster P450 genes shows (a) the absence of P450 genes on the chromosome 4 and Y, (b) more than half of the P450 genes are found on chromosome 2, and (c) the largest cluster contains nine genes. Sequence alignments were used to draw phylogenetic trees and to analyze the intron-exon organization of each functional P450 gene. Only five P450 genes are intronless. We found 57 unique intron positions, of which 23 were phase zero, 19 were phase one and 15 were phase two. There was a relatively good correlation between intron conservation and phylogenetic relationship between members of the P450 subfamilies. Although the function of many P450 proteins from vertebrates, fungi, plants and bacteria is known, only a single P450 from D. melanogaster, CYP6A2, has been functionally characterized. Gene organization appears to be a useful tool in the study of the regulation, the physiological role and the function of these P450s.     

Transgenic rice plants expressing human p450 genes involved in xenobiotic metabolism for phytoremediation

Phytoremediation is the use of plants to remove xenobiotic compounds from the environment. Plants have the inherent ability to detoxify xenobiotic pollutants, but they are generally poor at degrading them. The introduction of genes involved in xenobiotic degradation is aimed at enhancing plants' potential further. Rice (Oryza sativa) is a good candidate for this purpose and has been transformed with genes encoding cytochrome P450 monooxygenases CYP1A1, CYP2B6, and CYP2C19. The transgenic plants were more tolerant to various herbicides than nontransgenic Nipponbare rice plants, owing to enhanced metabolism by the introduced P450 enzymes. Transgenic plants were able to remove atrazine and metolachlor from soil. Field testing and risk assessment are very important for developing transgenic plants for phytoremediation. Transgenic rice plants should become useful as herbicide-tolerant crops and for phytoremediation of xenobiotic pollutants in future.     

Cytochromes P450: a success story

Cytochrome P450 proteins, named for the absorption band at 450 nm of their carbon-monoxide-bound form, are one of the largest superfamilies of enzyme proteins. The P450 genes (also called CYP) are found in the genomes of virtually all organisms, but their number has exploded in plants. Their amino-acid sequences are extremely diverse, with levels of identity as low as 16% in some cases, but their structural fold has remained the same throughout evolution. P450s are heme-thiolate proteins; their most conserved structural features are related to heme binding and common catalytic properties, the major feature being a completely conserved cysteine serving as fifth (axial) ligand to the heme iron. Canonical P450s use electrons from NAD(P)H to catalyze activation of molecular oxygen, leading to regiospecific and stereospecific oxidative attack of a plethora of substrates. The reactions carried out by P450s, though often hydroxylation, can be extremely diverse and sometimes surprising. They contribute to vital processes such as carbon source assimilation, biosynthesis of hormones and of structural components of living organisms, and also carcinogenesis and degradation of xenobiotics. In plants, chemical defense seems to be a major reason for P450 diversification. In prokaryotes, P450s are soluble proteins. In eukaryotes, they are usually bound to the endoplasmic reticulum or inner mitochondrial membranes. The electron carrier proteins used for conveying reducing equivalents from NAD(P)H differ with subcellular localization. P450 enzymes catalyze many reactions that are important in drug metabolism or that have practical applications in industry; their economic impact is therefore considerable.     

Cytochromes P450 in gibberellin biosynthesis

The gibberellins (GAs) are an important class of plant growth regulators that are active in many aspects of plant growth and development. GAs are synthesized by a complex pathway involving three enzyme classes spanning different subcellular compartments. One of these enzyme classes is the cytochrome P450s which catalyze a number of oxidation steps in the middle part of the pathway. Mutants in these cytochrome P450-mediated steps in a number of species have been crucial in isolating the genes encoding these enzymes and have also played an important role in understanding GA physiology. GAs are also synthesized by fungi, in a biosynthesis pathway largely catalyzed by cytochrome P450s. The fungal pathway appears to have evolved independently to that of higher plants.

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Plant cytochrome P450-mediated herbicide metabolism

In the last two decades it has become apparent that enzymes of the P450 monooxygenase (P450) superfamily are responsible for the Phase I metabolism of numerous herbicides representing several classes of organic compounds. The majority of experimental evidence for P450 involvement in herbicide metabolism has been derived from in vitro studies in which the catalytic activity of plant microsomes towards herbicidal substrates was measured in the presence of various P450 inhibitors and activators. While the studies with microsomes elicited much appreciation for the pivotal roles of plant P450s in herbicide metabolism, detailed characterization of these enzymes only became possible after the isolation of genes encoding specific isoforms responsible for herbicide conversion. Several lines of evidence suggest that the development of herbicide resistance in weeds by enhanced detoxification is frequently associated with elevated levels of P450 activity. Enhanced detoxification-based herbicide resistance is particularly difficult to control, because it can involve resistance to multiple, chemically unrelated classes of herbicides. Continued research efforts are aimed at elucidating the role of P450s in the metabolic fates of herbicides in plants and the development of herbicide resistance in weeds. Recent advances made in the isolation and genetic manipulation of P450 enzymes have created new opportunities for their application in engineering herbicide tolerance and bioremediation.     

Plant Cytochrome P450 Monooxygenases

Plant systems utilize a diverse array of cytochrome P450 monooxygenases (P450s) in their biosynthetic and detoxification pathways. The classic forms of these enzymes are heme-dependent mixed function oxidases that utilize NADPH or NADH and molecular oxygen to produce functionalized organic products. The nonclassical forms are monooxygenases that either do not utilize flavoproteins for dioxygen activation or fail to incorporate molecular oxygen into their final product. Biosynthetic P450s play paramount roles in the synthesis of lignin intermediates, sterols, terpenes, flavonoids, isoflavonoids, furanocoumarins, and a variety of other secondary plant products. Other catabolic P450s metabolize toxic herbicides and insecticides into nontoxic products or, conversely, activate nontoxic substances into toxic products. Biochemical and molecular characterizations on a number of plant P450s have indicated that the relationships between these heme proteins and their substrates are at least as complex as those that exist in mammalian systems. Examples now exist of plant P450s that metabolize: a narrow range of substrates to yield different products, a single substrate to yield different products, multiple substrates to yield the same product, or a single substrate sequentially to yield discrete intermediates in the biosynthesis of a single product. Extensive divergence of catalytic site as well as noncatalytic site residues accounts for the high degree of primary structure variation in the P450 gene superfamily and the diverse array of substrates synthesized and/or detoxified by these proteins. Classic P450s still retain a highly conserved F-G-R-C-G motif in their catalytic site and conserved amino acids in their oxygen binding pocket; nonclassical P450s diverge at several of these positions. A broad range of cloning and transient expression strategies are suitable for plant P450 studies and these have allowed for the isolation and characterization of a number of P450 cDNAs and genes. Because many of these sequences have been cloned only recently, much remains to be learned about the substrate specificities of P450 reactions in plants and the mechanisms by which their genes are regulated.