In the Medicago genus, triterpenic saponins are bioactive secondary metabolites constitutively synthesized in the aerial and subterranean parts of plants via the isoprenoid pathway. Exploitation of saponins as pharmac...In the Medicago genus, triterpenic saponins are bioactive secondary metabolites constitutively synthesized in the aerial and subterranean parts of plants via the isoprenoid pathway. Exploitation of saponins as pharmaceutics, agrochemicals and in the food and cosmetic industries has raised interest in identifying the enzymes involved in their synthesis. We have identified a cytochrome P450 (CYP72A67) involved in hemolytic sapogenin biosynthesis by a reverse genetic TILLING approach in a Medicago truncatula ethyl- methanesulfonate (EMS) mutagenized collection. Genetic and biochemical analyses, mutant complementation, and expression of the gene in a microsome yeast system showed that CYP72A67 is responsible for hydroxylation at the C-2 position downstream of oleanolic acid synthesis. The affinity of CYP72A67 for substrates with different substitutions at multiple carbon positions was investigated in the same in vitro yeast system, and in relation to two other CYP450s (CYP72A68) responsible for the production of medicagenic acid, the main sapogenin in M. truncatula leaves and roots. Full sib mutant and wild-type plants were compared for their sapogenin profile, expression patterns of the genes involved in sapogenin synthesis, and response to inoculation with Sinorhizobium meliloti. The results obtained allowed us to revise the hemolytic sapogenin pathway in M. truncatula and contribute to highlighting the tissue specificities (leaves/roots) of sapogenin synthesis.展开更多
TILLING技术(Targeting Induced Local Lesions IN Genomes)经过十多年的快速发展,已经应用到多种植物点突变高通量筛选与基因功能研究中,检测方法也逐渐趋于多样化,直接测序、毛细管电泳、高分辨熔解曲线等检测方法均得到了较多的应用...TILLING技术(Targeting Induced Local Lesions IN Genomes)经过十多年的快速发展,已经应用到多种植物点突变高通量筛选与基因功能研究中,检测方法也逐渐趋于多样化,直接测序、毛细管电泳、高分辨熔解曲线等检测方法均得到了较多的应用。与此同时EcoTILLING、DeTILLING及iTILLING等相关技术的发展扩大了TILLING技术的应用范围。本文着重介绍了目前TILLING分析中用到的不同检测方法及相关生物信息学工具,详细评述了TILLING技术在模式植物、主要粮食作物、油料作物、蔬菜等其它作物中的应用与研究进展。展开更多
TILLING(Targeting induced local lesions in genomes,定向诱导基因组局部突变技术)是一种高通量的等位变异创制和突变体快速鉴定技术,其实质是将传统的化学诱变方法和突变的高效筛选有效结合的反向遗传学研究方法。其技术原理是将传...TILLING(Targeting induced local lesions in genomes,定向诱导基因组局部突变技术)是一种高通量的等位变异创制和突变体快速鉴定技术,其实质是将传统的化学诱变方法和突变的高效筛选有效结合的反向遗传学研究方法。其技术原理是将传统的酶切技术与PCR技术相结合后采用红外双色荧光系统进行结果鉴定,从而筛选出相应的突变体。传统的TILLING技术主要用于筛选由人工诱导产生的突变体。Ecotilling技术由TILL-ING技术延伸而来,主要用于鉴定自然界中已经存在的突变体,其与传统的TILLING技术的区别主要为构建DNA池时略有差异。随着该项技术在拟南芥等模式植物中的成功应用,越来越多的人开始将其用于基因组较大的植物之中。本文对近年来TILLING技术在麦类作物中的应用进行了分析,并通过比较不同植物突变体库中的突变频率发现,经EMS处理的小麦等麦类作物突变体库中的突变频率显著高于其他植物,因此相信,TILLING技术将会作为一种常规手段在麦类作物尤其是普通小麦改良中得到越来越广泛的应用。展开更多
定向诱导基因组局部突变(targeting induced local lesions in genomes,TILLING)技术将化学诱变与高通量突变检测技术相结合,可高效、快速地从突变群体中鉴定出目标基因突变位点。本文在概述TILLING技术应用于水稻、小麦、玉米、大豆等...定向诱导基因组局部突变(targeting induced local lesions in genomes,TILLING)技术将化学诱变与高通量突变检测技术相结合,可高效、快速地从突变群体中鉴定出目标基因突变位点。本文在概述TILLING技术应用于水稻、小麦、玉米、大豆等作物突变研究现状的基础上,重点综述了TILLING分析群体构建与突变位点检测方法的技术改进与发展,探讨了TILLING技术目前存在的问题与前景。展开更多
The balance between the supply and demand of the major food crops is fragile, fueling concerns for long-term global food security. The rising population, increasing wealth and a proliferation of non- food uses (e.g. ...The balance between the supply and demand of the major food crops is fragile, fueling concerns for long-term global food security. The rising population, increasing wealth and a proliferation of non- food uses (e.g. bioenergy) has led to growing demands on agricul- ture, while increased production is limited by greater urbanization, and the degradation of land. Furthermore, global climate change with increasing temperatures and lower, more erratic rainfall is projected to decrease agricultural yields. There is a predicted need to increase food production by at least 70% by 2050 and there- fore an urgent need to develop novel and integrated approaches, incorporating high-throughput phenotyping that will both increaseproduction per unit area and simultaneously improve the resource use efficiency of crops. Yield potential, yield stability, nutrient and water use are all complex multigenic traits and while there is genetic variability, their complexity makes such traits difficult to breed for directly. Nevertheless molecular plant breeding has the potential to deliver substantial improvements, once the component traits and the genes underlying these traits have been identified. In addition, interactions between the individual traits must also be taken into account, a demand that is difficult to fulfill with traditional screening approaches. Identified traits will be incorporated into new cultivars using conventional or biotechnological tools. In order to better understand the relationship between genotype, component traits, and environment over time, a multidisciplinary approach must be adopted to both understand the underlying processes and identify candidate genes, QTLs and traits that can be used to develop improved crops.展开更多
Zebrafish(Danio rerio) is a well-established vertebrate animal model.A comprehensive collection of reverse genetics tools has been developed for studying gene function in this useful organism.Morpholino is the most ...Zebrafish(Danio rerio) is a well-established vertebrate animal model.A comprehensive collection of reverse genetics tools has been developed for studying gene function in this useful organism.Morpholino is the most widely used reagent to knock down target gene expression post-transcriptionally.For a long time,targeted genome modification has been heavily relied on large-scale traditional forward genetic screens,such as ENU(N-ethyl-N-nitrosourea) mutagenesis derived TILLING(Targeting Induced Local Lesions IN Genomes) strategy and pseudo-typed retrovirus mediated insertional mutagenesis.Recently,engineered endonucleases,including ZFNs(zinc finger nucleases) and TALENs(transcription activator-like effector nucleases),provide new and efficient strategies to directly generate site-specific indel mutations by inducing double strand breaks in target genes.Here we summarize the major reverse genetic approaches for loss-of-function studies used and emerging in zebrafish,including strategies based on genome-wide mutagenesis and methods for site-specific gene targeting.Future directions and expectations will also be discussed.展开更多
文摘In the Medicago genus, triterpenic saponins are bioactive secondary metabolites constitutively synthesized in the aerial and subterranean parts of plants via the isoprenoid pathway. Exploitation of saponins as pharmaceutics, agrochemicals and in the food and cosmetic industries has raised interest in identifying the enzymes involved in their synthesis. We have identified a cytochrome P450 (CYP72A67) involved in hemolytic sapogenin biosynthesis by a reverse genetic TILLING approach in a Medicago truncatula ethyl- methanesulfonate (EMS) mutagenized collection. Genetic and biochemical analyses, mutant complementation, and expression of the gene in a microsome yeast system showed that CYP72A67 is responsible for hydroxylation at the C-2 position downstream of oleanolic acid synthesis. The affinity of CYP72A67 for substrates with different substitutions at multiple carbon positions was investigated in the same in vitro yeast system, and in relation to two other CYP450s (CYP72A68) responsible for the production of medicagenic acid, the main sapogenin in M. truncatula leaves and roots. Full sib mutant and wild-type plants were compared for their sapogenin profile, expression patterns of the genes involved in sapogenin synthesis, and response to inoculation with Sinorhizobium meliloti. The results obtained allowed us to revise the hemolytic sapogenin pathway in M. truncatula and contribute to highlighting the tissue specificities (leaves/roots) of sapogenin synthesis.
文摘TILLING技术(Targeting Induced Local Lesions IN Genomes)经过十多年的快速发展,已经应用到多种植物点突变高通量筛选与基因功能研究中,检测方法也逐渐趋于多样化,直接测序、毛细管电泳、高分辨熔解曲线等检测方法均得到了较多的应用。与此同时EcoTILLING、DeTILLING及iTILLING等相关技术的发展扩大了TILLING技术的应用范围。本文着重介绍了目前TILLING分析中用到的不同检测方法及相关生物信息学工具,详细评述了TILLING技术在模式植物、主要粮食作物、油料作物、蔬菜等其它作物中的应用与研究进展。
文摘TILLING(Targeting induced local lesions in genomes,定向诱导基因组局部突变技术)是一种高通量的等位变异创制和突变体快速鉴定技术,其实质是将传统的化学诱变方法和突变的高效筛选有效结合的反向遗传学研究方法。其技术原理是将传统的酶切技术与PCR技术相结合后采用红外双色荧光系统进行结果鉴定,从而筛选出相应的突变体。传统的TILLING技术主要用于筛选由人工诱导产生的突变体。Ecotilling技术由TILL-ING技术延伸而来,主要用于鉴定自然界中已经存在的突变体,其与传统的TILLING技术的区别主要为构建DNA池时略有差异。随着该项技术在拟南芥等模式植物中的成功应用,越来越多的人开始将其用于基因组较大的植物之中。本文对近年来TILLING技术在麦类作物中的应用进行了分析,并通过比较不同植物突变体库中的突变频率发现,经EMS处理的小麦等麦类作物突变体库中的突变频率显著高于其他植物,因此相信,TILLING技术将会作为一种常规手段在麦类作物尤其是普通小麦改良中得到越来越广泛的应用。
文摘定向诱导基因组局部突变(targeting induced local lesions in genomes,TILLING)技术将化学诱变与高通量突变检测技术相结合,可高效、快速地从突变群体中鉴定出目标基因突变位点。本文在概述TILLING技术应用于水稻、小麦、玉米、大豆等作物突变研究现状的基础上,重点综述了TILLING分析群体构建与突变位点检测方法的技术改进与发展,探讨了TILLING技术目前存在的问题与前景。
基金funded by the Biotechnology and Biological Research Council of the UKDEFRA for the WGIN project
文摘The balance between the supply and demand of the major food crops is fragile, fueling concerns for long-term global food security. The rising population, increasing wealth and a proliferation of non- food uses (e.g. bioenergy) has led to growing demands on agricul- ture, while increased production is limited by greater urbanization, and the degradation of land. Furthermore, global climate change with increasing temperatures and lower, more erratic rainfall is projected to decrease agricultural yields. There is a predicted need to increase food production by at least 70% by 2050 and there- fore an urgent need to develop novel and integrated approaches, incorporating high-throughput phenotyping that will both increaseproduction per unit area and simultaneously improve the resource use efficiency of crops. Yield potential, yield stability, nutrient and water use are all complex multigenic traits and while there is genetic variability, their complexity makes such traits difficult to breed for directly. Nevertheless molecular plant breeding has the potential to deliver substantial improvements, once the component traits and the genes underlying these traits have been identified. In addition, interactions between the individual traits must also be taken into account, a demand that is difficult to fulfill with traditional screening approaches. Identified traits will be incorporated into new cultivars using conventional or biotechnological tools. In order to better understand the relationship between genotype, component traits, and environment over time, a multidisciplinary approach must be adopted to both understand the underlying processes and identify candidate genes, QTLs and traits that can be used to develop improved crops.
基金partially supported by the grants from National Program on Key Basic Research Project(973 program)(Nos.2012CB945101 and 201 ICBAO 1000)National Natural Science Foundation of China(NSFC)(Nos. 31110103904 and 30730056)
文摘Zebrafish(Danio rerio) is a well-established vertebrate animal model.A comprehensive collection of reverse genetics tools has been developed for studying gene function in this useful organism.Morpholino is the most widely used reagent to knock down target gene expression post-transcriptionally.For a long time,targeted genome modification has been heavily relied on large-scale traditional forward genetic screens,such as ENU(N-ethyl-N-nitrosourea) mutagenesis derived TILLING(Targeting Induced Local Lesions IN Genomes) strategy and pseudo-typed retrovirus mediated insertional mutagenesis.Recently,engineered endonucleases,including ZFNs(zinc finger nucleases) and TALENs(transcription activator-like effector nucleases),provide new and efficient strategies to directly generate site-specific indel mutations by inducing double strand breaks in target genes.Here we summarize the major reverse genetic approaches for loss-of-function studies used and emerging in zebrafish,including strategies based on genome-wide mutagenesis and methods for site-specific gene targeting.Future directions and expectations will also be discussed.
基金This work was supported the National High Technology Research and Development Program of China (863 Program) (No. 2002AA2Z1003), Key Project of Chinese National Programs for Fundamental Research and Development (No.2005CB120801).