基因组靶向定位诱导损伤技术(Targeting Induced Local Lesions In Genomes,TILLING)是在化学诱变和PCR定向筛选基础上发展起来的检测点突变的反向遗传学研究方法,在多种重要农作物上都有应用。本研究使用化学诱变剂甲基磺酸乙酯(Ethyl ...基因组靶向定位诱导损伤技术(Targeting Induced Local Lesions In Genomes,TILLING)是在化学诱变和PCR定向筛选基础上发展起来的检测点突变的反向遗传学研究方法,在多种重要农作物上都有应用。本研究使用化学诱变剂甲基磺酸乙酯(Ethyl methane sulfonate,EMS)处理大麦品种‘Tamalpais’,获得了2154个M2株系,同时开发了基于芹菜内切酶CEL I(celery juice extract)的酶切筛选体系。针对植物水杨酸抗病途径相关的两个重要基因EDR1和NPR1,检测到5个M2突变株系。序列分析表明,2个突变发生在内含子、1个NPR1基因的同义突变、2个EDR1基因的错义突变(His351Tyr和Pro556Ser)。本项研究为麦类作物反向遗传学研究奠定了基础。展开更多
Barley（Hordeum vulgare L.） is one of the oldest domesticated crops, showing dramatic adaptation to various climate and environmental conditions. As a major cereal crop, barley ranks the 4th after wheat, maize and ri...Barley（Hordeum vulgare L.） is one of the oldest domesticated crops, showing dramatic adaptation to various climate and environmental conditions. As a major cereal crop, barley ranks the 4th after wheat, maize and rice in terms of planting area and production all over the world. Due to its diploid nature, the cultivated barley is considered as an ideal model to study the polyploid wheat and other Triticeae species. Here, we reviewed the development, optimization, and application of transgenic approaches in barley. The most efficient and robust genetic transformation has been built on the Agrobacterium-mediated transfer in conjunction with the immature embryo-based regeneration. We then discussed future considerations of using more practical technologies in barley transformation, such as the T-DNA/transposon tagging and the genome editing. As a cereal crop amenable to genetic transformation, barley will serve as the most valuable carrier for global functional genomics in Triticeae and is becoming the most practical model for generating value-added products.展开更多
Plastid localized acetyl-CoA carboxylase(ACCase;EC 6.4.1.2)is a target for aryloxyphenoxypropionates(APPs)and cyclohexanediones(CHDs),two groups of selective herbicides used in controlling grassy weeds.Wheat and barle...Plastid localized acetyl-CoA carboxylase(ACCase;EC 6.4.1.2)is a target for aryloxyphenoxypropionates(APPs)and cyclohexanediones(CHDs),two groups of selective herbicides used in controlling grassy weeds.Wheat and barley are important cereal crops in the grass(Poaceae or Gramineae)family,and thus sensitive for those herbicides.Characterization of this form of ACCase(or ACC1)in wheat and barley is essential if these agents are used in the sustainable agriculture.In this study,it was confirmed that a single ACC1 gene presented on the second chromosome per homologous group in common wheat,wild emmer wheat,goat grass and barley.Using CRISPR/Cas9,the barley ACC1 gene was edited,specifically in the carboxyl transferase(CT)domain that was critical for herbicide responses in grass species.Two new alleles were generated,one with a 3-bp deletion leading to ACC1:p.Ile1878del and one with a 26-bp deletion causing ACC1:p.Ser2099_Lys2311del.Both were recovered as heterozygotes in the T0 generation.All the seven T0 plants harboring the 3-bp deletion grew normally,but the only T0 plant with 26-bp deletion died at the extension stage(Zadoks 32),probably because there was inadequate ACC1 activity when the plant was big.In the T1 generation,the 3-bp deletion(or Ile1878del)did not impact the edited plants in tiller numbers,tiller height,spike length and spikelet numbers,when compared to the wild-type allele in the non-edited segregants.This study demonstrated that CRISPR/Cas9 was practical to generate single amino acid deletions in the ACC1 protein and the Ile1878 deletion did not compromise plant growth.Unfortunately,the ACC1:p.Ile1878del protein did not confer resistance to the currently tested APP herbicides,including clethodim,haloxyfop,quizalofop-Pethyl and sethoxydim.展开更多
文摘基因组靶向定位诱导损伤技术(Targeting Induced Local Lesions In Genomes,TILLING)是在化学诱变和PCR定向筛选基础上发展起来的检测点突变的反向遗传学研究方法,在多种重要农作物上都有应用。本研究使用化学诱变剂甲基磺酸乙酯(Ethyl methane sulfonate,EMS)处理大麦品种‘Tamalpais’,获得了2154个M2株系,同时开发了基于芹菜内切酶CEL I(celery juice extract)的酶切筛选体系。针对植物水杨酸抗病途径相关的两个重要基因EDR1和NPR1,检测到5个M2突变株系。序列分析表明,2个突变发生在内含子、1个NPR1基因的同义突变、2个EDR1基因的错义突变(His351Tyr和Pro556Ser)。本项研究为麦类作物反向遗传学研究奠定了基础。
基金supported by the Natural Science Foundation of Shandong Province, China (JQ201107)the National Natural Science Foundation of China (31110103917)the Cooperative Innovation Center of Efficient Production with High Annual Yield of Wheat and Corn, Shandong Province, China
文摘Barley（Hordeum vulgare L.） is one of the oldest domesticated crops, showing dramatic adaptation to various climate and environmental conditions. As a major cereal crop, barley ranks the 4th after wheat, maize and rice in terms of planting area and production all over the world. Due to its diploid nature, the cultivated barley is considered as an ideal model to study the polyploid wheat and other Triticeae species. Here, we reviewed the development, optimization, and application of transgenic approaches in barley. The most efficient and robust genetic transformation has been built on the Agrobacterium-mediated transfer in conjunction with the immature embryo-based regeneration. We then discussed future considerations of using more practical technologies in barley transformation, such as the T-DNA/transposon tagging and the genome editing. As a cereal crop amenable to genetic transformation, barley will serve as the most valuable carrier for global functional genomics in Triticeae and is becoming the most practical model for generating value-added products.
基金Supported by China Research and Development Initiative on Genetically Modified Plants(2016ZX08009003-001-006)Hatch Project IDA01587 from the USDA National Institute of Food and Agriculture。
文摘Plastid localized acetyl-CoA carboxylase(ACCase;EC 6.4.1.2)is a target for aryloxyphenoxypropionates(APPs)and cyclohexanediones(CHDs),two groups of selective herbicides used in controlling grassy weeds.Wheat and barley are important cereal crops in the grass(Poaceae or Gramineae)family,and thus sensitive for those herbicides.Characterization of this form of ACCase(or ACC1)in wheat and barley is essential if these agents are used in the sustainable agriculture.In this study,it was confirmed that a single ACC1 gene presented on the second chromosome per homologous group in common wheat,wild emmer wheat,goat grass and barley.Using CRISPR/Cas9,the barley ACC1 gene was edited,specifically in the carboxyl transferase(CT)domain that was critical for herbicide responses in grass species.Two new alleles were generated,one with a 3-bp deletion leading to ACC1:p.Ile1878del and one with a 26-bp deletion causing ACC1:p.Ser2099_Lys2311del.Both were recovered as heterozygotes in the T0 generation.All the seven T0 plants harboring the 3-bp deletion grew normally,but the only T0 plant with 26-bp deletion died at the extension stage(Zadoks 32),probably because there was inadequate ACC1 activity when the plant was big.In the T1 generation,the 3-bp deletion(or Ile1878del)did not impact the edited plants in tiller numbers,tiller height,spike length and spikelet numbers,when compared to the wild-type allele in the non-edited segregants.This study demonstrated that CRISPR/Cas9 was practical to generate single amino acid deletions in the ACC1 protein and the Ile1878 deletion did not compromise plant growth.Unfortunately,the ACC1:p.Ile1878del protein did not confer resistance to the currently tested APP herbicides,including clethodim,haloxyfop,quizalofop-Pethyl and sethoxydim.
