Resveratrol is an important phytoalexin that adapts to and responds to stressful conditions and plays various roles in health and medical therapies.However,it is only found in a limited number of plant species in low ...Resveratrol is an important phytoalexin that adapts to and responds to stressful conditions and plays various roles in health and medical therapies.However,it is only found in a limited number of plant species in low concentrations,which hinders its development and utilization.Chalcone synthase(CHS)and stilbene synthase(STS)catalyze the same substrates to produce flavonoids and resveratrol,respectively.However,it remains unclear how CHS and STS compete in metabolite synthesis.In this study,two CHS2 mutant cell lines(MT1 and MT2)were generated using CRisPR/Cas9 genome editing.These CHS2 mutant cell lines exhibited abundant mutations in CHS2,leading to the premature termination of protein translation and subsequent CHS2 knockout.Amplicon sequencing confirmed comprehensive CHS2 knockout in MT1,whereas the wild-type sequence remained predominant in the MT2 cell line.Transcriptome and RT-qPCR results showed a significant downregulation of genes involved in flavonoid biosynthesis,including CHS2,CHS3,F3H,F3'H,DFR,FLS,LDOX,among others,resulting in decreased flavonoid accumulation,such as anthocyanins,proanthocyanidins,quercetin,and kaempferol.Conversely,STS genes involved in stilbenoid biosynthesis were upregulated competing with the flavonoid pathway.Consequently,there was a marked increase in stilbenoids,including resveratrol,piceatannol,piceid,and pterostilbene,with a 4.1-fold increase in resveratrol and a 5.3-fold increase in piceid(a derivative of resveratrol)observed in CHS2 mutant cell lines.This research demonstrates that CHS2 mutation induces a shift from flavonoid biosynthesis towards stilbenoid biosynthesis,offering new insights into metabolite biosynthesis and regulation,as well as an alternative solution for natural resveratrol production,and a novel breeding approach for eliminating non-target agronomic traits using CRISPR-Cas9.展开更多
为探讨我国芸薹属蔬菜的起源及遗传多样性,克隆、测序芸薹属不同种的Chs基因序列。A基因组二倍体、A基因组多倍体、B基因组多倍体和C基因组二倍体的Chs基因突变位点数分别为120、172、194和25个,Chs基因多态性表现为:B基因组多倍体>...为探讨我国芸薹属蔬菜的起源及遗传多样性,克隆、测序芸薹属不同种的Chs基因序列。A基因组二倍体、A基因组多倍体、B基因组多倍体和C基因组二倍体的Chs基因突变位点数分别为120、172、194和25个,Chs基因多态性表现为:B基因组多倍体>A基因组二倍体>A基因组多倍体>C基因组多倍体。Tajima'D、Fu and Li'D和Fu and Li'F检验表明A基因组二倍体、C基因组二倍体Chs基因是中性进化基因。HKA平衡检验及误配分析表明A基因组多倍体和B基因组多倍体Chs基因进化中存在选择作用。A基因组和B基因组间存在较低的共有差异和较高的共有多态性,C基因组与A、B基因组存在较高的共有差异和较低的共有多态性。系统发育树将芸薹属Chs基因序列分成4个亚支、10个支系。网状分析表明,白菜可能是四倍体A基因组的供体,黑芥可能是四倍体B基因组的供体,甘蓝可能是四倍体C基因组的供体。中国芸薹属蔬菜在Chs基因位点有较高的遗传多态性,不同基因组间分化程度不一样,B基因组分化较大,A和C基因组分化较小。A和B基因组的亲缘关系较A和C基因组以及B和C基因组更为接近。建议根据基因组的不同将中国芸薹蔬菜分成白菜组、芥菜组和甘蓝组,研究结果支持芸薹属进化的禹式三角模型。展开更多
基金founded by National Natural Science Foundation of China(32302284)Natural Science Foundation of Fujian Province,China(2021 J05091)+1 种基金High Quality Development"5511"Collaborative Innovation Project between Fujian and Chinese Academy of Agricultural Sciences(XTCXGC2021014)Fujian Provincial Department of Science and Technology of Special Public-funded Projects(2021R1032009).
摘要Resveratrol is an important phytoalexin that adapts to and responds to stressful conditions and plays various roles in health and medical therapies.However,it is only found in a limited number of plant species in low concentrations,which hinders its development and utilization.Chalcone synthase(CHS)and stilbene synthase(STS)catalyze the same substrates to produce flavonoids and resveratrol,respectively.However,it remains unclear how CHS and STS compete in metabolite synthesis.In this study,two CHS2 mutant cell lines(MT1 and MT2)were generated using CRisPR/Cas9 genome editing.These CHS2 mutant cell lines exhibited abundant mutations in CHS2,leading to the premature termination of protein translation and subsequent CHS2 knockout.Amplicon sequencing confirmed comprehensive CHS2 knockout in MT1,whereas the wild-type sequence remained predominant in the MT2 cell line.Transcriptome and RT-qPCR results showed a significant downregulation of genes involved in flavonoid biosynthesis,including CHS2,CHS3,F3H,F3'H,DFR,FLS,LDOX,among others,resulting in decreased flavonoid accumulation,such as anthocyanins,proanthocyanidins,quercetin,and kaempferol.Conversely,STS genes involved in stilbenoid biosynthesis were upregulated competing with the flavonoid pathway.Consequently,there was a marked increase in stilbenoids,including resveratrol,piceatannol,piceid,and pterostilbene,with a 4.1-fold increase in resveratrol and a 5.3-fold increase in piceid(a derivative of resveratrol)observed in CHS2 mutant cell lines.This research demonstrates that CHS2 mutation induces a shift from flavonoid biosynthesis towards stilbenoid biosynthesis,offering new insights into metabolite biosynthesis and regulation,as well as an alternative solution for natural resveratrol production,and a novel breeding approach for eliminating non-target agronomic traits using CRISPR-Cas9.
摘要为探讨我国芸薹属蔬菜的起源及遗传多样性,克隆、测序芸薹属不同种的Chs基因序列。A基因组二倍体、A基因组多倍体、B基因组多倍体和C基因组二倍体的Chs基因突变位点数分别为120、172、194和25个,Chs基因多态性表现为:B基因组多倍体>A基因组二倍体>A基因组多倍体>C基因组多倍体。Tajima'D、Fu and Li'D和Fu and Li'F检验表明A基因组二倍体、C基因组二倍体Chs基因是中性进化基因。HKA平衡检验及误配分析表明A基因组多倍体和B基因组多倍体Chs基因进化中存在选择作用。A基因组和B基因组间存在较低的共有差异和较高的共有多态性,C基因组与A、B基因组存在较高的共有差异和较低的共有多态性。系统发育树将芸薹属Chs基因序列分成4个亚支、10个支系。网状分析表明,白菜可能是四倍体A基因组的供体,黑芥可能是四倍体B基因组的供体,甘蓝可能是四倍体C基因组的供体。中国芸薹属蔬菜在Chs基因位点有较高的遗传多态性,不同基因组间分化程度不一样,B基因组分化较大,A和C基因组分化较小。A和B基因组的亲缘关系较A和C基因组以及B和C基因组更为接近。建议根据基因组的不同将中国芸薹蔬菜分成白菜组、芥菜组和甘蓝组,研究结果支持芸薹属进化的禹式三角模型。