Rice is a poor source of folate,an essential micronutrient for the body.Biofortification offers an effective way to enhance the folate content of rice and alleviate folate deficiencies in humans.In this study,we confi...Rice is a poor source of folate,an essential micronutrient for the body.Biofortification offers an effective way to enhance the folate content of rice and alleviate folate deficiencies in humans.In this study,we confirmed that OsADCS and OsGTPCHI,encoding the initial enzymes necessary for folate synthesis,positively regulate folate accumulation in knockout mutants of both japonica and indica rice backgrounds.The folate content in the low-folate japonica variety was slightly increased by the expression of the indica alleles driven by the endosperm-specific promoter.We further obtained co-expression lines by stacking OsADCS and OsGTPCHI genes;the folate accumulation in brown rice and polished rice reached 5.65μg/g and 2.95μg/g,respectively,representing 37.9-fold and 26.5-fold increases compared with the wild type.Transcriptomic analysis of rice grains from six transgenic lines showed that folate changes affected biological pathways involved in the synthesis and metabolism of rice seed storage substances,while the expression of other folate synthesis genes was weakly regulated.In addition,we identified Aus rice as a high-folate germplasm carrying superior haplotypes of OsADCS and OsGTPCHI through natural variation.This study provides an alternative and effective complementary strategy for rice biofortification,promoting the rational combination of metabolic engineering and conventional breeding to breed high-folate varieties.展开更多
Genome editing by clustered regularly interspaced short palindromic sequences(CRISPR)/CRISPRassociated protein 9(Cas9)has revolutionized functional gene analysis and genetic improvement.While reporter-assisted CRISPR/...Genome editing by clustered regularly interspaced short palindromic sequences(CRISPR)/CRISPRassociated protein 9(Cas9)has revolutionized functional gene analysis and genetic improvement.While reporter-assisted CRISPR/Cas systems can greatly facilitate the selection of genome-edited plants produced via stable transformation,this approach has not been well established in seed crops.Here,we established the seed fluorescence reporter(SFR)-assisted CRISPR/Cas9 systems in maize(Zea mays L.),using the red fluorescent Ds RED protein expressed in the endosperm(En-SFR/Cas9),embryos(Em-SFR/Cas9),or both tissues(Em/En-SFR/Cas9).All three SFRs showed distinct fluorescent patterns in the seed endosperm and embryo that allowed the selection of seeds carrying the transgene of having segregated the transgene out.We describe several case studies of the implementation of En-SFR/Cas9,Em-SFR/Cas9,and Em/En-SFR/Cas9 to identify plants not harboring the genomeediting cassette but carrying the desired mutations at target genes in single genes or in small-scale mutant libraries,and report on the successful generation of single-target mutants and/or mutant libraries with En-SFR/Cas9,Em-SFR/Cas9,and Em/En-SFR/Cas9.SFR-assisted genome editing may have particular value for application scenarios with a low transformation frequency and may be extended to other important monocot seed crops.展开更多
基金supported by the Central Public-Interest Scientific Institution Basal Research Fund,China(Grant No.CPSIBRF-CNRRI-202403)。
文摘Rice is a poor source of folate,an essential micronutrient for the body.Biofortification offers an effective way to enhance the folate content of rice and alleviate folate deficiencies in humans.In this study,we confirmed that OsADCS and OsGTPCHI,encoding the initial enzymes necessary for folate synthesis,positively regulate folate accumulation in knockout mutants of both japonica and indica rice backgrounds.The folate content in the low-folate japonica variety was slightly increased by the expression of the indica alleles driven by the endosperm-specific promoter.We further obtained co-expression lines by stacking OsADCS and OsGTPCHI genes;the folate accumulation in brown rice and polished rice reached 5.65μg/g and 2.95μg/g,respectively,representing 37.9-fold and 26.5-fold increases compared with the wild type.Transcriptomic analysis of rice grains from six transgenic lines showed that folate changes affected biological pathways involved in the synthesis and metabolism of rice seed storage substances,while the expression of other folate synthesis genes was weakly regulated.In addition,we identified Aus rice as a high-folate germplasm carrying superior haplotypes of OsADCS and OsGTPCHI through natural variation.This study provides an alternative and effective complementary strategy for rice biofortification,promoting the rational combination of metabolic engineering and conventional breeding to breed high-folate varieties.
基金supported by the National Science Foundation of China(Nos 31771808 and 32001551)National Key R&D Program of China(No.2020YFE0202300)+1 种基金the Key Area Research and Development Program of Guangdong Province(No.2018B020202008)the Agricultural Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences(S2021ZD03)。
文摘Genome editing by clustered regularly interspaced short palindromic sequences(CRISPR)/CRISPRassociated protein 9(Cas9)has revolutionized functional gene analysis and genetic improvement.While reporter-assisted CRISPR/Cas systems can greatly facilitate the selection of genome-edited plants produced via stable transformation,this approach has not been well established in seed crops.Here,we established the seed fluorescence reporter(SFR)-assisted CRISPR/Cas9 systems in maize(Zea mays L.),using the red fluorescent Ds RED protein expressed in the endosperm(En-SFR/Cas9),embryos(Em-SFR/Cas9),or both tissues(Em/En-SFR/Cas9).All three SFRs showed distinct fluorescent patterns in the seed endosperm and embryo that allowed the selection of seeds carrying the transgene of having segregated the transgene out.We describe several case studies of the implementation of En-SFR/Cas9,Em-SFR/Cas9,and Em/En-SFR/Cas9 to identify plants not harboring the genomeediting cassette but carrying the desired mutations at target genes in single genes or in small-scale mutant libraries,and report on the successful generation of single-target mutants and/or mutant libraries with En-SFR/Cas9,Em-SFR/Cas9,and Em/En-SFR/Cas9.SFR-assisted genome editing may have particular value for application scenarios with a low transformation frequency and may be extended to other important monocot seed crops.
