非特异性脂质转移蛋白(non-specific lipid transfer protein,nsLTPs)广泛存在于植物界中,参与许多关键的生物学过程,如花粉的发育、种子发育及细胞壁的延伸等。OsLTPL166基因编码区长度是465 bp,其编码的蛋白质由154个氨基酸组成;根据...非特异性脂质转移蛋白(non-specific lipid transfer protein,nsLTPs)广泛存在于植物界中,参与许多关键的生物学过程,如花粉的发育、种子发育及细胞壁的延伸等。OsLTPL166基因编码区长度是465 bp,其编码的蛋白质由154个氨基酸组成;根据跨膜结构域及信号肽预测,OsLTPL166在13~35氨基酸处含有跨膜结构域,N端存在一个32个氨基酸残基的信号肽,切割位点位于氨基酸残基32和33之间,产生具有122个氨基酸残基的成熟蛋白质。组织表达分析发现,OsLTPL166仅在种子中特异性表达,且种子发育后期表达量较高。为了研究水稻Os LTPL166在种子发育中的功能,本研究以‘浙辐粳83’为遗传背景构建了敲除载体,利用CRISPR/Cas9技术对OsLTPL166进行定点编辑,共获得24个独立的转基因株系,其中纯合突变株系有7株,编辑方式主要为碱基的插入和缺失,导致氨基酸序列发生移码,成功获得OsLTPL166功能缺陷型突变体。本研究为进一步探究该基因在种子发育进程中的生物学功能提供了遗传材料。展开更多
CRISPR/Cas9-based gene editing research has advanced greatly and shows broad potential for practical application in life sciences,but the Cas9 system is often constrained by the requirement of a protospacer adjacent m...CRISPR/Cas9-based gene editing research has advanced greatly and shows broad potential for practical application in life sciences,but the Cas9 system is often constrained by the requirement of a protospacer adjacent motif(PAM)at the target site.While xCas9,a variant derived from Streptococcus pyogenes Cas9(SpCas9),can recognize a broader range of PAMs,its application in non-model insects is lacking.In this study,we explored xCas9 activity in gene editing by selecting corazonin(Crz)and the target sites with various PAMs in Locusta migratoria,a destructive insect pest worldwide.We found that xCas9 could cleave the target site with AG PAM while SpCas9 could not,although xCas9 appeared to have lower activity than SpCas9 at the canonical NGG PAMs.The heritable homozygous Crz^(-/-)locust strain was generated by the application of xCas9.The Crz^(-/-)strain showed an albino body color,with significantly downregulated expression of several body color-related genes including Pale,Vermilion,Cinnabar,White andβ-carotene-binding protein.In addition,Crz^(-/-)mutants exhibited significantly reduced expression of Chitin synthase 1,along with a markedly lower chitin content as well as compact and rigid cuticles.Furthermore,Crz^(-/-)mutants displayed impaired performance under low-temperature stress,including prolonged lifespan,reduced body weight and smaller body size.Our results suggest that xCas9 is effective for insect genome editing,and Crz plays essential roles in insect body color,cuticle development and adaptation to low-temperature stress.The findings of this study extend the application of xCas9 in non-model insects and provide new insights into our understanding of the regulation of insect cuticle development and environmental adaptation.展开更多
基金supported by the Key Research and Development Project of Henan Province,China(221111112200)the National Natural Science Foundation of China(32070502 and 32072419)+1 种基金the Fundamental Research Program of Shanxi Province,China(202303021224005)the Natural Science Foundation of Henan Province,China(232300420185).
文摘CRISPR/Cas9-based gene editing research has advanced greatly and shows broad potential for practical application in life sciences,but the Cas9 system is often constrained by the requirement of a protospacer adjacent motif(PAM)at the target site.While xCas9,a variant derived from Streptococcus pyogenes Cas9(SpCas9),can recognize a broader range of PAMs,its application in non-model insects is lacking.In this study,we explored xCas9 activity in gene editing by selecting corazonin(Crz)and the target sites with various PAMs in Locusta migratoria,a destructive insect pest worldwide.We found that xCas9 could cleave the target site with AG PAM while SpCas9 could not,although xCas9 appeared to have lower activity than SpCas9 at the canonical NGG PAMs.The heritable homozygous Crz^(-/-)locust strain was generated by the application of xCas9.The Crz^(-/-)strain showed an albino body color,with significantly downregulated expression of several body color-related genes including Pale,Vermilion,Cinnabar,White andβ-carotene-binding protein.In addition,Crz^(-/-)mutants exhibited significantly reduced expression of Chitin synthase 1,along with a markedly lower chitin content as well as compact and rigid cuticles.Furthermore,Crz^(-/-)mutants displayed impaired performance under low-temperature stress,including prolonged lifespan,reduced body weight and smaller body size.Our results suggest that xCas9 is effective for insect genome editing,and Crz plays essential roles in insect body color,cuticle development and adaptation to low-temperature stress.The findings of this study extend the application of xCas9 in non-model insects and provide new insights into our understanding of the regulation of insect cuticle development and environmental adaptation.
