Vernalization is a physiological process in which prolonged cold exposure establishes flowering competence in winter plants. In hexaploid wheat, TaVRN1 is a cold-induced key regulator that accelerates floral transitio...Vernalization is a physiological process in which prolonged cold exposure establishes flowering competence in winter plants. In hexaploid wheat, TaVRN1 is a cold-induced key regulator that accelerates floral transition. However, the molecular mechanism underlying the gradual activation of TaVRN1 during the vernalization process remains unknown. In this study, we identified the novel transcript VAS (TaVRN1 alternative splicing) as a non-coding RNA derived from the sense strand of the TaVRN1 gene only in winter wheat, which regulates TaVRN1 transcription for flowering. VAS was induced during the early period of vernalization, and its overexpression promoted TaVRN1 expression to accelerate flowering in winter wheat. VAS physically associates with TaRF2b and facilitates docking of the TaRF2b-TaRF2a complex at the TaVRN1 promoter during the middle period of vernalization. TaRF2b recognizes the Sp1 motif within the TaVRN1 proximal promoter region, which is gradually exposed along with the disruption of a loop structure at the TaVRN1 locus during vernalization, to activate the transcription of TaVRN1. The tarf2b mutants exhibited delayed flowering, whereas transgenic wheat lines overexpressing TaRF2b showed earlier flowering. Taken together, our data reveal a distinct regulatory mechanism by which a long non-coding RNA facilitates the transcription factor targeting to regulate wheat flowering, providing novel insights into the vernalization process and a potential target for wheat genetic improvement.展开更多
MAD7 is an engineered nuclease of the Class 2 type V-A CRISPR-Cas(Cas12 a/Cpf1)family with a low level of homology to canonical Cas12 a nucleases.It has been publicly released as a royalty-free nuclease for both acade...MAD7 is an engineered nuclease of the Class 2 type V-A CRISPR-Cas(Cas12 a/Cpf1)family with a low level of homology to canonical Cas12 a nucleases.It has been publicly released as a royalty-free nuclease for both academic and commercial use.Here,we demonstrate that the CRISPR-MAD7 system can be used for genome editing and recognizes T-rich PAM sequences(YTTN)in plants.Its editing efficiency in rice and wheat is comparable to that of the widely used CRISPR-Lb Cas12 a system.We develop two variants,MAD7-RR and MAD7-RVR that increase the target range of MAD7,as well as an M-AFID(a MAD7-APOBEC fusion-induced deletion)system that creates predictable deletions from 50-deaminated Cs to the MAD7-cleavage site.Moreover,we show that MAD7 can be used for multiplex gene editing and that it is effective in generating indels when combined with other CRISPR RNA orthologs.Using the CRISPR-MAD7 system,we have obtained regenerated mutant rice and wheat plants with up to 65.6%efficiency.展开更多
基金We gratefully ack no wledge funding from the NSFC for the Basic Scie nee Center Program(31788103)the National Natural Science Foundation of China(31970529)the China Postdoctoral Science Foundation(2019M650892).
文摘Vernalization is a physiological process in which prolonged cold exposure establishes flowering competence in winter plants. In hexaploid wheat, TaVRN1 is a cold-induced key regulator that accelerates floral transition. However, the molecular mechanism underlying the gradual activation of TaVRN1 during the vernalization process remains unknown. In this study, we identified the novel transcript VAS (TaVRN1 alternative splicing) as a non-coding RNA derived from the sense strand of the TaVRN1 gene only in winter wheat, which regulates TaVRN1 transcription for flowering. VAS was induced during the early period of vernalization, and its overexpression promoted TaVRN1 expression to accelerate flowering in winter wheat. VAS physically associates with TaRF2b and facilitates docking of the TaRF2b-TaRF2a complex at the TaVRN1 promoter during the middle period of vernalization. TaRF2b recognizes the Sp1 motif within the TaVRN1 proximal promoter region, which is gradually exposed along with the disruption of a loop structure at the TaVRN1 locus during vernalization, to activate the transcription of TaVRN1. The tarf2b mutants exhibited delayed flowering, whereas transgenic wheat lines overexpressing TaRF2b showed earlier flowering. Taken together, our data reveal a distinct regulatory mechanism by which a long non-coding RNA facilitates the transcription factor targeting to regulate wheat flowering, providing novel insights into the vernalization process and a potential target for wheat genetic improvement.
基金supported by grants from the Strategic Priority Research Program of the Chinese Academy of Sciences(Precision Seed Design and Breeding,XDA24020101 and XDA24020310)the National Natural Science Foundation of China(31672015,31788103)the Youth Innovation Promotion Association of the Chinese Academy of Sciences(2020000003)。
文摘MAD7 is an engineered nuclease of the Class 2 type V-A CRISPR-Cas(Cas12 a/Cpf1)family with a low level of homology to canonical Cas12 a nucleases.It has been publicly released as a royalty-free nuclease for both academic and commercial use.Here,we demonstrate that the CRISPR-MAD7 system can be used for genome editing and recognizes T-rich PAM sequences(YTTN)in plants.Its editing efficiency in rice and wheat is comparable to that of the widely used CRISPR-Lb Cas12 a system.We develop two variants,MAD7-RR and MAD7-RVR that increase the target range of MAD7,as well as an M-AFID(a MAD7-APOBEC fusion-induced deletion)system that creates predictable deletions from 50-deaminated Cs to the MAD7-cleavage site.Moreover,we show that MAD7 can be used for multiplex gene editing and that it is effective in generating indels when combined with other CRISPR RNA orthologs.Using the CRISPR-MAD7 system,we have obtained regenerated mutant rice and wheat plants with up to 65.6%efficiency.