Base editing using CRISPR technologies is an invaluable tool for crop breeding. One of the major base editors, the adenine base editor(ABE), has been successfully used in both model plants and many crops.However, owin...Base editing using CRISPR technologies is an invaluable tool for crop breeding. One of the major base editors, the adenine base editor(ABE), has been successfully used in both model plants and many crops.However, owing to limited editing efficiency, the ABE has been difficult to apply in polyploid crops such as allohexaploid bread wheat that often require simultaneous mutation of multiple alleles for fast breeding. We have designed a wheat high-efficiency ABE(Whie ABE), using the newly developed high-activity adenosine deaminase Tad A8 e. In vivo and in vitro analysis demonstrated the improved applicability of Tad A8 e over the commonly used Tad A7.10. Dinitroaniline is a widely used herbicide with high effectiveness and low toxicity to animals. However, wheat cultivars with tolerance to dinitroaniline are rare, limiting the application of dinitroaniline in wheat planting. Using A-to-G editing with Whie ABE, we found that a Met-to-Thr mutation in wheat tubulin alleles located on chromosomes 1 A, 1 B, 1 D, 4 A, and 4 D increased the resistance of wheat to dinitroaniline, revealing a dosage effect of edited tubulins in resistance. The Whie ABE promises to be a valuable editing tool for accelerating crop improvement and developing herbicide-resistant wheat germplasm.展开更多
Wheat high-molecular-weight glutenin subunits(HMW-GS) determine dough elasticity and play an essential role in processing quality. HMW-GS are encoded by Glu-1 genes and controlled primarily at transcriptional level, i...Wheat high-molecular-weight glutenin subunits(HMW-GS) determine dough elasticity and play an essential role in processing quality. HMW-GS are encoded by Glu-1 genes and controlled primarily at transcriptional level, implemented through the interactions between cis-acting elements and trans-acting factors. However, transcriptional mechanism of Glu-1 genes remains elusive. Here we made a comprehensive analysis of cis-regulatory elements within 1-kb upstream of the Glu-1 start codon(-1000 to-1) and identified 30 conserved motifs. Based on motif distribution pattern, three conserved cis-regulatory modules(CCRMs), CCRM1(-300 to-101), CCRM2(-650 to-400), and CCRM3(-950 to-750), were defined, and their functions were characterized in wheat stable transgenic lines transformed with progressive 5′ deletion promoter::GUS fusion constructs. GUS staining, qP CR and enzyme activity assays indicated that CCRM2 and CCRM3 could enhance the expression level of Glu-1, whereas the 300-bp promoter(-300 to-1), spanning CCRM1 and core region(-100 to-1), was enough to ensure accurate Glu-1 initiation at 7 days after flowering(DAF) and shape its spatiotemporal expression pattern during seed development. Further transgenic assays demonstrated that CCRM1-2(-300 to-209) containing Complete HMW Enhancer(-246 to-209) was important for expression level but had no effect on expression specificity in the endosperm. In contrast, CCRM1-1(-208 to-101) was critical for both expression specificity and level of Glu-1. Our findings not only provide new insights to uncover Glu-1 transcription regulatory machinery but also lay foundations for modifying Glu-1 expression.展开更多
基金funded by the Agricultural Variety Improvement Project of Shandong Province(2019LZGC015)the National Natural Science Foundation of China(31901432)。
文摘Base editing using CRISPR technologies is an invaluable tool for crop breeding. One of the major base editors, the adenine base editor(ABE), has been successfully used in both model plants and many crops.However, owing to limited editing efficiency, the ABE has been difficult to apply in polyploid crops such as allohexaploid bread wheat that often require simultaneous mutation of multiple alleles for fast breeding. We have designed a wheat high-efficiency ABE(Whie ABE), using the newly developed high-activity adenosine deaminase Tad A8 e. In vivo and in vitro analysis demonstrated the improved applicability of Tad A8 e over the commonly used Tad A7.10. Dinitroaniline is a widely used herbicide with high effectiveness and low toxicity to animals. However, wheat cultivars with tolerance to dinitroaniline are rare, limiting the application of dinitroaniline in wheat planting. Using A-to-G editing with Whie ABE, we found that a Met-to-Thr mutation in wheat tubulin alleles located on chromosomes 1 A, 1 B, 1 D, 4 A, and 4 D increased the resistance of wheat to dinitroaniline, revealing a dosage effect of edited tubulins in resistance. The Whie ABE promises to be a valuable editing tool for accelerating crop improvement and developing herbicide-resistant wheat germplasm.
基金funded by the National Key Research and Development Program of China (2016YFD0100500)the National Natural Science Foundation of China (31571663, 31371623)Genetically Modified Organisms Breeding Major Project (2016ZX08009003-004)
文摘Wheat high-molecular-weight glutenin subunits(HMW-GS) determine dough elasticity and play an essential role in processing quality. HMW-GS are encoded by Glu-1 genes and controlled primarily at transcriptional level, implemented through the interactions between cis-acting elements and trans-acting factors. However, transcriptional mechanism of Glu-1 genes remains elusive. Here we made a comprehensive analysis of cis-regulatory elements within 1-kb upstream of the Glu-1 start codon(-1000 to-1) and identified 30 conserved motifs. Based on motif distribution pattern, three conserved cis-regulatory modules(CCRMs), CCRM1(-300 to-101), CCRM2(-650 to-400), and CCRM3(-950 to-750), were defined, and their functions were characterized in wheat stable transgenic lines transformed with progressive 5′ deletion promoter::GUS fusion constructs. GUS staining, qP CR and enzyme activity assays indicated that CCRM2 and CCRM3 could enhance the expression level of Glu-1, whereas the 300-bp promoter(-300 to-1), spanning CCRM1 and core region(-100 to-1), was enough to ensure accurate Glu-1 initiation at 7 days after flowering(DAF) and shape its spatiotemporal expression pattern during seed development. Further transgenic assays demonstrated that CCRM1-2(-300 to-209) containing Complete HMW Enhancer(-246 to-209) was important for expression level but had no effect on expression specificity in the endosperm. In contrast, CCRM1-1(-208 to-101) was critical for both expression specificity and level of Glu-1. Our findings not only provide new insights to uncover Glu-1 transcription regulatory machinery but also lay foundations for modifying Glu-1 expression.
