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Tissue-Specific CRISPR/Cas9 System of Cotton Pollen with GhPLIMP2b and GhMYB24 Promoters

한국식물학회지 2021년 64권 1호 p.13 ~ 21
Lei Jianfeng, Dai Peihong, Li Jiyang, Yang Mi, Li Xiuqin, Zhang Wanqi, Zhou Guantong, Guo Wangzhen, Liu Xiaodong,
소속 상세정보
 ( Lei Jianfeng ) - Xinjiang Agricultural University College of Agriculture
 ( Dai Peihong ) - Xinjiang Agricultural University College of Agriculture
 ( Li Jiyang ) - Xinjiang Agricultural University College of Agriculture
 ( Yang Mi ) - Xinjiang Agricultural University College of Agriculture
 ( Li Xiuqin ) - Xinjiang Agricultural University College of Agriculture
 ( Zhang Wanqi ) - Xinjiang Agricultural University College of Agriculture
 ( Zhou Guantong ) - Xinjiang Agricultural University College of Agriculture
 ( Guo Wangzhen ) - Nanjing Agricultural University State Key Laboratory of Crop Genetics and Germplasm Enhancement
 ( Liu Xiaodong ) - Xinjiang Agricultural University College of Agriculture

Abstract


CRISPR/Cas9 technology is a powerful tool for improving crop genetic traits. However, CRISPR/Cas9 system for efficient expression of cotton germ cells has still not been established. In this study, we developed a tissue-specific vectors to drive Cas9 expression with GhPLIMP2b and GhMYB24 promoters and established the effective method to transform cotton pollen by Agrobacterium vacuum infiltration. GhPLIMP2b and GhMYB24 promoters of cotton pollen were cloned into Cas9 expression vectors. The sgRNAs targetting to CLA1, ERA1 and GGB (drought-resistant negative regulation genes) were designed and constructed into GhPLIMP2b and GhMYB24 promoter vectors. Cotton pollens were tranformed by Agrobacterium vacuum infiltration with GhPLIMP2b and GhMYB24 promoter vectors. The results of clone sequencing shown that mutation types of the sequence were mainly base substitutions wth the frequency from 3.29 to 6.45%. Eleven potential off-target sites were chosen and two sites were observed. Our results indicated that GhPLIM2bP::Cas9 and GhMYB24P::Cas9 editing vectors achieved targeted edition of endogenous genes in cotton pollen, but there were a few off-target effects. This study provides an effective gene-editing system for the rapid acquisition of cotton mutants using pollen as a transgenic receptor.

키워드

Cotton pollen; CRISPR/Cas9; Agrobacterium vacuum infiltration; Mutation; Off-target

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