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Improvement of Chloroplast Transformation Using CRISPR/Cas9

文献类型： 外文期刊

作者： Tang, Ning 1 ; Xia, Yumei 1 ; Zhan, Yijie 1 ; Dan, Junhao 1 ; Yu, Mulan 1 ; Bu, Xiaolan 1 ; Cao, Mengliang 1 ;

作者机构： 1.Hunan Univ, Grad Sch, Longping Branch, State Key Lab Hybrid Rice, Changsha 410082, Hunan, Peoples R China

2.Hunan Acad Agr Sci, Hunan Hybrid Rice Res Ctr, Changsha 410125, Hunan, Peoples R China

关键词： Plastid Transformation; Homologous Recombination; CRISPR/Cas9; Transformation Efficiency

期刊名称：JOURNAL OF BIOBASED MATERIALS AND BIOENERGY （ 影响因子：0.708； 五年影响因子：0.739 ）

ISSN： 1556-6560

年卷期： 2020 年 14 卷 3 期

页码：

收录情况： SCI

摘要： Chloroplasts are organelles that contain genetic materials (DNA) in higher plant cells. The special genetic characteristics of chloroplasts mean that plasmid transformation has important research value, so it has become an important research direction second to nuclear transformation. Although the techniques of chloroplast genome modification have been successfully applied in tobacco and extended to other high plants, there are still many limitations. Exogenous genes are integrated into the chloroplast genome through homologous recombination. Therefore, the low efficiency of homologous recombination directly limits transformation efficiency. Gene editing with fixed-point cutting function and DNA damage repair mechanism may effectively improve the efficiency. In the present study, we aimed to use CRISPR/Cas9 to cut the site between two homologous recombinant fragments in chloroplast transformation to improve the efficiency by activating the DNA damage repair mechanism. The Cas9 gene and gRNA were added to the chloroplast transformation system of tobacco by co-transformation or integration into a transformation vector. The acquired resistant plants were screened by multiple selection of spectinomycin and chloroplast DNA was isolated for molecular detection by PCR. The results showed that the efficiency of chloroplast transformation increased by 6-10 times with the addition of gene editing technology. Although the transformation efficiency was still far below the level of nuclear transformation, this study may help to increase the efficiency of the plant chloroplast transformation system, and expand the types of plant receptors.

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