Effective editing for lysophosphatidic acid acyltransferase 2/5 in allotetraploid rapeseed (Brassica napus L.) using CRISPR-Cas9 system
Effective editing for lysophosphatidic acid acyltransferase 2/5 in allotetraploid rapeseed (Brassica napus L.) using CRISPR-Cas9 system
复制标题
使用 CRISPR-Cas9 系统对异源四倍体油菜 (Brassica napus L.) 中的溶血磷脂酸酰基转移酶 2/5 进行有效编辑
DOI：
10.1186/s13068-019-1567-8
复制
发表时间：
2019-09
影响因子：
6.3
通讯作者：
Li Maoteng
中科院分区：
文献类型：
--
作者：
Zhang Kai;Nie Liluo;Cheng Qiqi;Yin Yongtai;Chen Kang;Qi Fuyu;Zou Dashan;Liu Haohao;Zhao Weiguo;Wang Baoshan;Li Maoteng
背景
Background
Brassica napus is one of the most important oilseed crops, and can supply considerable amounts of edible oil as well as provide raw materials for the production of biodiesel in the biotechnology industry. Lysophosphatidic acid acyltransferase (LPAT), a key enzyme in the Kennedy pathway, catalyses fatty acid chains into 3-phosphoglycerate and promotes further production of oil in the form of triacylglycerol. However, because B. napus is an allotetraploid with two subgenomes, the precise genes which involved in oil production remain unclear due to the intractability of efficiently knocking out all copies with high genetic redundancy. Therefore, a robust gene editing technology is necessary for gene function analysis.
Results
An efficient gene editing technology was developed for the allotetraploid plant B. napus using the CRISPR-Cas9 system. Previous studies showed poor results in either on-target or off-target activity in B. napus. In the present study, four single-gRNAs and two multi-gRNAs were deliberately designed from the conserved coding regions of BnLPAT2 which has seven homologous genes, and BnLPAT5, which has four homologous genes. The mutation frequency was found to range from 17 to 68%, while no mutation was observed in the putative off-target sites. The seeds of the Bnlpat2/Bnlpat5 mutant were wizened and showed enlarged oil bodies, disrupted distribution of protein bodies and increased accumulation of starch in mature seeds. The oil content decreased, with an average decrease of 32% for Bnlpat2 lines and 29% for Bnlpat5 lines in single-gRNA knockout lines, and a decline of 24% for Bnlpat2 mutant lines (i.e., g123) and 39% for Bnlpat2/Bnlpat5 double mutant lines (i.e., g134) in multi-gRNA knockout lines.
Conclusions
Seven BnLPAT2 homologous genes and four BnLPAT5 homologous genes were cleaved completely using the CRISPR-Cas9 system, which indicated that it is effective for editing all homologous genes in allotetraploid rapeseed, despite the relatively low sequence identities of both gene families. The size of the oil bodies increased significantly while the oil content decreased, confirming that BnLPAT2 and BnLPAT5 play a role in oil biosynthesis. The present study lays a foundation for further oil production improvement in oilseed crop species.
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DOI：
10.1007/s11032-013-9954-5
发表时间：
2014
期刊：
Molecular breeding : new strategies in plant improvement
影响因子：
--
作者：
Wells R;Trick M;Soumpourou E;Clissold L;Morgan C;Werner P;Gibbard C;Clarke M;Jennaway R;Bancroft I
通讯作者：
Bancroft I
影响因子：
46.9
作者：
Kim YB;Komor AC;Levy JM;Packer MS;Zhao KT;Liu DR
通讯作者：
Liu DR
影响因子：
3.7
作者：
Wang X;Wang H;Long Y;Li D;Yin Y;Tian J;Chen L;Liu L;Zhao W;Zhao Y;Yu L;Li M
通讯作者：
Li M
影响因子：
5.3
作者：
Wang X;Long Y;Yin Y;Zhang C;Gan L;Liu L;Yu L;Meng J;Li M
通讯作者：
Li M
影响因子：
13.8
作者：
Jia H;Zhang Y;Orbović V;Xu J;White FF;Jones JB;Wang N
通讯作者：
Wang N