基于CRISPR/Cas9基因编辑技术靶向敲除OsFAD2创制高油酸水稻突变体

Creation of High Oleic Acid Rice Mutant by Targeted Knockout of OsFAD2 via CRISPR/Cas9 Genome Editing Technology

【目的】水稻OsFAD2编码脂肪酸脱饱和酶,调节油酸(C18∶1)向亚油酸(C18∶2)转化,该基因突变后可提高水稻的油酸含量。为了创制高油酸水稻突变体并助力稻米油发展,利用CRISPR/Cas9技术对OsFAD2进行定点编辑。【方法】以日本晴(粳稻)愈伤组织为材料,利用含有CRISPR/Cas9-FAD2-sgRNA载体的农杆菌介导获得转基因植株,Sanger测序与荧光定量PCR鉴定阳性植株,最后利用气相色谱质谱(GC-MS)检测稻米籽粒脂肪酸含量变化。【结果】靶点序列测序检测表明,有3株OsFAD2敲除植株在T0代产生了碱基变异,但是仅有1株不存在脱靶现象。随后阳性植株种子被扩繁至T1代,使得OsFAD2突变序列纯和、稳定,荧光定量PCR检测表明OsFAD2表达量比野生型植株显著减少。利用T1代转基因植株进行种子脂肪酸组分检测表明,敲除OsFAD2导致籽粒油酸含量显著上升约30%。【结论】利用CRISPR/Cas9技术对水稻OsFAD2编码区序列进行靶向敲除并获得了高油酸水稻突变体,为后续高油酸稻米育种提供种质资源。

【Objective】Rice Fatty Acid Desaturase 2 gene(FAD2)encodes the fatty acid desaturase and converts oleic acid(C18∶1)into linoleic acid(C18∶2),the mutant of which is capable of improving the content of oleic acid in rice.To obtain the high oleic acid rice mutant and facilitate the development of rice bran oil,site-specific editing of OsFAD2 is conducted by using the CRISPR/Cas9 technology.【Method】Nipponbare callus was used as explants to generate the transgenic plants by agrobacterium-mediated transformation protocol with CRISPR/Cas9-FAD2-sgRNA vector.Sanger sequencing and fluorescent quantitative PCR were used to identify the positive transgenic plants.Finally,gas chromatography-mass spectrometer(GC-MS)was employed to detect the content variation of fatty acids.【Result】Gene target sequencing suggested that three OsFAD2 knockout plants contained the genome bases variation in T0 generation,but only one positive transgenic line did not present off-target phenotype.Further,positive transgenic plant seeds were reproduced to T1 generation,as a result,OsFAD2 mutation generated genetic homozygosis and stable sequence,and qRT-PCR detection indicated that the relative expression of OsFAD2 decreased significantly compared with that of wild-type plant.Fatty acid composition detection suggested that the oleic acid increased by 30% approximately in the T1 seeds of positive knockout lines.【Conclusion】High oleic acid rice mutants were obtained by targeted knockout of the coding sequence of OsFAD2 via CRISPR/Cas9 technology,providing germplasm resources for subsequent high-oleic rice bran oil breeding.