利用CRISPR/Cas9技术创制大豆高油酸突变系

Creation of high oleic acid soybean mutation plants by CRISPR/Cas9

大豆是重要的油料作物,其种子脂肪酸中油酸含量是评价大豆油脂品质的重要指标之一。本研究设计了分别由AtU3d、AtU3b和AtU6-1启动子驱动、长20 bp的guide RNA(gRNA)靶点以靶向编辑GmFAD2-1A基因的外显子区,首先将这3个靶点一起组装到pYLCRISPR/Cas9-DB载体上,然后利用农杆菌介导的方法转化大豆材料华夏3号。通过PCR技术及测序分析对T1代转基因大豆植株靶点编辑情况进行检测,获得纯合GmFAD2-1A大豆突变体。GmFAD2-1A突变大豆植株在株高、主茎节数、单枝分枝数、叶形、花色、种皮色、种脐色、生育期等方面与对照大豆植株没有显著差异;而GmFAD2-1A突变体大豆种子油酸含量显著高于对照大豆品种华夏3号,说明GmFAD2-1A是油酸代谢过程中的关键基因。本研究利用CRISPR/Cas9技术成功对控制大豆油酸基因GmFAD2-1A进行编辑,获得稳定的纯合GmFAD2-1A大豆突变体材料,为高油酸育种提供了新的种质资源并创建了方法。

Oleic acid content is one of the essential indicators to evaluate quality of oil in soybean.Three sites of 20 nt guide RNA(gRNA)targeted to the exon of GmFAD2-1A were designed and transcribed from the AtU3d,AtU3b,and AtU6-1 promoters,respectively.The three target sites of gRNA were ligated to the vector pYLCRISPR/Cas9-DB,and then the recombinant plasmid was transformed into a soybean cultivar Huaxia 3 by Agrobacterium-mediated transformation.The sequences near the editing site were analyzed by the PCR method and sequencing from T1 transgenic soybean plants,homozygous GmFAD2-1A mutants were obtained using CRISPR/Cas9 technology.The agronomic traits such as plant height,main stem number,branching number per plant,leaf shape,flower color,seed coat color,hilum color and growth period were no significant difference between the transgenic soybeans and non-transformed controls.However,the content of oleic acid in the transgenic soybean seed was significantly higher than that of the control cultivar Huaxia 3,indicating that GmFAD2-1A was a key gene during synthesis of oleic acid.We succeeded in editing the GmFAD2-1A by CRISPR/Cas9 technology in soybean and obtained homozygous mutant materials,which provides new germplasm resources and method for the breeding of high oleic acid.