CRISPR/Cas9编辑Badh2基因改良优质粳稻品种香味性状

Development of Fragrant Japonica Rice by CRISPR/Cas9-targeted Editing on Badh2

【目的】利用基因编辑技术编辑水稻香味基因,改良优质粳稻香味性状。【方法】构建CRISPR/Cas9-BADH基因编辑载体,转化优质粳稻品种龙稻18、龙稻24和秀水134,测序鉴定3个优质粳稻品种的香味基因Betaine aldehyde dehydrogenase 2(Badh2)突变体并分析潜在脱靶效应,利用气相色谱-质谱联用技术测定不同遗传背景badh2突变体稻米的2-乙酰-1-吡咯啉(2-acetyl-1-pyrroline,2AP)含量。【结果】转化获得的30株T0代中有24株为badh2突变体,其中53.33%为杂合型突变,16.67%为纯合型突变,10%为双等位突变类型。T1代非转基因植株内共鉴定获得7种纯合badh2突变基因型。在5个预测位置上未检测到脱靶事件的发生,说明设计的sgRNA具有高度特异性。所有Badh2移码突变体稻米的2AP含量都达到或高于稻花香的水平,但不同品种来源的badh2突变体间2AP含量差异极显著。【结论】本研究提供了一个能够高效诱导水稻Badh2突变的CRISPR/Cas9定向编辑靶点,改良了生产上大面积推广的3个优质水稻品种龙稻18、龙稻24和秀水134的香味性状,发现了不同遗传背景的水稻badh2突变体间2AP含量差异显著,为基于定向编辑Badh2基因的方法培育适合生产应用的香稻品种、提高育种效率提供科学依据。

【Objective】The CRISPR/Cas9 technology was applied to edit the Badh2 in rice to enrich the fragrance of current japonica varieties for marketing enhancement.【Method】A CRISPR/Cas9-BADH vector was constructed to transform 3 elite japonica rice,Longdao 18,Longdao 24,and Xiushui 134.The mutant badh2 was identified by sequencing,and potential offtarget mutations examined.Contents of the aromatic 2-acetyl-1-pyrroline(2 AP)in the mutant rice cultivars was determined by gas chromatography-mass spectrometry.【Result】Of the 30 T0 transgenic plants,24 were found to contain the target mutant badh2,of which,53.3% were heterozygous,16.67% homozygous,and 10% bi-allelic.Seven homozygous mutation genotypes were obtained in the T1 non-transgenic mutant plants.There was no off-target mutation detected at all 5 potential sites indicating that a high specificity of the designed sgRNA for the predicted site.Interferences by the varied genome backgrounds of the rice varieties might have caused the significantly varied amounts of the aromatic 2 AP detected in all badh2 frame-shift mutant rice.【Conclusion】The CRISPR/Cas9 technology could effectively induce desired Badh2 mutation in rice.Improvement on the fragrance for 3 japonica rice could lead to wide applications in cultivating new varieties with added commercial value.More interestingly,the significant variations on 2 AP content among the mutant rice as discovered in this study would help further the understanding on the genetics associated with aroma rice breeding.