摘要
在低硫苷低芥酸的双低甘蓝型油菜基础上,培育稳定的高油酸油菜是当前重要的育种目标之一,本研究通过利用CRISPR/Cas9基因编辑对湖南农业大学选育的品种湘油15号(XY15)进行品种改良。BnaFAD2和BnaFAE1是油酸合成不饱和脂肪酸和超长链脂肪酸的两个关键基因。利用CRISPR/Cas9基因编辑系统与高效油菜转化技术相结合,对甘蓝型油菜XY15的BnaFAD2.A05、BnaFAD2.C05、BnaFAE1.A08和BnaFAE1.C03同时进行基因编辑,获得了7株T0代转基因植株,通过靶基因克隆测序,发现均未发生基因编辑。然而,在T1代植株中发现并检测到了基因编辑的发生,BnaFAD2.A05、BnaFAD2.C05、BnaFAE1.A08和BnaFAE1.C03的总基因编辑效率分别为38.89%、27.78%、22.22%、30.56%。在T2代植株中,筛选到了5株4个靶基因均发生了突变,且无外源基因的突变体材料Cas9-1-12-4、Cas9-1-12-5、Cas9-2-7-11、Cas9-5-5-3、Cas9-6-6-1,它们的相对油酸含量为68.69%、80.16%、75.82%、75.97%、74.37%,相比于野生型XY15(相对油酸含量为62.04%)均有显著提高。
On the basis of low-glucosinolate and low-erucic rapeseed(Brassica napus),to obtain stable high-oleic rapeseed is one of the current important breeding objectives. In this study,CRISPR/cas9 system was used to improve the variety XY15 selected by Hunan Agricultural University. BnaFAD2 and BnaFAE1 are two key genes for the conversion of oleic acid to unsaturated fatty acids and ultra-long-chain fatty acids. The CRISPR/Cas9 system was introduced into XY15 by using highly efficient rapeseed transformation technology. with BnaFAD2.A05,BnaFAD2.C05,BnaFAE1.A08 and BnaFAE1.C03 simultaneously edited. Through target gene cloning and sequencing,it was found that no gene editing occurred in the 7 T0 transgenic plants. However,gene editing was found and detected in T1 plants. The total gene editing efficiency of BnaFAD2.A05,BnaFAD2.C05,BnaFAE1.A08 and BnaFAE1.C03 were 38.89%,27.78%,22.22% and 30.56%,respectively. In the T2 plants,5 mutant plants with no exogenous genes and 4 target genes edited were selected,named Cas9-1-12-4,Cas9-1-12-5,Cas9-2-7-11,Cas9-5-5-3 and Cas9-6-6-1,and their relative oleic acid content was 68.69%,80.16%,75.82%,75.97% and 74.37%,respectively,which were significantly higher than that of the wild type XY15(62.04%).
作者
高谢旺
谭安琪
胡信畅
祝孟洋
阮颖
刘春林
GAO Xie-wang;TAN An-qi;HU Xin-chang;ZHU Meng-yang;RUAN Ying;LIU Chun-lin(Hunan Provincial Key Laboratory of crop Epigenetic Regulation and Development/Agricultural College of Hunan Agricultural University/Laboratory of Crop Metabolism Control and Metabolism Engineering,Changsha 410128)
出处
《植物遗传资源学报》
CAS
CSCD
北大核心
2020年第4期1002-1008,共7页
Journal of Plant Genetic Resources
基金
国家自然基金面上项目(31571707)。
