利用CRISPR/Cas9技术创制小麦耐低肥Taaap3突变体

Creating of Wheat Taaap3 Mutant with Low Fertilizer Tolerance Using CRISPR/Cas9 Technology

为了创制耐低肥、高产小麦(Triticum aestivum L.)新种质,利用CRISPR/Cas9技术编辑小麦中氨基酸透性酶基因TaAAP3,明确突变体类型,并分析突变体在低肥条件下的产量,筛选耐低肥、高产小麦新材料。结果表明,共获得了1个小麦TaAAP3基因单亚基因组纯合突变体和2个三亚基因组纯合突变体,主要发生的突变是单个碱基的插入和小片段DNA的插入、缺失,其导致了移码突变,使翻译提前终止,造成蛋白质序列完全被改变。突变体Taaap3幼苗叶片中TaAAP3基因的表达水平显著低于野生型,且TaAAP3在三突变类型中的表达量显著低于单突变类型。低肥条件下,3个Taaap3突变体产量均显著高于野生型,且TaAAP3基因表达水平越低,增产幅度越大。综上,在小麦中敲除TaAAP3基因后,其表达水平显著降低,且低肥条件下籽粒产量显著增加。

In order to create new wheat(Triticum aestivum L.)germplasm with low fertilizer tolerance and high yield,the CRISPR/Cas9 technology was utilized to edit the amino acid permeability enzyme gene TaAAP3 in wheat,the types of mutants were determined,the yields of mutants under low fertilizer condition were analyzed,and new wheat materials with low fertilizer tolerance and high yield were screened.The results showed that three mutants of TaAAP3 gene were obtained,including one monogenomic homozygous mutant and two trigenomic homozygous mutants.The main mutations were insertion of single base and insertion or deletion of small fragment of DNA,resulting in frameshift mutations that terminated translation prematurely and caused the protein sequence completely altered.The expression level of TaAAP3 gene in the leaves of Taaap3 mutants seedlings was significantly lower than that of wild type,and the expression level of TaAAP3 in mutants with trigenomic mutations was significantly lower than that with monogenomic mutation.The grain yields of the three Taaap3 mutants were significantly higher than that of wild type under low fertilizer condition,and the lower expression level of TaAAP3 gene,the greater increase of grain yield.Overall,knocking out the TaAAP3 gene in wheat significantly reduces its expression level,and significantly increases grain yield under low fertilizer condition.