图1 二穗短柄草CRISPR/Cas9-bdfls2基因编辑载体示意图 OsU3：sgRNA-Cas9-pHUE411 载体示意图。浅蓝色部分OsU3p表示水稻U3启动子;黄色和绿色部分表示sgRNA序列;其中黄色部分表示基因的靶序列;灰色部分表示水稻OsU3t终止子

Fig. 1 Illustration of CRISPR/Cas9-bdfls2 gene editing vector in B. distachyon OsU3：sgRNA-Cas9-pHUE411 vector. Light blue indicates the Oryzae U3 promoter. Yellow and green portions indicate the sgRNA sequence. The yellow portion indicates the target sequence of the gene. The grey portion indicates the Oryzae U3 terminator

图2 编辑载体和编辑后靶基因的鉴定 A：CRISPR/Cas9-bdfls2基因编辑载体的鉴定,1-4为检测的已转化CRISPR/Cas9-bdfls2的农杆菌菌株;B：靶基因扩增鉴定,1-5为由转基因植株提取的基因组DNA扩增的含有靶基因编辑位点的片段。M为DNA marker;WT为二穗短柄草Bd21野生型对照;-为阴性对照

Fig. 2 Identification of gene editing vector and edited target gene A：Identification of CRISPR/Cas9-bdfls2 gene editing vector. 1-4 are the assayed Agrobacterium tumefaciens strains for transformed CRISPR/Cas9-bdfls2. B：Amplification and identification of target gene. 1-5 are genome-DNA-amplified fragments containing target gene editing sites extracted from transgenic plants. M is the DNA marker. Bd21 is the wild type（WT）. - indicates the negative control

图3 二穗短柄草bdfls2 突变体基因型检测 A：bdfls2突变体的基因型。红色下划线表示sgRNA：Cas9编辑的靶序列,蓝色代表对应的前间隔序列邻近基序（PAM）,1#、2#分别代表不同的突变体植株,A1、A2分别代表Allele 1和Allele 2,序列中红色 - 表示碱基缺失;B：bdfls2突变体的的测序图。WT为作为对照的Bd21野生型

Fig. 3 Genotyping of bdfls2 mutants in B. distachyon A：Genotyping of bdfls2 mutants. Red underline refers to sgRNA：Cas9-edited bdfls2 mutation. Blue underline refer to schematic illustration of the sgRNA：Cas9 targets and the corresponding protospacer-adjacent motif（PAM）（blue）of the targeted genes. The 1# and 2# stands for different mutant plants,A1 and A2 for Allele 1 and Allele 2,and the red lines in sequences stands for base deletion. B：Sequencing of bdfls2 mutants. WT is the Bd21 wild type as control

图4 二穗短柄草野生型和bdfls2 突变体在flg22处理下的ROS检测

Fig. 4 ROS burst of wild-type B. distachyon and bdfls2 mutants under flg22 treatment

图5 实时荧光定量RT-PCR 对PAMPs诱导的防卫基因的检测 二穗短柄草叶片在5 μmol/L flg22（A）或 100 μg/mL chitin（B）未处理（0 h）及处理后1,3,6 h的 BdChit8和PR2 基因的相对表达量,通过实时荧光定量RT-PCR检测得出。图中各组数据均有3组生物学重复,各柱表示为平均值±标准误。图中柱子上的*或**分别代表P < 0.05 或P < 0.01

Fig. 5 Detection of defense gene expression induced upon different PAMPs treatment by real-time quantitative RT-PCR The relative BdChit8 and PR2 expression in the leaves of B. distachyon seedlings treated with 5 μmol/L flg22（A）or 100 μg/mL chitin（B）were determined by quantitative real-time PCR. Data are presented as the mean ± standard error of three biological replicates.* or ** above the bars indicate P < 0.05 or P < 0.01