水稻抗白叶枯病基因Xa47(t)a的敲除及抗性鉴定

Knockout and identification of rice bacterial blight resistance gene Xa47(t)a

为鉴定水稻Xa47(t)a基因对白叶枯病的抗性,以水稻种质L214为材料,通过构建针对Xa47(t)a基因的Xa47(t)a-Cas9敲除载体来获得敲除突变体,利用生物信息学技术对突变的Xa47(t)a基因进行突变类型分析,同时采用实时荧光定量PCR(quantitative real-time PCR,qPCR)技术分析突变体中Xa47(t)a及病程相关基因的表达情况,并于水稻孕穗期对突变体及其野生型植株接种11株白叶枯病菌Xanthomonas oryzae pv.oryzae菌株进行抗性鉴定。结果表明,在Xa47(t)a基因的第2外显子区域进行基因编辑后成功获得25株T0代突变体株系;测序分析发现T0代突变体株系中有13种不同的突变体类型,其中纯合突变体有3种类型,且突变位点均在靶标位点的11位碱基处缺失1~4个A碱基;氨基酸序列分析发现大部分突变体中Xa47a编码的蛋白翻译会提前终止,由原来的803个氨基酸变为144~166个氨基酸;qPCR分析结果表明突变体中Xa47(t)a及大部分病程相关基因的表达水平显著低于野生型株系;抗性鉴定结果显示,生长至孕穗期的T1代纯合突变体株系对白叶枯病菌Y8、C5、C9、PXO99A、T7147、YJdp-2和YJws-2菌株的抗性显著低于野生型株系。表明Xa47(t)a基因赋予了稻种材料L214良好的抗白叶枯病功能。

Rice germplasm L214 was used to create the Xa47(t)a-Cas9 knockout vector for the Xa47(t)a gene in order to create a mutant,which could be used to study the Xa47(t)a gene resistance to bacterial blight.The mutant Xa47(t)a genes mutation type was examined using bioinformatics technology.Xa47(t)a and pathogenesis-related genes were also examined using quantitative real-time PCR(qPCR)in the mutant lines.The mutants and their wild-type plants were injected with 11 strains of Xanthomonas oryzae pv.oryzae(Xoo)at the booting stage of the rice plant in order to identify their disease resistance.The results showed that 25 T0 mutant lines were effectively created by gene editing in the second exon region of the Xa47(t)a genes.In the T0 generation,13 distinct classes of mutant lines,including three homozygous mutants,were identified with sequencing analysis.Amino acid sequence analysis revealed that the translation of the Xa47(t)a-encoded protein in most mutants was terminated prematurely,ranging from 803 to 144-166 amino acids.qPCR analysis showed that Xa47(t)a and pathogenesis-related genes were expressed at much lower levels in the mutants than in the wild type.Inoculation experiments demonstrated that T1 homozygous mutants exhibited significantly lower resistance to Xoo strains Y8,C5,C9,PXO99A,T7147,YJdp-2,and YJws-2 at the booting stage compared to the wild type.These findings suggest that the rice material L214 has high resistance to bacterial blight due to the presence of the Xa47(t)a gene.