OsNAC2d基因编辑水稻突变体的创建及其对干旱胁迫的响应

Construction of rice mutants by gene editing of OsNAC2d and their response to drought stress

为探究转录因子OsNAC2d的生物学功能及其对水稻耐旱性的影响,本研究利用CRISPR/Cas9编辑技术对粳稻中花11中的OsNAC2d基因进行突变,并考察osnac2d突变体在田间种植下的农艺性状,以及突变体幼苗在干旱胁迫下的生长情况和OsNAC2d基因表达水平。结果表明,OsNAC2d基因主要在水稻成熟籽粒、叶片和花药中表达,在根系和茎中的表达量较低,并且受干旱胁迫诱导,在10株阳性OsNAC2d基因突变株系的T2代植株中,筛选出6种纯合osnac2d突变体,田间试验调查表明,osnac2d突变体与野生型中花11相比,株高、有效穗、穗长、穗粒数、结实率、千粒重和单株产量等农艺性状无显著差异。在干旱胁迫时,osnac2d突变体幼苗的根系与植株生长、根系和地上部生物量的积累均受到抑制,且OsNAC2d在osnac2d突变体中的表达量维持在较低水平,而野生型水稻的OsNAC2d表达则受干旱胁迫诱导而增强,植株生长与生物量积累未受到显著抑制,表明转录因子OsNAC2d正调控水稻响应干旱胁迫。创建的osnac2d突变体材料为进一步揭示OsNAC2d的生物学功能及其响应干旱胁迫的精细调控机制提供了优质种质资源。

The main objective of this study is to explore the biological function of transcription factor OsNAC2d and its effect on drought resistance in rice.The sequence of OsNAC2d was firstly edited in japonica variety Zhonghua 11 through the CRISPR/Cas9 technology.The agronomic traits of osnac2d mutants were investigated in normal field cultivation,and the growth status and relative expression level of OsNAC2d genes were performed in the young mutant seedlings under the drought condition.There was the higher expression level of OsNAC2d in grain,leaf,and anther than those in root and stem,and the relative expression of OsNAC2d was enhanced by drought stress.Six homozygous T2 osnac2d lines were identified in two editing target sites of OsNAC2d genomic sequence through the nucleotide sequencing technology.There was no significant difference between osnac2d mutants and wild type in the agronomic traits including plant height,effective panicle number per plant,panicle length,grain number per panicle,seed setting rate,1000-grain weight,and grain yield per plant.Under the drought condition,the growth of roots and seedling of osnac2d mutants were depressed,and the biomass of roots and above-ground were also decreased.Meanwhile,the relative expression of OsNAC2d in the osnac2d mutant retained as extremely low level as those in the normal cultivating condition.However,the relative expression of OsNAC2d in wild type was enhanced by drought stress,and the growth and biomass accumulation of wild type were not evidently hindered by drought stress.These results indicated that OsNAC2d positively regulated the drought response in rice.The osnac2d mutants in this present study provided a precious genetic resource for deeply revealing the biological function of OsNAC2d and its fine regulating mechanism in response to drought stress.