摘要： 为了阐明水稻膜联蛋白基因家族成员OsAnn8在干旱和低温胁迫条件下的功能作用机制，通过荧光定量PCR技术对不同干旱和低温处理下的水稻叶片中OsAnn8基因进行转录水平上的定量分析，结果表明，OsAnn8基因在干旱胁迫处理前后表达量呈现出高-低-高的变化趋势，而在冷胁迫处理前后表达量则呈现出低-高-低-低的变化。随后，利用CRISPR/Cas9基因编辑技术对OsAnn8基因进行定点编辑，并借助农杆菌介导将OsAnn8基因靶位点的CRISPR/Cas9基因编辑植物表达载体导入转基因受体品种TP309，经潮霉素筛选后共获得32株转基因阳性植株，靶位点扩增测序峰图分析表明其中的2个单株出现了重叠峰，进一步的T载体克隆分别测序表明，这2个单株为单等位基因敲除，说明本研究已经成功获得了OsAnn8基因靶位点敲除的单等位突变体，不同类型的水稻OsAnn8基因突变体的创建，为水稻膜联蛋白基因家族成员OsAnn8在非生物胁迫条件下的功能研究奠定了材料基础。

关键词: 水稻, 膜联蛋白, OsAnn8, 荧光定量PCR, CRISPR/Cas9

Abstract: In order to clarify the functional mechanism of rice annexin OsAnn8 under drought and low temperature stress, the transcription level of OsAnn8 in rice leaves was analysed by fluorescence quantitative PCR technology. The results showed that the expression of OsAnn8 gene presented a high-low-high trend before and after drought stress, but a low-high-low-low changes before and after cold stress. Subsequently, the knocking out of OsAnn8 was also performed using the CRISPR/Cas9 gene editing techniques, and the CRISPR/Cas9 gene editing plant expression vector was transformed into the transgenic receptor variety TP309 by the agrobacterium-mediated method. A total of 32 transgenic positive plants were obtained by selection for hygromycin resistance. The analysis of sequencing map in target locus showed that overlapping peaks existed in two individual plants. Furthermore, T vector clones were sequenced separately, indicating that the monoallelic mutation happened in the two individual plants. The results indicated that monoallelic mutants had been obtained at OsAnn8 gene locus successfully. The creation of different types of mutants at OsAnn8 gene locus of rice laid the material base for gene function research under abiotic stress.

Key words: Rice, Annexin, OsAnn8, Fluorescence quantitative PCR, CRISPR/Cas9

中图分类号: 

• S511.03