摘要
【目的】植物特异性IQD家族蛋白质是一类植物特有的钙调素结合靶蛋白,在植物的生物防御和发育调节中发挥重要作用。前期酵母双杂筛选试验表明,Pik-H4与OsIQD1存在互作关系,为进一步揭示Os IQD1在水稻抗稻瘟病中的生物学功能,利用CRISPR/Cas9编辑技术对OsIQD1基因进行定点编辑。【方法】通过序列比对获得水稻OsIQD1。在OsIQD1第1外显子和第5外显子(DUF4005结构域)区域设计2个20 bp的编辑靶点,将2个靶点核苷酸片段克隆至pRGEB32载体,获得p RGEB32-OsIQD1-gRNA载体,利用农杆菌介导法转化水稻Pik-H4 NIL愈伤组织,经再生培养、潮霉素检测获得转基因阳性植株,并对T0代转基因植株靶点区域序列进行PCR和测序,分析osiqd1的突变类型。【结果】转基因再生植株经过对抗潮霉素基因HPTⅡ的PCR鉴定,获得30株阳性株系。对T0代植株靶点附近序列的测序结果表明,OsIQD1基因被成功编辑,两个位点的编辑效率分别为21.67%和26.67%,其中,靶点2区域有1个纯合突变株系。【结论】研究结果为进一步利用osiqd1突变体开展其参与钙离子/钙调素信号转导通路的机制研究提供了遗传材料,初步推定OsIQD1参与植物的基础免疫反应。
【Objective】IQD gene family proteins are plant specific calmodulin-binding target proteins,which play an important role in plant defense and development regulation.The interaction between Pik-H4 and OsIQD1 was found in the yeast double hybrid screening experiment in laboratory.In order to reveal the specific biological functions of OsIQD1 in rice blast resistance,OsIQD1 gene was edited by using CRISPR/Cas9 editing technique.【Method】OsIQD1 was obtained by sequence alignment.Two 20 bp editing targets were designed in exon 1 and exon 5 of OsIQD1(DUF4005 domain),and the nucleotide fragments of the two targets were cloned into pRGEB32 vector to obtain pRGEB32-OsIQD1-gRNA vector.The rice Pik-H4 NIL callus was transformed by agrobacterium-mediated method.Transgenic positive plants were obtained by regeneration culture and hygromycin resistance screening.PCR and sequencing were performed on the target region sequences of transgenic plants of T0 generation to analyze the mutation types of osiqd1.【Result】The transgenic regenerated plants were identified by PCR with hygromycin resistance gene HPT Ⅱ,and 30 positive lines were obtained.Sequencing analysis of the sequences near the target site of T0 generation plants showed that OsIQD1 gene was successfully edited,and the editing efficiency of the two sites was 21.67% and 26.67%,respectively.There was one homozygous mutant line in target 2 region.【Conclusion】The study results provide genetic material for further studies on the mechanism of osiqd1 mutation involved in calcium ion/calmodulin signaling pathway,and OsIQD1 is preliminarily presumed to be involved in the basic immune response of plants.
作者
林春姿
谢华斌
黄其伟
高上
王加峰
LIN Chunzi;XIE Huabin;HUANG Qiwei;GAO Shang;WANG Jiafeng(College of Agriculture,South China Agricultural University/National Engineering Research Center of Plant Space Breeding,Guangzhou 510642,China)
出处
《广东农业科学》
CAS
2022年第8期1-10,共10页
Guangdong Agricultural Sciences
基金
广东省自然科学基金(2019A1515011825)。
