利用CRISPR/Cas9基因编辑技术构建水稻ospin9突变体  

Generation of ospin9 Mutants in Rice by CRISPR/Cas9 Genome Editing Technology

作　　者：吴世洋 杨晓祎 张艳雯 侯典云[1] 胥华伟[1] WU ShiYang;YANG XiaoYi;ZHANG YanWen;HOU DianYun;XU HuaWei(College of Agriculture,Henan University of Science and Technology,Luoyang 471000,Henan)

机构地区：[1]河南科技大学农学院,河南洛阳471000

出　　处：《中国农业科学》2021年第18期3805-3817,共13页Scientia Agricultura Sinica

基　　金：国家自然科学基金(182300410012);河南省自然科学基金(82300410012、202300410151);棉花生物学国家重点实验室开放课题(CB2020A24)。

摘　　要：【目的】生长素输出载体蛋白(PIN-FORMED,PIN)是控制生长素极性运输的关键蛋白,水稻OsPIN9是单子叶植物特有的PIN基因,但其生物学功能仍有待研究。利用CRISPR/Cas9基因编辑技术对OsPIN9进行编辑,获得OsPIN9发生突变的基因编辑株系,对进一步深入研究OsPIN9功能提供依据。【方法】根据OsPIN9序列设计特异性编辑位点,构建OsPIN9编辑载体,以日本晴愈伤组织为受体,通过农杆菌介导法获得抗性植株,通过PCR鉴定转基因植株。转基因植株通过PCR和测序明确OsPIN9的突变类型,获得ospin9纯合突变体并分析突变蛋白与野生型蛋白的差异。qRT-PCR分析突变体幼苗根部OsPINs的表达,进一步明确突变体与野生型对照植株之间的表型差异。以0.05μmol·L-1的萘乙酸(1-naphthaleneacetic acid,NAA)处理幼苗7 d,分析NAA对植株表型的影响。【结果】在水稻OsPIN9第1外显子处设计靶点并构建表达载体,通过遗传转化成功获得18株T0代转基因植株,测序分析发现转基因株系中有3种不同的突变方式,均为在靶位点的18位碱基处插入不同的单碱基,其中,3株插入T碱基,3株插入G碱基,1株插入C碱基,共获得基因编辑株系7株,进一步鉴定获得2种纯合突变体。序列比对分析表明,这两种类型的突变均造成移码突变和蛋白翻译提前终止,由原来的426个氨基酸缩短为172个氨基酸,跨膜螺旋结构域分析表明突变体中OsPIN9蛋白的跨膜结构完全消失。qRT-PCR分析表明,2个突变株系的OsPIN9转录水平显著降低,OsPIN1a和OsPIN5b表达上调,而OsPIN5a表达受到抑制。幼苗期的表型分析表明,突变体的株高显著低于野生型,不定根数显著少于野生型,但根长没有显著变化。NAA处理下,植株的生长受到抑制,ospin9突变体的不定根数仍少于野生型,但差异已不显著。【结论】利用CRISPR/Cas9技术对水稻生长素输出载体蛋白OsPIN9进行定向编辑,可获得无转基因成分的基【Objective】Auxin efflux protein family PIN-FORMED(PIN)is a key protein family in controlling polar auxin transport(PAT).OsPIN9 is one of the monocot-specific PIN genes in rice,while its biological function still needs to be further elucidated.In this study,OsPIN9 was edited and ospin9 homozygous mutants were obtained using CRISPR/Cas9 genome editing technology.The resultant ospin9 mutant lines could provide a basis for further research on the function of OsPIN9.【Method】The specific target sequence was designed according to OsPIN9 genome sequence and OsPIN9 genome editing vector was constructed.Nippobare(Oryza sativa japonica)was used as the material and the hygromycin-resistant rice was obtained by Agrobacterium-mediated transformation.The positive transgenic lines were screened by PCR.The mutation sites were confirmed by the combination of PCR and subsequent analysis of sequencing results,the homozygous mutants were obtained and the difference of amino acid sequence and tertiary structure of OsPIN9 protein was analyzed between WT and ospin9 mutants.The expression of OsPINs genes in mutant roots was performed by quantitative real-time PCR(qRT-PCR),and the phenotype of ospin9 mutants was analyzed at the seedling stage.The effects of 1-naphthaleneacetic acid(NAA)treatment on seedling development were also analyzed under 0.05μmol·L-1 NAA for 7 d.【Result】The target site sequence was designed based on the sequence of exon1 of OsPIN9 and,subsequently,the OsPIN9 genome editing recombinant vector was constructed.A total of 18 independent transgenic lines were obtained by transformation.Sequencing analysis revealed that three different mutation types were present in 7 T0 generation lines,including 3 lines with T insertion,3 lines with G insertion and 1 line with C insertion,and all the mutation sites happened at the 18th base of the target sequence.Two homozygous mutation lines were further identified in the T1 generation.BLAST analysis showed that the two types of OsPIN9 mutations caused frame-shift mutatio

关 键 词：水稻 OsPIN9 生长素极性运输 CRISPR/Cas9 

分 类 号：S511[农业科学—作物学]