利用CRISPR/Cas9技术编辑DTH8基因改良水稻99-25的抽穗期  

Editing DTH8 Gene Using CRISPR/Cas9 Technology to Improve Heading Date of Rice 99-25

作　　者：张浩[1,2] 柳絮 宣宁[2] 张华[2] 高瑞钰[1,2] 赵倩倩 姚方印 ZHANG Hao;LIU Xu;XUAN Ning;ZHANG Hua;GAO Ruiyu;ZHAO Qianqian;YAO Fangyin(Shandong Normal University,Jinan 250014,China;Biotechnology Research Center,Shandong Academy of Agricultural Sciences,Jinan 250100,China)

机构地区：[1]山东师范大学,山东济南250014 [2]山东省农业科学院生物技术研究中心,山东济南250100

出　　处：《华北农学报》2020年第6期58-66,共9页Acta Agriculturae Boreali-Sinica

基　　金：国家转基因重大专项(2016ZX08001001-001-007);山东省农业良种工程(2017LZ029;2019LZGC017,2019LZGC003);山东省农业科学院创新工程(CXGC2016A02);山东省自然科学基金(ZR2019BC105);山东省现代农业产业技术体系(水稻)项目(SDAIT-17-03)。

摘　　要：CRISPR/Cas9基因编辑技术已经成为水稻育种的重要手段。为培育早熟、丰产、优质的种质资源,以农艺性状优良、迟熟粳稻品种香糯99-25为试验材料,构建了CRISPR/Cas9双靶点表达载体对抽穗期基因DTH8进行编辑,采用农杆菌介导法将构建好的表达载体转化农杆菌EHA105。用携带重组质粒的农杆菌菌液侵染水稻愈伤组织,成功获得了30株T_(0)转基因苗。对T_(0)植株利用潮霉素特异引物进行分子检测,其中阳性植株29株,阳性率高达96.7%。设计特异性引物对29株阳性苗的靶位点上下游600 bp进行PCR扩增测序,结果表明,7株转基因阳性苗在第2个靶点附近发生了碱基替换或插入突变。对这7株突变株系的抽穗期进行调查,发现DTH8-2、DTH8-5、DTH8-9、DTH8-10、DTH8-17、DTH8-21的抽穗期提前。利用实时荧光定量PCR检测发现DTH8-2、DTH8-9、DTH8-17与香糯99-25相比DTH8基因表达显著降低。成功利用CRISPR/Cas9基因编辑技术对香糯99-25的DTH8基因进行了编辑,获得了抽穗期提前的DTH8突变体材料,为培育早熟、丰产、优质的水稻品种提供了种质资源。CRISPR/Cas9 gene editing technology has become an important means for rice breeding.For cuttivating early maturity,high yield and high quality germplasm resources,Xiangnuo 99-25 which is a late-maturing japonica rice with excellent agronomic traits was used as the test material.The CRISPR/Cas9 dual-target expression vector was constructed to edit the heading gene DTH8 and transformed into Agrobacterium EHA105.EHA105 was transformed into rice callus via Agrobacterium-mediated transformation method,and 30 T_(0)transgenic seedlings were obtained.All the T_(0)seedlings were detected by PCR using hygromycin specific primer.Among them,29 strains were detected as positive plants and the positive rate was 96.7%.Specific primers were designed to amplify 600 bp upstream/downstream of the target site in the 29 positive seedlings.Sequence results showed that 7 seedlings had mutations,such as base substitution and insertion around the second target site.The heading date of DTH8-2,DTH8-5,DTH8-9,DTH8-10,DTH8-17,DTH8-21 in these 7 seedlings were detected early heading date compared with Xiangnuo 99-25.And DTH8 expression in DTH8-2,DTH8-9,DTH8-17 were down-regulated compared with Xiangnuo 99-25.CRISPR/Cas9 gene editing technology was successfully used to edit DTH8 gene in Xiangnuo 99-25.The DTH8 mutant rice with early heading stage was obtained,which provided germplasm resources for cultivating early maturing,high-yield and high-quality rice varieties.

关 键 词：水稻 抽穗期 基因编辑 DTH8 CRSPR/Cas9 

分 类 号：S511.03[农业科学—作物学] Q78[生物学—分子生物学]