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

doi:10.7668/hbnxb.20191109

摘要/Abstract

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T₀转基因苗。对T₀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。

关键词: 水稻, 抽穗期, 基因编辑, DTH8, CRSPR/Cas9

Abstract: 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 T₀ transgenic seedlings were obtained. All the T₀ 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.

Key words: Rice, Heading stage, Gene editing, DTH8, CRISPR/Cas9

中图分类号:

S511.03
Q78

张浩, 柳絮, 宣宁, 张华, 高瑞钰, 赵倩倩, 姚方印. 利用CRISPR/Cas9技术编辑DTH8基因改良水稻99-25的抽穗期[J]. 华北农学报, 2020, 35(6): 58-66. doi: 10.7668/hbnxb.20191109.

ZHANG Hao, LIU Xu, XUAN Ning, ZHANG Hua, GAO Ruiyu, ZHAO Qianqian, YAO Fangyin. Editing DTH8 Gene Using CRISPR/Cas9 Technology to Improve Heading Date of Rice 99-25[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2020, 35(6): 58-66. doi: 10.7668/hbnxb.20191109.

http://www.hbnxb.net/CN/Y2020/V35/I6/58

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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

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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

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