CRISPR/Cas9 技术编辑淀粉合成基因PUL
网络出版日期: 2019-09-20
基金资助
转基因生物新品种培育重大专项（2016ZX08001006）
Construction of PUL Mutants in Rice Based on CRISPR/Cas9 Technology
利用CRISPR/Cas9技术对调控稻米脱支酶基因PUL定点编辑，获得了具有重要育种价值的PUL 等位变异。首先设计2个guide RNA（gRNA）靶位点，构建OsPUL，基因定点编辑载体pC1300-Cas9。 然后利用农杆菌介导侵染水稻材料中花11，提取T0代转基因植株的基因组 DNA，并对编辑位点附近的DNA片段进行 PCR 检测及测序分析。结果表明，T0代材料中PUL的突变频率高达90%, 其中纯合缺失突变率约占51%。对T1代纯合缺失突变体的千粒重性状的调查分析结果表明，部分pul的缺失突变能显著提高千粒重（大于5%）。通过RT-PCR分析T0代突变体中相关基因表达表明，目的基因PUL、支链淀粉相关基因和粒重负调控相关基因（GS3和GW8）表达降低，而粒重正调控相关基因（GL7）表达增加。不同类型pul突变体的成功创建丰富了PUL的变异类型，为水稻遗传改良提供了重要的遗传材料。
关键词： 水稻; 基因编辑; CRISPR/Cas9; PUL; 千粒重
奉宝兵,魏祥进,焦桂爱,胡时开,邬亚文,谢黎虹,圣忠华,邵高能,唐绍清* . CRISPR/Cas9 技术编辑淀粉合成基因PUL[J]. 中国稻米, 2019 , 25(5) : 99 -104 . DOI: 10.3969/j.issn.1006-8082.2019.05.022
A set of pul （pullanase） mutants were constructed using CRISPR/Cas9 technology in this study. Two sites of 20nt guide RNA（gRNA） targeted to the exon of OsPUL, there ligated to the vector pC1300-Cas9 based on golden gate cloning strategy. The recombinant plasmid was transferred to a rice cultivar, ZH11, by Agrobacterium-mediated transformation. Sequencing for the genomic DNA of PUL locus in T0 rice showed the mutagenesis frequency for PUL was more than 90%, including 51% of homozygous deletion mutations. Further analysis for the T1 mutants showed that almost all the homozygous deletion mutants improved the thousand-grain weight significantly（more than 5%）. Quantification of the genes related expression of T0 by RT-PCR, the results showed that the transcription level of the amylopectin starch synthesis gene and grain weight negative regulatory gene（GS3, GW8） were decreased. However, the grain weight positive regulatory gene（GL7）was increased. The successful pul editing not only provided a series of pul mutants for high and stable yield of rice, but also proved that CRISPR/Cas9 is a facile and powerful means of rice genetic engineering for scientific and agricultural applications, which has important theoretical and practical significance for rice breeding.
Key words： rice; genome editing; CRISPR/Cas9; pullanase; thousand-grain weight
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