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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2020, Vol. 40 Issue (12): 49-57    DOI: 10.13523/j.cb.2008119
技术与方法     
利用CRISPR/Cas9鉴定玉米发育相关基因ZmCen*
雷海英1,赵青松1,白凤麟1,宋慧芳1,王志军2,**()
1长治学院生物科学与技术系 长治 046011
2长治学院化学系 长治 046011
Identification of Developing-related Gene ZmCen Using CRISPR/Cas9 in Maize
LEI Hai-ying1,ZHAO Qing-song1,BAI Feng-lin1,SONG Hui-fang1,WANG Zhi-jun2,**()
1 Faculty of Biology Sciences and Technology, Changzhi University, Changzhi 046011, China
2 Department of Chemistry, Changzhi University, Changzhi 046011, China
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摘要：

目的: 利用CRISPR/Cas9(clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) 系统构建玉米中心蛋白(Centrin)的表达载体,经转化后分析其对玉米生长发育的影响。方法: 针对ZmCen基因的第一个外显子设计sgRNA,将其连入pOMS01-Cas9-ZmCen-sgRNA表达载体,转化农杆菌GV3101后,侵染玉米自交系材料B104的愈伤组织,经继代、诱导、分化成苗,筛选出转基因后代。对T0代和T1代基因组DNA进行PCR验证、测序及表型分析。结果: 成功构建ZmCen的表达载体。侵染农杆菌后,PCR测序显示,T0 代和T1 代突变率分别为 20.13% 和 64.52%,其中T1 代的纯合缺失突变率为5%。序列分析表明,ZmCen基因的编辑靶点附近发生了碱基的替换、插入或缺失。经与野生型表型比对发现,ZmCen 突变体T1代植株出现发育缓慢且雄花序不完全发育表型,纯合突变体植株雄花序则完全不发育。结论: 通过 CRISPR/Cas9技术成功地对玉米ZmCen基因进行了编辑,ZmCen突变体的获得为玉米雄性器官发育相关基因的研究奠定了基础。
关键词： CRISPR/Cas9技术;  玉米;  ZmCen雄花序    
Abstract:

Objective: To construct a ZmCen gene expressing vector using CRISPR/Cas9 system and analyze its effect on growth and development in maize after transformation. Methods: A sgRNA in the first exon of ZmCen gene was designed targeting it. The sgRNA was inserted into the pOMS01-Cas9-ZmCen-sgRNA vector, and its transgenic lines B104 were obtained via Agrobacterium-mediated transformation, subcultured, induced and differentiated into seedlings. The T0 and T1 generations genomic DNA was amplified and analyzed, and the plants were phenotype comparison screening. Results: The ZmCen expressing vector was successfully constructed. The genomic DNA of T0 and T1 generations was detected by PCR and sequenced. The mutagenesis frequency for ZmCen was 20.13% and 64.52% in T0 and T1 transgenic lines, respectively. The frequency of homozygous deletion mutation was 5% in T1 transgenic lines. Sequence analysis showed that base substitutions, insertions, or deletions occurred near the editing target of the ZmCen gene. Compared with the wild-type phenotype, it was found that the T1 generation plants of ZmCen mutant showed incomplete male inflorescence phenotype, while the male inflorescence of homozygous mutant plants was sterility. Conclusion: The ZmCen gene was succeed editing by CRISPR/Cas9 technical in maize, and the successful construction of ZmCen mutants lays a foundation for the related-genes study of maize male organ development.
Key words: CRISPR/Cas9    Maize    ZmCen male inflorescence
收稿日期: 2020-08-11 出版日期: 2021-01-14
ZTFLH:  Q789  
基金资助: * 国家自然科学基金(21201024);山西省应用基础研究项目(201801D121065);山西省高校科技创新资助项目(201802106);山西省“1331工程”重点创新研究团队项目资助项目(2018018)
通讯作者: 王志军     E-mail: czxywzj@163.com
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引用本文:

雷海英,赵青松,白凤麟,宋慧芳,王志军. 利用CRISPR/Cas9鉴定玉米发育相关基因ZmCen*[J]. 中国生物工程杂志, 2020, 40(12): 49-57.

LEI Hai-ying,ZHAO Qing-song,BAI Feng-lin,SONG Hui-fang,WANG Zhi-jun. Identification of Developing-related Gene ZmCen Using CRISPR/Cas9 in Maize. China Biotechnology, 2020, 40(12): 49-57.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2008119    或    https://manu60.magtech.com.cn/biotech/CN/Y2020/V40/I12/49

引物名称 引物(5'→3') 用途
T-F GGCGAGGCCTAGGGCTCGCCCTCATGG 合成sgRNA
T-R CCATGAGGGCGAGCCCTAGGCCTCAAA
pSgA-T GACCATAGCACAAGACAGGCGT 中间载体检测
pOSCas9-F GATGGGTTTTTATGATTAGAGTCC 表达载体检测
pOSCas9-R GGCTCGTATGTTGTGTGG
ZmCenT-F CAAGAAAATATCGGTCCATACGC 基因编辑验证
ZmCenT-R TGAGGTAAGGCAGGCATAACAA
ZmCenT-S ATCATTAGGTTCAGTTTTGTTTTT 基因测序验证测定
表1  所用的 PCR 引物序列
药品 用量
pOSCas9/ Asc I + Xba I 30ng
pSgA-T/Asc I + Xba I 10ng
T4 ligase buffer 1μl
T4 ligase 0.5μl
ddH2O 至10μl
表2  表达载体构建体系
图1  ZmCen基因结构、靶标导向RNA的设计及pOMS01-Cas9-ZmCen-gRNA载体构建及鉴定
图2  部分T0代编辑靶位点的试纸条及PCR扩增产物的琼脂糖凝胶电泳图
图3  部分T0 代与 T1 代植株基因编辑靶点序列比对结果
Target gene Number of sgRNA plants with different mutation types Number of
plants tested		 Percent of
mutation
Insert Delete Change
T0 generation 5 (3.24%) 9 (5.84%) 17 (11.04%) 154 20.13%
T1 generation 10 (16.13%) 11 (17.74%) 19 (30.64%) 62 64.52%
表3  sgRNA基因编辑突变类型检测
图4  T0和T1代植株中Cas9基因PCR琼脂糖凝胶电泳图
图5  野生型与T1代基因突变株系的雄花序表型及花药、花粉发育状态对比
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