中国农业科学 ›› 2017, Vol. 50 ›› Issue (7): 1294-1301.doi: 10.3864/j.issn.0578-1752.2017.07.012

• 园艺 • 上一篇    下一篇

香蕉CRISPR/Cas9基因编辑技术体系的建立

胡春华,邓贵明,孙晓玄,左存武,李春雨,邝瑞彬,杨乔松,易干军   

  1. 广东省农业科学院果树研究所/农业部南亚热带果树生物学和种质资源利用重点实验室,广州 510640
  • 收稿日期:2016-11-08 出版日期:2017-04-01 发布日期:2017-04-01
  • 通讯作者: 易干军,Tel:020-87596278;E-mail:yiganjun@vip.163.com
  • 作者简介:胡春华,Tel:13660062169;E-mail:huchunhua007@126.com。
  • 基金资助:
    农业部“948”项目(2016-X22)、广州市科技计划项目(201707010154)、国家香蕉产业体系(CARS-32-01)、广东省科技计划项目(2014B070706018,2015A030302046)、农业部物质资源保护项目(16RZZY-13)

Establishment of an Efficient CRISPR/Cas9-Mediated Gene Editing System in Banana

HU ChunHua, DENG GuiMing, SUN XiaoXuan, ZUO CunWu, LI ChunYu, KUANG RuiBin, YANG QiaoSong, YI GanJun   

  1. Fruit Tree Research Institute, Guangdong Academy of Agricultural Science/Key Laboratory of Biology and Genetic Resource Utilization of Fruit Trees in South Subtropics, the Ministry of Agriculture, Guangzhou 510640
  • Received:2016-11-08 Online:2017-04-01 Published:2017-04-01

摘要: 【目的】建立香蕉CRISPR/Cas9基因编辑技术体系,为在香蕉上利用CRISPR/CAS9技术开展香蕉基因功能研究和香蕉育种工作开辟新的路径。【方法】根据香蕉A基因组八氢番茄红素脱氢酶(phytoene dehydrogenase,PDS)基因组序列,利用在线工具ZiFiT Targeter Version 4.2确定合适的CRISPR/Cas9靶标序列,选择其中一个位点作为靶标位点,设计包含靶标基因MaPDS序列的sgRNA。利用一套改良的CRISPR/Cas9多靶点载体系统,以pYLgRNA-LacZ-U6a质粒为模版,Overlapping PCR法构建U6a-sgRNA表达盒,再利用Golden Gate Cloning法将U6a-sgRNA表达盒克隆到pYLCRISPR/Cas9载体中,构建以MaPDS为靶标基因的pYLCRISPR/Cas9-sgRNA载体。构建的质粒含Cas9psgRNA表达盒,其中Cas9pPUbi启动子驱动,sgRNA水稻来源的RNA启动子U6a驱动。将构建好的载体转入农杆菌EHA105,转化香蕉主栽品种巴西蕉胚性细胞悬浮系,获得抗性再生植株。设计PCR引物扩增包含靶标序列的MaPDS序列片段,检测和分析再生植株MaPDS被编辑的情况。【结果】试验选择MaPDS作为CRISPR/Cas9靶标基因,设计一个靶标位点,利用Overlapping PCR法获得了U6a-sgRNA表达盒,利用Golden Gate Cloning法将其克隆到pYLCRISPR/Cas9的Bsa I位点,成功构建了针对MaPDS的pYLCRISPR/Cas9-sgRNA载体。经过农杆菌浸染、抗性筛选、抗性胚诱导、萌发及生根,最终获得抗性独立转化株系129个。其中,71个株系出现白化表型,产生白化表型的几率达55%。失绿突变体的出现意味着MaPDS蛋白功能丧失。随机取转化株系中的白化表型株系33个和正常表型株系14个,提取其叶片基因组DNA,扩增含有MaPDS的靶位点片段,序列分析结果表明,白化表型株系的MaPDS靶位点序列发生了基因编辑。主要是在靶位点附近增加1个碱基T或A,或是在靶位点附近或下游发生碱基颠换或转换,出现非靶标位点突变。这些突变形式均能导致MaPDS蛋白翻译错误,从而使MaPDS蛋白丧失功能,表现为白化。转化株系中表型正常植株的MaPDS靶位点序列与野生型一致,未检测到变异。【结论】成功在香蕉体内实现了对内源MaPDS的定点敲除,获得了基因定点敲除的突变体株系,为进一步利用基因编辑技术在香蕉上的应用奠定了基础。

关键词: 香蕉, CRISPR/Cas9, MaPDS, 基因编辑

Abstract: Objective The objective of this study is to establish a CRISPR/Cas9-mediated gene editing system in order to find a new way for revealing gene function and breeding of banana. Method According to the Musa genomic (A genome) sequence of phytoene desaturase gene 8 (MaPDS), online tools ZiFiT Targeter Version 4.2 was used to determine the appropriate designing of CRISPR/Cas9 target sites, one of the output target sites was selected for designing the sgRNA. The sgRNA and U6a promoter sequences were linked by overlapping PCR to produce U6a-sgRNA expression cassette, then the fragment was cloned into the binary pYLCRISPR/Cas9 multiplex genome targeting vector system based on Golden Gate Cloning, a pYLCRISPR/Cas9 vector carrying sgRNA cassette named pYLCRISPR/Cas9-sgRNA was generated. The plant expression binary vector, containing a Cas9 (Cas9p) expression cassette, driven by the maize ubiquitin promoter (Pubi), and the sgRNA with target sequence of MaPDS was transcribed under the control of the U6a promoter from rice. The constructed plasmid was then transformed into EHA105 strain of Agrobacterium tumefaciens, which was subsequently used to infect embryogenic cell suspensions (ECS) of banana to obtain resistant plantlets. PCR amplifications were carried out using primer pairs flanking the designed target site. The PCR products were sequenced for detection of mutation.ResultMaPDS was selected as the target of Cas9 endonuclease, and a sgRNA was designed as the target site for CRIPSR/Cas9. The sgRNA with target sequence was firstly inserted into the sgRNA expression cassette by using overlapping PCR, then the cassette was combined with the pYLCRISPR/Cas9 in a single plant binary vector. The results of identification by enzyme digestion and sequencing showed that the pYLCRISPR/Cas9-sgRNA vector aiming at MaPDSa Cas9 expression cassette driven by Pu was successfully constructed, and the vector harbors bi, and a sgRNA expression cassette under the U6a promoter. Using this system and Agrobacterium-mediated transformation,129 independently transformed lines with visible albino, chimerism mutant phenotypes and normal phenotypes were regenerated from ECS after transformation, resistance screening, resistant embryo induction, germination and rooting process. The ratio of albino and chimerism phenotype occurred in transgenic banana was more than 55% (71 out of 129).Albino phenotype in these transgenic plants indicates the loss of PtoPDS function. To further verify whether the loss of green in the transgenic banana was caused by generation of mutations in the MaPDS by the CRISPR/Cas9 system, 33mutant phenotypes and 14 normal phenotypes from 129 independent transgenic plants were randomly selected for sequencing. The results confirmed that all of these transgenic plants with albino phenotype contained mutants in the MaPDS, insertion (a Aor T) and displacement were found in the target sites of MaPDS in the mutants. And it was also found the displacement in downstream of the target sites of MaPDS in the experiments, no mutation was detected in the normal phenotypes of transgenic and control plants.Conclusion The CRISPR/Cas9 system was successfully applied to knock out the endogenous phytoene dehydrogenase (PDS) gene in banana. The results demonstrate that the Cas9/sgRNA system can be exploited to targeted mutagenesis and effectively create knockout mutations in banana.

Key words: banana, CRISPR/Cas9, MaPDS, gene editing