基于CRISPR/Cas9技术创制木薯MeSTP7和MeSTP15双基因突变体

木薯（Manihot esculenta Crantz）是一种热带、亚热带重要的粮食与能源作物,提高木薯的产量对木薯产业发展至关重要。木薯块根的发育直接影响其产量情况,而不同逆境胁迫会影响木薯块根的发育情况。因此解析木薯块根的发育机制有助于通过分子育种手段实现木薯高产,以及获得具备一定抗逆品质的优良种质,增强木薯的适应性,从而扩大木薯种植的推广面积。己糖转运蛋白（STPs）是一类MSTs亚家族蛋白,通过转运糖类物质调节植物生长发育及非生物胁迫。本实验室前期鉴定出MeSTP7和MeSTP15基因在块根发育时期的糖分累积、应对非生物胁迫过程中起到重要作用,因此获得木薯MeSTP7和MeSTP15双突变体将有助于解析木薯块根发育机制及创制抗逆新种质。为了得到MeSTP7和MeSTP15木薯双突变体,利用在线软件CRISPR-P v2.0同时设计靶标基因MeSTP7和MeSTP15的sgRNA,成功构建了MeSTP7和MeSTP15的CRISPR/Cas9双基因编辑载体。将编辑载体转化农杆菌LBA4404后,侵染‘华南8号’（SC8）木薯脆性胚性愈伤组织,经过Sanger测序分析MeSTP7和MeSTP15成功发生编辑,而潜在的脱靶位点未发生编辑,说明双编辑载体可对MeSTP7和MeSTP15基因进行编辑,且不造成脱靶现象,然后将侵染后的脆性胚性愈伤组织诱导出子叶,经过15 mmol/L的潮霉素B筛选出生根植株,生根植株再经过分子检测,成功获得带有双编辑载体表达框的转基因阳性植株。以阳性植株的基因组作为模板,分别用PCR扩增2个基因的靶位点前后各100 bp核苷酸序列,经过Hi-TOM测序后,结果显示有26个株系发生突变,其中MeSTP7/15双基因突变体有23个,MeSTP7单基因突变体2个,MeSTP15单基因突变体1个,编辑类型多为单碱基缺失或插入,大片段碱基的缺失所占比率小。初步在组培瓶中观察突变体的变型,发现单突变体和双突变体的根系生长均受抑制,植株矮小,且MeSTP7和MeSTP15双突变体受损更严重。该研究结果不仅为进一步解析木薯块根发育机制奠定基础,还为获得木薯抗病、抗逆新种质提供材料。

Construction of MeSTP7 and MeSTP15 Double Mutants in Cassava Based on CRISPR/Cas9 Technology

Cassava (Manihot esculenta Crantz) is an important food and energy crop in the tropics and subtropics, and improving cassava production is essential for the development of the cassava industry. The development of cassava storage root directly affects its yield, and different adversity stresses can affect the development of cassava storage root. Therefore, the analysis of cassava storage root development mechanism can help to achieve high cassava yield through molecular breeding, as well as to obtain excellent germplasm with certain resistance quality and enhance the adaptability of cassava, so as to expand the extension of cassava cultivation. Hexose transport proteins (STPs) are a subfamily of MSTs that regulate plant growth and development as well as abiotic stresses by transporting sugars. The MeSTP7 and MeSTP15 genes have been identified to play an important role in sugar accumulation during tuber development and in response to abiotic stresses. To obtain MeSTP7 MeSTP15 cassava double mutants, CRISPR/Cas9 double gene editing vectors for MeSTP7 and MeSTP15 were successfully constructed by designing the sgRNAs of both target genes, MeSTP7 and MeSTP15, using the online software CRISPR-P v2.0. After transformation of the editing vector into Agrobacterium tumefaciens LBA4404, ‘SC8’ cassava brittle embryonic healing tissues were infiltrated, and MeSTP7 and MeSTP15 were successfully edited by Sanger sequencing analysis, while the potential off-target sites were not edited, indicating that the dual editing vector could edit both MeSTP7 and MeSTP15 genes without causing off-target phenomenon. The rooted seedlings were then screened with 15 mmol/L Hygromycin B to produce rooted seedlings, which were then characterized detection to obtain positive transgenic cassava plants with the expression frame of the double-edited vector. The genomes of positive plants were used as the templates for PCR amplification of 100 bp nucleotide sequences before and after each target site of the two genes, and after Hi-TOM sequencing, the results showed that there were 26 strains with mutations, including 23 MeSTP7/15 double mutants, 2 MeSTP7 single mutants and 1 MeSTP15 single mutant, and the editing types were mostly single. The types of editing were mostly single base deletions or insertions, with a small percentage of deletions of large segments of bases. Our preliminary observations of the mutant variants in histoponic flasks revealed that root growth was inhibited and plants were dwarfed in both the single and double mutants, and that the MeSTP7 and MeSTP15 double mutants were more severely damaged. These results not only lay the foundation for further analysis of the mechanism of cassava tuber development, but also provide material for obtaining new germplasm for cassava disease and stress resistance.