木薯MeSSⅢ基因的CRISPR/Cas9基因编辑载体构建及验证

Construction and Verification of CRISPR/Cas9 Gene Editing Vector for Cassava MeSSⅢGene

木薯块根淀粉葡聚糖链结构是决定淀粉品质的关键因素,可溶性淀粉合成酶Ⅲ(SSⅢ)是调控植物支链淀粉葡聚糖长链合成的关键酶,木薯有两个MeSSⅢ的同源基因MeSSⅢ-1和MeSSⅢ-2。为了研究木薯MeSSⅢ对木薯块根淀粉品质形成的影响,根据MeSSⅢ-1和MeSSⅢ-2的保守区段,本研究构建了MeSSⅢ-1和MeSSⅢ-2的双基因编辑载体。利用在线软件CRISPR-P v2.0同时设计靶标MeSSⅢ-1和MeSSⅢ-2的sg RNA,通过酶切连接构建重组pCAMBIAP1301-Cas9-MeSSⅢ-gRNA质粒。将基因编辑载体转化农杆菌LBA4404后侵染木薯脆性胚性愈伤组织,筛选阳性脆性胚性愈伤组织,并提取其总DNA。通过PCR扩增靶点MeSSⅢ-1和MeSSⅢ-2的区段,并将扩增片段进行Sanger测序,分析其编辑的靶标位点。结果发现MeSSⅢ-1和MeSSⅢ-2靶标位点被成功编辑,本研究有助于进一步获得MeSSⅢ基因突变体,以深入解析该基因在木薯淀粉合成通路中的作用。

Starch glucan chain structure of cassava root is the key factor to determine starch quality.Soluble starch synthaseⅢ(SSIII)is the key enzyme to regulate the synthesis of long chain in plant amylopectin glucan.Cassava has two MeSSⅢhomologous genes MeSSⅢ-1 and MeSSⅢ-2.To study the effect of cassava MeSSⅢon the quality formation of cassava root starch,a double gene editing vector for MeSSⅢ-1 and MeSSⅢ-2 was constr-ucted.The sgRNA target for MeSSⅢ-1 and MeSSⅢ-2 was designed simultaneously by online software CRISPR-Pv2.0 based on the conserved segments,and the recombinant pCAMBIAP1301-Cas9-MeSSⅢ-gRNA plasmid was constructed by digestion and ligation.The gene editing vector was transformed into LBA4404 Agrobacterium competent cells and used to infect the friable embryogenic callus of cassava,and the their DNA was extracted.The target segments of MeSSⅢ-1 and MeSSⅢ-2 were amplified by PCR for Sanger sequencing,and analyzed the editing of target position.The results showed that the target sites of MeSSⅢ-1 and MeSSⅢ-2 were successfully edited.This study helps to further obtain mutants of the MeSSⅢgene to analyze the role of this gene in the cassava starch synthesis pathway.