Abstract
Starch glucan chain structure of cassava root is the key factor to determine starch quality. Soluble starch synthase III (SSIII) is the key enzyme to regulate the synthesis of long chain in plant amylopectin glucan. Cassava has two MeSSIII homologous genes MeSSIII-1 and MeSSIII-2 . To study the effect of cassava MeSSIII on the quality formation of cassava root starch, a double gene editing vector for MeSSIII-1 and MeSSIII-2 was constructed. 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 MeSSIII gene to analyze the role of this gene in the cassava starch synthesis pathway.
Highlights
Cassava (Manihot esculenta Crantz) is a tropical and sub-tropical tuberous cash crop with starch as 85% of the dry matter in the tuber and is the sixth most important food crop in the world after wheat, rice, corn, potatoes and barley
Analysis of the sgRNA secondary structure, the prediction results showed that the target sequence secondary structure is relatively loose, which is conducive to binding the target position (Figure 2). 1.2 Construction of pCAMBIA1301-Cas9-MeSSIII-gRNA gene editing vector Nuclear localization signals were added on both sides of the Cas9 protein of the pCAMBIA1301-Cas9-gRNA gene editing vector and expressed by 35S promoters. sgRNA expression driven by Arabidopsis AtU6-26 promoter
The results showed that the amplified obtained fragments were about 350 bp, consistent with the expected fragment size, which is indicating that the plasmid pCAMBIA1301-Cas9MeSSIII-gRNA gene editing vector was successfully transformed into LBA4404 Agrobacterium (Figure 5)
Summary
Cassava (Manihot esculenta Crantz) is a tropical and sub-tropical tuberous cash crop with starch as 85% of the dry matter in the tuber and is the sixth most important food crop in the world after wheat, rice, corn, potatoes and barley (da Silva et al, 2017). The structure of cassava starch, like other plant starch, is composed of two dextran polymers, which can be divided into two categories: amylose and amylopectin. The ratio of amylose to amylopectin is an important factor to determine the physicochemical properties of starch. The mechanism of complex formed by starch synthesis-related enzymes regulating tapioca root starch synthesis is unclear. Analysis of the mechanism of cassava starch synthesis by using CRISPR/Cas technology to create mutants related to starch synthesis pathway is an effective means to improve starch quality. Molecular Plant Breeding 2020, Vol., No.17, 1-8 http://genbreedpublisher.com/index.php/mpb CRISPR/Cas technology to construct two encoded gene MeSSIII-1 and MeSSIII-2 dual mutant vectors of cassava soluble starch synthase SSIII, and verified the editing effect of the vectors. It is helpful to further explore the function of SSIII regulating cassava starch synthesis
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