Editing of the starch branching enzyme gene SBE2 generates high-amylose storage roots in cassava
Editing of the starch branching enzyme gene SBE2 generates high-amylose storage roots in cassava
复制标题
编辑淀粉分支酶基因 SBE2 可在木薯中产生高直链淀粉贮藏根
DOI：
10.1007/s11103-021-01215-y
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发表时间：
2021-11-18
影响因子：
5.1
通讯作者：
Zhang, Peng
中科院分区：
文献类型：
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作者：
Luo, Shu;Ma, Qiuxiang;Zhang, Peng
关键信息 首次实现了通过 CRISPR/Cas9 介导的淀粉分支酶基因 SBE2 诱变生产高直链淀粉木薯。高直链淀粉木薯（Manihot esculenta Crantz）非常适合淀粉工业应用和生产供人类消费的更健康的加工食品。在这项研究中，我们报告了通过 CRISPR/Cas9 介导的淀粉分支酶 2 (SBE2) 诱变生产高直链淀粉木薯。在所有再生植物中均发现了 SBE2 两个目标外显子的突变；这些突变包括 SBE2 基因中的核苷酸插入和短或长缺失，被分为八个突变系。在SBE2的第二个外显子中具有长片段缺失的三个突变体M6、M7和M8显示没有SBE2蛋白的积累。从田间收获后，与野生型相比，在这些突变体中观察到显着更高的直链淀粉（表观直链淀粉含量高达 56%）和抗性淀粉（高达 35%），导致快速碘染色后淀粉颗粒呈深蓝色，并随着糊化温度和峰值时间的增加而改变淀粉粘度。进一步的H-1-NMR分析显示淀粉支化度显着降低，支链淀粉的短链（聚合度[DP] 15-25）减少，长链（DP>25，尤其是DP>40）增加，这表明木薯SBE2在支链淀粉生物合成过程中催化短链形成。在淀粉中也检测到从 A 型结晶度到 B 型结晶度的转变。我们的研究表明，CRISPR/Cas9 介导的木薯淀粉生物合成基因诱变是产生具有有价值的淀粉特性的新品种的有效方法，可用于食品和工业应用。
Key message The production of high-amylose cassava through CRISPR/Cas9-mediated mutagenesis of the starch branching enzyme gene SBE2 was firstly achieved. High-amylose cassava (Manihot esculenta Crantz) is desirable for starch industrial applications and production of healthier processed food for human consumption. In this study, we report the production of high-amylose cassava through CRISPR/Cas9-mediated mutagenesis of the starch branching enzyme 2 (SBE2). Mutations in two targeted exons of SBE2 were identified in all regenerated plants; these mutations, which included nucleotide insertions, and short or long deletions in the SBE2 gene, were classified into eight mutant lines. Three mutants, M6, M7 and M8, with long fragment deletions in the second exon of SBE2 showed no accumulation of SBE2 protein. After harvest from the field, significantly higher amylose (up to 56% in apparent amylose content) and resistant starch (up to 35%) was observed in these mutants compared with the wild type, leading to darker blue coloration of starch granules after quick iodine staining and altered starch viscosity with a higher pasting temperature and peak time. Further H-1-NMR analysis revealed a significant reduction in the degree of starch branching, together with fewer short chains (degree of polymerization [DP] 15-25) and more long chains (DP>25 and especially DP>40) of amylopectin, which indicates that cassava SBE2 catalyzes short chain formation during amylopectin biosynthesis. Transition from A- to B-type crystallinity was also detected in the starches. Our study showed that CRISPR/Cas9-mediated mutagenesis of starch biosynthetic genes in cassava is an effective approach for generating novel varieties with valuable starch properties for food and industrial applications.