Editing of Rice Isoamylase Gene ISA1 Provides Insights into Its Function in Starch Formation
Editing of Rice Isoamylase Gene ISA1 Provides Insights into Its Function in Starch Formation
复制标题
水稻异淀粉酶基因 ISA1 的编辑为其在淀粉形成中的功能提供了见解
DOI：
10.1016/j.rsci.2018.07.001
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发表时间：
2019-03-01
期刊：
影响因子：
4.8
通讯作者：
Wei Xiangjin
中科院分区：
文献类型：
--
作者：
Chao Shufen;Cai Yicong;Wei Xiangjin
异淀粉酶1（Isoamylase 1，ISA 1）是一种在支链淀粉合成中起主要作用的异淀粉酶型脱支酶。本研究利用成簇规则间隔短回文重复序列（CRISPR）/CRISPR相关核酸内切酶9（CRISPR/Cas9）系统，通过农杆菌介导转化水稻，对ISA 1基因进行编辑。我们从55个T-0转基因事件中鉴定了36个基因编辑株系，并将突变形式分为7种类型。其中，两个纯合突变体，cr-isa 1 -1（1型，带有腺嘌呤插入）和cr-isa 1 -2（3型，带有胞嘧啶缺失）被选择用于进一步分析。cr-isa 1 -1和cr-isa 1 -2的种子大小均受到影响，且两个突变体的胚乳皱缩，粒重显著降低。电镜分析表明，cr-isa 1 -1和cr-isa 1 -2胚乳细胞中存在异常淀粉粒和淀粉体。与野生型相比，cr-isa 1突变体胚乳中总淀粉、直链淀粉和支链淀粉含量显著降低，而糖含量和淀粉胶稠度显著增加。cr-isa 1突变体的糊化温度和淀粉链长度分布也发生了变化。此外，大多数淀粉合成相关基因的转录水平显着降低cr-isa 1突变体。研究结果表明，ISA 1基因的编辑影响淀粉合成和胚乳发育，对水稻品质育种具有潜在的指导意义。
Isoamylase 1 (ISA1) is an isoamylase-type debranching enzyme which plays a predominant role in amylopectin synthesis. In this study, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated endonuclease 9 (CRISPR/Cas9) system was used to edit ISA1 gene in rice via Agrobacterium-mediated transformation. We identified 36 genetic edited lines from 55 T-0 transgenic events, and classified the mutation forms into 7 types. Of those, two homozygous mutants, cr-isa1-1 (type 1, with an adenine insertion) and cr-isa1-2 (type 3, with a cytosine deletion) were selected for further analysis. Seed sizes of both cr-isa1-1 and cr-isa1-2 were affected, and the two mutants also displayed a shrunken endosperm with significantly lower grain weight. Electron microscopy analysis showed that abnormal starch granules and amyloplasts were found in cr-isa1-1 and cr-isa1-2 endosperm cells. The contents of total starch, amylose and amylopectin in the endosperm of the cr-isa1 mutants were significantly reduced, whereas sugar content and starch gel consistency were observably increased compared to the wild-type. The gelatinization temperature and starch chain length distributions of the cr-isa1 mutants were also altered. Moreover, transcript levels of most starch synthesis-related genes were significantly lower in cr-isa1 mutants. In conclusion, the results indicated that gene edition of ISA1 affected starch synthesis and endosperm development, and brought potential implications for rice quality breeding.