Editing of Rice Isoamylase Gene ISA1 Provides Insights into Its Function in Starch Formation

作     者：CHAO Shufen CAI Yicong FENG Baobing JIAO Guiai SHENG Zhonghua LUO Ju TANG Shaoqing WANG Jianlong HU Peisong WEI Xiangjin 

作者机构：State Key Laboratory of Rice Biology China National Rice Research Institute Southern Regional Collaborative Innovation Center for Grain and Oil Crops College of Agronomy Hunan Agricultural University 

出 版 物：《Rice science》 (水稻科学（英文版）)

年 卷 期：2019年第26卷第2期

页      面：77-87页

核心收录：

学科分类：09[农学] 

基　　金：supported by the National Nature Science Foundation of China (Grant Nos. 31471472 and 31521064) the National S&T Major Project of China (Grant No. 2016ZX08001006) the Central Level, Non-Profit, Scientific Research Institutes Basic R and D Operations Special Fund (Grant Nos.Y2017PT46 and 2017RG002-1) 

主　　题：ISA1 rice starch biosynthesis starch granule physicochemical property CRISPR Cas9 

摘      要：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.