Targeted Mutagenesis Of Amino Acid Transporter Genes For Rice Quality Improvement Using The CRISPR/Cas9 System

Rice is one of the most important food crops in China.With the increasing improvement of people’s living standards,there is an ever-growing demand for rice quality.The grain protein content plays an important role in rice quality.However,traditional breeding to reduce GPC takes a long time,and involves blindness.With the emergence and development of molecular biology,gene editing has been widely applied in the research and cultivation of high-quality rice.In this study,CRISPR/Cas9 system was used to edit the amino acid permeases(AAP)members to study the effect of their mutation on protein content and the taste quality.The results are as follows:1.Phylogenetic tree and expression analysis of 19 family members of the amino acid permeases were conducted.Five genes which exhibit high expression in endosperm and show distant relationship were selected.We edited them in YG158,WYG30,NG9108 and a japonica line 17GTM11 respectively.The mutation efficiency of To generation varied with the genetic background,and homozygous mutants with T-DNA free were successfully obtained in T1 generation.2.The mutations of these five genes plays a similar role in the reduction of protein content,of which OsAAP10,OsAAP11 and OsAAP13 showed a slightly stronger role.Further analysis showed that the loss-function of the selected AAPs led to a certain degree of reduction of all the four protein components.The reduction of gluten content was the main reason for the GPC decrease.3.Loss of function of selected AAPs not only affects the protein content,but also changes the starch content and starch composition.The amylose contents of some mutants were significantly reduced,however the total starch increased.Compared with wild type,the mutant had higher peak viscosity,breakdown viscosity and lower setback viscosity.The effect of OsAAP10,OsAAP11 and OsAAP13 on starch viscosity was greater than that of OsAAP3 and OsAAP6.4.The GPC of each mutants were decreased both in the T1 and T2 generation.According to the decreasing extent on protein,the regulatory effects of the five genes were OsAAP11,OsAAP10,OsAAP13,OsAAP3 and OsAAP6.5.Taste value analysis showed that AAPs could improve the rice taste quality.Most of the mutants had higher taste value than the wild type in which OsAAP11 showed the most significant improvement.The improvement of lower taste YG158 was better than NG9108 with higher taste value.Taken together,targeted mutagenesis of amino acid permeases,especially OsAAP11 through CRISPR/Cas9 system can rapidly reduce the GPC and improve the ECQ,providing a new strategy for the breeding of cultivars with desire ECQ.