| Rice is one of the most important food crops in the world.More than 1/2 of the world’s population depends on rice.It has become an important content of rice breeding to improve the yield and quality of rice by molecular biology technology.Rice panicle especially grain number per panicle,as an important complex agronomic trait,has always been the main breeding target in rice yield improving.The QTL of Rice Panicle Grain number character,GNP1(Grain Number per Panicle 1)shows increasing effect on total spikes and grains number in rice high yield breeding.Genetic analysis shows that the OsGNP1 gene encodes a gibberellin synthesis related oxidase,it may be involved in gibberellin and cytokinin regulation which mediated by the KNOX family transcription factors.The dynamic balance can regulate the development of rice panicle primordia thus contribute to the grain number phenotype of rice.In order to further clarify OsGNP1 gene function,as well as its influence on panicle growth and development of rice under different expression level,this study used the CRISPR-Cas9 system,constructed three vectors targeted OsGNP1 gene named pZJP064,pZJP065 and pZJP066 repestively.Through the genetic transformation method mediated by agrobacterium,we transformed vectors into Nipponbare,and detected OsGNP1 mutation in order to obtained T0 transgenic rice plants.In addition,we selected homozygous mutation of OsGNP1 to conduct gene function and agronomic traits related analysis.The main research results are as follows:1.To analysis rice OsGNP1 gene,we constructed three genetic editing vectors pZJP064,pZJP065 and pZJP066.Among those,pZJP064 and pZJP065 knockout OsGNP1 gene encoding area with two different targeting sites sgRNA1 and sgRNA2,in order to obtain the gene encoding area knockout mutant.Vector pZJP066 targeted sgRNA3 and sgRNA4 sites simultaneously in OsGNP1 gene promoter area which controls gene expression level in order to generate large fragment deletion mutation.2.By agrobacterium mediated genetic transformation methods we obtained pZJP064 mutant in amount of 37 strains,pZJP065 mutant in amount of 45 strains and pZJP066 mutant in amount of 11 strains respectively which all them were transgenic positive rice plants.Then we compared transgenic plants and wild type controls in the process of growth and development,and it showed no significant difference.3.We detected the mutation efficiency of pZJP064 and pZJP065 transgenic positive plants respectively which targeting OsGNP1 gene coding region.The sgRNA1 site mutation rate was 37.5%,of which the efficiency of the homozygous mutation was 6.25%.The efficiency of sgRNA2 site targeting mutation was 90%,and the homozygous mutation efficiency was 78%.Sequencing results showed that 90% of the two loci mutation genotypes were insertion mutation,and sgRNA2 single-base homozygous A/T single base insertion rate reached 96.3%,which suggests there may be a propensity in gene repairing at particular gene locus.In addition,we obtained 17 strains and 20 strains of T1 rice plants respectively with OsGNP1 gene mutation,showed obvious phenotype which is shortened spike time and these materials could be used for further functional analysis.4.The detection mutant of pZJP066 transgenic rice plants showed that sgRNA3 and sgRNA4 which targeted the promoter region of OsGNP1 were simultaneously mutated,and the efficiency of large fragments deletion was 18.2%.In OsGNP1 gene promoter region,the size of large fragment detection mutant was smaller than wild type,and the plant growth was slightly slow,and it withered before reach the heading stage.Therefore,it is necessary to analysis the changed OsGNP1 gene expression level influence on endogenous GA content in rice,as well as the effects on grain development,grain shape and grain size. |
