Genetic Improvement Of Several Important Agronomic Traits In Rice Using Genome Editing Techniques

Rice(Oryza sativa L.)is staple food for nearly half of the world’s poppulation.With the continuous increase of the population in China,and the continuous improvement of living standards,there is a higher demand for the diversification of rice quality,which has brought huge challenges to the majority of breeding workers.The period of traditional breeding is qiute long and the process is cumbersome.Gene editing technology offers the possibility of rapid improvement of important agronomic traits,especially quality traits and can be used for rapid breeding improvement.In this study,we took the rice grain length gene(GS3),fragrance gene(BADH2),thermo-sensitive male sterility gene(TGMS5)and OsGABA-1 gene that affect the in vivo seeds gamma-aminobutyric as target genes,four elite rice varieties were used as transformed varieties,and CRISPR/Cas9 gene editing technology was used to knock out the four target genes to acquire artificially modified rice materials to shorten the breeding process and widely accelerate conventional rice breeding.The main results of this experiment were as follows:(1)GS3 and BADH2 gene editingGS3 is a major gene that controls the grain length of rice and has a negative regulation effect on grain length.BADH2 regulates rice fragrance.Through construction of bi-gene CRISPR/Cas9 knockdown vector,we obtained the positive transgenic plants under background of R573 and Guang 8B,Liao 39 B and CNJ1,respectively,verified by PCR amplication and sequencing identification.Further,phenotypic analysis of GS3 and BADH2 co-knockout positive plants in the T2 generation revealed that the the grain length of mutant lines became shorter and was fragrant compared with wild plant under Liao 39 B and CNJ1 background,fragrant mutants were also acquired under R573 and Guang 8B background,no significant differences of other agronomic traits were detected between mutant lins and wild type.(2)Creation of functional rice with high γ-aminobutyric acidOsGABA-1 affects the expression of γ-aminobutyric acid aminotransferase in rice,which negatively regulates the content of r-aminobutyric acid(GABA)in rice.We knocked out the OsGABA-1 gene in two indica rice varieties,CNJ1 and CNXJ.Phenotype analysis were carried out at T1 generation,we found that grain length of mutant lines become longer and chalkiness is significantly increased,the detection of various amino acids and GABA in mature seeds of wild plant and mutant lines revealed that the content of GABA was significantly increased compared with the wild type,which can be directly used for utilization of hypertension functional rice.(3)Materialinnovation of thermo-sensitive male sterility lineFertility of two-line CMS was controlled by nuclear gene,which is not restricted by restoration relationship and has the advantages of free apolegamy,simplified seed production procedure.In this study,TGMS5 was knockout under the CNXJ and LNYJ background and obtained homozygous positive mutants.T2 homozygous mutant lines were planted in Hainan in different stages to encounter high and low temperature respectively.Our results found that the knockout mutant lines were partial male fertile under low temperature conditions,while were sterile under high temperature environment.This shows that the temperature-sensitive CMS can be artificially created through mutation of TGMS5 function.It can fully expand the germplasm resources of rice two-line in fertility and promote two-line breeding research.