Construction And Identification Of Cytokinin Oxidases/Dehydrogenase(OsCKXs) Gene Family Mutant Library In Rice

Rice(Oryza satica L.)is not only an important food crop but also an typical model organism in monocotyledonous plants.With the in-depth study of rice functional genomics and the maturity of molecular operation technology,it is increasingly possible to effectively reveal the biological functions of rice endogenous genes and then people attempt to achieve molecular breeding.Cytokinin(CTK)is an essential hormone ingredient for plant growth and development.Cytokinin oxidases/dehydrogenase(CKX)regulates the concentration of cytokinin in plants by degrading cytokinin,and it is a key enzyme in the degradation pathway of CTK hormone pathway.Eleven CKX genes were annotated in the whole genome of rice.Researches have revealed the regulatory effect of some genes among OsCKXs for rice growth.But due to the lack of clear mutant materials,the knowledge of biological function of OsCKX gene family is limited.In this research,we knockout OsCKXs gene family members of Nipponbare,creating OsCKX1~OsCKX11 single gene mutant materials and multi-gene polymerization mutant materials using CRISPR-Cas9 and CRISPR-Cas12 a gene-editing technique.This will lay the foundation for systematically studing OsCKXs gene biological function and effective molecular breeding.The main research contents and results are as follows:1.Basing on the design standard of single guide RNA(sgRNA)of CRISPR-Cas9 gene-editing technique,the 5'-NGG-3' trinucleotide PAM sites scanned OsCKX1~OsCKX11 gene coding regions,and 20 bp upstream of PAM were selected.We constructed directed knockout vectors targeting OsCKX1~OsCKX11,and then introduced into the rice receptor materials based on Agrobacterium-mediated genetic transformation.Finally,the targeted knockout mutant materials were screened and identified from the resistant regeneration materials.The results revealed that the CRISPR-Cas9 system showed clear editing activities for different OsCKXs gene family members except OsCKX1-sgRNA1 site.The editing efficiencies of different sites were from 27.3%(OsCKX1-sgRNA2)to 100%(OsCKX3-sgRNA1).Considering the potential inactive sgRNA site,the experiment designed double sgRNA strategy to effectively ensure the creation of OsCKXs knockout mutant materials;2.Basing on the design standard of crRNA of CRISPR-Cas12 a gene-editing technique,the 5'-TTTV-3' tetranucleotide PAM sites scanned OsCKX1~OsCKX11 gene coding regions,and 23 bp DNA sequence downstream of PAM were selected.We constructed directed knockout vectors targeting OsCKX1~OsCKX11,and genetic transformation,regeneration and mutant screening were performed.The results showed that the stability of the CRISPR-Cas12 a system was somewhat better than that of the CRISPR-Cas9.All the constructed CRISPR-Cas12 a editing vectors showed clear editing activities with an editing efficiencies from 23.1%(OsCKX5-crRNA)to 100%(OsCKX8-crRNA);3.Targeted knockout T0 mutant individuals of different members of the OsCKX gene family were screened and identified.Their self-crossing T1 materials were harvested and identified.The statistical results showed that the genotype of T1 segregation materials accorded with Mendelian inheritance law,and obtained OsCKXs mutation could be stably inherited.Further analysis of the phenotype of T1 knockout mutants revealed that OsCKX1,OsCKX6,and OsCKX7 knockout mutant materials showed larger grain size and showed a yield increasing potential.4.The rice OsCKX gene family members have high similarity.In order to effectively avoid gene redundancy interference and further clarify the biological functions of OsCKX gene family,gene editing vectors were constructed to create OsCKXs polymerized mutant materials.The vectors were based on CRISPR-Cas12 a gene editing system targeting the closer related genes in OsCKX gene family.