Creation And Analysis Of Rice OsCKX4/5/9 Multiple Gene Editing Mutant

Cytokinin(CTK)is a kind of plant hormone that contains adenine derivatives and promotes cell division.It is mainly distributed in shoot tips,root tips,and germinated seeds.Its main physiological function is to promote cell division and regulate its differentiation,as well as delay the degradation of protein and chlorophyll,and delay aging.The synthesis of cytokinins in plants is mainly carried out in the root tips,and isopentenyl pyrophosphate and adenosine monophosphate produce isopentenyl adenosine phosphate under the action of isopentenyl transferase(IPT),And further produce different types of cytokinins.The degradation of cytokinin is mainly catalyzed by cytokinin oxidases/dehydrogenase(CKX).CKX is the only enzyme that can irreversibly inactivate cytokinins.It affects the growth and development of plants by regulating the dynamic balance of synthesis and degradation of different cytokinins in plants.Eleven members of the rice OsCKX gene family have been discovered,and some members such as OsCKX2,OsCKX4,OsCKX11,etc.have undergone related functional studies.However,due to the lack of systematic and comprehensive single-gene and multi-gene mutant materials,key issues such as the function of specific gene members in the OsCKX gene family,the way of interaction between gene members,and whether there are functional redundancy or complementarity among gene members are still unclear.clear.Based on the single-gene mutants of OsCKX4,OsCKX5 and OsCKX9 obtained in the early stage of this experiment,this study further created the double-gene and triple-gene aggregate mutant materials of OsCKX4/5/9,and compared and analyzed single-gene,double-gene and triple-gene mutations.The similarities and differences of the body,the preliminary clarification of the functions of the OsCKX4,OsCKX5 and OsCKX9 genes and the way of interaction between the three genes laid the foundation for the functional research of the OsCKX gene family.The main research contents and results of this paper are as follows:(1)Based on the homology analysis of the members of the OsCKX gene family,based on the OsCKX4,OsCKX5 and OsCKX9 single-gene mutants obtained in the early stage of this experiment,the CRISPR-Cas12 a technology was used to construct a targeted multi-gene knockout editing of OsCKX4/5/9 Carrier.Agrobacterium tumefaciens-mediated genetic transformation was introduced into rice,and OsCKX4/5/9double-gene and triple-gene mutant materials were screened and identified in resistant regenerated plants.The experimental results showed that 27 T0 generation plants were obtained,2 OsCKX4/9 double-gene mutants,2 OsCKX5/9 double-mutant materials,and4 OsCKX4/5/9 triple-gene mutants.(2)The OsCKX4,OsCKX5 and OsCKX9 single-gene and multi-gene mutant materials obtained in the early stage were planted and progeny screened and identified,and finally obtained stable genetic material without a carrier: 1 strain of OsCKX4 mutant,3 strains of OsCKX5 mutant There are 5 strains of OsCKX9 mutant,4 strains of OsCKX4/9 double gene mutation,and 3 strains of OsCKX4/5/9 triple gene mutation material.(3)The obtained single-gene,double-gene,and triple-gene mutant materials were compared and analyzed for growth and development and field agronomic traits.The results showed that osckx4,osckx5 and osckx9 single-gene mutants were similar to wild-type strains in seed grain types.There was no significant difference in the ratio,and the seed grain length of osckx4/9 double protrusion material and osckx4/5/9 triple protrusion material was significantly shorter than WT,and the thousand-grain weight was also significantly lower than WT.The seed germination of osckx4,osckx4/9 and osckx4/5/9 was significantly slower than that of WT,and the root length of 7 days of germination was also significantly shorter than that of WT.There was no significant difference between osckx5 and osckx9 and WT,osckx4/9 and osckx4/5/9 The plant height at the tillering stage was significantly lower than that of WT,and the leaves and stems were also smaller than WT,while the number of tillers increased significantly,which was 2 to 3 times that of WT.In addition to the plant height slightly lower than that of WT,the single-projecting material was not compared with WT in other respects.Significant differences.Compared with WT,the panicle type of osckx4/9 and osckx4/5/9 at the maturity stage was significantly shortened and smaller,and the number of grains and branches per stalk were significantly reduced.There was no significant change in the panicle type of the single-gene mutants.OsCKX4,OsCKX5 and OsCKX9 may be involved in seed germination and early growth of young roots,and also have an effect on plant height,ear type and seed grain type,and the functions of the three are redundant.(4)The expression level of the cytokinin signal response gene OsRR was detected in the seedling roots and leaves of OsCKX4,OsCKX5 and OsCKX9 single,double,and triplex materials.The results showed that the OsRR5 and 6 genes in the seedlings were in the singlets.The expressions in the double-sudden and triple-sudden materials all increased significantly,and the expression of OsRR1-6 genes in the double-suddens material in the young leaves increased significantly,while the changes in the triple-sudden materials were not obvious,which may be due to the three-sudden materials.Excessive accumulation of CK content in plants caused an emergency response in plants,and other signal pathways were used to reduce endogenous CK levels.Mutations in OsCKX4,OsCKX5 and OsCKX9 genes may affect the endogenous CK content of plants,thereby causing the up-regulation of cytokinin response gene expression.