Expression And Preliminary Functional Analysis Of Fructokinase Family Genes In Rice

Fructokinase(FRK),which can phosphorylate fructose,is an essential enzyme in the fructose catabolic pathway.It is widely found in bacteria,higher plants,and animals.Studies have shown that plant FRK family genes have important biological functions in maintaining normal growth and development of plants as well as stress adaptation.However,the expression and function of OsFRKs in rice remain obscure.In this study,five OsFRKs(OsFRK1～OsFRK5)were identified by bioinformatics analysis,and its structure,cis-acting elements,phylogenetic trees,and expression had been analyzed.We successfully cloned OsFRKl～OsFRK3 by RT-PCR and analyzed its expression in different tissues of ZH11 by qRT-PCR.Additionally,Ti homozygous knockout mutants of OsFRKl-OsFRK4 were successfully created by CRISPR/Cas9 technology.Besides,the phenotypes of them were analyzed.Overall,the above analysis preliminarily revealed the expression characteristics and the possible biological functions of OsFRKs,as well as laid an important theoretical foundation for the molecular breeding of rice in the future.Our main findings are as follows:1.The main structural characteristics of the OsFRKs.Base on bioinformatics analysis,we found that all of the OsFRKs protein contain ATP binding site and substrate recognition site.In addition,OsFRK1～OsFRK3 can be divided into a subfamily,and OsFRK4～OsFRK5 belong to another subfamily.These OsFRKs were distributed on chromosome 1,3,6 and 8,with the gene size ranging from 2770 bp to 6214 bp,including 2～7 exons,which encodes 323～589 amino acids.Its molecular weight of the protein is 34.72～65.55 kDa,and the isoelectric point is 5.02～6.60.The subcellular predictive analysis revealed that they are all located in plasmids,and OsFRK1 may also be located in cytoplasm and mitochondria.Besides,the promoter sequence of OsFRKs contains multiple components related to development,stress and hormone response.It suggests that they should play an important role in the development and the adaptation to adversity of rice.2.Expression profiling of OsFRKs in different tissues of rice under normal conditions.The results of tissue-specific expression profiles of OsFRKs suggested that they might all be constitutive expressions,in which the expression patterns of OsFRK4 and OsFRK5 were very similar.However,the result of qRT-PCR shows that OsFRK1,OsFRK2,OsFRK4,and OsFRK5 were expressed constitutively,but OsFRK3 was expressed specifically(only in rhizome,stem,and leaves).OsFRKl and OsFRK3 have the highest expression levels in the L3 leaf,while OsFRK2 is more significantly expressed in the seedling roots.3.The T1 generation single-gene knockout homozygous mutants of OsFRK1～OsFRK4 were successfully obtained.In this study,CRISPR/Cas9 technology was used to successfully create To knockout mutants of OsFRK1～OsFRK4,and its mutation rates were 46.4%,92.9%,93.3%,and 66.7%.After breeding and identification,the T1 generation knockout homozygous mutants with different mutation types of 6(OsFRK1),6(OsFRK2),10(OsFRK3)and 6(OsFRK4)were successfully acquired.The amino acid functional domain encoded by these four OsFRKs genes were destroyed or prematurely terminated in translation.However,there were off-targets in some mutants of OsFRK2 and OsFRK3.We will choose eligible mutants in subsequent experiments,or by designing new target sites and recreating the knock-out homozygous mutants without off-target.4.Partial phenotypic analysis of homozygous mutants of OsFRKs in rice.The investigation of T1 knockout homozygous mutants about agronomic characteristics showed that OsFRKl might have no influence on plant height,panicle length,seed setting rate,and 1000-grain weight.But OsFRK2 and OsFRK3 positively regulate the seed setting rate and 1000-grain weight of the main ear of rice,which indicates that OsFRK1 and OsFRK3 may play an important role in the development of panicle characters in rice.Besides,the T1 knockout homozygous mutants of OsFRK4 had a significant albino lethal phenotype,which indicates that OsFRK4 is important for the normal development of chloroplasts.Furthermore,it was found that the root development of T2 knockout homozygous mutant of OsFRK2 was much slower than ZH11,and the growth state of the upper part of the root was also inhibited,indicating that OsFRK2 may play a very important role in the growth and development of rice seedlings,but it needs to be confirmed by further experiments.