Function Analyses Of The PPR And SPL Gene Family And OsSPL4 Gene In Rice

PPR(pentatricopeptide repeats protein)gene family and SPL(SBP proteins-like)gene family are two important gene families,which participate in plants growth and development.It has been reported that PPR proteins are RNA binding proteins,and involved in post-transcription regulation,including RNA splicing,editing,stability and translation.PPR proteins play important roles in leaves development,cytoplasmic male sterility regulation,seeds development and response to different stresses in plants.SPL proteins are plant-specific transcription factors,which binding promoter sequence and regulate gene expression with mi R156/157.In plants,SPL proteins regulate many processes,including embryonic development,flower development,stage transition,plant architecture construction and plant hormone signal transduction.The function of many PPR and SPL genes had been elucidated,but studies of those genes in rice are still limited.In the present study,a whole genome-wide analysis of PPR and SPL genes in rice(Oryza sativa L.)were performed.In addition,some transgenic lines of rice were constructed for further function studies of OsSPL4.The main finding of this study are as follow:1.A total of 491 PPR was found in the rice genome,of which 246 PPR genes belong to the P subfamily and 245 genes belong to the PLS subfamily.Gene sequence analysis showed that 65% of PPR genes lack intron and 16% of PPR genes only contain one intron,while many PPR genes have a syntenic relationship with others.It was indicated that PPR genes family might be derived from amplification by transposition.The expression of PPR genes was tissue-specific,and many PPR proteins were predicted to target chloroplasts or mitochondria,where they participated in post-transcription regulation.2.In the present study,49 miRNAs were predicted to target 54 PPR genes,and three PPR genes were verified as corresponding miRNA targets.Meanwhile,RNA-seq data of WXS(F)and WXS(S)was analyzed.The results showed that miRNAs with their PPR target might be related to anther development during fertility transition.Besides,the expression pattern analysis showed that many PPR genes could be induced under biotic and abiotic stresses.Using q RT-PCR,seven PPR genes were confirmed that their expression was up-regulated under salinity or drought stress,which indicated that these PPR genes might participate in response to different stresses in rice.3.There are 19 SPL genes that distributed in ten rice chromosomes,except 10 th and 12 th chromosomes.Genes sequence analysis showed that some SPL genes located in different chromosomes had a syntenic relationship and contained similar gene structure and protein structure.The result revealed that chromosomal segment duplications were involved in the expansion of this gene family.In rice,the amino acid sequence of SBP-box was highly conserved.Only OsSPL9 proteins contained two zinc fingers that consisted of CCCC-CCHC,while others had a CCCH-CCHC motif.Phylogenetic relationship analysis revealed that plant SPL proteins may be derived from a common ancestor,and a number of them may have undergone further differentiation in monocotyledon and dicotyledon lineages.Expression pattern of 12 SPL genes was regulated by osa-mi R156,while the target sites of SPL4 and SPL13 transcripts were located in 3’-UTR.SPL genes had different expression in various tissues and could be induced under biotic and abiotic stresses,which indicated that these SPL genes participated in rice development and regulated the response processes of rice under different stresses.4.Some transgenic lines were constructed for further function research of OsSPL4 in this study.We finally obtained 23 spl4 transgenic lines using the CRISPR/Cas9 system.The DNA sequence of SPL4 was changed and the normal function was lost in spl4 lines.Compared to wild type,spl4 lines showed a different phenotype in the development stage.The mutants had an increased height of plants and length of leaves.Meanwhile,the number of panicle primary branches were up-regulated,which could improve grain yield.In OE-SPL4 lines,the expression of SPL4 was increased,while the phenotype was opposite to spl4 lines.Besides,RNAi and target mimicry vectors were constructed for new transgenic lines,and expected traits also showed up.The results indicated that SPL4 was involved in leaves and panicle development of rice.Analysis of RNA-Seq data also showed that SPL4 was important for rice development via regulating the expression of downstream genes and participating in different pathways.