Screening And Valida Ation Of Proteins Interacting With FhRWP Of Citrus

Polyembryony is extremely recognizable in citrus,and nucellar embryos occurring in nucellar tissue outside the embryo sac,belongs to the sporophyte apomixis.Currently,polyembryony seriously hinders the breeding process.Previous studies have identified the CitRWP,a key transcription factor involving in the development of nucellar.Transgenic poly embryonic ’Hamlin’ sweet orange showed monoembryonic trait and gained progeny of sexual reproduction when the expression of CitRWP is suppressed by RNAi.Meanwhile,there is a MITE(miniature inverted repeat transposable element)at 133 bp upstream of the CitRWP gene,associating with the expression of the CitRWP.However,the mechanism of nucellar embryo initiation is less known.There were a F1 segregating population constructed in peculiar short juvenile period(～8 months)materials of Hongkong kumquat(Fortunella hindsii),and also presents the MITE(named FhpMITE)insertion in the promoter region of FhRWP.In this study,ovules of polyembryonic Hongkong kumquat in flowering were obtained to construct yeast library,and the candidate transcription factor FhARID was obtained by screening the yeast library and luciferase assay.The structure and function of FhARID was analyzed via bioinformatics and gene-edited Hongkong kumquat individuals were obtained by CRISPR/spCas9 system.This study provides insights into the potential functions of FhARID,which contributes to parsing the relationship between FhARID and the polyembryonic phenotype in citrus,and revealing the regulatory mechanism of FhARID in initial process of nucellar embryony.The main results were as follows:1.Ovules of polyembryonic Hongkong kumquat in flowering were obtained to construct yeast library,and FhpMITE was gained to construct yeast bait strain.Five related transcription factors was obtained via screening yeast library,including FhARID、FhDUF155、FhSPIL1、FhATG8f and FhTFIIB1.Based on gene function annotations and literature reports,FhARID was selected as a candidate transcription factor for further verification;2.The result of YlH assay revealed that the transcription factor FhARID specifically binds a 91bp AT-rich structure of FhpMITE in 133bp upstream to the coding region of FhRWP.Luciferase assay indicated that FhARID significantly increased the expression of FhRWP.Subcellular localization analysis shows FhARID is located in the nucleus.The result of qRT-PCR reveal FhARID was highly expressed in ovule tissue.3.Conservation analysis of amino acids and phylogenetic analysis revealed that transcription factor FhARID had the ARID domain and HMG-box domain.As a member of ARID-HMG subfamily,FhARID has two other homologous genes in Hongkong kumquat.The analysis of amino acid sequence showed that ARID proteins had no obvious structural differences in citrus genus.Promoter analysis revealed that the promoter region of FhARID contained twenty cis-acting elements,of which the most are light-responsive elements.The result of interaction proteins prediction showed that FhARID could participates in the formation of chromatin remodeling complex,which can regulate and regulate chromatin structure and affect gene expression.Six individuals transformants were obtained and required further identification in T1 generation.In conclusion,we indentified a novol transcription factor FhARID via Y1H screen,which can bind the promoter of FhRWP involving in the initiation of nucellar embryos.Furthermore,we cleared the process and mechanism how FhARID activated FhRWP.Finally,we hope to observe the related phenotype through the citrus genetic transformation system.