Molecular Mechanism Of PpBBX24-like And PpWRKY22L Transcription Factors Regulating Anthocyanin Biosynthesis In Red Pear

The coloration of pear skin is mainly caused by anthocyanin accumulation,which is important determinant of fruit quality.Genetic material determines whether anthocyanins can be synthesized.The structural genes of anthocyanin synthesis has been well studied.The regulating roles of transcription factors,such as MYBs,on anthocyanin biosynthesis has been deeply studied.However,in pear,there is still few research on location and molecular mechanism of key genes for red peel coloring.‘Hongzaosu’,which can accumulate anthocyanins in its peel,is a bud sport of a green pear‘Zaosu’.In this study,we used a F1 hybrid population of‘Yuluxiang’בHongzaosu’as experimental materials,and mapped key genes for the peel coloring of‘Hongzaosu’,the divergences of both sequence and function among alleles of a candidate gene PpBBX24-like were investigated.Moreover,based on the previous study of our lab,PpWRKY22L may participate in the coloring process of red pear.Using‘Hongzaosu’pear as material,this study conducted a detailed exploration of the molecular mechanism underlying how PpWRKY22L regulates pear anthocyanin synthesis.The main results of this study are as follows.1.PpBBX24-like is associated with the red peel coloring of‘Hongzaosu’A total of 180 individuals in the F1 hybrid population of‘Yuluxiang’בHongzaosu’were used as materials.Using GBS technology,the average raw data sequenced for each individual was 788.62 Mb,and the total sequencing amount was 146.68 Gb.After comparing the sequencing data with the reference genome of‘Cuiguan’pear,population SNP detection and SNP molecular marker development are carried out.We screened out 13972 SNP markers,which were used to construct a high-density genetic linkage map,including 17 linkage groups(LG),with a total genetic distance of 3158.01 cM,an average distance of 0.43 cM and a maximum gap of 12.74 cM.Among them,there are 5009 SNP markers on the male map(‘Hongzaosu’),with a total genetic distance of 3600.91 cM,an average distance of 0.72 cM and a maximum gap of 39.31 cM.Using the genetic map,we finally mapped the key genes determining coloration to the segment between 4514414 bp and 4695749 bp of Chr 4,which is about 180 Kb and contains a total of 21 genes.The 21 genes were annotated and analyzed,and 5genes were screened out.The qPCR analysis showed that only PpBBX24-like can be induced by light,meanwhile showing the same trend as PpCHS,PpANS,PpF3H,PpMYB10.In‘Hongzaosu’and‘Zaosu’,we cloned three alleles of PpBBX24-like designated as PpBBX24-1,PpBBX24-2 and PpBBX24-1~m,respectively.Compared with other genotypes of PpBBX24-like,we found that PpBBX24-1~monly exists in‘Hongzaosu’and contains a 14 bp deletion,causing frameshift mutations and prematurely stopping of translation.Above all,we considered PpBBX24-like as a candidate gene causing the coloration of‘Hongzaosu’.Using F1 hybrid population of‘Hongzaosu’בKorla Xiangli’as materials,we confirmed that PpBBX24-1~monly exists in the individuals of red phenotype by sequence alignment analysis,PAGE and HRMA.Thus PpBBX24-like can be used as a molecular marker.Through analysing the conserved domains,we found PpBBX24-like contains two B-box domains at the N-terminus.In addition,PpBBX24-1 and PpBBX24-2 were located in the nucleus,while PpBBX24-1~mlocated in both nucleus and cytoplasm.Dual luciferase assay show that PpBBX24-like can regulate the expression of anthocyanin structural genes.2.PpWRKY22L promotes anthocyanin biosynthesisBased on previous transcriptome data of our lab,PpWRKY22L was identified.Through analysing conserved domains,PpWRKY22L belongs to the group IIe of WRKY family.PpWRKY22L can respond to light,and the expression pattern is similar to PpMYB10 and PpCHS.Transgenic callus from Pyrus communis‘Clapp’s Favorite’and transient expression assay showed that PpWRKY22L can promote the accumulation of anthocyanins.PpWRKY22L-VIGS fruits were unable to accumulate anthocyanins near the injection hole.Furthermore,PpWRKY22L can upregulate the expression of PpCHS,PpDFR and PpUFGT.Yeast two-hybrid assay showed that PpWRKY22L could not interact with PpHY5,PpbHLH3and PpMYB10,while we found that PpWRKY22L can interact with PpbHLH64 and PpMYB114 by BIFC,which provided evidence for our subsequent exploration of MBWW complex.In summary,using the F1 hybrid population of‘Yuluxiang’בHongzaosu’,we mapped a segment closely linked to the coloration of red pear peel,with a size of about 180 Kb.Within the segment,PpBBX24-like was identified.It was found that there were sequence variations of PpBBX24-like between‘Zaosu’and‘Hongzaosu’,which caused changes of subcellular location and function.In addition,we also identified a light-induced transcription factor PpWRKY22L.PpWRKY22L can promote the biosynthesis of anthocyanin in red pear and upregulate the expression of structure genes of anthocyanin biosynthesis.On this basis,we found that PpWRKY22L can interact with PpbHLH64 and PpMYB114,laying the foundation for the follow-up study of the regulation mode of PpWRKY22L-mediated anthocyanin biosynthesis.We preliminarily explored the molecular mechanism of PpBBX24-like and PpWRKY22L in regulating anthocyanin accumulation,and lay a foundation for clarifying the coloring mechanism of‘Hongzaosu’and molecular marker-assisted breeding.